Note: Descriptions are shown in the official language in which they were submitted.
CA 02743477 2011-05-12
INSTRUMENTS AND METHOD FOR EXPOSING A RECEPTACLE TO
MULTIPLE THERMAL ZONES
FIELD OF THE INVENTION
[00002] The present invention relates to multi-chambered receptacles and
associated
instruments and detection devices for use in performing complex processes.
BACKGROUND OF THE INVENTION
[00004] Highly sophisticated instruments have been developed for performing
complex
assays requiring multiple process steps to be performed simultaneously and
independent of
each other. Such instruments can be used to perform chemical analyses,
immunoassays,
molecular-based tests, and the like. The most advanced of these instruments
are capable of
performing sample-to-result, nucleic acid-based amplification tests ("NAAT")
that allow for
walk-away testing. See Friedenberg et al., "Developing a Fully Automated
Instrument for
Molecular Diagnostic Assays," IVD Technology (2005) 11(6):47-53; Hill,
"Automating
Nucleic Acid Amplification Tests," IVD Technology (2000) 6(7):36-45. Fully
automated
NAAT testing reduces the chances for contamination or user error and is
increasingly
important because of a national shortage of medical technologists trained to
conduct more
complex assays, such as NAAT tests. With full automation, the instrument
performs all the
necessary steps of an assay with minimal human intervention. For NAAT assays,
these steps
include processing of raw samples to extract one or more nucleic acids of
interest and to
separate the nucleic acids from potentially interfering materials; performing
an amplification
reaction, such as polymerase-based extension reaction, to increase the
sensitivity of the assay
(e.g., TMA, SDA or PCR); and detection of the nucleic acids of interest. In
general, however,
1
CA 02743477 2011-11-09
instruments used to perform NAAT assays are not easily portable and their
usefulness is
typically limited to large-scale testing in controlled environments.
Therefore, a need
currently exists for a compact system capable of performing sample-to-result,
NAAT assays
in point-of-use testing, such as in field testing or bedside medical
applications.
SUMMARY OF THE INVENTION
[00005] The present invention provides compact instruments, detectors and
associated
receptacles and processes for performing complex procedures, such as sample-to-
result
NAAT assays, that permit point-of-use testing at substantial cost savings to
conventional,
large-scale instrument systems. The receptacles include interconnected
chambers that can be
prepackaged in unit dose form with all of the reagents needed to perform an
assay. The
receptacles are closed systems that minimize opportunities for contamination.
[00005A] Various embodiments of this invention provide an instrument for
processing a
sample in a receptacle having a plurality of interconnected chambers, the
instrument being
constructed and arranged to support the receptacle in an operative position
during the
processing and comprising: one or more multi-chamber thermal elements each
defining a
multiple chamber thermal zone disposed to be in thermal communication with the
receptacle
and constructed and arranged to transmit thermal energy between each multiple
chamber
thermal zone and an associated multi-chamber region of the receptacle, wherein
the
associated multi-chamber region of the receptacle encompasses all or a portion
of each of two
or more but less than all chambers of the receptacle; and a controller
programmed to control
operation of the one or more multi-chamber thermal elements to selectively
heat or cool the
chambers encompassed within the multi-chamber regions associated with the
multiple
chamber thermal zones.
[00005B] Various embodiments of this invention provide a method for heating or
cooling substances within a receptacle having a plurality of interconnected
chambers, the
method comprising the steps of. positioning the receptacle in thermal
communication with
one or more multiple chamber thermal zones contained in an analyzer, each
multiple chamber
thermal zone being associated with a region of the receptacle encompassing all
or a portion of
each of two or more but less than all chambers of the receptacle; and
transmitting thermal
energy between each multiple chamber thermal zone and the chambers encompassed
by the
region associated with the multiple chamber thermal zone to selectively heat
or cool
2
CA 02743477 2011-05-12
substances contained within the encompassed chambers to a temperature
different than the
temperature of the chambers encompassed by at least one other region.
[00006] In a first embodiment, a multi-chambered receptacle is provided that
permits
multiple process steps or processes to be performed independently and/or
simultaneously. In
one embodiment, the receptacle comprises: (i) a first linear path of chambers
interconnected
by a plurality of openable connections that includes: first and second
chambers connected by
a first openable connection, where the first and second chambers and the first
openable
connection are configured to permit substance movement from the first chamber
to the
second chamber when a substance-moving force is applied to the contents of the
first
chamber and the first openable connection has been altered from a closed state
to an open
state; third and fourth chambers connected by a second openable connection,
where the third
and fourth chambers and the second openable connection are configured to
permit substance
movement from the third chamber to the fourth chamber when a substance-moving
force is
applied to the contents of the third chamber and the second openable
connection has been
altered from a closed state to an open state; and an intermediate chamber
between the second
and fourth chambers; and (ii) a sample inlet port for receiving sample into a
sample receiving
chamber, provided that if the sample receiving chamber is a chamber of the
first linear path,
then the sample receiving chamber is between the second and fourth chambers.
The sample
inlet port may be closed with, for example, a Luer connection. The
intermediate chamber is
directly or indirectly connected to the second chamber by a third openable
connection, and
the second and intermediate chambers and the third openable connection are
2a
CA 02743477 2011-05-12
configured to permit substance movement from the second chamber to or toward
the intermediate
chamber when a substance-moving force is applied to the contents of the second
chamber and the
third openable connection has been altered from a closed state to an open
state. The intermediate
chamber is also directly or indirectly connected to the fourth chamber by a
fourth openable
connection, and the fourth and intermediate chambers and the fourth openable
connection are
configured to permit substance movement from the fourth chamber to or toward
the intermediate
chamber when a substance-moving force is applied to the contents of the fourth
chamber and the
fourth openable connection has been altered from a closed state to an open
state. The first linearpath
of chambers is configured so that if a substance-moving force is applied to
the contents of the first
chamber, then the third openable connection is not altered from a closed state
to an open state. The
first linear path of chambers is also configured so that if a substance-moving
force is applied to the
contents of the third chamber, then the fourth openable connection is not
altered from a closed state
to an open state. The second and fourth chambers are not interconnected by any
arrangement of
chambers that does not include the intermediate chamber. In a preferred
aspect, each of the plurality
of interconnected chambers is adjacent to at least one other chamber of the
receptacle (seals alone
separate chambers). Additionally, the chambers of the receptacle may have a
radial arrangement,
where the end chambers (i.e., outermost chambers of a linear path of chambers)
have a non-circular
arrangement.
[00007] In one aspect, the receptacle comprises second and third linear paths
of chambers,
where the chambers of each of the second and third linear paths are
interconnected by a plurality of
openable connections and comprise a sixth chamber connected to a first process
chamber by a fifth
openable connection, where the first process chamber is any chamber of the
first linear path that is
located between the first and third chambers, and where the sixth and first
process chambers and the
fifth openable connection are configured to permit substance movement from the
sixth chamber to
the first process chamber when a substance-moving force is applied to the
contents of the sixth
chamber and the fifth openable connection has been altered from a closed state
to an open state. The
second linear path of this aspect comprises the first chamber but not the
third chamber of the first
linear path, and the third linear path comprises the third chamber but not the
first chamber of the first
linear path. The intermediate chamber may be the first process chamber or the
sample receiving
chamber. Alternatively, the sixth chamber may be the sample receiving chamber.
3
CA 02743477 2011-05-12
[00008] In another aspect, the receptacle comprises a seventh chamber
connected to the sixth
chamber by a sixth openable connection, where the sixth and seventh chambers
and the sixth
openable connection are configured to permit substance movement frorn the
seventh chamber to the
sixth chamber when a substance-moving force is applied to the contents of the
seventh chamber and
the sixth openable connection has been altered from a closed state to an open
state. For this aspect,
the sixth chamber may be the sample receiving chamber.
[00009] One or more chambers of the second and third linear paths may comprise
a solid
support for immobilizing an analyte in the sample. The solid support may be
any material, in a
natural or modified form, which is capable of immobilizing an analyte of
interest. A preferred solid
support is a magnetically-responsive particle or bead that can be manipulated
by an applied magnetic
field. The solid support may be provided to, for example, any of the sixth,
seventh and first process
chambers. To concentrate the solid support within a chamber (i.e., increase
the density of solid
support material within a region of a chamber without increasing the total
amount of solid support
material provided to the chamber), the solid support can be provided to the
chamber with an
immiscible liquid which is non-reactive with the other components of the
chamber (i.e., inert). The
immiscible liquid may be an oil, preferably a mineral oil.
[00010] In yet another aspect, the receptacle comprises a fourth linear path
of chambers
interconnected by a plurality of openable connections and includes: an eighth
chamber connected to a
second process chamber of at least one of the second and third linear paths by
a seventh openable
connection, where the eighth and second process chambers and the seventh
openable connection are
configured to permit substance movement from the eighth chamber to the second
process chamber
when a substance-moving force is applied to the contents of the eighth chamber
and the seventh
openable connection has been altered from a closed state to an open state; and
a ninth chamber
connected to the second process chamber by an eighth openable connection,
where the ninth and
second process chambers and the eighth openable connection are configured to
permit substance
movement from the second process chamber to the ninth chamber when a substance-
moving force is
applied to the contents of the second process chamber and the eighth openable
connection has been
altered from a closed state to an open state, and where the fourth linear path
does not include a
chamber of the second or third linear paths other than the second process
chamber. The second
4
CA 02743477 2011-05-12
process chamber may be adjacent the sample receiving chamber or it may be the
sample receiving
chamber. To purify one or more analytes in the sample, the eighth chamber may
contain a wash
solution for removing unwanted material from the sample and the ninth chamber
may be
substantially void, so that it can function as a waste chamber for spent wash
solution.
[00011] In a further aspect, the receptacle includes a tenth chamber connected
to the
intermediate chamber by a ninth openable connection, where the tenth and
intermediate chambers
and the ninth openable connection are configured to permit substance movement
from the tenth
chamber to the intermediate chamber when a substance-moving force is applied
to the contents of the
tenth chamber and the ninth openable connection has been altered from a closed
state to an open
state. The first linear path of this aspect does not include the tenth
chamber.
[00012] Substance movement between chambers can be facilitated by chambers
having
flexible portions that yield to moderate external forces (i.e., forces that do
not rupture chamber-
defining members or otherwise damage a receptacle in a way that renders it
inoperative for its
intended purpose). Thus, the receptacle may include top and bottom or opposed
members, with at
least one of the members being a flexible sheet. The flexible sheet may have a
plurality of layers
(including one or a plurality of plastic layers selected to have desired
bonding characteristics) which
exhibit acceptable light, water and/or oxygen transmission properties. Each of
the opposed members
may be formed from flexible sheets. At least one of the flexible sheets may
include a foil layer.
[00013] Depending on the types of materials being bonded, the boundaries of
the
interconnected chambers may be defined by any sealing means, including
adhesive or heat sealing,
ultrasonic welding or radio frequency ("RF") welding. When one of the members
of a receptacle is a
flexible sheet having an exposed plastic layer, heat seals may be used to
define the boundaries of the
interconnected chambers. Each openable connection may be blocked with one or a
combination of
barriers, including a seal, valve, or external force (e.g., actuator) applied
to the connection, when in a
closed state to prevent substance movement between chambers. The seal may be a
burstable seal
(e.g., peelable heat seal, such as chevron or V-shaped seal). Seals blocking
the connections between
chambers and chamber-defining seals are preferably formed under different
conditions so that the
chamber-defining seals resist peeling or rupturing when forces are applied to
the openable
connections to alter them from closed states to open states. In this respect,
the chamber-defining
CA 02743477 2011-05-12
seals are referred to as "permanent seals."
[00014] At least one of the openable connections of the receptacle may be
configured so that it
can be altered from the closed state to the open state by a substance-moving
force applied to an
adjacent chamber. The substance-moving force may be in the form of, for
example, an internal
compressor, vacuum, or a roller or actuator that presses against a flexible,
at least partially
compressible portion of the adjacent chamber. An example of an external
actuator is a pneumatic
actuator or group of actuators having compression pads that are shaped to
generally conform to the
shape of the chamber or a flexible portion thereof. Alternatively, the
substance-moving force maybe
a manual, digital force
[00015] To process large samples, the volume capacity of the sample receiving
chamber may
be greater than the volume capacity of any chamber directly connected to the
sample receiving
chamber that is other than an end chamber. By sequentially processing portions
of the sample,
unwanted sample and process materials can be removed to a waste chamber or to
a chamber that has
already been vacated of a process material, and analyte in the sample can be
concentrated to a more
manageable size for analyzing. Being able to concentrate the analyte will
limit the required
dimensions of the receptacle, which is particularly advantageous for field
applications since larger
receptacles require larger and heavier instruments for processing samples.
Analyte concentration
may be carried out using a receptacle having a flexible member and a
cooperating array of actuators
which allows aliquots of sample to be incrementally moved and processed.
[00016] For processes having a detection component, at least one of the
chambers may be
configured to enable detection of a characteristic of a sample. What is
detected maybe, for example,
the existence of an analyte, a chemical reaction, or a change in a property of
a sample or sample
component. In one aspect, detection may include determining the existence or
amount of a signal
indicative of the characteristic of the sample. Examples of such signals
include light (e.g.,
luminescence or fluorescence), turbidity, radioactivity, and electrical
currents. For light detection, at
least a portion of a detection chamber needs to be formed of optically
transmissive materials (e.g.,
transparent or translucent).
[00017] A process material for use in preparing, modifying, reacting with or
otherwise
6
CA 02743477 2011-05-12
affecting a sample or component of a sample may be provided to any chamber of
the receptacle. The
process material may be provided to at least one of the first and second
chambers. The same or
different process materials may be provided to the first and second chambers,
such as a dried reagent
(e.g., lyophilized or tableted reagent) provided to the second chamber and a
reconstitution reagent
provided to the first chamber for reconstituting the dried reagent. With this
particular combination
of process materials, it maybe desirable to further include an immiscible
liquid (e.g., an oil, such as
a mineral oil) to the first chamber in an amount sufficient to facilitate
reconstitution of the dried
reagent. (The reconstitution reagent and the immiscible liquid may be provided
together from the
second chamber or they may be provided to the first chamber from different
chambers.) In this
aspect, the ratio of the immiscible liquid to the reconstitution reagent is
preferably from about 1:10 to
about 10: 1, and more preferably from about 1:3 to about 10: 1. The immiscible
liquid should not be
reactive with the dried reagent or the reconstitution reagent. Similarly, the
third and fourth chambers
may be provided with a reconstitution reagent and dried reagent, respectively,
where the
reconstituted forms of the dried reagents can be united to achieve a combined
effect. For example,
the dried reagents of the second and fourth chambers may be amplification and
enzyme reagents,
respectively, having components needed for a nucleic acid-based amplification
reaction. An
immiscible liquid may also be combined with the reconstitution reagent of the
third chamber to
facilitate reconstitution of the dried reagent contained in the fourth
chamber.
[00018] One of the dried reagents present in the second and fourth chambers
may include a
binding agent, such as a probe for forming a probe:target complex with a
product of the nucleic acid-
based amplification reaction. The probe may have an oligonucleotide component
that hybridizes
with specificity (i.e., does not detectably hybridize to non-target nucleic
acid in a sample) to an
amplification product of the nucleic acid-based amplification reaction. For
detection, the probe may
be associated with a label, such as a fluorescent, luminescent or radioactive
moiety. Alternatively,
the reaction may include a label which recognizes the formation of the
probe:target complex, such as
an intercalating dye (e.g., ethidium bromide or SYBR Green), or detection may
occur without the
aid of a label, such as by detecting electrical signals or mass changes
associated with the formation
of the probe:target complex. To enable real-time detection in a nucleic acid-
based amplification
reaction, the probe may assume a different and detectable conformation when it
is in a hybridized
state than when it is in an unhybridized state. Such probes may include
interacting labels that
7
CA 02743477 2011-05-12
undergo a detectable signal change when the probe complexes with an
amplification product.
[00019] For chambers containing dried reagents, all or a portion of the
materials used to
construct the chambers may exhibit a greater water vapor transmission rate
("WVTR") than the
materials used to construct at least one other chamber of the receptacle,
particularly a directly
connected chamber containing a liquid. By way of example, the directly
connected chambers maybe
formed with one or more flexible plastic layers and the liquid-containing
chamber may further
include a foil layer or layers which have a lower water vapor transmission
rate than the plastic layers
used to form each of the directly connected chambers. In one aspect, at least
a portion of a chamber
containing a dried reagent is constructed of optically transmissive materials
so that the contents of
the chamber can be interrogated by an optical sensor (e.g., fluorometer or
luminometer).
[00020] To further control the moisture level in chambers containing dried
reagents, the
receptacle may be stored in a sealed container until use. The sealed container
may include a
desiccant for drawing moisture from the receptacle and maximizing the
stability of the dried
reagents.
[000211 The receptacles of this embodiment may be used or adapted for use in
any of the other
embodiments described herein.
[00022] In another embodiment, a first method of processing a sample in a
receptacle having a
plurality of interconnected chambers is provided, where the method includes
the steps of providing a
sample to a first chamber of the receptacle; independently combining the
following in separate
chambers of the receptacle: (i) at least a portion of the sample contained in
the first chamber and at
least a portion of a sample processing reagent contained in a second chamber
of the receptacle; (ii) at
least a portion of a substance contained in a third chamber and at least a
portion of a substance
contained in a fourth chamber; and (iii) at least a portion of a substance
contained in a fifth chamber
and at least a portion of a substance contained in a sixth chamber; and, after
performing substeps (i)-
(iii), combining in a chamber of the receptacle a component of the sample with
at least a portion of
each of the resulting combinations of substeps (ii) and (iii). In a preferred
aspect, each of the plurality
of interconnected chambers is adjacent to at least one other chamber of the
receptacle (seals alone
separate chambers). Additionally, the chambers of the receptacle may have a
radial arrangement,
8
CA 02743477 2011-05-12
where the end chambers (i.e., the outermost chambers of a linear path of
chambers) of the receptacle
have a non-circular arrangement.
[00023) In one aspect, the first and second chambers are directly connected to
each other, the
third and fourth chambers are directly connected to each other, and the fifth
and sixth chambers are
directly connected to each other. In another aspect, the method further
includes the step of mixing
the resulting combination of at least one of substeps (i)-(iii) by alternately
moving the combination
between a pair of directly connected chambers in the receptacle. In still
another aspect, the first
chamber is intermediate between the fourth and sixth chambers, and the
receptacle comprises a
sample inlet port for receiving the sample into the first chamber. In yet
another aspect, the resulting
combination of substep (i) is moved to a seventh chamber intermediate between
the first chamber
and at least one of the fourth and sixth chambers prior to combining a
component of the sample with
at least a portion of each of the resulting combinations of substeps (ii) and
(iii). In a further aspect,
the fourth and sixth chambers are each directly connected to the first chamber
but not to each other.
In a still further aspect, no component of the sample is moved into the third
or fourth chamber or,
alternatively, the fifth or sixth chamber during the method. The sample
processing chamber may be
the first chamber or it may be directly connected to the first chamber.
[00024] A solid support may be included in the sample processing reagent for
immobilizing an
analyte present in the sample. While immobilized on the solid support and
contained in the sample
processing chamber, one or more non-analyte components of the sample may be
removed to a waste
chamber of the receptacle, which may be an eighth chamber directly connected
to the sample
processing chamber. Preferred solid supports are dispersible in a liquid
medium, such as
magnetically-responsive particles or beads that are exposed to magnetic forces
during removal of the
non-analyte components.
[00025] In another aspect, the method further includes the steps of: providing
a wash solution
to the sample processing chamber; mixing the solid support and the wash
solution in the sample
processing chamber; and removing the wash solution from the sample processing
chamber to the
eighth chamber while the analyte remains immobilized by the solid support in
the sample processing
chamber. The wash solution may be a buffered, non-reactive solution that is
provided from a ninth
chamber directly connected to the sample processing chamber. Additionally, a
rinse solution may be
9
CA 02743477 2011-05-12
provided from a tenth chamber directly connected to the sample processing
chamber when it is
needed or useful for removing residual wash solution in the sample processing
chamber, such as
when the wash solution contains one or more components known to be inhibitory
to a desired
reaction in the receptacle (e.g., detergents that inhibit nucleic acid-based
amplification reactions).
[00026] A dried reagent may be reconstituted in at least one of substeps (ii)
and (iii), where at
least one of the chambers of these two substeps includes a solution formulated
for reconstituting a
corresponding dried reagent. The dried reagent may be in, for example, a
lyophilized or tableted
form.
[00027] In still another aspect, the method of this embodiment further
includes, after
combining a component of the sample with at least portions of the resulting
combinations of
substeps (ii) and (iii), the step of detecting a characteristic of the
contents of a detection chamber of
the receptacle. The detecting step may include determining the existence or
amount of a signal
indicative of the presence or amount of a component of the sample. At least a
portion of the
detection chamber may be constructed of optically transmissive materials,
thereby permitting the
contents of the detection chamber to be interrogated by an optical sensor
located or moved to a
position adjacent the detection chamber. The detection chamber may be the
third, fourth, fifth or
sixth chamber.
[00028] What is detected in the detection chamber may be a product of an
amplification
reaction. The amplification reaction may be a nucleic acid-based amplification
reaction (e.g., target
or signal amplification). For this use, a dried enzyme reagent may be
reconstituted in substep (ii). In
addition to reconstituting a dried enzyme reagent in substep (ii), a dried
amplification reagent may be
reconstituted in substep (iii). At least one of the amplification and enzyme
reagents may include a
probe capable of forming a detectable probe:target complex with a product of
the nucleic acid-based
amplification reaction (e.g., a hybridizing probe). The probe may include one
or more labels to
facilitate detection. The product of the nucleic acid-based amplification
reaction may be detected at
the conclusion of the amplification reaction or in real-time using, for
example, a probe that assumes
a differently detectable conformation when it is hybridized to a product of
the reaction.
[00029] Receptacles of this embodiment may include a flexible portion that
yields to a
CA 02743477 2011-05-12
compressive force, thereby facilitating substance movement between chambers.
The receptacle may
include opposed members, where at least one of the members includes a flexible
sheet. The
chambers, and any associated interconnections, may be defined by a sealing
engagement between the
opposed members, which, depending on the materials being joined, may be formed
by any sealing
means, including, as described above, adhesives or heat sealing, ultrasonic
welding or RF welding.
Each of the opposed members may include a flexible sheet. The flexible sheet
may have a plurality
of layers (including one or a plurality of plastic layers selected to have
desired bonding
characteristics) which exhibit acceptable light, water and/or oxygen
transmission properties. At least
one of the flexible sheets may include a foil layer.
[00030] The chambers of the receptacle may be connected by a plurality of
openable
connections, and each openable connection is configured to permit substance
movement between
directly connected chambers when a substance-moving force is applied to the
contents of at least one
of the directly connected chambers and the openable connection has been
altered from a closed state
to an open state. Connections in the closed state may be blocked by one or a
combination of
obstructions, including a seal, valve, or external force (e.g., actuator)
applied to the connection. The
seal may be a burstable seal (e.g., peelable heat seal, such as a chevron or V-
shaped seal).
[00031) In yet another aspect, the method of this embodiment further includes
the step of
applying a substance-moving force to each of a plurality of the chambers,
where the substance-
moving force includes one or more actuators, and where each of the plurality
of chambers is adapted
to cooperate with the actuators in substance movement between the plurality of
chambers. The
actuators may include compression pads generally conforming to the shapes of
the flexible portions
of the chambers. The one or more actuators may be pneumatic actuators.
Alternatively, the
substance-moving force may include an external roller or a positive or
negative force applied within
the receptacle.
[00032] In still another embodiment, a second method is provided for
processing a sample in a
receptacle having a plurality of interconnected chambers, where the method
includes the steps of:
providing a sample to a first chamber of the receptacle; independently
combining the following in
separate chambers of the receptacle: (i) at least a portion of the sample
contained in the first chamber
and at least a portion of a sample processing reagent contained in a second
chamber of the receptacle;
11
CA 02743477 2011-05-12
and (ii) at least a portion of a substance contained in a third chamber and at
least a portion of a
substance contained in a fourth chamber; and, after performing substeps (i)
and (ii), combining in a
chamber of the receptacle a component of the sample with at least a portion of
the resulting
combination of substep (ii) and at least a portion of a substance or
combination of substances
contained in a fifth chamber, provided that no component of the sample is
moved into at least one of
the third and fifth chambers during the method if the fifth chamber is
directly connected to the fourth
chamber, also provided that no component of the sample is moved into at least
one of the third and
fifth chambers during the method if the fifth chamber is not directly
connected to either of the third
and fourth chambers, and further provided that no component of the sample is
moved into the fifth
chamber during the method if the fifth chamber is directly connected to the
third chamber but not to,
the fourth chamber. The chambers of the receptacle may have a radial
arrangement, where the end
chambers of the receptacle have a non-circular arrangement.
[00033] In one aspect, the first and second chambers are directly connected to
each other and
the third and fourth chambers are directly connected to each other. In another
aspect, the method of
this embodiment further includes the step of mixing the resulting combination
of at least one of
substeps (i) and (ii) by alternately moving the combination between a pair of
directly connected
chambers in the receptacle. In still another aspect, the first chamber is
intermediate between the
fourth and fifth chambers, and the receptacle includes a sample inlet port for
receiving the sample
into the first chamber. In yet another aspect, the fourth and fifth chambers
are each directly
connected to the first chamber. In a further aspect, the resulting combination
of substep (i) is moved
to a sixth chamber intermediate between the first and fourth chambers. In a
still further aspect, the
fourth and fifth chambers are directly connected to the seventh chamber. In
another aspect, the fifth
chamber is directly connected to the third chamber but not to the fourth
chamber. In still another
aspect, each of the third and fifth chambers is directly connected to the
fourth chamber. The sample
processing chamber may be the first chamber or directly connected to the first
chamber.
[00034] In another aspect, the method of this embodiment further includes the
step of
removing one or more non-analyte components of the sample to a waste chamber
of the receptacle
while an analyte present in the sample remains immobilized by a solid support
contained in a sample
processing chamber of the receptacle. The solid support may be provided to the
receptacle in the
12
CA 02743477 2011-05-12
sample processing reagent. The solid support is preferably dispersible in a
liquid medium, such as
magnetically-responsive particles or beads that are exposed to a magnetic
field during removal of the
non-analyte components. The waste chamber may be a seventh chamber that is
directly connected to
the sample processing chamber.
[00035] In yet another aspect, the method of this embodiment further includes
the steps of
providing a wash solution to the sample processing chamber; mixing the solid
support and the wash
solution in the sample processing chamber; and removing the wash solution from
the sample
processing chamber to the seventh chamber while the analyte remains
immobilized by the solid
support in the sample processing chamber. The wash solution may be provided
from an eighth
chamber that is directly connected to the sample processing chamber.
[00036] A first dried reagent may be reconstituted in substep (ii), and the
fifth chamber may
contain a reconstituted form of a second dried reagent. The first dried
reagent may contain a probe
capable of forming a detectable probe:target complex with a product of a
nucleic acid-based
amplification reaction. The first dried reagent maybe an enzyme reagent and
the fifth chamber may
contain an amplification reagent. The dried reagents may be in lyophilized
forms.
[00037] Other process steps and particulars of the receptacles that maybe used
in the method
of this embodiment are set forth above in the description of the first
embodiment of a method of
processing a sample in a receptacle having a plurality of interconnected
chambers.
[00038] In yet another embodiment, an instrument programmed to process a
sample in
accordance with any of the methods described herein is provided. The
instrument is adapted to
receive and align a receptacle having a non-linear arrangement of
interconnected chambers in a
stationary receptacle-receiving area associated with the instrument. The
instrument comprises an
actuator system operatively positioned with respect to the receptacle-
receiving area that includes a
plurality of actuators arrayed to conform to the arrangement of at least a
portion of the chambers and
to selectively apply pressure to flexible portions of the chambers, thereby
forcing fluid substances to
move between directly connected chambers. The instrument also comprises a
detector operatively
positioned adjacent the receptacle-receiving area so as to be in operative
proximity to a detection
chamber contained in a receptacle provided to the instrument, where the
detector is capable of
13
CA 02743477 2011-05-12
detecting a characteristic of the contents of the detection chamber. The
instrument further comprises
a controller programmed to control the operation of the instrument, including
the actuator system, the
detector, and thermal elements.
[00039] In further embodiment, a system is provided that comprises the above-
described
instrument for processing samples and a receptacle positioned in the
receptacle-receiving area. In a
preferred aspect, the receptacle is formed from first and second opposed
members joined to each
other so as to define the plurality of interconnected chambers. For certain
applications, the
receptacle includes a linear path having a minimum of five chambers. At least
one of the opposed
members comprises flexible portions, and at least a portion of the passages
include fluid barriers.
[00040] In a still further embodiment, a detector for detecting an optical
signal that may be
indicative of the presence, amount, or state of one or more analytes in a
sample, includes one or more
excitation channels, each adapted to direct an excitation signal of a
prescribed excitation optical
characteristic toward the sample. Each excitation channel includes a light
emitting element adapted
to emit excitation light and excitation optical elements defining an
excitation optical path having an
excitation optic axis. The excitation optical elements are constructed and
arranged to transmit at
least a portion of the light emitted by the light-emitting element having the
prescribed excitation
optical characteristic toward the sample. The detector further comprises one
or more emission
channels, each adapted to receive an emission signal from the sample. Each
emission channel
comprises emission optical elements defining an emission optical path having
an emission optic axis,
and the emission optical elements are constructed and arranged to transmit at
least a portion of any
light emitted by the sample having a prescribed emission optical
characteristic. Each emission
channel further includes a light-detecting element and associated circuitry
adapted to detect light
transmitted by the emission optical elements and to convert the detected light
to an electronic signal
indicative of at least one of the presence and strength of the detected light.
The light-emitting and
light detecting elements are operably connected to a single circuit board. The
detector further
includes one or more optic elements constructed and arranged to receive an
excitation signal from
each excitation channel and direct at least a portion of each excitation
signal at a prescribed location
and to receive emission signals emitted from the sample at the prescribed
location and to direct at
least a portion of the received emission signals into each emission channel.
The detector does not
14
CA 02743477 2011-05-12
include a reflective element for redirecting all light impinging on the
reflective element in a direction
different from an incidence direction of the impinging light, and the detector
does not include a light
characteristic separating element for redirecting a portion of the light
impinging on the separating
element having a first light characteristic in a direction different from an
incidence direction of the
impinging light and for transmitting a portion of the light impinging on the
separating element
having a second light characteristic.
[00041) In another aspect, the excitation optic axis of each excitation
channel and the emission
optic axis of each emission channel are parallel to one another throughout
their extents.
[00042] In a further aspect, the one or more optic elements are constructed
and arranged to (1)
receive an excitation signal from each excitation channel and direct at least
a portion of the
excitation signal at a prescribed location on a container within which the
sample is processed, and (2)
receive an emission signal emitted from the sample within the container and to
direct at least a
portion of the received emission signals into each emission channel.
[00043] In a further aspect, the detector comprises two or more excitation
channels and two or
more emission channels.
[00044] In a further aspect, the one or more optic elements consist of a
single, undivided lens.
[00045] In a still further embodiment, a detector for detecting an optical
signal from a sample
that may be indicative of the presence, amount, or state of one or more
analytes in the sample
comprises one or more excitation channels, each adapted to direct an
excitation signal of a prescribed
excitation wavelength or range of excitation wavelengths toward the sample and
one or more
emission channels, each adapted to receive an emission signal from the sample
and detect an
emission signal having a prescribed emission wavelength or range of emission
wavelengths. Each
excitation channel comprises a light emitting element adapted to emit
excitation light and excitation
optical elements defining an excitation optical path having an excitation
optic axis. The excitation
optical elements are constructed and arranged to transmit at least a portion
of the light emitted by the
light-emitting element having the prescribed excitation wavelength or range of
excitation
wavelengths toward the sample. Each emission channel comprises emission
optical elements
defining an emission optical path having an emission optic axis. The emission
optical elements are
CA 02743477 2011-05-12
constructed and arranged to transmit at least a portion of any light emitted
by the sample having the
prescribed emission wavelength or range of emission wavelengths. Each emission
channel further
includes a light-detecting element adapted to detect light transmitted by the
emission optical
elements and to convert the detected light to an electronic signal indicative
of at least one of the
presence and strength of the detected light. The excitation and emission optic
axes are parallel to
one another throughout their extents. And the detector further includes an
optic lens constructed and
arranged with respect to the excitation and emission channels-to (1) direct
excitation light transmitted
by each excitation channel and impinging on a different portion of the optic
lens at a prescribed
location and (2) receive emission signals emitted by the sample at the
prescribed location and to
direct at least a portion of the received emission signals into each emission
channel.
[00046]. In another aspect, the light emitting element comprises a light-
emitting diode.
[00047] In a further aspect, the excitation optical elements of each
excitation channel comprise
a lens and an excitation filter constructed and arranged to transmit only
light having the prescribed
excitation wavelength or range of excitation wavelengths.
[00048) In a further aspect, the light-detecting element comprises a
photodiode.
[00049] In a further aspect, the emission optical elements of each emission
channel comprise a
lens and an emission filter constructed and arranged to transmit only light
having the prescribed
emission wavelength or range of emission wavelengths.
[00050] In a further aspect, the light emitting elements of the excitation
channels and the light
detecting elements of the emission channels are mounted on the same plane.
(00051] In a further aspect, the detector further comprises a housing, wherein
each of the
excitation and emission channels is disposed within a different conduit
defined within the housing.
[00052] In a further aspect, the detector comprises two excitation channels
and two emission
channels, and the conduits of the excitation channels and the emission
channels are disposed in a
circular pattern, with the conduits being spaced by approximately 90 .
[00053] In a further aspect, the detector further comprises a base including
at least one printed
16
CA 02743477 2011-05-12
circuit board, the housing is mounted to the base, and the light emitting
elements of the excitation
channels and the light detecting elements of the emission channels are
operatively connected to the
printed circuit board.
[00054] In a further aspect, the detector further comprises ambient light
filtering circuitry
comprising excitation modulation circuitry and detection circuitry. The
excitation modulation
circuitry is constructed and arranged to modulate the excitation signal of
each excitation channel at a
predefined excitation frequency, and the detection circuitry is constructed
and arranged to identify
that portion of the detected light that is substantially at the excitation
frequency.
[00055] In a further aspect, the detector comprises two or more excitation
channels, and the
excitation frequency is different for the excitation signal of each excitation
channel.
[00056] In a further aspect, the detector comprises two or more excitation
channels, and the
excitation frequency is the same for the excitation signal of each excitation
channel.
[00057] In a further aspect, the excitation optical elements and the emission
optical elements
do not include optic fibers.
[00058] In a still further embodiment, a detector is provided that detects
optical emissions of
two or more different wavelengths or ranges of wavelengths from a sample,
wherein emissions of
two or more different wavelengths may be indicative of the presence, amount,
or state oftwo or more
analytes of interest in the sample, and wherein the emissions are detected
without moving
components of the detector with respect to the sample. The detector comprises
two or more
excitation channels fixed with respect to the sample and each other and two or
more emission
channels fixed with respect to the sample, the excitation channels, and each
other. Each excitation
channel is adapted to direct an excitation signal of a different prescribed
excitation wavelength or
range of excitation wavelengths toward the sample, and each excitation channel
comprises a light
emitting element adapted to emit excitation light and excitation optical
elements defining an
excitation optical path having an excitation optic axis. The excitation
optical elements are
constructed and arranged to transmit at least a portion of the light emitted
by the light-emitting
element having the prescribed excitation wavelength or range of excitation
wavelengths toward the
sample. Each emission channel is adapted to receive and detect an emission
signal of a different
17
CA 02743477 2011-05-12
prescribed emission wavelength or range of emission wavelengths from the
sample, and each
emission channel comprises emission optical elements defining an emission
optical path having an
emission optic axis, and the emission optical elements are constructed and
arranged to transmit at
least a portion of any light emitted by the sample having the prescribed
emission wavelength or range
of emission wavelengths. Each emission channel further includes a light-
detecting element adapted
to detect light transmitted by the emission optical elements and to convert
the detected light to an
electronic signal indicative of at least one of the presence and strength of
the detected light. The
detector is adapted to direct light having a unique excitation wavelength or
range of excitation
wavelengths at the sample with each of the excitation channels and detect
light having a unique
emission wavelength or range of emission wavelengths emitted from the sample
with each of the
emission channels without moving the excitation channels or the emission
channels with respect to
the sample or each other.
[00059] In another aspect the excitation optical elements are constructed and
arranged to
transmit at least a portion of the light emitted by the light-emitting element
having the prescribed
excitation wavelength or range of excitation wavelengths toward a container
within which the
sample is processed. The emission optical elements are constructed and
arranged to transmit at least
a portion of any light having the prescribed emission wavelength or range of
emission wavelengths
emitted by the sample from within the container. The detector is adapted to
direct light having a
unique excitation wavelength or range of excitation wavelengths at the
container with each of the
excitation channels and detect light having a unique emission wavelength or
range of emission
wavelengths emitted from the container with each of the emission channels
without moving the
excitation channels or the emission channels with respect to the container or
each other.
[00060] In a still further embodiment, a method for detecting an optical
signal that may be
indicative of the presence, amount, or state of one or more analytes in a
sample comprises generating
a first excitation signal and transmitting a portion of the first excitation
signal having a first
excitation wavelength or range of excitation wavelengths along a first
excitation path, focusing the
first excitation signal at the sample with a first portion of an optic lens,
and directing a portion of a
signal emitted by the sample, if any, into a first emission path using a
second portion of the optic
lens, transmitting light having a first emission wavelength or range of
emission wavelengths along
18
CA 02743477 2011-05-12
the first emission path, and detecting light having the first emission
wavelength or range of emission
wavelengths. The first excitation path and the first emission path are
parallel to each other
throughout their extents.
[00061] In another aspect, the method further comprises generating a second
excitation signal
and transmitting a portion of the second excitation signal having a second
excitation wavelength or
range of excitation wavelengths along a second excitation path, focusing the
second excitation signal
at the sample with a third portion of the optic lens, and directing a portion
of a signal emitted by the
sample, if any, into a second emission path using a fourth portion of the
optic lens, transmitting light
having a second emission wavelength or range of emission wavelengths along the
second emission
path, and detecting light having the second emission wavelength or range of
emission wavelengths.
The first and second excitation paths and the first and second emission paths
are parallel to each
other throughout their extents.
[00062] In a further aspect, focusing the first excitation signal at the
sample comprises
focusing the first excitation signal at the contents of a container within
which the sample is being
processed.
[00063] In a further aspect, directing a portion of a signal emitted by the
sample into a first
emission path comprises directing a signal emitted by the sample within the
container.
[00064] In a further aspect, the optic lens comprises a solid, undivided lens.
[00065] In a further aspect, the method comprises transmitting the first
excitation signal along
the first excitation path and transmitting light along the first emission path
without the use of (1) a
reflective element for redirecting all light impinging on the reflective
element in a direction different
from an incidence direction of the impinging light or (2) a light
characteristic separating element for
redirecting a portion of the light impinging on the separating element having
a first light
characteristic in a direction different from an incidence direction of the
impinging light and for
transmitting a portion of the light impinging on the separating element having
a second light
characteristic.
[00066] In a further aspect, the focusing and directing steps are accomplished
without any
19
CA 02743477 2011-05-12
optic element other than the single lens outside the first excitation path and
the first emission path.
[00067] In a further aspect, the generating step is performed by a light
emitting diode.
[00068] In a further aspect, transmitting the portion of the first excitation
signal having the
first excitation wavelength or range of excitation wavelengths along the first
excitation path
comprises filtering the first excitation signal to remove wavelengths of the
first excitation signal
other than the first excitation wavelength or range of excitation wavelengths,
and transmitting light
having the first emission wavelength or range of emission wavelengths along
the first emission path
comprises filtering the portion of the emission signal directed into the first
emission path to remove
wavelengths of the first emission signal other than the first emission
wavelength or range of emission
wavelengths.
[00069] In a further aspect, the method further comprises modulating the
excitation signal at a
predefined excitation frequency and identifying that portion of the detected
light that is substantially
at the excitation frequency.
[00070] Transmitting the portion of the first excitation signal having the
first excitation
wavelength and transmitting light having the first emission wavelength along
the first emission path
do not include transmitting light along an optic fiber.
[00071] The manufacture and special uses of receptacles in accordance with the
present
invention will now be described. For the described embodiments, the
receptacles may include
opposed members that are configured to define a plurality of interconnected
chambers, where the
chambers are joined by passages or openings, as between adjacently positioned
chambers, or by
channels or passageways, as between separated chambers. The plurality of
interconnected chambers
may be defined by seals formed between the opposed members by any sealing
means, such as, for
example, adhesives or heat sealing, ultrasonic welding or RF welding. At least
one of the opposed
members may include a flexible sheet that includes a flexible, at least
partially compressible portion
of a chamber. The flexible sheet may have a plurality of layers, including one
or more plastic layers
selected to have desired bonding characteristics, as well as acceptable light,
water and/or oxygen
transmission properties. Both opposed members may be formed from flexible
sheets, at least one of
which may include a foil layer.
CA 02743477 2011-05-12
[00072] The connections between directly connected chambers of these
receptacles may be
blocked by openable connections that can be altered from closed states to open
states when
substance-moving forces are applied to the contents of the chambers. Each
substance-moving force
may include, for example, an externally applied force pressing on a chamber,
such as one or more
actuators (e.g., pneumatic actuators) or a roller, or it may include a
positive or negative force applied
within the receptacle. If the substance-moving force is one or more actuators,
the actuators may
include compression pads generally conforming to the shape of the flexible
portion of a chamber,
such as a chamber wall. The connection may be blocked in the closed state by
one or a combination
of obstructions, including a seal, valve, or external force (e.g., actuator)
applied to the connection.
The seal may be a burstable seal, such as a peelable heat seal (e.g., chevron
or V-shaped seal).
[00073] In another embodiment, a method is provided for loading liquid
substances into a
receptacle having a plurality of interconnected chambers, where the method
includes the steps of:
sequentially providing an immiscible liquid and a liquid substance (the liquid
substance following
the immiscible liquid) to an open chamber of the receptacle, where the
immiscible liquid has a lower
density than the liquid substance, and where the immiscible liquid is provided
to the open chamber in
an amount sufficient to control wicking of the liquid substance within the
open chamber, and closing
the open chamber to provide a substantially fluid-tight seal. The open chamber
may be one of a
plurality of chambers opened to a side of the receptacle and adapted to
receive substances for use in
performing an assay. The lower density immiscible liquid floats on the liquid
substance, which was
found to control wicking in an open chamber that can lead to contamination of
the contents of an
adjacent chamber or affect the concentration of a process material used in a
reaction or other process.
The immiscible liquid is preferably non-reactive with the liquid substance,
and the amount of the
immiscible liquid provided to an open chamber will depend, at least in part,
on the volume capacity
of the chamber and the volume of the liquid substance provided to the open
chamber.
Notwithstanding, the ratio of the immiscible liquid to the liquid substance is
preferably up to about
10:1. Any immiscible liquid is contemplated, but oils (e.g., mineral oils) are
especially suitable.
[00074] The receptacle may be comprised of opposed members, at least one of
which includes
a flexible sheet. The flexible sheet may include a flexible portion of the
open chamber that is drawn
away from the opposed member during the providing step, when the immiscible
liquid and the liquid
21
CA 02743477 2011-05-12
substance are delivered to the open chamber. The opposed members may comprise
opposed flexible
sheets, where the opposed flexible sheets include opposed flexible portions of
the open chamber that
are drawn away from each other during the providing step. After delivering the
immiscible liquid
and the liquid substance to the open chamber, the open chamber may be closed
by any means
sufficient to provide a fluid-tight seal, such as a heat seal formed between
opposed members of the
open chamber. The steps of the method are preferably automated.
[00075] In still another embodiment, a method of manufacturing a receptacle
having a
plurality of interconnected chambers to separately contain liquid and dried
substances is provided,
where the method includes the steps of: providing at least one liquid
substance to one or more first
chambers opened to a first side of the receptacle; closing the first chambers
to provide substantially
fluid-tight enclosures; providing at least one dried substance to one or more
second chambers opened
to a second side of the receptacle; and closing the second chambers to provide
substantially fluid-
tight enclosures, where the chambers of the receptacle are separated from each
other by fluid barriers,
and where dried substances are not provided to chambers opened to the first
side and liquid
substances are not provided to chambers opened to the second side. The liquid
substances may be
provided to one or more chambers of the receptacle that are then closed off
prior to providing the
dried substances to one or more chambers or vice versa. The first and second
sides of the receptacle
are positioned adjacent to each other or, preferably, opposite each other.
[00076] An immiscible liquid may be provided to one or more of the first
chambers of the
receptacle prior to providing the liquid substances, where the immiscible
liquid is selected to have a
lower density than any of the liquid substances provided to the receptacle.
The immiscible liquid is
preferably non-reactive with any of the liquid substances and may be, for
example, an oil (e.g., a
mineral oil). The ratio of the immiscible liquid to any of the liquid
substances is preferably up to
about 10: 1.
[00077] The liquid and dried substances may be used to perform an assay. At
least a portion
of the liquid substances may be used to reconstitute, dissolve or rehydrate
the dried substances. In a
particularly preferred embodiment, the dried substances include amplification
and enzyme reagents
for performing a nucleic acid-based amplification reaction, and the liquid
substances include
corresponding reconstitution reagents.
22
CA 02743477 2011-05-12
[00078) The receptacle may be comprised of opposed members, at least one of
which includes
a flexible sheet. The flexible sheet may include flexible portions of the open
chambers that are
drawn away from the opposed member during the providing steps. The opposed
members may
comprise opposed flexible sheets, where the opposed flexible sheets include
opposed flexible
portions, such as chamber walls, of the open chambers that are drawn away from
each other during
the providing steps. Following each of the providing steps, the first chambers
and or the second
chambers are closed by any means sufficient to provide a fluid-tight seal,
such as a heat seal formed
between opposed members of the open chamber. The steps of the method are
preferably automated.
[00079] In yet another embodiment, a method is provided for concentrating an
analyte
contained in a sample delivered to a receptacle having a plurality of
interconnected chambers. The
method includes the steps of forming a first volume in a first chamber of the
receptacle, where the
first volume comprises the sample and a solid support; immobilizing the
analyte on the solid support;
removing non-analyte components of the sample from the analyte; and moving a
second volume
comprising the analyte to a partitioned section of the first chamber or to a
second chamber of the
receptacle, where the second volume and the volume capacity of the partitioned
section of the first
chamber or the second chamber are each less than the first volume. The first
chamber may be a
sample receiving chamber having a sample addition port. .
[00080] The solid support of the first volume may be present in the first
chamber when the
method of this embodiment is initiated or it may be moved to the first chamber
from a third chamber
prior to or during the forming step. Non-analyte components of the sample may
be removed from
the analyte while the analyte is immobilized on the solid support in the first
chamber or in a chamber
other than the first chamber. To facilitate movement of the second volume
between chambers or to
prevent the solid support from obstructing connections between chambers, the
first volume may
further include a non-reactive, immiscible liquid. The immiscible liquid may
be, for example, an oil
(e.g., a mineral oil). The ratio of the immiscible liquid to the remainder of
the first volume in the
first chamber is preferably about 1: 10 to about 10:1, and more preferably
about 1:3 to about 10:1.
[00081] Moving the second volume to the second chamber may be accomplished by
applying
a substance-moving force to a flexible portion of the first chamber, such as a
compressible chamber
wall, thereby forcing the second volume through a connection to the second
chamber. The
23
CA 02743477 2011-05-12
connection may be an openable connection, such as a seal, that can be altered
from a closed state to
an open state when the substance-moving force is applied to the flexible
portion of the first chamber.
[00082] Alternatively, or in combination with a seal, the first and second
chambers may be
isolated from each other by an external and retractable force applied to the
connection prior to the
moving step. The external closing force, alone or in combination with a seal,
provides a
substantially fluid-tight seal and may include one or more actuators, such as
pneumatic actuators,
having compression pads that extend across the connection.
[00083] A solid support of this embodiment may be in the form of a particle or
bead. The
solid support may be a magnetically-responsive material that is exposed to a
magnetic field when
non-analyte components of a sample are removed from the analyte.
[00084] The immobilizing step may be specific, partially specific, or non-
specific for the
analyte. For example, if the analyte is a target nucleic acid, the solid
support may be used to
immobilize essentially any nucleic acid present in the sample, a group of
nucleic acids -- such as
obtained from a phylogenetic grouping of organisms -- to which the target
nucleic acid belongs, or
the target nucleic acid but not other nucleic acids present in the sample. The
analyte may remain
immobilized by the solid support during the moving step, or it may be eluted
first and then moved
independent of the solid support. In the latter case, the solid support may
remain in the first chamber
while the analyte is being moved to the second chamber.
[00085] In a further embodiment, a method is provided for concentrating an
analyte contained
in a sample delivered to a receptacle having a plurality of interconnected
chambers. The method
includes the steps of. forming a first volume in a first chamber of the
receptacle, where the first
volume comprises the sample and a solid support; immobilizing the analyte on
the solid support;
moving an aliquot of the first volume from the first chamber to a second
chamber of the receptacle
directly connected to the first chamber, where the volume capacity of the
second chamber is less than
the first volume; isolating the solid support in the second chamber, removing
non-analyte
components of the sample to a waste chamber of the receptacle, where the waste
chamber is directly
connected to the second chamber; and repeating the moving, isolating and
removing steps one or
24
CA 02743477 2011-05-12
more times. Aliquots of the first volume may be moved to the second chamber by
applying
substance-moving forces to a flexible portion of the first chamber, such as a
compressible chamber
wall, so that the aliquots move through a connection to the second chamber.
The above-described
receptacles for use in concentrating an analyte present in a sample may be
adapted for use in the
method of this embodiment.
[00086] In a still further embodiment, a receptacle having a plurality of
interconnected
chambers is provided, where the chambers of the receptacle include a first
chamber directly
connected to a second chamber and containing a sample processing reagent which
includes
dispersible solid supports in a liquid medium. Also contained in the first
chamber is an immiscible
liquid in an amount sufficient to hinder the solid supports from obstructing
the connection. The
immiscible liquid is preferably non-reactive with the other components of the
sample processing
reagent and may be, for example, an oil (e.g., a mineral oil). The solid
supports may be particles or
beads, such as magnetically-responsive particles or beads that can be isolated
in the presence of a
magnetic force. When the sample processing reagent is contacted with a fluid
sample in the first
chamber, the ratio of the immiscible liquid to the liquid medium/fluid sample
combination is
preferably about 1:10 to about 10:1, and more preferably about 1:3 to about
10:1.
[00087] Where the receptacle is comprised of opposed members, the sides of the
receptacle
are formed by the joining of the opposed members. One of the sides, indicated
to a top end because
of its orientation during use, may have a sample receiving chamber with a
sample addition port for
receiving a sample into the sample receiving chamber. At least a portion of
the second chamber may
be positioned closer to a bottom end of the receptacle than the first chamber.
With this
configuration, the connection from the first chamber to the second chamber
preferably has a
generally downward orientation relative to the top end (i. e., the connection
joins a lower half of the
first chamber to an upper half of the second chamber). The sample receiving
chamber may be first
chamber.
[00088] The solid supports of this embodiment are small relative to the volume
capacities of
corresponding holding chambers. As such, the solid supports are dispersible in
the liquid contents of
the chambers, such that they are susceptible to manipulation in the
receptacle, as with charged solid
supports that are influenced by magnetic forces. The solid supports are
preferably adapted to
CA 02743477 2011-05-12
immobilize one or more analytes of interest that are suspected of being
present in a sample so that
the analytes can be isolated and non-analyte components can removed from the
sample, especially
interfering materials that can affect the results or performance of a
procedure. The solid supports
may be used in combination with capture agents, such as capture probes, to
immobilize analytes.
[00089] In another embodiment, a method of using the above receptacle is
provided to effect
movement of a fluid substance between chambers of the receptacle. In a first
step of this method, a
fluid sample is combined with the sample processing reagent and the immiscible
liquid in a first
process chamber of the receptacle, the immiscible liquid being provided in an
amount sufficient to
concentrate the solid supports in the first process chamber. Concentrating the
solid supports limits
their dispersion within a chamber, such that they are less disperse than they
would be in the absence
of the immiscible liquid. This, in turn, minimizes the presence of the solid
supports at peripheral
portions of the chamber, especially portions adjacent connections between
chambers. In a second
step, a fluid substance is moved from the first process chamber to a second
chamber of the receptacle
through a connection, such as a passage or channel between the chambers. The
fluid substance may
be the resulting combination of the first step or it maybe, for example, a
buffered solution containing
a purified form of the analyte, if present in the sample. The presence of the
immiscible liquid in the
first step hinders the solid supports from obstructing movement of the fluid
substance through the
connection.
[00090] In still another embodiment, a receptacle is provided for improving
the stability of
dried substances stored in the same receptacle as liquid substances. The
receptacle has a plurality of
interconnected chambers that include first and second chambers which are
directly connected to each
other. The first chamber contains a dried substance and the second chamber
contains a liquid
substance capable of altering the state or a characteristic of the dried
substance. The liquid substance
may be formulated to reconstitute, dissolve or rehydrate the dried substance
upon contact. The dried
substance may be, for example, a lyophilized or tableted reagent. To prevent
the dried and liquid
substances from prematurely coming into contact with each other, the
connection between the first
and second chambers is usually blocked by, for example, a seal, valve or other
obstruction, including
external forces. The connection may be blocked by an openable connection that
can be altered from
a closed state to an open state when a substance-moving force is applied to
the contents of the second
26
CA 02743477 2011-05-12
chamber. The substance-moving force may be one or more compressive forces,
such as one or more
pneumatic actuators, applied to a flexible portion of the second chamber.
[00091] The first and second chambers are constructed so that water vapor
passes from the
first chamber at a faster rate than it passes from the second chamber. In this
way, it is believed that
water vapor is drawn from the second chamber, through the first chamber and
into the ambient
environment without substantially hydrating the dried substance, which might
affect its stability and
performance in an assay. Where multiple chambers are directly connected to the
first chamber, water
vapor passes from the first chamber faster than it passes from any directly
connected, liquid-holding
chamber, and preferably from any directly connected chamber. To achieve this
effect, the first
chamber preferably includes at least one chamber wall having a greater water
vapor transmission rate
than any chamber wall of a directly connected, liquid-holding chamber of the
receptacle.
[00092] In one aspect, each of the first and second chambers includes a
flexible chamber wall.
The flexible walls may include a plurality of layers, one or more of which may
be a plastic layer. To
limit light, vapor and/or oxygen transmission through a chamber wall, at least
one of the layers may
include a foil layer. In another aspect, the flexible wall of the second
chamber fully comprises a foil
layer and the flexible wall of the first chamber does not fully comprise a
foil layer. The flexiblewall
of the first chamber may include one or more but less than all of the layers
of the flexible wall of the
second chamber. To facilitate detection of a light signal from the contents of
the first chamber, the
chamber wall of the first chamber may include an optically transmissive
region. Ina further aspect,
the second chamber is constructed so that it does not include a chamber wall
having an optically
transmissive region, which could affect the water vapor transmission rate of
the chamber wall and
the stability of the dried substance.
[00093] To limit exposure to ambient conditions (e.g., light, water and/or
oxygen) which
could affect the stability of the dried substance, the receptacle may be
contained within a sealed
container. A desiccant is preferably included in the sealed container.
[00094] In yet another embodiment, a method is provided for mixing substances
contained in
first and second chambers of a receptacle having a plurality of interconnected
chambers that relies
upon gravity to move the contents of one of the chambers. In this method, the
receptacle is first
27
CA 02743477 2011-05-12
oriented in an analyzer so that the first chamber is positioned substantially
above the second
chamber, thus permitting the contents of the first chamber to be drawn by
gravity (or negative
pressure) into the second chamber when a fluid communication is established
between the first and
second chambers. In a preferred embodiment, the fluid communication is
established when an
openable connection between the first and second chambers is altered from a
closed state to an open
state by a substance-moving force applied to the contents of the second
chamber, such as one or
more compressive forces applied to a flexible portion of the second chamber.
The same force used
to establish fluid communication between the first and second chambers may
also be used to move
the contents of the second chamber to the first chamber. After moving the
contents of the second
chamber into the first chamber, the substance-moving force is removed so that
the contents of the
first chamber can be drawn into the second chamber. This process may be
repeated one or more
times to achieve complete mixing of substances stored in the first and second
chambers prior to the
initiation of this procedure.
[00095] The first and second chambers may each contain a fluid substance or
the first chamber
may contain a dried substance, such as a lyophilized or tableted reagent. If a
dried substance is
contained in the first chamber, then the fluid substance of the second chamber
is provided to
reconstitute, dissolve or rehydrate the dried substance. It was discovered
that including a non-
reactive, immiscible liquid with the fluid substance of the second chamber
facilitates more complete
mixing of the substances to be combined, especially when a dried substance is
being hydrated. The
immiscible liquid may be an oil, such as a mineral oil, and the ratio of the
immiscible liquid to the
fluid substance is preferably about 1:10 to about 10:1, and more preferably
about 1:3 to about 10:1.
[00096) In a further embodiment, a system is provided for performing gravity-
assisted mixing
of substances contained in first and second chambers of a receptacle having a
plurality of
interconnected chambers. The system includes one of the above-described
receptacles and an.
analyzer which supports the receptacle in an operative position. The analyzer
includes one or more
actuators for applying a compressive force to the second chamber to thereby
displace at least a
portion of the contents of the second chamber into the first chamber. Removing
the compressive
force from the second chamber allows the contents of the first chamber to flow
by gravity into the
second chamber without applying a compressive force to the contents of the
first chamber. The
28
CA 02743477 2011-05-12
analyzer is programmed to control operation of the actuators and to cause the
contents of the second
chamber to move between the first and second chambers multiple times, thus
mixing the combined
contents of the first and second chambers. The actuators may be pneumatic
actuators and preferably
have compression pads that are arranged to generally conform to the shape of
the second chamber, or
at least a flexible portion of the second chamber.
[00097] In a preferred aspect, each of the chambers of the receptacle is an at
least partially
compressible chamber to facilitate substance movement between chambers.
According to this
aspect, the analyzer includes a plurality of actuators, each of which is
associated with at least one of
the chambers of the receptacle, and the controller is programmed to control
the operation of the
actuators and to cause the actuators to move substances among the various
chambers by selective
application of external force by the actuators. The controller is not
programmed to cause the
analyzer to apply a compressive force to the contents of the first chamber or,
alternatively, the
analyzer does not include an actuator associated with the first chamber. In
certain aspects, an
actuator plate provided to house the actuators includes an opening adjacent
the first chamber to
permit a detector to detect a characteristic of the contents of the first
chamber, such as an optical
signal.
[00098] In a still further embodiment, a receptacle having a plurality of
interconnected
chambers is provided that includes at least one chamber treated to limit
evaporation of a contained
liquid substance. The chamber has a flexible portion, such as a compressible
chamber wall, and
contains, in addition to a liquid substance, an immiscible liquid. The
immiscible liquid coats an
inner surface of the chamber, thereby, limiting evaporation of the liquid
substance from the chamber.
The immiscible liquid is preferably non-reactive with the other contents of
the chamber and may be,
for example, an oil (e-g., a mineral oil). The ratio of the immiscible liquid
to the other liquid
contents of the chamber is preferably about 1:10 to about 10:1, and more
preferably about 1:3 to
about 10:1.
[00099] In another embodiment, a method of mixing substances contained in a
receptacle
having a plurality of interconnected chambers is provided. In a first aspect
of this embodiment, the
method includes the steps of: forming a volume in a first chamber of the
receptacle, where the
volume comprises first and second substances and an immiscible liquid that is
preferably non-
29
CA 02743477 2011-05-12
reactive with the first and second substances; and moving the volume through a
connection between
the first chamber and a second chamber of the receptacle one or more times to
facilitate mixing of
the first and second substances in the presence of the immiscible liquid. The
first and second
chambers of this aspect may include flexible portions that yield to substance-
moving forces. In a
second aspect of this embodiment, the method includes the steps of. forming a
volume in a closed
chamber of the receptacle, where the volume comprises first and second
substances and a preferably
non-reactive, immiscible liquid, and where the closed chamber comprises a
flexible portion that
yields to substance-moving forces; closing the chamber to provide a
substantially fluid-tight seal; and
applying the substance-moving forces to different portions of the flexible
portion in an alternating
manner to mix the first and second substances in the presence of the
immiscible liquid within the
closed chamber. In preferred aspects, immiscible liquids were found to improve
mixing by, among
other things, generally concentrating the substances to be mixed toward the
centers of chambers.
The ratio of the immiscible liquid to other liquid substances in the chambers
is preferably from about
1:10 to about 10:1, and more preferably from about 1:3 to about 10:1.
[000100] To effect mixing in the first aspect of this embodiment, it is
preferred that substance-
moving forces are alternately applied to the flexible portions of the first
and second chambers during
the moving step. (The connection and/or second chamber may also be configured
to facilitate a
turbulent flow and mixing of the substances when moved from the first
chamber.) In this aspect, the
substance-moving forces are compressive forces that may be applied by one or
more actuators having
compression pads generally conforming to the shape of the flexible portions,
such as chamber walls,
of each of the first and second chambers. In the second aspect of this
embodiment, the substance-
moving forces are compressive forces that may include two or more actuators
having compression
pads that are arranged to generally conform to the shape of the flexible
portion of the closed
chamber. The actuators are preferably pneumatic actuators-
[000101] Alternatively, or in combination with an internal barrier (e.g.,
seal), the first and
second chambers may be isolated from each other by an external and retractable
compressive force
applied to the connection prior to the moving step. The external closing
force, alone or together with
an internal obstruction, provides a substantially fluid-tight seal and may
include one or more
actuators having compression pads that extend across the connection. Pneumatic
actuators are
CA 02743477 2011-05-12
particularly preferred.
[000102] The substances used to form the volume of this method of mixing are
typically
liquids, and the immiscible liquid may be, for example, an oil (e.g., a
mineral oil).
[000103] In still another embodiment, a receptacle having a plurality of
interconnected
chambers is provided, where one of the chambers is a first chamber containing
a process material
and a preferably non-reactive, immiscible liquid in an amount sufficient to
substantially coat an
interior surface of the first chamber, thereby hindering the process material
from sticking to the
interior surface. The process material is preferably a liquid, such as a
reconstitution reagent, and the
immiscible liquid is preferably an oil (e.g., a mineral oil). The ratio of the
immiscible liquid to the
process material is preferably about 1:10 to about 10:1, and more preferably
about 1:3 to about 10:1.
The first chamber is directly connected to a second chamber of the receptacle
and may comprise a
flexible portion, such as a compressible chamber wall, that yields to a
substance-moving force.
[000104] In yet another embodiment, a method of moving a process material in a
receptacle
having a plurality of interconnected chambers is provided, where the method
includes the steps of:
forming a volume in a first chamber of the receptacle, where the volume
comprises a process
material and an immiscible liquid in an amount sufficient to substantially
coat an interior surface of
the first chamber, thereby hindering the process material from sticking to the
interior surface of the
first chamber; and moving the volume from the first chamber to a second
chamber of the receptacle
through a connection between the first and second chambers. The above-
described receptacles for
use in coating the interior surface of a chamber may be used in this method.
[000105] In a further embodiment, a method of moving a liquid substance from a
first chamber
to a second chamber of a receptacle is provided, where the first and second
chambers are directly
connected to each other by an openable connection. The method includes the
steps of. forming a
volume in the first chamber comprising the liquid substance and a preferably
non-reactive,
immiscible liquid; and applying a substance-moving force to the volume of the
first chamber
sufficient to alter the openable connection from a closed state to an open
state and to move the
volume of the first chamber to the second chamber, where the volume in the
first chamber is
insufficient to alter the openable connection from the closed state to the
open state in the absence of
31
CA 02743477 2011-05-12
the immiscible liquid. The immiscible liquid may be, for example, an oil
(e.g., a mineral oil).
[000106] In a still further embodiment, a method of altering the temperature
of a liquid
substance contained in a chamber of a receptacle is provided. The method
includes a first step of
forming a volume in a first chamber of the receptacle so that a flexible
portion of a first chamber
wall expands to contact a stationary thermal element positioned adjacent the
first chamber. The
volume in the first chamber comprises a liquid substance and a corresponding
immiscible liquid,
where the presence of the immiscible liquid causes the flexible portion of the
first chamber to contact
the thermal element to a greater extent than if the immiscible liquid was
absent from the first
chamber. In a second step, the temperature of the liquid substance in the
first chamber is altered by
transmitting thermal energy between the thermal element and the volume. The
liquid substance of
this method may be a reagent for performing an assay, and the immiscible
liquid is preferably inert,
such that is does not react with any components of the assay. The ratio of the
immiscible liquid to
the remaining volume in the first chamber is preferably up to about 10:1.
Suitable immiscible liquids
include, for example, oils (e.g., a mineral oil).
[000107] One or more connections may be included for joining the first chamber
to one or more
other chambers of the receptacle, where the connections remain blocked at
least until a
predetermined temperature of the fluid volume is reached during the altering
step. At least one of the
connections may be an openable connection that can be altered from a closed
state to an open state
when a substance-moving force is applied to the contents of the process
chamber. In the closed state,
at least one connection may be blocked by an internal barrier and/or an
externally applied closing
force, such as an actuator.
[000108] The first chamber may also include a flexible portion of a second
chamber wall that
yields to a substance-moving force. The second flexible portion may be
positioned adjacent the
substance-moving force, which is located opposite the thermal element. The
substance-moving force
may include one or more actuators having compression pads generally conforming
to the shape of
the second flexible portion. The one or more actuators may be pneumatic
actuators. In this aspect,
the substance-moving force contacts the second flexible portion during the
method. The second
flexible portion may, but need not, expand during the fluid volume forming
step.
32
CA 02743477 2011-05-12
[000109] The thermal element may function alone or it may cooperate with the
substance-
moving force to alter the temperature of the fluid volume during the altering
step (e.g., the substance-
moving force can also function as a thermal conducting medium). See, e.g.,
Devaney et aL,
"Temperature Control Device and Reaction Vessel," U.S. Patent No. 5,460,780.
The thermal
element may include a heat transfer element formed from a thermally conductive
material, such as
aluminum or other metals or combinations of metals. The heat transfer element
may be placed in
thermal contact with a thermoelectric device to effect temperature changes. To
raise or lower the
temperature of the heat transfer element, the thermoelectric device may
operate on the Peltier effect.
The temperature of the fluid volume will generally be elevated during the
temperature altering step
and, for certain applications, may be cycled between different temperatures
(e.g., PCR thermal
cycling to effect binding and extension of primers in the presence of single-
stranded nucleic acid
templates and melting of double-stranded extension products).
[000110] In a still further embodiment, an instrument for processing a sample
in a receptacle
having a plurality of interconnected chambers is constructed and arranged to
support the receptacle in
an operative position during the processing. The instrument comprises one or
more thermal elements
defming one or more multiple chamber thermal zones. The multiple chamber
thermal zones are in
thermal communication with the receptacle and are constructed and arranged to
transmit thermal
energy between each multiple chamber thermal zone and an associated region of
the receptacle,
wherein the associated region of the receptacle encompasses all or a portion
of each of two or more
but less than all chambers of the receptacle. The instrument also includes a
controller programmed
to control operation of the one or more thermal elements defining the multiple
chamber thermal
zones to selectively heat or cool the chambers encompassed within the regions
associated with the
multiple chamber thermal zones-
[000111] In anther aspect, the instrument further comprises one or more
thermal elements
defining one or more single chamber thermal zones disposed to be in thermal
communication with
the receptacle and constructed and arranged to transmit thermal energy between
each single chamber
thermal zone and an associated region of the receptacle encompassing all or a
portion of one
chamber of the receptacle. The controller is also programmed to control
operation of the thermal
elements defining the single chamber thermal zones to selectively heat or cool
the chambers
33
CA 02743477 2011-05-12
encompassed within the regions associated with the single chamber thermal
zones.
[000112] The instrument may have anywhere from one to five thermal zones
including all
multiple chamber thermal zones or a combination of multiple chamber and single
chamber thermal
zones.
[000113] In anther aspect, the thermal elements comprise one or more Peltier'
devices
controlled by the controller to selectively heat or cool a body with which the
Peltier' device is in
thermal contact..
[000114] In a further aspect, the thermal elements comprise a heat transfer
element formed
from a thermally conductive material and having a peripheral shape
corresponding to a
predetermined shape of a multiple chamber thermal zone defined by the heat
transfer element. The
heat transfer element may be formed from aluminum.
[000115] In another aspect, the instrument further comprises a temperature
sensor in thermal
communication with the heat transfer element. The sensor is adapted to sense
the temperature of the
heat transfer element and communicate the sensed temperature to the
controller.
[000116] The heat transfer element may be held within the instrument in a
fixed position with
respect to the receptacle.
[000117] In another aspect, the thermal elements comprise one or more Peltier'
devices
controlled by the controller to selectively heat or cool a body with which the
Peltier' devices are in
thermal contact and a heat transfer element associated with each multiple
chamber thermal zone.
The heat transfer element is formed from a thermally conductive material and
has a generally flat
surface and a peripheral shape corresponding to a predetermined shape of a
multiple chamber
thermal zone defined by the heat transfer element, and the Peltier' devices
are in thermal contact with
the heat transfer element.
[000118] In another aspect, each multiple chamber thermal zone is separated
from other
multiple chamber thermal zones by isolating structure comprising a thermally
non-conductive
material. The heat transfer element and the isolating structure may be held
within the instrument in
34
CA 02743477 2011-05-12
fixed positions with respect to the receptacle.
[000119] The instrument may further include a heat dissipation element
constructed and
arranged to dissipate heat from the Peltier' devices. And the heat dissipation
element may comprise
a heat sink formed from a conductive material and including a block having one
side in thermal
communication with at least one Peltier' device and an opposite side from
which heat dissipation
fins extend from the block. In one embodiment, the heat sink may be made from
aluminum.
[000120] In another aspect, the heat dissipation element may further comprises
a fan disposed
adjacent the heat sink and configured to generate an air flow over the heat
dissipation fins of the heat
sink. Operation of the fan may be controlled by the controller.
[000121] In another aspect, the instrument may comprise one or more
temperature sensors for
sensing the temperature of each thermal zone and communicating the sensed
temperature to the
controller.
[000122] In another aspect, the controller is configured to control the
operation of the thermal
elements to establish an ambient temperature within a prescribed temperature
range. The prescribed
temperature range may be about 20 C to 40 C or about 25 C to 37 C.
[000123] In another aspect, the controller is configured to control operation
of the thermal
elements to heat one or more chambers to temperatures within a prescribed
temperature range. The
prescribed temperature range of the chambers may encompass temperatures
required to perform a
process requiring thermal cycling. The prescribed temperature range may be
about 5 C to 95 C.
[000124] In another aspect, the process may be a PCR amplification reaction.
[000125] In another aspect, the controller may be configured to control
operation of the thermal
elements to heat or cool the contents of chambers encompassed within a region
associated with the
multiple chamber thermal zone to a predetermined temperature for a
predetermined period of time.
[000126] In a further aspect, the when a receptacle is supported in the
operative position within
the instrument, filling a chamber encompassed within the region associated
with the multiple
chamber thermal zone with fluid will increase thermal communication between
the chamber and the
CA 02743477 2011-05-12
multiple chamber thermal zone.
[000127] In a still further embodiment, a method for heating or cooling
substances within a
receptacle having a plurality of interconnected chambers comprises the steps
of positioning the
receptacle in thermal communication with one or more multiple chamber thermal
zones contained in
an analyzer. Each multiple chamber thermal zone is associated with a region of
the receptacle
encompassing all or a portion of each of two or more but less than all
chambers of the receptacle.
The method further includes transmitting thermal energy between each multiple
chamber thermal
zone and the chambers encompassed by the region associated with the multiple
chamber thermal
zone to selectively heat or cool substances contained within the encompassed
chambers to a
temperature different than the temperature of the chambers encompassed by at
least one other region.
[000128] In another aspect, the method further comprising positioning the
receptacle in thermal
communication with one or more single chamber thermal zones contained in the
analyzer, each
single chamber thermal zone being associated with a region of the receptacle
encompassing all or a
portion of one chamber of the receptacle.
[000129] In another aspect, the transmitting step comprises heating or cooling
the thermal zone
with one or more Peltier' devices.
[000130] In another aspect, the ambient temperature of the analyzer is
different than the
temperature of substances contained within chambers encompassed by the regions
of the receptacle
associated with each thermal zone during the method-
[000131] In another aspect, the transmitting step comprises alternately
heating and cooling at
least one of the multiple chamber thermal zones.
[000132] In another aspect, the method further comprises thermally separating
each thermal
zone from other thermal zones.
[000133] In another aspect, the method further comprises dissipating heat from
the thermal
zone.
[000134] In another aspect, the method further comprises sensing the
temperature of each
36
CA 02743477 2011-05-12
thermal zone.
[000135] In another aspect, the method further comprises expanding a chamber
encompassed
by the region of the receptacle associated with a multiple chamber thermal
zone to increase the
thermal communication between the expanded chamber and the associated multiple
chamber thermal
zone.
[000136] In another aspect, the analyzer contains at least three multiple
chamber thermal zones.
[000137] In another aspect, each of the plurality of interconnected chambers
is adjacent to at
least one other chamber of the receptacle.
[000138) These and other features, aspects, and advantages of the present
invention will
become apparent to those skilled in the art after considering the following
detailed description,
appended claims and accompanying drawings.
BRIEF DESCRIPTION OF THE FIGURES
[000139] Figures lA-1 C are plan views illustrating a multi-chambered
receptacle embodying
aspects of the current invention.
[000140] Figure 2 is schematic block diagram of the functional architecture of
a system
embodying aspects of the present invention.
[000141] Figure 3 is an exploded perspective view of an automated instrument
embodying
aspects of the present invention.
[000142] Figure 4 is a schematic view illustrating an arrangement of
compression pads of a
pressure mechanism cluster of the instrument.
[000143] Figure 5 is a plan view of a front side of a door assembly of the
instrument.
(000144] Figure 6 is an exploded perspective view of a fluorometer embodying
aspects of the
present invention.
37
CA 02743477 2011-05-12
[000145] Figure 7 is a perspective view of a rear housing of the fluorometer.
[000146] Figure 8A is an end view of the rear housing of the fluorometer.
[000147] Figure 8B is a cross-section of the rear housing taken along the line
8B-8B of Figure
8A.
[000148] Figure 8C is a cross-section of the rear housing taken along the line
8C-8C of Figure
8B.
[000149] Figure 9A is an end view of the fluorometer.
[000150] Figure 9B is a cross-section of the fluorometer taken along the line
9B-9B of Figure
9A.
[000151] Figure 9C is a cross-section of the fluorometer taken along the line
9C-9C of Figure
9B.
[000152] Figures 1 OA and 1 OB are a side and top view, respectively, of an
embodiment of a
compression pad integrated with a signal detector.
[000153] Figure 11 is a perspective view of an alternative embodiment of a
compression pad
integrated with a signal detector.
[000154] Figure 12A is a plan view illustrating an alternative embodiment
multi-chambered
receptacle embodying aspects of the current invention.
[000155] Figure 12B is an exploded perspective view of the receptacle of
Figure 12A.
[000156] Figure 13 is a front perspective view of an alternative embodiment of
an automated
instrument embodying aspects of the present invention.
[000157] Figure 14 is a rear perspective view of the instrument of Figure 13,
with a top portion
of an exterior housing removed to show the interior of the housing.
[000158] Figure 15 is a front perspective view of the instrument of Figure 13,
with the top
38
CA 02743477 2011-05-12
portion of the exterior housing removed.
[000159] Figure 16 is a front view illustrating an arrangement of compression
pads of a
pressure mechanism cluster of the instrument of Figure 13.
[000160] Figure 17A is a rear perspective view of an air manifold and attached
components of
the instrument of Figure 13.
[000161] Figure 17B is a circuit diagram of the pneumatic system of the
instrument.
[000162] Figure 18 is a cross-section of an alternative embodiment of a
compression pad
integrated with a signal detector
[000163] Figure 19 is a cross-section of a compression pad integrated with a
magnet actuator.
[000164] Figure 20 is an exploded perspective view of a temperature control
system of the
instrument of Figure 13.
[000165] Figure 21 is a schematic view of interconnection circuitry and power
supplies for the
fluorometer of Figures 6-9C..
[000166] Figure 22 is a schematic view of control, processing and
communication circuitry for
the fluorometer.
[000167] Figure 23 is a schematic view of circuitry for voltage measurement
and LED control
for the fluorometer.
[000168] Figure 24 is a schematic view of LEDs, RF shielding, and power
filtering circuitry for
the fluorometer.
[000169] Figure 25A is a schematic view of a first front-end amplifier circuit
for the
fluorometer.
[000170] Figure 25B is a schematic view of a second front-end amplifier
circuit for the
fluorometer.
39
CA 02743477 2011-05-12
[000171] Figure 26 is a schematic view of a demodulation circuit for the
fluorometer.
[000172] Figure 27 is a graph showing relative fluorescent units detected
versus time for a set
of manually performed real-time amplification reactions.
[000173] Figure 28 is a graph showing relative fluorescent units detected
versus time for a set
of real-time amplification reactions carried out using liquid reagents and
receptacles and instruments
embodying aspects of the invention.
[000174] Figure 29 is a graph showing relative fluorescent units detected
versus time for a set
of real-time amplification reactions carried out using a urine sample, liquid
reagents and receptacles
and instruments embodying aspects of the invention.
[000175] Figure 30 is a graph showing relative fluorescent units detected
versus time for a set
of real-time amplification reactions carried out using dried reagents and
receptacles and instruments
embodying aspects of the invention.
[000176) Figure 31 is a graph showing relative fluorescent units detected
versus time for a set
of real-time amplification reactions carried out with and without oil in the
amplification and/or
enzyme reagents.
GENERAL OVERVIEW OF THE INVENTION
[000177] The present invention relates to multi-chambered receptacles that can
be used to
perform one or more manual or automated processes with a sample of interest,
such as determining
the chemical composition of a substance, measuring the activity of a group of
metabolic enzymes, or
testing for the presence of one or more analytes in a sample. Some or all of
the chambers of the
receptacles are interconnected by means of blocked or sealed passages that can
be permanently or
temporarily opened to permit the movement of substances between chambers. The
arrangement of
chambers within the receptacles permits complex processes to be performed by
allowing different
steps of a process or multiple processes to be performed non-sequentially
and/or simultaneously.
[000178] Receptacles of the present invention maybe constructed of flexible or
rigid materials,
as well as combinations thereof, provided the receptacles permit substances to
be forced or drawn
CA 02743477 2011-05-12
between chambers. At least some of the chambers can be pre-loaded with process
materials (e.g.,
reagents) and then enclosed or sealed off from the environment prior to
loading sample material.
The process and sample materials may be comprised of liquids, solids, gases,
or combinations
thereof. Once sample material is loaded into a chamber or chambers, the
receptacle may be sealed or
otherwise closed to maintain all materials within the receptacle during a
procedure. Alternatively, a
sample chamber may remain open after a procedure has been initiated so that
some or all of the
sample material is added to the receptacle after the procedure has begun.
[000179] The passages interconnecting the chambers are sized and arranged to
permit
substances to pass between adjacent chambers. The substances are preferably
fluids or fluidized
substances and may include, for example, gels, emulsions, suspensions and
solids, where the solids
may be transported through the passages alone or using, for example, a fluid
carrier, such as an inert
oil. Barriers are provided to block the movement of substances through the
passages until such
movement is desired. The receptacles, or the receptacles in cooperation with
an automated
instrument, can include one or multiple types of barriers. Such barriers maybe
constructed from the
materials of the receptacle (e.g., openable seals), or they may be fixed,
movable or alterable
components or substances positioned adjacent to or inserted into the passages
(e.g., valves,
magnetically-responsive particles, or heat-sensitive wax plugs), or they may
be components of the
automated instrument that apply a reversible, compressive force to the
passages (e.g., actuators).
[000180] Altering or removing a barrier between adjacent chambers allows a
substance present
in one chamber to be forced or drawn into an adjacent chamber. This movement
may be achieved
by, for example, the action of a pressure source, such as an actuator or a
group of actuators that are
adapted to apply pressure to the exterior of the chamber to thereby collapse
or partially collapse the
chamber to force all or a portion of the material between chambers. The
material may be moved
unidirectionally or bidirectionally between chambers where, for example, a
mixing of combined
materials is desired.
[0001811 The chambers are arranged in the receptacle so that there are at
least two non-linear
paths that allow for steps of a process to be performed independent of each
other. This provides a
tremendous advantage in that the substances of two or more sets of chambers
can be mixed or
combined before the resulting mixtures or combinations, or the underlying
substances of the separate
41
CA 02743477 2011-05-12
sets of chambers, are contacted with each other. By permitting process steps
to be performed
independent of each other, complex procedures having a series of steps that
cannot or are preferably
not performed linearly (i.e., steps are performed sequentially) can be
performed with the receptacles
of the present invention.
[000182] The compact design of the receptacles and systems of the present
invention makes
them especially suitable for use in point-of-care and field applications. By
sealing off chambers pre-
loaded with the process materials needed to carry out a process, contamination
and user error issues
are substantially minimized. The receptacles of the present invention are also
ideal for unit dose
testing, where chambers of the receptacles are pre-loaded with the precise
amounts of process
materials required to conduct a test.
DETAILED DESCRIPTION OF THE INVENTION
[000183] While the present invention may be embodied in a variety of forms,
the following
description and accompanying figures are merely intended to disclose some of
these forms as
specific examples of the present invention. Accordingly, the present invention
is not intended to be
limited to the forms or embodiments so described and illustrated.
Definitions
[000184] The following terms have the following meanings unless expressly
stated to the
contrary. It is noted that the term "a" or "an" entity refers to one or more
of that entity; for example,
"an analyte," is understood to represent one or more analytes. As such, the
terms "a" or "an," "one or
more," and "at least one" can be used interchangeably herein.
[000185] Adjacent/Adjacently. With reference to chambers, the term "adjacent"
or
"adjacently" means that the referred to chambers adjoin each other (i.e., the
chambers are positioned
directly next to each other in a receptacle). The only structure separating
adjacent chambers of a
receptacle when a process is initiated is a seal. Thus, adjacently positioned
chambers are not
connected to each other by channels or passageways.
[000186] Amplification/Amplification Reaction. By "amplification" or
"amplification
reaction" is meant a procedure for increasing the amount, concentration or
detestability of a
42
CA 02743477 2011-05-12
substance that is indicative of the presence of an analyte in a sample.
[000187] Amplification Conditions. By "amplification conditions" is meant
temperature
conditions adequate to effect an amplification reaction.
[000188] Amplification Oligonucleotide. By "amplification oligonucleotide" is
meant an
oligonucleotide that binds to a target nucleic acid, or its complement, and
participates in a nucleic
acid-based amplification reaction.
[000189] Amplification Product. By "amplification product" is meant a nucleic
acid
generated in a nucleic acid-based amplification reaction that contains a
target sequence for detection.
[000190] Amplification Reagent. By "amplification reagent" is meant a material
containing
one or more components needed for an amplification reaction. In a nucleic acid-
based amplification
reaction, such components may include amplification oligonucleotides (e.g.,
primers and/or
promoter-primers), nucleoside triphosphates, and/or cofactors needed for
amplification of a target
nucleic acid sequence (e.g., divalent cations such as Mg+ ).
(000191] Analyte. By "analyte" is meant a sample, or a component of a sample,
that is
undergoing analysis.
[000192] Assay. By "assay" is meant a qualitative or quantitative analysis of
one or more
analytes.
[000193] Barrier. By "barrier" is meant a structure or material that impedes
or prevents the
movement of substances between spaces.
[000194] Blocked. By "blocked" is meant closed to the movement of a substance.
[000195] Binding Agent. By "binding agent" is meant a molecule or molecular
complex
capable of binding to a component of a sample or reaction mixture. The binding
agent may be, for
example, an antibody, antigen, peptide, protein, nucleic acid or analog
thereof, organic molecule, or
complex of any of the foregoing (e.g., antibody:nucleic acid complex).
[000196] Burstable Seal. By "burstable seal" is meant a seal that ruptures or
peels when
43
CA 02743477 2011-05-12
sufficient pressure is applied to the seal.
[000197] Capture Agent. By "capture agent" is meant a binding agent capable of
binding to
an analyte and of being directly or indirectly bound to a solid support.
[000198] Capture Probe. By "capture probe" is meant a binding agent capable of
binding to a
nucleic acid analyte.
[000199] Chamber. By "chamber" is meant a distinct section or space within a
receptacle.
[000200] Chamber-Defining Member. By "chamber-defining member" is meant the
whole
(e.g., bladder) or a part (e.g., wall) of what determines the volume of a
chamber. The chamber-
defining member may consist of a single component (e.g., one layer) or
multiple components (e.g., a
plurality of layers bonded together).
[000201] Closed/Closing. With reference to a chamber, "closed" or "closing"
means that a
chamber of a receptacle is not in fluid communication, or the chamber is
placed in a condition in
which it is not in fluid communication, with any other chamber of the
receptacle. With reference to a
sample-holding receptacle, "closed" means that all chambers of a receptacle
are maintained in a
substantially airtight environment relative to the ambient environment. With
reference to a
receptacle prior to sample addition, "closed" means that all chambers of a
receptacle except a
sample-receiving chamber are maintained in a substantially airtight
environment relative to the
ambient environment.
[000202] Concentrate. By "concentrate" is meant to limit dispersion of one or
more
components within a chamber.
[000203] Contiguous Path of Chambers. By "contiguous path of chambers" is
meant a series
of adjacently connected chambers.
[000204] Directly Connected. By "directly connected" is meant that there are
no intervening
chambers in the connection between two referred to chambers.
[000205] Distinct Connection. By "distinct connection" is meant a connection
that is separate
44
CA 02743477 2011-05-12
from and non-overlapping with any other connection of a receptacle.
[000206] End Chambers. By "end chambers" is meant the outermost chambers of a
linear
path of chambers.
[000207] Enzyme Reagent. The phrase "enzyme reagent" refers to a material that
contains at
least one enzyme that participates in a process. In a nucleic acid-based
amplification reaction, the
enzyme reagent may contain one or more enzymes that catalyze the synthesis of
DNA and/or RNA
polynucleotides using an existing strand of DNA or RNA as a template. Examples
of such enzymes
include DNA-dependent DNA polymerases (e.g., DNA polymerase I from E. coli and
bacteriophage
T7 DNA polymerase), DNA-dependent RNA polymerases or transcriptases (e.g., DNA-
dependent
RNA polymerases from E. coli and bacteriophages T7, T3 and SP6), RNA-dependent
DNA
polymerases or reverse transcriptases, and RNA-dependent RNA polymerases
(e.g., an RNA
replicase).
[000208] Flexible. By "flexible" is meant a property of a material that allows
it yield to a
reasonable force without tearing or breaking.
[000209] Fluid. By "fluid" is meant a substance that tends to flow or to
conform to the shape
of its receptacle (e.g., a liquid or gas). The fluid may be a fluidized
substance or mixture of liquids
or gases, such as an emulsion. As used herein, the term "fluid" also refers to
a substance, such as a
paste, that yields to pressure by changing its shape.
[000210] Fluidized. By "fluidized" is meant a substance that has been altered
so that it is in a
form or medium that has fluid characteristics.
[000211] Immiscible Fluid. By "immiscible fluid" is meant a fluid that does
not mix with one
or more liquids contained in a receptacle.
[000212] Immunoassay. By "immunoassay" is meant an assay which involves an
antibody-
antigen interaction.
[000213) Independently Combining. The phrase "independently combining" means
separately combining two or more sets of substances in distinct chambers of a
receptacle, where the
CA 02743477 2011-05-12
separate combinations of substances do not come into contact with each other.
[000214] Indirectly Connected. By "indirectly connected" is meant that there
are one or more
intervening chambers in the connection between two referred to chambers-
[000215] Interconnected. The term "interconnected" refers to chambers that are
fluidly
connected or connectable, as in the case of an openable connection.
[000216] Intermediate Between. The phrase "intermediate between" means that
the
referenced chamber is located between and in the same linear path as each of
two other identified
chambers, or that the referenced chamber is located between and in a different
linear path with each
of two other identified chambers.
[000217] Intermediate Chamber. By "intermediate chamber" is meant a chamber
that is
connected by openable connections to at least two other chambers.
[000218] Isolated. By "isolated" is meant that one or more components of a
sample are
sequestered from one or more other components of the sample.
[000219] Label. By "label" is meant any substance having a detectable
property.
[000220] Linear Path. By "linear path" is meant a contiguous path of
consecutively ordered
chambers interconnected by a plurality of openable connections and defined by
a first end chamber, a
last end chamber, and one or more intermediate chambers disposed between the
first and last end
chambers.
[000221] Non-Circular Arrangement. The phrase "non-circular arrangement"
refers to an
arrangement of interconnected chambers that includes two or more linear paths,
where the end
chambers of the linear paths are not circularly arranged about a central
chamber.
[000222] Non-linear. By "non-linear" is meant at least two contiguous paths of
consecutively
ordered chambers that share less than all chambers in common.
[000223] Non-sequential. By "non-sequential" is meant that certain steps of a
process are
performed independent of each other rather than in sequence.
46
CA 02743477 2011-05-12
[000224] Nucleic Acid-Based Amplification. By "nucleic acid-based
amplification" is meant
an amplification reaction that is dependent upon the presence of a nucleic
acid.
[000225] Oligonucleotide. By "oligonucleotide" is meant a polymeric chain of
at least two,
generally between about five and about 100, chemical subunits, each subunit
comprising a nucleotide
base moiety, a sugar moiety, and a linking moiety that joins the subunits in a
linear spatial
configuration. Common nucleotide base moieties are guanine (G), adenine (A),
cytosine (C),
thymine (T) and uracil (U), although other rare or modified nucleotide bases
able to hydrogen bond
are well known to those skilled in the art. Oligonucleotides may optionally
include analogs of any of
the sugar moieties, the base moieties, and the backbone constituents.
Preferred oligonucleotides of
the present invention range in size from about 10 to about 100 residues.
Oligonucleotides maybe
purified from naturally occurring sources, but preferably are synthesized
using any of a variety of
well-known enzymatic or chemical methods.
[000226] Openable Connection. By "openable connection" is meant a passage that
can be
temporarily or permanently altered from a "closed" state in which the
connection is blocked by a
barrier to an "open" state in which the barrier has been modified or moved so
that a substance can
pass through an opening in the passage.
[000227] Optically Transmissive. The phrase "optically transmissive" is a
reference to
materials permitting the passage of light, so that light emitted on one side
of the materials is
detectable by an optical device positioned on the opposite side of the
materials.
[000228] Primer. By "primer" is meant an amplification oligonucleotide capable
of being
extended at its 3'-end in the presence of a polymerase in a template-dependent
manner.
[000229] Probe. By "probe" is meant a binding agent that binds to an analyte
or other
substance in a reaction mixture to form a detectable probe:target complex
indicative of the presence
of the analyte in a sample under the conditions of a process. For nucleic acid-
based reactions, the
probe comprises an oligonucleotide having a base sequence sufficiently
complementary to a nucleic
acid sequence indicative of the presence of a target nucleic acid to form a
detectable probe:target
complex therewith. A probe may also include non-complementary sequences, such
as a sequence for
47
CA 02743477 2011-05-12
immobilizing the probe on a solid support, a promotor sequence, a binding site
for RNA
transcription, a restriction endonuclease recognition site, or sequences which
will confer a desired
secondary or tertiary structure, such as a catalytic active site or a hairpin
structure, which can be used
to facilitate detection and/or amplification. Probes of a defined sequence may
be produced by
techniques known to those of ordinary skill in the art, such as by chemical
synthesis, and by in vitro
or in vivo expression from recombinant nucleic acid molecules.
[000230] Process. By "process" is meant a series of actions, changes or
functions performed
on or with a substance to bring about a result.
[000231] Purified. By "purified" is meant that one or more components of a
sample are
removed from one or more other components of the sample.
[000232] Reagent. By "reagent" is meant any non-sample substance used in a
process,
including reactants in a chemical, biochemical or biological reaction,
diluents, solvents, wash
materials, rinse materials, buffers and the like.
[000233] Real-Time. The phrase "real-time" means that a characteristic of a
reaction is or is
capable of being detected as the reaction is occurring.
[000234] Receptacle. By "receptacle" is meant a device having a plurality of
interconnected
chambers capable of receiving and/or holding substances.
[000235] Reconstitution Reagent. By "reconstitution reagent" is meant a
reagent used to alter
a non-fluid process material to a fluid or fluidized state.
[000236] Sample. By "sample" is meant a substance capable of being subjected,
in whole or in
part, to a process.
[000237] Sample Processing Reagent. By "sample processing reagent" is meant a
reagent that
alters or is useful for altering the original state of a sample.
[000238] Sample Receiving Chamber. By "sample receiving chamber" is meant a
chamber of
a receptacle that is open or openable for receiving a sample to be processed.
48
CA 02743477 2011-05-12
[000239] Seal. By "seal" is meant a barrier formed between adjacent chambers.
The seal may
be, for example, a heat seal formed between opposed thermoplastic sheets.
[000240] Target Nucleic Acid. By "target nucleic acid" is meant a nucleic acid
analyte.
[000241] Target Sequence. By "target sequence" is meant a nucleic acid
sequence contained
within a target nucleic acid or its complement that is amplified and/or
detected in a detection assay.
[000242] Water Vapor Transmission Rate. By "water vapor transmission rate" or
"WVTR"
is meant the steady state rate at which water vapor permeates through a
material at specified
conditions of temperature and relative humidity. Water vapor transmission rate
values are expressed
in g/m2/24 hrs. A PERMATRAN-W water vapor permeation instrument available
from MOCON,
Inc. of Minneapolis, MN (Model 3/33) can be used to measure WVTR in accordance
with ASTM F
1249.
Multi-Chambered Receptacles
[000243] Receptacles in accordance with the present invention include a
plurality of
interconnected, non-linearly arranged chambers arrayed to perform one or more
processes. The
precise dimensions of the receptacles will depend upon the number and
arrangement of the
chambers, as well as the volume of substances to be loaded or moved into the
chambers. The
receptacles preferably have relatively broad surface dimensions in relation to
their thicknesses,
although this is not a requirement. The receptacles are preferably formed from
top and bottom
portions, each of which may be made with rigid and/or flexible materials. In a
preferred mode, at
least one of the top and bottom portions of the preferred receptacles is at
least partially flexible.
[000244] The non-linear arrangement of chambers allows for non-sequential
processing of
samples in the receptacles. The exact number, configuration, sizes and
arrangement of chambers
will depend on the particular process or processes to be performed. Distinct
from the surrounding
receptacle materials, the chambers may be constructed of rigid or flexible
materials or combinations
thereof. Material selection will depend in part on whether the chambers must
yield to an external
pressure in order to move substances between chambers. The chambers may be of
any shape that
does not interfere with the movement of substances between chambers, which
includes generally
49
CA 02743477 2011-05-12
planar or bubble-like shapes, including hemispherical and spherical shapes. In
a preferred
embodiment, the chambers are generally flat and have a tear-drop shape that
funnels substances
through an open connecting passage and into an adjacent chamber. Tear-drop
shaped chambers also
advantageously focus pressure on connecting passages, thereby more readily
opening barriers such as
seals and valves used to temporarily block access to adjacent chambers. The
chambers may have the
same or different shapes and/or sizes.
[000245] Passages used to connect the chambers of a receptacle maybe, for
example, portals or
passageways that are dimensioned to permit substances used in a process to
move between chambers.
In the case of a portal, a seal or other barrier may be substantially all that
separates adjacent
chambers. A passageway on the other hand comprises a conduit extending between
chambers.
Portals are preferred because they permit a more compact receptacle design and
require materials to
travel shorter distances between the various chambers. Some of the passages
may remain open
throughout a process, such as a passage leading to a waste chamber, while
others are blocked by
barriers until it is desired to move a substance between chambers. The
barriers can be selectively
altered from "closed" to "open" states to create substance-transferring
connections, which are
preferably fluid-transferring connections for moving fluids and substances in
a fluidized form. A
barrier may be an external force, such as a compressive force provided by, for
example, a clamping
device (e.g. pneumatically driven actuator having a clamping pad), or it may
be a seal or valve that
yields to pressure, is mechanically operated, or is altered by, for example,
heat, laser ablation, or a
chemical or biochemical reaction to provide an opening between chambers, or it
may be an external
force/seal combination. In a preferred embodiment, the passages are blocked
with a heat seal, such
as a V-shaped or chevron seal, that is reinforced with a compressive force
during use. While the
blocking properties of some barriers are designed to be affected by conditions
such as heat (e.g., wax
plugs), or may be affected by a chemical agent moved into or formed in a
chamber, barriers formed
or positioned in a receptacle should not otherwise be influenced by
environmental conditions or
substances that are contained within the chambers.
[000246] Once a barrier separating adjacent chambers has been removed or
altered to create an
opening, a substance may be pushed, pressed, drawn or otherwise moved, such as
by gravity, into a
neighboring chamber. If the chambers are to be acted upon by a pressing force,
such as a roller or an
CA 02743477 2011-05-12
actuator-driven compression pad, to move substances between chambers, then the
chambers are
formed to have at least one flexible surface that yields to the pressure of
the pressing force.
Otherwise, the chambers may be constructed of a rigid material, as in the case
of pads or vacuums
used to push or draw substances into adjacent chambers. This is also true
where gravity draws a
substance from a chamber that is positioned above a receiving chamber,
although in some
applications it is desirable for both chambers to have at least one flexible
surface so that substances
can be moved back-and-forth between chambers in order to mix two substances.
[000247] The chambers are arranged in the receptacles so that there are at
least two distinct
linear paths. Each of the paths includes at least three contiguously connected
chambers, with at least
one of the paths preferably including five or more contiguously connected
chambers. As used
herein, the phrase "contiguously connected chambers" refers to a series of
directly connected
chambers in which the chambers are successively arranged, with one chamber
coming after another.
Each of the paths may share at least one but less than all chambers in common
with any other path in
the receptacle. This arrangement of chambers allows certain steps of a process
to be performed
independently and/or simultaneously. In this way, substances used in a complex
process can be
prepared and kept segregated until it desirable to combine them. Such
independent activities may
include, for example, dissolving, diluting, mixing, combining or reacting
substances. By way of
example only, one set of adjacent chambers may contain a dried, primer-
containing amplification
reagent for use in amplifying a target nucleic acid sequence present in a
sample and a reagent for
reconstituting the amplification reagent, while another set of adjacent
chambers may contain a dried
enzyme/probe reagent for use in amplifying and detecting the target nucleic
acid sequence and a
reagent for reconstituting the enzyme reagent. If, in this particular example,
the amplification and
enzyme/probe reagents are prematurely combined in their reconstituted forms,
there is some risk of
target-independent amplification that could interfere with the amplification
of the target or consume
scarce reagents for amplification. See, e.g., Adams et al., "Decoy Probes,"
U.S. Patent No.
6,297,365. Therefore, it is desirable to separately reconstitute these
reagents and then to combine
them in the presence of the target nucleic acid.
[000248] For illustration purposes only, Figure 1 C shows a receptacle 10
having a non-linear
arrangement of chambers that defines a number of distinct linear paths. Some
of the possible linear
51
CA 02743477 2011-05-12
paths of this receptacle are identified with letter designations, each chamber
of a linear path having
the same letter designation (i.e., A, B, C or D), and each chamber of a linear
path being assigned a
distinct number. The arrangement of the paths is such that the steps of a
process can be performed
non-sequentially. For example, a sample material can be added to chamber A2
and mixed with a
binding agent provided from chamber A l before isolating and purifying an
analyte in chamber A3.
At the same time or different times, a first dried or solid process material
in chamber B2 can be
reconstituted with a first reconstitution reagent (e.g., a first solvent)
provided from chamber BI.
Also, at the same or different times, a second dried or solid process material
in chamber B4 can be
reconstituted with a second reconstitution reagent (e.g., a second solvent)
provided from chamber
B5. Finally, the purified analyte and the reconstituted first and second
process materials can be
combined in chamber B3 or B4 for detection of the analyte. While there are
only four distinct linear
paths identified in Figure I C, it is readily apparent that other possible
linear paths and combinations
of linear paths could be utilized.
[000249] The non-linear arrangement of chambers in the receptacles can
facilitate their use in
performing multiple processes of the same or different kind. This is because
the chambers can be
arranged so that substances and/or chambers that are not shared between
processes remain isolated
during a process. Thus, the present invention also relates to "universal"
receptacles, where a single
receptacle can be designed and manufactured, including pre-loading of process
materials, for
multiple applications. In this way, the end-user does not need to have a
different receptacle for each
process to be performed or to predict the volume requirements for any
particular process in advance.
Materials used to form the chambers and barriers of the present invention
should be selected to
maintain acceptable stability and reactivity levels of the various substances
used to perform a
process. Such materials may provide, for example, a moisture barrier for
substances that are altered,
degraded or otherwise affected by moisture. In an alternative or complementary
approach, a
desiccant, such as calcium oxide, may be loaded with a dried process material
to minimize the affects
of moisture in a chamber. When a process is being performed in a receptacle,
it may be desirable to
sequester the desiccant to prevent it from interfering with or altering a
reaction involving the dried
process material. The desiccant can be sequestered by, for example, locating
it in a section of a
chamber containing the dried process material or by placing the desiccant in
an adjacent chamber
having an open connection with the chamber holding the dried process material.
It maybe desirable
52
CA 02743477 2011-05-12
to block this open connection after reconstituting the dried process material
to prevent unwanted
interactions with the reconstituted process material. Yet another approach
would be to store the
receptacle in a vessel formed from a material or materials that provide a
moisture barrier and/or
which includes a desiccant. Common desiccants include clay, silica gels,
calcium oxide and
synthetic molecular sieves. An example of a molecular sieve is a Type 4A
Molecular Sieve
MultiformTM Tablet having a 0.45" diameter and a 0.125" height, where "Type
4A" indicates a pore
size of 4 angstroms (Multisorb Technologies, Inc., Buffalo, NY; Product No. 02-
00674AH01).
[000250] Similarly, receptacle materials may be selected to protect substances
from the
environmental affects of exposure to oxygen or electromagnetic radiation.
Alternatively, the
materials used to form the receptacles may be selected to prevent stored
substances from adversely
interacting with each other by selecting materials that provide a barrier
against transmission of any
liquid, solid or gas intended for use in the receptacle: It may also be
desirable to use materials in
constructing the receptacles that protect against evaporation of substances to
prevent the activity of
those substances from being altered. Further, the materials selected for use
should not significantly
alter the intended functions of the stored substances, nor should they adhere
to or otherwise bind
reactants in a manner that significantly affects their ability to participate
in a process or processes.
[000251] Where a procedure involves sample manipulations that require
concentrating or
moving magnetic particles within or between chambers, at least a portion of
the receptacle will need
to be constructed of materials that do not substantially interfere with the
influence of magnetic fields
generated by adjacently positioned magnets. For processes requiring heating
and/or cooling of all or
some of the chambers, either continuously or for precise periods of time, the
receptacles must be
capable of an energy transfer on at least one side of the receptacles that is
capable of affecting the
thermal conditions of the contents of chambers requiring heating and/or
cooling. Additionally, the
receptacles may include optically transparent portions capable of transmitting
light of the visible,
infrared and/or ultraviolet spectrum to detect changes in the physical
characteristics of a sample, such
as color or turbidity, or to enable the detection of labels that are
indicative of the presence of analytes
of interest.
[000252] Receptacles of the present invention may be constructed from such
materials as
polymers, glass, silicon, metals, ceramics and combinations thereof. The
materials used will depend,
53
CA 02743477 2011-05-12
in part, on the means selected for moving substances between chambers, whether
a physical change
in the sample must be visualized or a light signal detected, and the manner in
which substances
present in the receptacles, such as magnetic particles, are to be manipulated.
The materials used to
construct receptacles should be stable under the expected transportation,
storage and use conditions.
Such conditions include temperature, pressure, humidity and duration of
storage. Also, materials
used to form flexible chambers of the receptacles should yield to the selected
pressure forces for
moving substances between chambers without being torn, punctured or ruptured.
[000253] In a preferred embodiment, the receptacles are formed from flexible
top and bottom
sheets that are of the same or different materials and may be multilaminates.
Each sheet preferably
has at least one liquid-impervious layer, and the sheets preferably have a
relatively uniform
thickness, which may be between about 0.05 mm and 2.0 mm. Also, select regions
of one of the
multilaminates are preferably optically transparent or translucent. The sheets
may be formed from,
for example, foils - with one or more holes cut in the foil to provide
detection windows as necessary
- and/or thermoplastic materials such as polypropylene (e.g., Reflex
polyolefins available from the
Rexene Corporation, Dallas, TX), polyester, polyethylene (e.g., polyethylene
teraphthalate ("PET")
and polyethylene naphthalate ("PAN")), polyvinyl chloride, polyvinylidene
chloride, polycarbonate
resins (e.g., polyvinyl fluoride films) and polyurethane. In a particularly
preferred embodiment, the
top and bottom sheets are each multilaminates, an example of which is a
Scotchpak film layer (3M
Corporation, St. Paul, MN; Cat. No. ES-48) bonded to a Perflex foil layer
(Perfecseal, Oshkosh,
WI; Product No. 35786). Other suitable materials for forming the flexible
sheets of this embodiment
will be appreciated by those skilled in the art. See, e.g., Burke (1992) WAAC
Newsletter 14(2):13-
17.
[000254] Exemplary laminates include: foil coated PET with Surlyn blend peel
layer (4.5
mils), clear double AlOx coated PET on low-density polyethylene ("LPDE") with
coextruded peel
layer (4.5 mils), foil coated LDPE with coextruded peel layer (4.5 mils), foil
coated LDPE seal layer
(3.5 mils), clear single AlOx coated PET on Biaxial Oriented Polyamide with
peel layer (4 mils),
clear AlOx coated PET on LDPE seal layer with zone coat defined frangible seal
(2.5 mil), foil
barrier with peel sealant. (3.5 mil), clear AIOx coated PET on LDPE seal layer
(2.5 mil), clear AlOx
coated PET on LDPE seal layer, (4 mil), PET coated Foil with HDPE seal layer,
clear AIOx coated
54
CA 02743477 2011-05-12
PET with EVA based peel layer, (3 mil), and foil coated PET with Surlyn blend
peel layer (4.5
mils), as well as laminates of opaque polyethylene terephthalate ("OPET"),
ink, white LDPE,
aluminum foil, polyethylene ("PE"), linear low-density polyethylene ("LLDPE"),
and nylon and
OPET, white PE, foil, adhesive, and EZ Peel Sealant.
[000255] Opposed inner heat sealing layers of the top and bottom sheets in the
preferred
receptacles are bonded to form the walls of the chambers and openable seals
(e.g., chevron seals) that
separate adjoining chambers using heat sealing techniques well known in the
art. The bonds defining
the walls of the chambers are stronger than the openable seals separating
chambers so that when
pressing forces are applied to the chambers, materials are forced between
chambers rather than
peeling apart the walls of the chambers. Target seal strengths for chamber
seals may be on the order
of about 9-10 lb/inch, and target seal strengths for peelable seals may be on
the order of 2.2 - 2.3
lb/inch.
[000256] More specifically, flexible or semi-rigid receptacles, or pouches -
including features
of the receptacles, such as chambers, passages, permanent and semi-permanent
(e.g., ruptureable,
burstable, peelable, frangible, etc.) seals - can be formed by welding two
films together using heated
filaments, a heat sealing die, impulse welder, or ultrasonic welder or other
known techniques.
Alternatively, adhesives, or other bonding techniques, capable of forming
bonds of differential seal
strengths, can be used.
[000257] A receptacle constructed for implementation within the present
invention preferably
includes chambers defined by permanent inter-film bonds formed around the
peripheries of the
chambers to avoid peeling or creep of the bonds. Semi-permanent seals which
are used to initially
block passages or portals interconnecting adjacent chambers are constructed
and arranged to rupture,
or burst, when subject to a predetermined, preferably consistent force to
provide fluid
communication between the adjacent chambers. Application of a compression
force to the chamber
causes lateral expansion of the fluid or other substance contained within the
chamber in a direction
that is transverse to the direction of the force. The permanent and semi-
permanent inter-film bonds
defining the chamber preferably have bursting pressures, or seal strengths,
such that the expanding
fluid will generate a sufficient force to hydraulically peel, or rupture, the
semi-permanent seal, but
will not generate force sufficient to peel the permanent bond defining the
remainder of the periphery
CA 02743477 2011-05-12
of the chamber. As well known in the art, the burst pressure, or seal
strength, of a seal or bond
formed by known techniques is a function of a number of factors, including,
the nature of the
materials being bonded together, the temperature at the interface of the
materials, the pressure
applied to the materials, and the dwell time or period of time during which
the assembled films are
exposed to elevated temperatures and pressures.
[000258] Methods for forming such receptacles having bonds of differential
seal strengths are
well-known in the art. Exemplary disclosures can be found in Johnson et al.,
"Analytical Test Pack
and Process for Analysis," U.S. Patent No. 3,476,515 at col. 3, lines 36-56;
Freshour et al., "Flexible
Packages Containing Nonfusible High Peel Strength Heat Seals," U.S. Patent No.
3,496,061 at col. 2,
lines 6-52, and col. 7, lines 50-59; Farmer, "Packaging Device," U.S. Patent
No. 5,131,760 at col. 4,
lines 25-34; Robinson et al., "Diagnostics Instrument," U.S. Patent No.
5,374,395 at col. 31, lines
27-58; and Rees et al., "Method and Apparatus for Forming Heat Seals with
Films," U.S. Patent No.
6,342,123 (disclosure is directed to the formation of differential seals with
a single die).
[000259] Surprisingly, it was discovered that there are advantages to
constructing chambers
designated for holding moisture-sensitive materials, such as dried process
materials, to include
regions that permit a greater degree of moisture transmission than surrounding
portions of the
receptacles. For example, the sheets of the flexible receptacle described
above may include
thermoplastic and foil layers, where at least one of the sheets includes cut-
outs in the foil layer
around the chambers containing moisture-sensitive materials. (Cut-outs may
also be needed for
chambers requiring light transmission for detection or other purposes.) To
keep the moisture-
sensitive materials dry, the receptacles are placed in sealed, desiccant-
containing vessels, where
moisture is drawn from chambers holding moisture-sensitive materials and into
the vessels where it
is absorbed by the desiccants. The desiccant-containing vessels should be
constructed of materials
having low moisture vapor transmission rates, such as a Mylar OBI2 polyester
packaging film
available from Dupont Packaging and Industrial Polymers of Wilmington, DE.
[000260] To register the receptacles in an instrument, the receptacles may be
provided with
attachment holes that are aligned with corresponding mounting posts in the
instrument..
Alternatively, the receptacles may be precisely positioned in an instrument
using hooks, loops,
adhesives and other like attachment materials. Where an instrument includes a
slot for receiving and
56
CA 02743477 2011-05-12
registering receptacles, those receptacles constructed of flexible materials
are preferably supported
by a rigid frame about their peripheries for precisely positioning the
receptacles within the
instrument. It is also contemplated that in some embodiments no positioning
structures will be
required.
[000261] One or more labels or devices providing information that is human
and/or machine
readable or recognizable may be affixed to or otherwise associated with the
receptacles in regions
that do not interfere with the processing of samples. The labels and/or
devices may provide
information relating to the sample type or source and/or the testing protocol
or other process to be
run. Markings on labels preferably include scannable barcodes. Such labels may
be, for example,
peel-off labels that can be transferred to an associated chart or file.
Alternatively, the information
may be printed on or formed in a material used to construct the receptacles.
Substances Used in the Receptacles
[000262] The chambers can be loaded with reagents, compounds, compositions or
other
substances for use in a single process, multiple applications of the same
process, or multiple
processes of the same or different kind (e.g., nucleic acid-based tests and/or
immunoassays). The
types of substances that can be loaded into the chambers include liquids,
solids, gases, and various
combinations thereof. For some processes, it may also be desirable to leave
one or more chambers
initially empty so that they may serve as, for example, sample, waste,
venting, mixing or detection
chambers within a receptacle. Receptacles having arrangements of chambers that
can be used to
perform any of multiple different procedures may have additional empty
chambers depending on the
number of process materials loaded to perform any particular procedure.
Liquids that may be loaded
and moved between chambers include aqueous and non-aqueous substances,
combinations of liquid
substances, such as mixtures of liquid substances and emulsions (with and
without an emulsifier or
emulsifiers), and liquefied substances, such as solids melted by heating or
gases condensed by
cooling. Solids that may be loaded and moved between chambers include waxes,
mixtures of solids,
and solids in liquids, such as suspensions (e.g., colloids, including gels)
and slurries. Solids can be
in a wide variety of forms, including their natural elemental or molecular
forms.
[000263] Liquid, partially liquid and/or solid substances can be prepared so
that they are in a
57
CA 02743477 2011-05-12
dried or altered solid form when loaded into a chamber. Such substances may be
the product of, for
example, encapsulation, lyophilization, pelletizing, powderizing,
tabletization, drying, spotting,
including spotting of the same or different substances within a chamber
(including multiple spots of
the same and/or different substances in array patterns), the formation of
particles, fibers, networks or
meshes, and absorption and/or drying onto a carrier, including an inner
surface of a chamber. These
substances mayprovide advantages, such as improved stability or durability,
enhanced effectiveness,
convenience of manufacturing and handling, precise amounts of substances,
protection against
environmental and other stresses, including temperature, moisture, oxygen and
electromagnetic
radiation, loading into spatially separated sections of a chamber, and
protection against premature
and adverse interactions of different substances within a receptacle,
including unintended
interactions between a sample and process materials.
[000264] Solids loaded in the chambers could be useful for such functions as
filtration,
immobilization, collection, drying, detection (e.g., probe reagents,
chromatography, electrophoresis,
etc.), and amplification (e.g., amplification oligonucleotides and enzyme
reagents). A solid may
remain unchanged during a process or it may be altered prior to or after
initiating a process. Types of
alterations may include dissolution, the formation of a suspension, slurry or
gel, melting, or a
chemical, biochemical or biological reaction. Such alterations may be caused
by, for example, an
interaction with a fluid loaded or formed in an adjacent chamber, heating,
cooling, irradiating,
sonicating and/or subjecting the solid or solids of a chamber to an electrical
current or magnetic
field.
[000265] Substances may be loaded into the receptacles using manual, semi-
automated or
automated methods. Particular process materials that may be loaded into the
receptacles include, for
example, dried and/or liquid reagents, including binding reagents (e.g.,
nucleic acids, antibodies,
antigens, receptors and/or ligands) and signal generators, solvents, diluents,
suspensions, solutions,
including wash and rinse reagents, and solid supports, including particles,
beads and filters. Loading
may be accomplished by such means as pouring, pipetting, injecting, spotting,
drawing (e.g., applied
vacuum or syringe), evacuating, exchanging atmospheres, and the like. Keeping
the amount of air
present in a chamber to a minimum is generally preferred and, for
reproducibility, the air/material
ratios in like chambers should be kept substantially constant across
receptacles prepared for identical
58
CA 02743477 2011-05-12
uses. When loading substances into a flexible receptacle, the substances are
preferably loaded from
access openings extending from an edge or edges of the receptacle into the
chambers to be loaded.
Figure lB provides an illustration of access openings 19, 21, 23, 25, 31, 33,
35 in an exemplary
receptacle 10 described more fully below. The opposed sides of the receptacle
are preferably drawn
apart by suctioning to facilitate loading of substances and to control wicking
of liquid substances up
the sides of partially sealed chambers. Dried or solid substances are
preferably loaded first, and the
chambers so loaded are temporarily sealed with a tack seal, which provides a
substantially fluid-tight
seal to protect substances that are sensitive to or altered by the presence of
moisture. Liquid
substances are then loaded, and all openings leading to chambers with loaded
substances are sealed
with a heat seal.
[000266] Some process materials exhibit a strong tendency to wick up the sides
of the
chambers during loading and, in some instances, migrate into adjacent chambers
where they can alter
the nature, concentration and/or performance of other process materials. By
way of example, if
amplification and enzyme reagents co-mingle prematurely, unintended
interactions could occur
which consume a portion of these reagents prior to contacting them with a
target nucleic acid. To
address this problem, it was discovered that providing oil (e.g., light
mineral oil) to the chambers
prior to loading process materials significantly reduces the wicking effect
and improves the
performance of processes. Also, when a light mineral oil is included in
chambers filled near their
capacities, any loss of material during the sealing or closing process is
typically limited to the inert,
inactive oil rather than active process materials. Further, an oil layer
situated above a heat-labile
process material (e.g., enzyme reagents) will insulate the process material
from the high temperatures
used in sealing closed the receptacle.
[000267] The use of oil was found to have other benefits as well. For example,
if a process
material contains particles or beads (e.g., magnetically-responsive particles)
that tend to settle along
the sides of a chamber, or adjacent passages between chambers, the use of oil
helps to concentrate
the particles or beads toward the center of the chamber. Otherwise, it might
be difficult to fully re-
suspend the particles or beads or they could clog a passage, thereby
preventing or interfering with the
movement of substances between chambers. Additionally, oil can be used to
increase the fluid
volume of a chamber that otherwise has an insufficient amount of a process
material to fully or
59
CA 02743477 2011-05-12
adequately open a barrier in an adjoining passage when pressure is applied to
the chamber. And,
because oil is inert, it will not affect the relative concentrations of
combined process materials.
Another benefit of oil is that it interferes with the evaporation of liquid
substances from the
chambers. For receptacles including rigid portions, substances may be provided
to cavities formed in
the rigid portions by such means as spraying, spotting or otherwise bonding or
adhering substances to
surfaces of the cavities, or by pouring or pipetting. Alternatively, process
materials are provided to
the receptacles, in whole or in part, through resealable openings, such as
Luer connections, septums
or valves. In this latter embodiment, substances maybe added to receptacles
while procedures are in
progress.
[000268] All substances, except sample material, are preferably provided to
the receptacle and
sealed to the environment prior to shipping for use. By doing so, processes
carried out with the
receptacles are easier to perform, opportunities for operator error are
minimized, and there is less risk
that a receptacle or associated process materials will become contaminated.
Substances loaded in
advance must be provided in a form and kept under conditions such that the
substances remain stable
prior to use. Among other things, this means that the materials used to
construct a chamber cannot
adversely affect a loaded substance, thereby altering its intended function or
performance. Likewise,
a loaded substance should not substantially affect the function or performance
of the chamber it is
stored in.
[000269] Types of sample materials that can be tested with the receptacles of
the present
invention include both fluid and solid samples. Fluid samples that may be
tested with the receptacles
include, for example, urine, blood, saliva, mucus, seminal fluid, amniotic
fluid, cerebrospinal fluid,
synovial fluid, cultures, liquid chemicals, condensed gases and water. Solid
samples that can be
tested with the receptacles include, for example, tissues, stool, soil,
plants, powders, crystals, food
and filters. Sample materials may be provided to the receptacles in a raw or
processed form. A
processed sample is one that has been modified in any manner, such as by
removing components of a
raw sample or by otherwise altering the material from its original state. For
example, with a solid
sample, it may be necessary to alter the sample either prior to or after
adding the solid material to a
sample chamber so that an analyte of interest is free to move between chambers
of the receptacle. In
an altered state, the solid sample may form part of, for example, a
suspension, slurry or homogenate
CA 02743477 2011-05-12
or it may be a liquefied or dissolved form of the solid sample.
[000270] Sample materials are preferably introduced into one or more sample
chambers of a
receptacle through an inlet port immediately prior to initiating a process,
although some of the steps
of a process may be initiated or completed prior to adding sample material to
the receptacle. The
inlet port may be an access opening or it may include, for example, the female
portion of a Luer
connection for insertion of a syringe or other having a male Luer connection.
If the sample material
is stable in the receptacle, then the sample material may not need to be added
to the receptacle
immediately prior to use. For automated uses of the receptacles, it is
generally preferable to load
sample material into the sample chamber or chambers of a receptacle manually
or in a separate
loading device. If sample material is directly loaded into receptacles being
held by an associated
instrument, there is an increased chance of carry-over contamination between
receptacles that could
lead to a false positive or altered result. One such loading device that can
be used or adapted for use
with flexible receptacles of the present invention is the FastPack Sample
Dispenser (Qualigen, Inc.,
Carlsbad, CA). For some applications, a relatively large volume of sample
material maybe required
to ensure that there is a detectable amount of an analyte, if present in the
sample. Receptacles of the
present invention can be designed to include sample chambers that are larger
than subsequent
chambers that are employed to process a sample. Using manually or
automatically activated pressure
means, aliquots of a sample can be sequentially treated in a neighboring
chamber or chambers to
remove unwanted components of the sample and to reduce the volume or size of
the sample being
moved between chambers. The unwanted components can be transferred to a
designated waste
chamber in the receptacle. Separating an analyte from other components of a
sample will generally
involve immobilizing the analyte within a chamber, removing unbound material,
and further
purifying the immobilized analyte by washing it one or more times with a wash
reagent.
Instruments for Manipulating the Contents of the Receptacles
[000271] Receptacles of the present invention are preferably adapted for use
with an automated
instrument capable of acting on all or a portion of the chambers of a
receptacle to affect the location
or state of substances contained therein. Such actions may include moving
substances between or
within chambers, opening and closing interconnections between chambers,
reinforcing barriers
between chambers, localized or generalized heating or cooling, and screening
for one or multiple
61
CA 02743477 2011-05-12
signals or other physical, chemical, biochemical or biological events that may
be indicative of, for
example, the presence, amount or state of one or more analytes of interest.
Other effects of such
actions may be to mix, combine, dissolve, reconstitute, suspend, isolate, wash
or rinse substances of
a process, to manipulate dried or solid substances, to remove waste
substances, and/or to reduce the
volume of a substance, such as a sample substance, to facilitate processing in
a receptacle. The
instrument may be used to process substances in a single receptacle or it may
be adapted to process
substances in multiple receptacles independently and in any desired order,
including simultaneously
(i.e., parallel processing). The instrument, alone or as part of an overall
system, preferably has the
capability of collecting, analyzing, and/or presenting data during and/or
after a process has been
performed. All or 'a portion of the actions of the instrument are governed by
a controller.
[000272] The instrument is designed to cooperate with a receptacle to move
substances
between and/or within chambers. Substances may be moved in a receptacle by
applying an external
pressing force or forces to a flexible surface or surfaces of a chamber, such
as by the use of linear
actuators or rollers, or by applying an internal pressing force, such as by
the use of pistons contained
in piston chambers that are in air and/or fluid communication with the
contents of selected chambers.
Alternatively, a vacuum may be created to draw substances between chambers or
to different regions
of a chamber. Magnetic fields may be used to direct the movement of magnetized
substances within
and between chambers, as well as substances associated therewith. Other means
for moving
substances between or within chambers may include centrifugal forces,
gravitational forces,
electrical forces, capillarity, convection, sonication, irradiation and the
like. In a particularly
preferred embodiment, one or a combination of actuator pads are used to press
substances into
adjacent chambers or to move substances within chambers. The use of a
combination of actuators
can facilitate serial movement of substances into adjacent chambers or the
mixing of substances
within a chamber.
[000273] In a preferred mode, burstable heat seals interrupt connecting
passages between at
least a portion of the adjacent chambers of a receptacle and provide a barrier
against the movement
of substances between chambers. Actuator-driven compression pads of a
cooperating instrument
may be used to apply pressure to the chambers, thereby peeling the seals apart
(e.g., bursting) and
allowing some or all of the substances of the chambers to pass into adjacent
chambers. Where an
62
CA 02743477 2011-05-12
opened seal allows for a bidirectional flow of substances, it may be desirable
to use at least one
actuator as a clamping device to prevent a backflow of substances. The
actuators can also be used as
clamps to prevent seals from prematurely opening. As an example, a chamber may
be directly
connected to multiple other chambers. To focus the flow of a substance into a
desired chamber,
those chambers that are not being utilized are sealed-off by using the
actuators as clamps to reinforce
sealed interconnections with the undesired chambers. Actuators may also be
used to control the flow
of substances when no seals are provided.
[000274] Substances in a chamber, or in multiple chambers, can be mixed in an
instrument by
various active and passive means. "Active" methods of mixing substances
involve the application of
a mechanical force, such as a pressing force, whereas "passive" methods of
mixing substances do not
involve the application of a mechanical force, such as by gravitation. In one
method, substances are
mixed by force of flow as a substance of one chamber is moved into and
contacts a substance of a
second chamber. By another method, substances are mixed by turbulence when one
of the
substances is forced through the restricted space of a passage joining two
chambers. Alternatively,
the substances of two chambers can be mixed by forcing the combined substances
back-and-forth
between the two chambers. Yet another method of mixing involves the use of
multiple actuators
adjacent a chamber to force combined substances between different regions of a
chamber. In one
application of this embodiment, the actuator is a movable optical element of a
light detecting device
(e.g., fluorometer) that is in sliding engagement with a corresponding ring
member, where the optical
element and the ring member generally conform to the shape of an associated
chamber and move
into engagement with the chamber in an alternating fashion to achieve mixing.
In another approach,
mixing is carried out by forcing a first substance upward from a first chamber
into a second chamber,
where it is combined with a second substance, and then allowing for gravity
assisted movement of
the combined substances back into the first chamber. This procedure can be
repeated until the
desired degree of mixing is achieved. Other procedures for mixing may involve,
for example,
heating and/or cooling to produce convection mixing or sonication, with or
without solid particles.
[000275] The instrument and receptacle can also cooperate to manipulate
components of a
substance. For example, one or more chambers of the receptacle may include a
filter, or series of
filters, for removing constituents of a substance as the instrument actively
or passively causes the
63
CA 02743477 2011-05-12
substance to pass through the filter. The constituents removed from a
substance may include
components of a sample material that can interfere with a process or solid
supports that are used for
processing a sample material, such as beads, particles, rods, fibers and the
like. These solid supports
may be used, for example, to bind an analyte, either directly or indirectly,
or components of a
sample. In another approach, the instrument may be adapted to cause solid
supports or solid
substances in a material to be concentrated in a chamber by imposing a
centrifugal force on the
receptacle. In an alternative approach, magnetically responsive particles
present in a chamber can be
manipulated by magnetic forces exerted by a component of the instrument. By
isolating the
magnetic particles in a specific chamber, substances bound to the particles
remain in the chamber
while unbound substances can be removed from the chamber. In addition to
separating wanted from
unwanted materials, solid supports, such as beads and particles, can also be
used in the receptacles of
the present invention to facilitate a reduction in the volume of a sample
material. The initial volume
of the sample material may be relatively large to ensure that there is an
adequate quantity of the
analyte of interest for detection and/or quantification. However, in some
cases this initial volume is
too large for practical processing of the sample material in the receptacle.
Instruments and
receptacles in accordance with the present invention can address this problem
by immobilizing the
analyte on a solid support (e.g., magnetically-responsive particle) in, for
example, a sample receiving
chamber, isolating the solid support within the chamber (e.g., exposing the
particles to a magnetic
field), and then generating forces that remove the remainder of the sample
material from the sample
receiving chamber and dispose of it in a designated waste chamber.
Alternatively, this same
procedure can be performed in an adjoining chamber of a receptacle having a
smaller volume
capacity than the sample receiving chamber by incrementally moving, isolating
and separately
processing aliquots of a sample material. By having or moving the solid
support into a smaller
chamber, processing of the sample material may be more efficient and the
consumption of process
materials lower.
[000276] To purify an analyte, the solid support can be washed one or more
times with a wash
reagent in a designated sample processing chamber. When performing a wash
procedure, a force or
forces may be imposed by the instrument which cause the solid support to
remain isolated or
otherwise concentrated in the sample processing chamber or which cause it to
be resuspended in the
wash reagent. Resuspending the solid support in the wash reagent may be
accompanied by mixing,
64
CA 02743477 2011-05-12
such as by agitating the receptacle, a turbulent movement of wash reagent from
the wash reagent
chamber into the sample processing chamber, or the use ofpressing forces to
mix the contents of the
sample processing chamber. After an appropriate dwell time, the solid support
can again be isolated
or otherwise concentrated and the wash reagent moved from the sample
processing chamber into a
waste chamber. This process can be repeated as needed.
[000277] The instrument may include elements for controlling the thermal
conditions of one or
more chambers or for providing a uniform temperature within the instrument.
Factors to be
considered in selecting thermal control elements for use in performing a
particular process include
determining the desired temperature range, the rate of change of temperature,
the accuracy, precision
and stability of temperature, whether zonal heating and/or cooling or a
uniform temperature is
required, and the effect of external conditions, as well as heat-producing
components of the
instrument (e.g., motors), on temperature control capabilities. The heating
and/or cooling of the
chambers or subsets of chambers may be accomplished with thermal control
elements that use, for
example, electrical changes, radiation, microwaves, sonication, convection,
conduction, forced air,
chemical reactions (e.g., exothermic and endothermic reactions), biological
activities (e.g., heat-
generating growth), circulating fluids (e.g., heated water or freon), and the
like to alter the thermal
conditions of a chamber or chambers. Alternatively, the instrument may be
placed in a temperature-
controlled environment, such as an incubator or refrigerator, to maintain a
uniform temperature.
[000278] A preferred instrument includes thermal conducting plates, such as
copper or
aluminum plates, that align with a collection of chambers, or a particular
chamber or region of a
chamber, when the receptacle is properly loaded in the instrument. The
temperature of each plate
can be controlled by, for example, the use of a thermoelectric device.
Depending on the direction of
current flow in a thermoelectric device, the junction of dissimilar conductors
in the thermoelectric
device will either absorb or release heat. Thus, thermoelectric devices can be
used for the heating
and/or cooling of chambers or regions of chambers. Other advantages of
thermoelectric devices
include their size, the absence of any moving parts or vibration, rapid
temperature changes, precision
temperature control, and no CFCs or moving fluids.
[000279] In practice, the thermal conducting plates are positioned in the
instrument so that they
will be in the general proximity of, and preferably contacting, the chambers
or regions of chambers
CA 02743477 2011-05-12
to be heated or cooled in a properly loaded receptacle. The plates are
separated from each other
using a non-conductive material, such as Ultem polyimde thermoplastic resin
or Deirin acetyl
resin. Using the thermoelectric devices, heat is transferred by conduction,
convection or radiation.
[000280] In an alternative embodiment, all or a portion of the thermal control
elements may be
associated with actuators which provide localized heating or cooling of
chambers.
(000281] The instrument preferably includes at least one detector for
detecting a signal or other
physical, chemical, biochemical or biological event. The detector may be used
to detect whether an
analyte or multiple analytes are present in a sample material, or present in
an altered state. The
detector, in cooperation with a microprocessor, may also provide information
about the quantity of
an analyte or analytes present in a sample material. Detectors contemplated by
the present invention
include fluorometers, luminometers, spectrophotometers, infrared detectors and
charged-coupled
devices. Each of these types of detectors, or signal receiving components
associated with these
detectors, can be positioned adjacent a detection chamber for detecting a wide
variety of signal types.
A detector may be mounted on a movable platform so that the detector can be
positioned adjacent
different chambers of a stationary receptacle. Alternatively, multiple types
of detectors may be
movably mounted on a platform to facilitate different detection methods for
different processes. The
instrument may also include multiple detectors of the same or different types
for detecting signals
emitted from different chambers simultaneously. Fiber optics may also be used
to collect signals
from different locations and transmit this information to a detector or
detectors at stationary sites
removed from the receptacle. Also contemplated is a fiber optic arrangement in
combination with a
movable detector. Other possible detectors might be used to detect, for
example, radioactive,
magnetic or electronic labels, Raman scattering, Surface Plasmon Resonance,
gas, turbidity, or a
mass, density, temperature, electronic or color change.
Uses of the Receptacles
[000282] The receptacles of the present invention can be used, alone or in
combination with a
cooperating instrument, to perform a variety of processes. Such processes may
include, for example,
separating or isolating an analyte of interest from other components of a
sample, exposing a sample
or component of a sample to reagents and conditions needed to analyze the
sample, and/or
66
CA 02743477 2011-05-12
performing a chemical, biochemical or biological reaction which effects a
detectable change, such as
a change in composition, sequence, volume, quantity, mass, conductivity,
turbidity, color,
temperature or the like. As discussed above, the receptacles are particularly
suited for use in
applications requiring or benefiting from actions that are performed non-
sequentially. These types of
applications include, but are not limited to, complex tests or assays
involving detectable binding
interactions such as antigen-antibody, nucleic acid-nucleic acid, and receptor-
ligand interactions.
[000283] Nucleic acid based assays that can be performed in the receptacles of
the present
invention may rely upon direct detection of a target nucleic acid or detection
of an amplification
product indicative of the presence of the target nucleic acid in a sample.
Direct detection requires
that there be a sufficient quantity of the target nucleic acid in a sample to
sensitively determine the
presence of a target sequence associated with, for example, gene expression, a
chromosomal
abnormality or a pathogenic organism. Because of the large cellular quantities
of ribosomal RNA
(rRNA) in non-viral organisms, and the sequence conservation that enables
phylogenetically
coherent groupings of organisms to be distinguished from each other, rRNA is
an ideal target for a
direct detection assay designed to determine the presence of a pathogenic
organism (e.g., bacterium,
fungus or yeast). See, e.g., Kohne, "Method for Detecting, Identifying, and
Quantitating Organisms
and Viruses," U.S. Patent No. 5,288,611; and Hogan et al., "Nucleic Acid
Probes for Detection
and/or Quantitation of Non-Viral Organisms," U.S. Patent No. 5,840,488.
Regardless of the type of
nucleic acid being targeted, the sensitivity of a direct detection assay can
be improved by a signal
amplification procedure in which a probe or probe complex binding to a target
nucleic acid has
multiple labels for detection, thereby increasing the signal of an assay
without affecting the amount
of target in the sample. See, e.g., Hogan et al., "Branched Nucleic Acids,"
U.S. Patent No.
5,424,413; and Urdea et al., "Nucleic Acid Multimers and Amplified Nucleic
Acid Hybridization
Assays Using the Same," U.S. Patent No. 5,124,246. Another form of
amplification that does not
require increasing the copy number of a target nucleic acid sequence is probe
amplification, which
includes procedures such as the Ligase Chain Reaction (LCR). LCR relies upon
repeated cycles of
probe hybridization and ligation to generate multiple copies of a nucleic acid
sequence. See, e.g.,
Birkenmeyer et al., "Amplification of Target Nucleic Acid Using Gap Filling
Ligase Chain
Reaction," U.S. Patent No. 5,427,930. Other contemplated signal amplification
procedures include
those utilizing Third Wave Technology's Invader chemistry. See, e.g.,
Kwiatkowski et al.,
67
CA 02743477 2011-05-12
"Clinical, Genetic, and Pharmacogenetic Applications of the Invader Assay,"
Mol. Diagn. (1999)
4(4):353-64.
[000284] Target nucleic acid amplification involves the use of amplification
oligonucleotides
(e.g., primers) and polymerases to enzymatically synthesize nucleic acid
amplification products
(copies) containing a sequence that is either complementary or homologous to
the template nucleic
acid sequence being amplified. The amplification products may be either
extension products or
transcripts generated in a transcription-based amplification procedure. The
amplification
oligonucleotides may be provided to a reaction mixture free in solution or one
or more of the
amplification oligonucleotides maybe immobilized on a solid support, including
the inner surface of
a chamber or chambers within a receptacle. See, e.g., Adams et al., "Method
for Performing
Amplification of Nucleic Acid with Two Primers Bound to a Single Solid
Support," U.S. Patent No.
5,641,658; and Browne, "Nucleic Acid Amplification and Detection Method," U.S.
Patent
Application Publication No. US 2005-0287591 Al. Examples of nucleic acid
amplification
procedures practiced in the art include the polymerase chain reaction (PCR),
strand displacement
amplification (SDA), helicase dependent amplification (HDA), loop-mediated
isothermal
amplification (LAMP), and a variety of transcription-based amplification
procedures, including
transcription-mediated amplification (TMA), nucleic acid sequence based
amplification (NASBA),
and self-sustained sequence replication (3SR). See, e.g., Mullis, "Process for
Amplifying, Detecting,
and/or Cloning Nucleic Acid Sequences," U.S. Patent No. 4,683,195; Walker,
"Strand Displacement
Amplification," U.S. Patent No. 5,455,166; Kong et al., "Helicase Dependent
Amplification of
Nucleic Acids," U.S. Patent No. 7,282,328, Notomi et al., "Process for
Synthesizing Nucleic Acid,"
U.S. Patent No. 6,410,278; Kacian et al., "Nucleic Acid Sequence Amplification
Methods," U.S.
Patent No. 5,399,491; Becker et al., Single-Primer Nucleic Acid Amplification
Methods," U.S.
Patent No. 7,374,885; Malek et al., "Enhanced Nucleic Acid Amplification
Process," U.S. Patent
No. 5,130,238; and Lizardi et al. (1988) BioTechnology 6:1197. With some
procedures, the
formation of detectable amplification products depends on an initial
antibody/antigen interaction.
See, e.g., Cashman, "Blocked-Polymerase Polynucleotide Immunoassay Method and
Kit," U.S.
Patent No. 5,849,478. Nucleic acid amplification is especially beneficial when
the amount of analyte
(e.g., targeted nucleic acid, antigen or antibody) present in a sample is very
low. By amplifying a
target sequence associated with the analyte and detecting the synthesized
amplification product, the
68
CA 02743477 2011-05-12
sensitivity of an assay can be vastly improved, since less analyte is needed
at the beginning of the
assay to ensure detection of the analyte.
[000285] The conditions of a target nucleic acid amplification reaction may be
substantially
isothermal or they may require periodic temperature changes, as with PCR
thermal cycling. The
instrument described supra may provide a constant or ambient temperature or it
maybe modified and
programmed to fluctuate the overall temperature within the instrument or,
alternatively, particular
zones of the instrument which affect specific chambers of a receptacle. Target
nucleic acid
amplification reactions may be either "real-time" or "end-point" assays. A
compact, lightweight,
multi-channel fluorometer that is particularly suited for use in performing
real-time assays in an
instrument of the present invention is described' below. Real-time
amplification assays involve
periodically determining the amount of targeted amplification products as the
amplification reaction
is taking place, thereby making it easier to provide quantitative information
about an analyte (e.g.
target nucleic acid) present in a sample, whereas end-point amplifications
determine the amount of
targeted amplification products after the amplification reaction has occurred,
generally making them
more useful for providing qualitative information about an analyte present in
a sample. Algorithms
for calculating the quantity of target nucleic acid or other analyte
originally present in a sample based
on signal information collected during or at the completion of an
amplification reaction include those
disclosed by Wittwer et al., "PCR Method for Nucleic Acid Quantification
Utilizing Second or Third
Order Rate Constants," U.S. Patent No. 6,232,079; Sagner et al., "Method for
the Efficiency-
Corrected Real-Time Quantification of Nucleic Acids," U.S. Patent No.
6,691,041; McMillan et al.,
"Methods for Quantitative Analysis of a Nucleic Acid Amplification Reaction,"
U.S. Patent No.
6,911,327; Light et al., "Method for Determining the Amount of an Analyte in a
Sample," U.S.
Patent Application Publication No. US 2006-0276972 Al; Chismar et al., "Method
and Algorithm
for Quantifying Polynucleotides," U.S. Patent Application Publication No. US
2006-0292619 Al;
and Ryder et al., "Methods for Determining Pre-Amplification Levels of a
Nucleic Acid Target
Sequence from Post-Amplification Levels of Product," U.S. Patent No.
5,710,029. Also, to confirm
that the amplification conditions and reagents were appropriate for
amplification, it is generally
desirable to provide an internal control sequence at the start of a nucleic
acid amplification reaction.
See, e.g., Wang et al., "Quantitation of Nucleic Acids Using the Polymerase
Chain Reaction," U.S.
Patent No. 5,476,774.
69
CA 02743477 2011-05-12
[000286] Detection of a target nucleic acid may be in situ or in vitro. See,
e.g., Gray et al.,
"Methods for Chromosome-Specific Staining," U.S. Patent No. 5,447,841. For in
vitro assays, it
may be necessary to lyse or permeabilize cells to first release the targeted
nucleic acid and make it
available for hybridization with a detection probe. See, e.g., Clark et al.,
"Method for Extracting
Nucleic Acids from a Wide Range of Organisms," U.S. Patent No. 5,786,208. If
the cells are lysed,
the contents of the resulting lysate may include, in addition to nucleic
acids, organelles, proteins
(including enzymes such as proteases and nucleases), carbohydrates, and
lipids, which may
necessitate further purification of the nucleic acids. Additionally, for
pathogenic organisms,
chemical or thermal inactivation of the organisms may be desirable. The cells
may be lysed or
permeabilized prior to loading sample into a receptacle of the present
invention, or a sample or other
chamber of the receptacle may be pre-loaded with an agent for performing this
function. Cells may
be lysed or permeabilized by a variety of means well known to those skilled in
the art, including by
chemical, mechanical (e.g., sonication) and/or thermal means. One preferred
lytic agent in described
in the Examples section below.
[000287] Released nucleic acids can be isolated or separated in the
receptacles from other
sample components that may act as inhibitors which interfere with the
detection and/or amplification
of a target sequence. The presence of potentially interfering components will
vary depending on the
sample type and may include components of the cell lysate, such as nucleases
that can digest the
released and targeted nucleic acids. Some unwanted sample components may be
separated from the
target nucleic acid through precipitation or solid phase capture by providing
to a chamber such
materials as filters, beads, fibers, membranes, glass wool, filter paper,
polymers or gels. Suitable
filters include glass, fiberglass, nylon, nylon derivatives, cellulose,
cellulose derivatives, and other
polymers. Alternatively, a solid phase material may be used to capture sample
components for
lysing, such as cells, spores or microorganisms, where the components may be
captured by physical
retention (e.g., size exclusion, affinity retention, or chemical selection).
[000288] Various solid phase methods for capturing nucleic acids are known in
the art and can
be readily adapted for use in the receptacles of the present invention. These
methods maybe specific
or non-specific for the targeted nucleic acid. One such method is Solid Phase
Reversible
Immobilization, which is based on the selective immobilization of nucleic
acids onto magnetic
CA 02743477 2011-05-12
microparticles having carboxyl group-coated surfaces. See Hawkins, "DNA
Purification and
Isolation Using Magnetic Particles," U.S. Patent No. 5,705,628. In another
method, magnetic
particles having poly(dT) sequences derivatized thereon bind to capture probes
having 5' poly(dA)
tails and 3' target binding sequences. See, e.g., Weisburg et al., "Two-Step
Hybridization and
Capture of a Polynucleotide," U.S. Patent No. 6,534,273. Yet another commonly
used method binds
nucleic acids to silica or glass particles in the presence of guanidinium
thiocyanate, which is a known
agent for lysing cells and inactivating nucleases. Still another approach is
based on the
ChargeSwitch Technology, which is a magnetic bead-based technology that
provides a switchable
surface that is charge dependent on the surrounding buffer pH to facilitate
nucleic acid purification
(Invitrogen Corporation, Carlsbad, CA; Cat. No. CS 12000). In low pH
conditions, the
ChargeSwitch Magnetic Beads have a positive charge that binds the negatively
charged nucleic
acid backbone. Proteins and other contaminants that are not bound can be
washed away. By raising
the pH to 8.5, the charge on the surface is neutralized and the bound nucleic
acids are eluted.
[000289] In another approach, capture probes that are capable of binding to
the targeted nucleic
acid (or to intermediate oligonucleotides that also bind to the targeted
nucleic acids) are covalently or
non-covalently attached to an inner surface of a designated sample processing
chamber during
manufacture of the receptacle. Attachment chemistries are well known to
skilled artisans and
include amine and carboxylic acid modified surfaces for covalent attachment of
oligonucleotides and
biotin-labeled oligonucleotides and avidin- or streptavidin-coated surfaces
for non-covalent
attachments. With this approach, targeted nucleic acids introduced into the
sample processing
chamber can be immobilized on the surface of the chamber and liquid and other
unbound materials
can be removed without having to immobilize or trap particles or beads.
Following separation from
other materials of the sample, the targeted nucleic acids may remain
immobilized on the surface for
amplification and detection or they may be first eluted from the capture
probes. Alternatively, the
capture probes could be immobilized on a porous solid support, such as a
sponge, that is located in
the sample processing chamber.
[000290] Capture probes suitable for use in the present invention may be
specific or non-
specific for the targeted nucleic acids. A specific capture probe will include
a target binding region
that is selected to bind to a target nucleic acid under a predetermined set of
conditions and not to
71
CA 02743477 2011-05-12
non-target nucleic acids which may be present in a sample. A non-specific
capture probe does not
discriminate between target and non-target nucleic acids under the conditions
of use. Wobble
capture probes are an example of a non-specific capture probe and may include
at least one random
or non-random poly(K) sequence, where "K" can represent a guanine, thymine or
uracil base. See
Becker et al., "Methods of Nonspecific Target Capture of Nucleic Acids," U.S.
Patent Application
No. 11/832,367, which enjoys common ownership herewith. In addition to
hydrogen bonding with
cytosine, its pyrimidine complement, guanine will also hydrogen bond with
thymine and uracil.
Each "K" may also represent a degenerate nucleoside such as inosine or
nebularine, a universal base
such as 3-nitropyrrole, 5-nitroindole or 4-methylindone, or a pyrimidine
orpurine base analog such
as dP or dK. The poly(K) sequence of a wobble capture probe is of sufficient
length to non-
specifically bind the target nucleic acid, and is preferably 6 to 25 bases in
length.
[000291] Formats for detecting a target nucleic acid or related amplification
product can be
divided into two basic categories: heterogeneous and homogeneous. Both of
these detection formats
can be adapted for use in the receptacles of the present invention.
Heterogeneous assays include a
step to separate bound from unbound probe, while no such physical separation
step is used in
homogeneous assays. Numerous heterogeneous and homogeneous detection methods
are known in
the art. See, e.g., Jung, P. et al. 1997. "Labels and Detection Formats in
Amplification Assays." In
Nucleic Acid Amplification Technologies, eds. Lee, H. et al., 135-150. Natick,
MA: BioTechnique
Books.
[000292] Assay methods utilizing a physical separation step include methods
employing a
solid-phase matrix, such as glass, minerals or polymeric materials, in the
separation process. The
separation may involve preferentially binding the probe:analyte complex to the
solid phase matrix,
while allowing the unassociated probe molecules to remain in a liquid phase.
Such binding may be
non-specific, as, for example, in the case of hydroxyapatite, or specific, for
example, through
sequence-specific interaction of the target nucleic acid with a capture probe
that is directly or
indirectly immobilized on the solid support. In any such case, the amount
ofprobe remaining bound
to the solid phase support after a washing step is proportional to the amount
of analyte in the sample.
[000293] Alternatively, the assay may involve preferentially binding the
unhybridized probe
while probe:analyte complexes remain in the liquid phase. In this case the
amount of probe in the
72
CA 02743477 2011-05-12
liquid phase after a washing step is proportional to the amount of analyte in
the original sample.
When the probe is a nucleic acid or oligonucleotide, the solid support can
include, without limitation,
an adsorbent such as hydroxyapatite, a polycationic moiety, a hydrophobic or
"reverse phase"
material, an ion-exchange matrix, such as DEAF, a gel filtration matrix, or a
combination of one or
more of these solid phase materials. The solid support may contain one or more
oligonucleotides, or
other specific binding moiety, to capture, directly or indirectly, probe,
target, or both. In the case of
media, such as gel filtration, polyacrylamide gel or agarose gel, the
separation is not due to binding
of the oligonucleotide but is caused by molecular sieving of differently sized
or shaped molecules.
In the latter two cases, separation may be driven electrophoretically by
application of an electrical
current through the gel causing the differential migration through the gel of
nucleic acids of different
sizes or shapes, such as double-stranded and single-stranded nucleic acids.
[000294] A heterogeneous assay method may also involve binding the probe to a
solid-phase
matrix prior to addition of a sample suspected of containing the analyte of
interest. The sample can
be contacted with the label under conditions that would cause the desired
nucleic acid to be labeled,
if present in the sample mixture. The solid phase matrix may be derivatized or
activated so that a
covalent bond is formed between the probe and the matrix. Alternatively, the
probe maybe bound to
the matrix through strong non-covalent interactions, including, without
limitation, the following
interactions: ionic, hydrophobic, reverse-phase, immunobinding, chelating, and
enzyme-substrate.
After the matrix-bound probe is exposed to the labeled nucleic acid under
conditions allowing the
formation of a hybrid, the separation step is accomplished by washing the
solid-phase matrix free of
any unbound, labeled analyte. Conversely, the analyte can be bound to the
solid phase matrix and
contacted with labeled probe, with the excess free probe washed from the
matrix before detection of
the label.
[000295] As noted above, homogenous assays take place in solution, without a
solid phase
separation step, and commonly exploit chemical differences between free probe
and probe:analyte
complexes. An example of an assay system that can be used in a homogenous or
heterogeneous
format is the hybridization protection assay (HPA). See Arnold et at,
"Homogenous Protection
Assay," U.S. Patent 5,283,174. In HPA, a probe is linked to a chemiluminescent
moiety, contacted
with a sample and then subjected to selective chemical degradation or a
detectable change in stability
73
CA 02743477 2011-05-12
under conditions which alter the chemiluminescent reagent linked to
unhybridized probe without
altering the chemiluminescent reagent linked to a probe:analyte complex.
Subsequent initiation of a
chemiluminescent reaction causes the hybrid-associated label to emit light.
[000296] Other homogeneous assays rely upon a physical alteration to a
detection probe or
amplification primer to provide a detectable signal change indicative of the
presence of a target
nucleic acid. Probes and primers capable of undergoing detectable physical
alterations include, but
are not limited to, self-hybridizing probes, such as molecular beacons or
molecular torches, bi-
molecular probes, TagMan probes that are commercially available from Applied
Biosystems,
LuxTM primers that are commercially available from Invitrogen Corporation, and
signal primers.
See, e.g., Tyagi et al. (1996) Nature Biotechnology 14(3):303-308; Becker el
at., "Molecular
Torches," U.S. Patent No. 6,849,412; Morrison, "Competitive Homogeneous
Assay," U.S. Patent
No. 5,928,862; Tapp et al. (2000) BioTechniques 28(4):732-738; Nazarenko
(2006) Methods Mol.
Biol. 335:95-114; and Nazarenko (1997) Nucleic Acids Res. 25(12):2516-2521.
Each of these
probes and primers relies upon a conformational change in the probe or primer
upon hybridization to
a target nucleic acid to render a detectable change in an associated reporter
moiety (e.g., fluorescent
molecule). Prior to hybridization, signal from the reporter moiety may be
altered by an associated
quencher moiety which, in the case of LuxTM primers, is a guinine located near
the 3' end of the
primer sequence.
[000297] Particularly preferred detection probes for use in real-time
amplification reactions are
self-hybridizing probes that emit differentially detectable signals, depending
on whether the probes
remain self-hybridized or bind to amplification products. The probes may be
provided to a reaction
mixture free in solution or immobilized on solid supports. See, e.g., Cass et
al., "Immobilized
Nucleic Acid Hybridization Reagent and Method," U.S. Patent No. 6,312,906.
Advantageously, they
may also be provided to a reaction mixture before, after or at the time an
amplification reaction has
been initiated. If the probes are provided on a solid support, then the solid
support may additionally
include one or more immobilized amplification oligonucleotides for amplifying
a target nucleic acid
sequence. Preferred self-hybridizing probes include molecular beacons and
molecular torches.
[000298] Molecular beacons comprise nucleic acid molecules or analogs thereof
having a target
complementary sequence, an affinity pair (or nucleic acid or nucleic acid
analog arms or stems)
74
CA 02743477 2011-05-12
holding the probe in a closed conformation in the absence of a target nucleic
acid sequence, and a
label pair that interacts when the probe is in a closed conformation. See
Tyagi et al., "Detectably
Labeled Dual Conformation Oligonucleotide Probes, Assays and Kits," U.S.
Patent No. 5,925,517.
Hybridization of the target nucleic acid and the target complementary sequence
separates the
members of the affinity pair, thereby shifting the probe to an open
conformation. The shift to the
open conformation is detectable due to reduced interaction of the label pair,
which may be, for
example, a fluorophore and a quencher (e.g., DABCYL and EDANS).
[000299] Molecular torches have distinct regions of self-complementarity,
described as the
"target binding" and "target closing" domains. These domains are linked by a
joining region and are
sufficiently complementary to hybridize to each other under predetermined
hybridization assay
conditions. When exposed to denaturing conditions, the complementary regions
melt, leaving the
target binding domain available for hybridization to a target sequence when
the predetermined
hybridization assay conditions are restored. And when exposed to strand
displacement conditions, a
portion of the target sequence binds to the target binding domain, thereby
displacing the target
closing domain from the target binding domain. Molecular torches are designed
so that the target
binding domain favors hybridization to the target sequence over the target
closing domain. The
target binding domain and the target closing domain of a molecular torch
include interacting labels
positioned so that a different signal is produced when the molecular torch is
self-hybridized than
when it is hybridized to a target nucleic acid, thereby permitting detection
of probe:target complexes
in a test sample in the presence of unhybridized probe having a viable label
or labels associated
therewith.
[000300] Different types of interacting moieties can be used to determine
whether a probe has
undergone a conformational change. For example, the interacting moieties may
be a
luminescent/quencher pair, a luminescent/adduct pair, a Forrester energy
transfer pair or a dye dimer.
More than one type of label may be present on a particular molecule.
[000301] A luminescent/quencher pair is made up of one or more luminescent
moieties, such as
chemiluminescent or fluorescent moieties, and one or more quenchers.
Preferably, a
fluorescent/quencher pair is used to detect a probe that has undergone a
conformational change. A
fluorescent moiety absorbs light of a particular wavelength, or wavelength
range, and emits light
CA 02743477 2011-05-12
with a particular emission wavelength, or wavelength range. A quencher moiety
dampens, partially
or completely, signal emitted from an excited fluorescent moiety. Quencher
moieties can dampen
signal production from different fluorophores. For example, DABCLY ([4-(4'-
dimethylaminophenylazo) benzoic acid]) can quench about 95% of the signal
produced from
EDANS (5-(2'-aminoethyl)aminoaphthaline-I-sulfonic acid), rhodamine and
fluorescein.
[000302] Different numbers and types of fluorescent and quencher moieties can
be used. For
example, multiple fluorescent moieties can be used to increase signal
production from an opened
molecular beacon or torch, and multiple quencher moieties can be used to help
ensure that, in the
absence of a target sequence, an excited fluorescent molecule produces little
or no signal. Examples
of fluorophores include acridine, fluorescein, sulforhodamine 101, rhodamine,
EDANS, Texas Red,
Eosine, Bodipy and lucifer yellow. See, e.g., Tyagi et al. (1998) Nature
Biotechnology 16:49-53.
Examples of quenchers include DABCYL, Thallium, Cesium, and p-xylene-bis-
pyridinium bromide.
[000303] A luminescent/adduct pair is made up of one or more luminescent
moieties and one or
more molecules able to form an adduct with the luminescent molecule(s) and,
thereby, diminish
signal production from the luminescent molecule(s). The use of adduct
formation to alter signals
from a luminescent molecule using ligands free in solution is disclosed by
Becker et al., "Adduct
Protection Assay," U.S. Patent No. 5,731,148.
[000304] Forrester energy transfer pairs are made up of two moieties, where
the emission
spectra of a first moiety overlaps with the excitation spectra of a second
moiety. The first moiety can
be excited and emission characteristic of the second moiety can be measured to
determine if the
moieties are interacting. Examples of Forrester energy transfer pairs include
pairs involving
fluorescein and rhodamine; nitrobenz-2-oxa-1,3-diazole and rhodamine;
fluorescein and
tetramethylrhodamine; fluorescein and fluorescein; IAEDANS and fluorescein;
and BODIPYFL and
BIODIPYFL.
[000305] Dye dimers comprise two dyes that interact upon the formation of a
dimer to produce
a different signal than when the dyes are not in a dimer conformation. See,
e.g., Packard et al. (1996)
Proc. Natl. Acad. Sci. USA 93:11640-11645.
[000306] While homogeneous assays are generally preferred, essentially any
labeling and
76
CA 02743477 2011-05-12
detection system that can be used for monitoring specific nucleic acid
hybridization can be used in
conjunction with the receptacles of the present invention. Included among the
collection of useful
labels are radiolabels, intercalating dyes, enzymes, haptens, linked
oligonucleotides,
chemiluminescent molecules and redox-active moieties that are amenable to
electronic detection
methods. Preferred chemiluminescent molecules include acridinium esters of the
type disclosed by
Arnold et al. in "Homogeneous Protection Assay," U.S. Patent No. 5,283,174 for
use in connection
with hybridization protection assays (HPA), and of the type disclosed by
Woodhead et a!. in
"Detecting or Quantifying Multiple Analytes Using Labeling Techniques," U.S.
Patent No.
5,656,207 for use in connection with assays that quantify multiple targets in
a single reaction.
Preferred electronic labeling and detection approaches are disclosed by Meade
et al., "Nucleic Acid
Mediated Electron Transfer," U.S. Patent Nos. 5,591,578, and Meade, "Detection
of Analytes Using
Reorganization Energy," U.S. Patent No. 6,013,170. Redox active moieties
useful as labels include
transition metals such as Cd, Mg, Cu, Co, Pd, Zn, Fe and Ru.
[000307] Synthetic techniques and methods of bonding reporter moieties to
nucleic acids and
detecting reporter moieties are well known in the art. See, e.g., J. SAMBROOK
ETAL., MOLECULAR
CLONING: A LABORATORY MANUAL, Chapter 10 (2d ed. 1989); Becker et al., U.S.
Patent No.
6,361,945; Tyagi et a!., U.S. Patent No. 5,925,517, Tyagi et a!., "Nucleic
Acid Detection Probes
Having Non-FRET Fluorescence Quenching and Kits and Assays Including Such
Probes," U.S.
Patent No. 6,150,097; Nelson et a!., U.S. Patent No. 5,658,737; Woodhead et
a!., U.S. Patent No.
5,656,207; Hogan et a!., "Nucleic Acid Probes for Detection and/or
Quantitation of Non-Viral
Organisms," U.S. Patent No. 5,547,842; Arnold et al., U.S. Patent No.
5,283,174; Kourilsky et a!.,
"Method of Detecting and Characterizing a Nucleic Acid or Reactant for the
Application of this
Method," U.S. Patent No. 4,581,333; and Becker et a!., U.S. Patent No.
5,731,148.
[000308] Process materials may be provided to the chambers of a receptacle in
a dried or liquid
form. Providing process materials in a dried form can be especially beneficial
where the process
materials are, in their liquid form, unstable, biologically or chemically
active, temperature sensitive
or chemically reactive with each other. Drying inhibits the activity of
microorganisms and enzymes
and can improve the shelf-life and storage conditions of process materials
(room temperature as
opposed to cold storage). Dried process materials, in addition to their
reactive components, may
77
CA 02743477 2011-05-12
include a cryoprotectant (e.g., disaccharides such as sucrose, maltose,
lactose or trehalose) to help
preserve the biological activity of a material as it is being frozen, dried
and/or reconstituted and a
stabilizing agent (e.g., various sugars including sucrose and trehalose, sugar
alcohols and proteins) to
prevent or delay the loss of a materialfls biological activity over time. The
suitability of any given
cryoprotectant or stabilizing agent will depend on the nature of the material
being dried. After
drying, the process materials should be sealed to prevent reabsorption of
moisture. Methods and
devices for drying process materials are well known in the art and include
lyophilization or freeze-
drying. See, e.g., Price et al., "Pelletized Pregnancy Test Reagents," U.S.
Patent No. 3,862,302;
Temple et al, "Process for Freezing or Chilling," U.S. Patent No. 4,655,047;
Milankov et al.,
"Cryogenic Apparatus," U.S. Patent No. 4,982,577; Shen et al., "Stabilized
Enzyme Compositions
for Nucleic Acid Amplification," U.S. Patent No. 5,834,254; Buhl et al.,
"Dried Chemical
Compositions," U.S. Patent No. 6,251,684; and McMillan, "Universal and Target
Specific Reagent
Beads for Nucleic Acid Amplification," U.S. Patent Application Publication No.
US 2006-0068398
Al.
Illustrative Embodiments
[000309] An exemplary embodiment of a multi-chambered receptacle embodying
aspects of the
invention is designated by reference number 10 in Figure 1 A. In this
embodiment, the receptacle 10
is a generally planar vessel having flexible top and bottom sheets formed from
thin flexible
materials, such as foils and/or plastics, and an upper edge 12 and a lower
edge 14 that indicate the
preferred orientation of the receptacle during use and define an upper
direction and a lower direction.
The exemplary receptacle 10 has dimensions of about 7.5 inches by about 3.2
inches and is less than
about 1 /4 inch thick (when filled with sample and process materials), but
maybe of any dimensions
suitable for manual manipulation or for use with an automated system, such as
the one described
herein. In general, the dimensions of the receptacle must accommodate the
substances needed to
conduct a process or set of processes. Persons of skill will recognize that a
wide variety of sizes,
conformations, shapes, and the like are compatible with various processes and
are contemplated for
use.
[000310] The receptacle may include one or more attachment or alignment holes
74 that
register with structures, such as hooks or pins, of an automated instrument
for mounting and/or
78
CA 02743477 2011-05-12
alignment of the receptacle with respect to the instrument. One or more labels
for identification of
the sample, patient or any other information of interest, including test
conditions and parameters
optionally may be provided on a receptacle surface or embedded in material
used to construct the
receptacle. Such labels can include indicia that are human readable, machine
readable (e.g.,
barcodes), Optical Character Recognition (OCR), Radio Frequency Identification
(RFID), or some
combination thereof. Referring to Figure IA, receptacle 10 comprises a number
of chambers that
form part of an integrated system, where the chambers collectively define a
plurality of non-linear
pathways punctuated with selectively openable connections. Inlet port 52 and
neck portion 51 serve
as a channel for receiving a sample or other material for processing, testing
or subjecting to reactants
and may have any suitable configuration for admission of the sample or other
material. Sample
material may be transferred to the receptacle 10 by any suitable means, for
example, by using a
syringe with needle that punctures the inlet port 52. The inlet port 52
alternatively may comprise the
female portion of a Luer connection for insertion of a syringe or other
container having a male Luer
connection or an unsealed opening in the top of the receptacle through which
the sample material
may be poured, pipetted or otherwise inserted. The inlet port 52 is preferably
located at or near the
upper edge 12 of the receptacle 10 to reduce the potential for spillage of
sample material upon its
transfer or prior to placement of the receptacle into an automated instrument.
However, the inlet port
52 may be located at any edge of the receptacle 10 or located more centrally,
as is convenient, for
example as a slot or other opening, which optionally is reversibly sealed. For
example, the inlet port
may be closed by heat sealing the opposed sheets of the receptacle 10 after
admission of sample
material.
[0003111 The integrated chamber system of the illustrated receptacle 10
includes eleven
chambers C16, C18, C20, C22, C24, C26, C28, C30, C32, C34 and C36. The
chambers are
generally enclosed compartments that may be connected (selectively,
temporarily, or permanently)
with one or more adjacent chambers so as to permit substances to flow between
at least a portion of
the adjacent chambers, as well as between various chambers of the integrated
system. Each chamber
may contain a substance used to perform a process within the receptacle 10
such as, for example,
sample material, sample processing reagents for preparing a sample material
for further analysis,
reactants, solvents, diluents, wash reagents and the like. Furthermore,
chambers may function, either
through manual manipulation or in cooperation with various elements of an
instrument, as the locus
79
CA 02743477 2011-05-12
for performing one or more process steps, such as an analyte purification
procedure, mixing,
heating/cooling, detection of a signal or visual characteristic (e.g. color
change), waste storage and
removal, etc. Some chambers may be pre-loaded with substances, e.g., sample,
reaction reagents,
buffers, etc., when the receptacle 10 is placed in an instrument and other
chambers may be initially
empty, but one or more substances may be moved into or through the initially-
empty chamber when
performing a process. Some chambers may not be used at all, depending on the
requirements of the
particular process being performed within the receptacle 10.
[000312] In an exemplary use of the receptacle 10, the chambers can be filled
with substances
needed to perform a binding reaction, such as an immunoassay or a nucleic acid-
based reaction. In
such an application of the receptacle 10, chamber C 16 may be loaded with
sample material, chamber
C 18 may be loaded with a sample processing reagent for binding and
immobilizing an analyte
present in the sample material on a solid support, chamber C26 may function as
a sample processing
chamber for separating the immobilized analyte from other components of the
sample material,
chamber C22 may be loaded with a dried, first process material, chamber C20
may be loaded with a
reagent for reconstituting the first process material, chamber C28 may be
loaded with a dried, second
process material, chamber C30 may be loaded with a reagent for reconstituting
the second process
material, chamber C34 may be loaded with a wash reagent, and chamber C36 may
function as a
waste chamber into which waste substances are moved when performing a process
and in which
those waste substances are stored in relative isolation from other aspects of
the process. In addition
to containing the second process material, chamber C28 may also function as a
detection chamber for
detecting a signal or change in a reaction mixture that is indicative of the
presence of the analyte in
the sample material. In an alternative implementation of the receptacle 10,
chamber C32 could
contain a reagent for reconstituting a dried, second process material
contained in chamber C30,
which then could be used to reconstitute a dried, third process material
contained in chamber C28.
Alternatively, a dried, second process material could be loaded in chamber C30
and a reagent for
reconstituting the second process material could be loaded in chamber C32,
where reconstituted
forms of the first and second process materials could be combined with the
separated analyte in
chamber C28.
[000313] Other non-limiting uses of the receptacle 10 will be described in the
Examples section
CA 02743477 2011-05-12
of the disclosure.
[000314] In the illustrated embodiment, chamber C34 is specially designed to
contain a wash
reagent and includes an upper portion 38, a lower neck 40, a vertical section
42, and a lateral section
44 extending toward chamber C26.
[000315] Also, in the illustrated embodiment of the receptacle 10, chamber C36
is
advantageously configured to function as a waste chamber for receiving waste
materials from
chamber C26 and includes an initial vertical inlet 48 extending from chamber
C26, an upper neck 46,
and a collection region 50. Vertical inlet 48 is positioned above chamber C26
and is connected to
chamber C26 by means of portal 70 positioned at the top of chamber C26. When
an analyte
separation procedure, such as a magnetic separation procedure, is performed in
chamber C26,
bubbles may be formed in or moved into chamber C26 by, for example, a
detergent present in the
reaction mixture (e.g., a detergent-based lytic agent provided to the sample
or detergent present in a
sample processing reagent).
[000316] As illustrated in Figure IA, the chambers of the receptacle 10 are
interconnected as
follows: chamber C 18 is connected to chamber C 16 by portal 54; chamber C 16
is connected to
chamber C26 by portal 62; chamber C20 is connected to chamber C22 by portal
56; chamber C22 is
connected to chamber C26 by portal 58; chamber C24 is connected to chamber C26
by portal 60;
chamber C32 is connected to chamber C30 by portal 68; chamber C30 is connected
to chamber C28
by portal 66; chamber C28 is connected to chamber C26 by portal 64; chamber
C34 is separately
connected to chamber C26 by portal 72; and, as noted above, chamber C26 is
connected to chamber
C36 by portal 70. In some embodiments, one or more of portals 54, 56, 58, 60,
62, 64, 66, 68, 70,
and 72 is temporarily closed to prevent fluid flow therethrough by an openable
seal, such as a heat
seal that peels open when pressure is applied to a connected chamber.
[000317] In the embodiment of Figure IA, chamber C16 of the receptacle 10 is
configured to
hold a suitable sample volume. Generally, the sample will be a fluid or
fluidized sample, such as a
fluid sample taken from a human or other animal, which may include blood or a
blood product (i.e.,
plasma or serum), cerebral spinal fluid, a conjunctiva specimen, a respiratory
specimen, a
nasopharyngeal specimen, or a genitourinary tract specimen, or it may be, for
example, an
81
CA 02743477 2011-05-12
environmental, industrial, food, beverage or water sample. Solid or viscous
sample materials (e.g.,
food, fecal matter and sputum) will generally need to be at least partially
solubilized prior to adding
the sample material to chamber Cl 6 (although it is also possible to
solubilize such sample materials
directly in the receptacle as well). The sample material may be organic or
inorganic, and it may be a
material for processing or analysis or a reactant in a chemical, biochemical
or biological reaction.
[000318] The volume capacity of chamber C 16 is preferably from about 10 ,uL
to about 1 mL,
more preferably up to about 850 ML, and most preferably about 625 .iL. This
volume capacity is
intended to accommodate the total volume of substance expected to be placed in
chamber C16,
which in the exemplary application described herein, includes a volume of
sample material combined
with the sample processing reagent from chamber C 18. At the most preferred
volume amounts, this
would be a 500.,uL sample combined with 12512L of a sample processing reagent.
At the low end of
the expected volume range there needs to be enough fluid in the chamber C16 to
force open the seal
at portal 62 upon the application of external pressure to chamber C16. At the
upper end of the
expected volume range, the volume placed in chamber C 16 cannot be so great
that there is stretching
of the receptacle and perhaps peeling or rupturing of a wall of the chamber.
[000319] Portal 54 connects chamber C18 to chamber C 16 and is temporarily
closed by a
selectively openable seal. Upon application of sufficient compressive force to
chamber C 18 by a
pressure application mechanism, an example of which is described below, the
seal closing portal 54
is opened and the sample processing reagent is moved from chamber C l 8 to
chamber CI 6, where it
is mixed with the sample material provided to chamber C16. The sample
processing reagent
preferably includes a binding agent and a solid support, such as magnetically-
responsive particles,
for immobilizing the analyte.
[000320] The first process material is contained in chamber C22, and the
reconstitution reagent
for the first process material is contained in chamber C20. In one embodiment,
the first process
material is an amplification reagent. Dried or solid amplification reagents
are preferred because they
are more stable than liquid amplification reagents. Suitable carriers for the
amplification reagent
include any chemically inert or compatible material and may optionally
include, for example,
diluents, binding agents, lubricants, dissolution aids, preservatives and the
like. In one embodiment,
amplification reagents are frozen in a cryogenic fluid to form uniformly sized
pellets, which are then
82
CA 02743477 2011-05-12
lyophilized for use in unit dose applications. Solid forms of the
amplification reagents can also be
compressed into pellets or tablets, but may be in the form of powders,
granules, or any other
convenient and stable solid form. Dried amplification reagents are also
preferred because there is
less chance of an accidental rupture with dried reagents than with liquid
reagents, and solid materials
provide for very precise dosing of reagents.
[000321] If the first process material is an amplification reagent, then the
amplification reagent
may contain at least one amplification oligonucleotide, such as a primer, a
promoter-primer, and/or a
non-extendable promoter-provider oligonucleotide, nucleoside triphosphates,
and cofactors, such as
magnesium ions, in a suitable buffer. The specific components of the
amplification reagent will
depend on the amplification procedure to be practiced. An exemplary
amplification reagent for
performing a transcription-based amplification reaction is described in the
examples portion of this
disclosure.
[000322] In some embodiments, chamber C20 is left empty or is omitted
altogether if the first
process material is omitted or is provided in a fluid form in chamber C22.
Alternatively, chamber
C22 could be left empty and liquid loaded in chamber C20.
[000323] Upon application of a sufficient compressive force to chamber C20 by
a pressure
application mechanism, such as the one described below, the seal closing
portal 56 is opened and the
reconstitution reagent contained in chamber C20 is transferred to the first
process material contained
in chamber C22.
[000324] In embodiments in which the reconstitution reagent is not provided
(e.g., chamber
C20 is empty or omitted in the illustrated receptacle 10), the first process
material may be a liquid or
a solid that is pre-dissolved prior to loading in chamber C22. If the first
process material is an
amplification reagent, then the amplification oligonucleotides are preferably
present in great excess.
Appropriate amounts of these and other reagents can be determined by the
skilled artisan and will
depend on the assay parameters and the amount and type of target to be
detected.
[000325] After the reconstitution reagent is transferred from chamber C20 to
chamber C22, a
pressure application mechanism applies an external pressure to chamber C22
that opens the seal
closing portal 58 between chamber C22 and chamber C26, thereby causing a
reconstituted form of
83
CA 02743477 2011-05-12
the first process material to flow from chamber C22 to chamber C26. In some
embodiments, the
contents of chambers C20 and C22 are mixed, such as by moving the combined
materials between
chambers C20 and C22 several times, prior to transferring the reconstituted
form of the first process
material to chamber C26.
[000326] A rinse, if used, follows a wash step and is intended to remove
substances present in
the wash reagent that might interfere with processing of the analyte. The
rinse reagent is contained
in chamber C24 and preferably comprises an aqueous buffered solution
containing detergent or
functionally similar material. The rinse reagent could be a reconstituted form
of the first process
material (e.g., an amplification reagent without nucleoside triphosphates).
Alternatively, the rinse
reagent may be a buffer containing no detergents, no anionic detergents, a
lower concentration of
anionic detergents than the wash reagent, or a nonionic detergent to
counteract the effect of an
anionic detergent present in the wash reagent. The volume of rinse reagent
contained in chamber
C24 in preferred embodiments is from about 150 L to about 500 L. At the
appropriate time, a
pressure application mechanism applies pressure on chamber C24 and produces
fluid pressure that
opens the seal closing the portal 60 between chamber C24 and chamber C26,
allowing the rinse
reagent to flow from chamber C24 to chamber C26.
[000327] Chamber C30 contains a reagent for reconstituting the second process
material. In
preferred embodiments, the volume of the reconstitution reagent contained in
chamber C30 is from
about 20 to about 125 L, and more preferably from about 25 to about 100 L.
If the receptacle is
used to perform a nucleic acid-based amplification reaction, then the second
process material may
contain one or more enzymes, such as polymerases for effecting extension
and/or transcription of a
target sequence and, optionally, a probe that specifically and detectably
binds to an amplification
product containing the target sequence or its complement. Suitable carriers
for solid enzyme and/or
probe reagents include any chemically inert or compatible material and may
optionally include, for
example, diluents, binding agents, lubricants, dissolution aids, preservatives
and the like. The
enzyme and/or probe reagents can be frozen in a cryogenic fluid to form
uniformly sized pellets,
which are then lyophilized for use in unit dose applications. Solid forms of
the enzyme and probe
reagents can also be compressed into pellets or tablets with suitable carriers
for ease of handling, but
may be in the form of a powder, granules, or any convenient and stable solid
form. The two solid
84
CA 02743477 2011-05-12
compositions may be formulated as separate pellets or combined as a single
solid form.
Furthermore, a dried probe reagent may be loaded into a chamber, such as
chamber C28, in, for
example, pellet or granule form, or it may be sprayed, printed or otherwise
applied to the walls of a
chamber.
[000328] In reconstituting dried process materials, it was observed that the
reconstitution
reagents have a tendency to concentrate along the perimeters of the chambers
(e.g., heat sealed
regions defining the chamber walls), such that dried process materials that
are more centrally located
in the chambers either do not dissolve or do not fully dissolve. To overcome
this problem, the
inventors discovered that by providing a light oil, such as a mineral oil
(e.g., silicon oil), with
reconstitution reagents, they were able to direct the reconstitution reagents
toward the centers of
chambers, thus improving reconstitution of dried process materials. The oil
was also found to have a
"squeegee" effect, in which the oil essentially sweeps along the walls of a
chamber, thereby causing
all or substantially all of a substance to be moved into an adjacent chamber.
This is particularly
critical in unit dose applications that are sensitive to changes in the
amounts or concentrations of
process materials. Oil was also found to contribute to better mixing of
substances by concentrating
aqueous substances near the centers of chambers, which, in combination with
the squeegee effect,
ensures that more of the substances being mixed are transferred between
chambers. An additional
benefit of oil is its coating ability, which prevents or interferes with
substances sticking to the
surfaces of chambers. As an alternative to oil, other inert, immiscible
liquids having similar
advantages may be used.
[000329] It should be mentioned here that an advantage of the receptacle 10
embodying aspects
of the present invention is the ability, due to the non-linear arrangement of
the chambers, to
reconstitute process materials in a non-sequential manner. That is, it is not
necessary for the first
process material to be fully reconstituted before reconstituting the second
process material. The first
process material can be reconstituted in chamber C22 and the second process
material can be
reconstituted in chamber C28 at any time that is required or convenient for
performing a process,
including simultaneously.
[000330] A pressure application mechanism physically presses upon chamber C30
to produce
fluid pressure that opens the seal closing portal 66 between chamber C30 and
chamber C28, allowing
CA 02743477 2011-05-12
fluid flow between the chambers and transferring the reconstitution reagent
from chamber C30 to
chamber C28 to dissolve the second process material contained therein. If the
process material
contained in chamber C28 is already in a liquid form - for example, if the
enzyme reagent and
detection probe are prepared in a liquid form or are reconstituted prior to
loading them into the
receptacle - chamber C30 may be empty. For certain liquid process materials,
it may be desirable to
include a detergent, such as TRITON X- 100
(octylphenolpoly(ethyleneglycolether)õ), to prevent
components of the process materials from sticking to the walls of the chamber.
Furthermore, if the
process materials provided in a liquid or reconstituted form are sensitive to
electromagnetic
radiation, then the chamber holding these process materials (e.g., chamber
C28) may be constructed
using light-shielding materials.
[000331] It is often desirable and necessary to effect a mixing between
substances moved from
one chamber into an adjacent chamber. For example, when moving a
reconstitution reagent from
chamber C30 into chamber C28 to reconstitute a dried process material
contained in chamber C28, it
is desirable to mix the reconstitution reagent and dried process material. In
the illustrated
embodiment, chamber C30 and chamber C28 are positioned and oriented with
respect to each other
to facilitate gravity assisted mixing of the combined contents of the two
chambers. With gravity
assisted mixing, a pressure mechanism is used to force a substance (e.g.,
reconstitution reagent) from
a lower chamber (e.g., chamber C30) to an upper chamber (e.g., chamber C28).
Gravity assisted
mixing generally depends on at least one of the following mechanisms: (i)
turbulence generated
when a substance is forced through a relatively narrow passage connecting
adjacently positioned
upper and lower chambers (or upper and lower regions of a chamber connected by
a restricted
section), where the substances in both the upper and lower chambers (or
sections of a chamber)
contain liquids; (ii) movement of the combined liquids about the periphery of
the upper chamber;
and (iii) gravitational movement of the substance through the passage
connecting the upper and
lower chambers. One advantage of gravity assisted mixing is that a pressure
mechanism does not
have to be associated with the upper chamber (or upper region of a chamber).
[000332] Chamber C34 contains a wash reagent which is used to remove unwanted
materials
from the sample processing procedure performed in chamber C26. The volume of
wash reagent
contained in chamber C34 in preferred embodiments is from about 400 .tL to
about 5,000 pL and
86
CA 02743477 2011-05-12
most preferably is from about 700 gL to about 2,000 L. A pressure application
mechanism presses
selected portions of chamber C34, thereby producing a fluid pressure that
opens the seal closing
portal 72, and forces wash reagent into chamber C26, the sample processing
chamber. As described
above, chamber C34 includes an upper portion 38, a lower neck 40, a vertical
section 42, and a
lateral section 44 extending toward chamber C26. Due to the arrangement of
chamber C34, it is
believed that gravitational forces assist in moving the wash reagent from the
upper portion 38
through the lower neck 40. In some embodiments, the instrument may include
passive means, such
as a sponge or other compressible body positioned adjacent upper portion 38,
to apply a continuous
and relatively mild pressure to the upper portion to further assist in forcing
substance toward the
lower neck 40. From the lower neck, pressure mechanisms, examples of which are
described below,
are used to force the wash reagent - usually a portion at a time - through the
vertical and lateral
sections 42, 44 and then through portal 72 into chamber C26. Another pressure
mechanism, an
example of which is described below, is positioned at lower neck 40 to
function as a clamp for
selectively stopping further movement of wash reagent.
[000333] Chamber C36, when used for waste collection, is empty prior to
performing a
procedure and is designed to contain the total waste material volume required
by a procedure,
including, for example, waste materials containing sample material, wash
reagent, rinse reagent, and
other expended process materials (e.g., reagents). In general, the preferred
capacity for chamber C36
is about 2 mL when used as a waste chamber.
[000334] As explained above, portal 70 connects chamber C26 and chamber C36
and has an
upper orientation relative to chamber C26. This orientation was discovered to
be advantageous
because bubbles which may be formed in chamber C26 during a separation
procedure, possibly due
to the presence of detergent-based solutions, will naturally tend to rise and
accumulate adjacent to
portal 70 near the top of chamber C26. Thus, bubble-containing waste materials
are more easily and.
efficiently transferred to chamber C36 and, therefore, less likely to
interfere with subsequent signal
detection steps. The location of portal 70 adjacent the top of chamber C26
also helps to retain solid
support particles in chamber C26 when waste material is removed during a
sample processing
procedure, especially one involving the use of magnetically-responsive
particles. As discussed more
fully below, during a preferred sample processing procedure, magnetically-
responsive particles used
87
CA 02743477 2011-05-12
to bind analyte are immobilized when a magnetic field is applied to the
contents of chamber C26.
Bubbles that form in chamber C26 during sample processing will tend to collect
near the top of
chamber C26 and will generally not come into contact with the more centrally
located magnetically-
responsive particles when the waste material is moved from chamber C26 to
chamber C36. If the
connection between a sample processing chamber and a waste chamber is other
than at the top of
sample processing chamber, then at least some bubbles will remain in the
sample processing
chamber when the waste materials are moved from the sample processing chamber
to the waste
chamber. Additionally, at least some of the bubbles that form will likely pass
over the immobilized
particles and could impart a force strong enough to dislodge some particles,
thereby causing some of
the particles to be transferred to the waste chamber with the waste materials.
The sensitivity and
repeatability of processes are thereby improved by the design of the chambers
in the exemplified
receptacle 10 because solid support particles are more likely to be retained
in the designated sample
processing chamber during a separation procedure.
[000335) In the illustrated embodiment employing receptacle 10, chamber C26
(the sample
processing chamber) is connected to six chambers, including chamber C16 (the
sample chamber),
chamber C22 (a first process material chamber), chamber C24 (the rinse reagent
chamber), chamber
C28 (a second process material chamber), chamber C34 (the wash reagent
chamber), and chamber
C36 (the waste chamber). Prior to performing an assay or other process in the
receptacle, chamber
C26 can be empty.
[000336] When receptacle 10 is placed in an automated instrument (described
below), chamber
C26 is oriented so that a removable magnetic field can be applied to the area
of chamber C26. In one
embodiment, the magnetic field is applied by an actuator which moves a
permanent magnet to a
position adjacent to the chamber C26. Suitable magnets are those having a
holding force of about
4.0 lbs. each, such as those available from Bunting Magnets Co. of Newton, KS
as Catalog No.
N50P250250. In a preferred aspect of this embodiment, the magnet is moved into
position by a
magnet actuation mechanism (described in more detail below) along the plane of
the receptacle when
the receptacle is placed into the automated instrument. The magnet may be
moved from any
direction relative to the receptacle. The magnet applies a magnetic field to
chamber C26 and its
contents of sufficient strength to retain magnetic particles in chamber C26
while the field is being
88
CA 02743477 2011-05-12
applied. As will be appreciated by the person of ordinary skill in the art,
when a permanent magnet
is used, the magnet must be movable to a location sufficiently distant from
chamber C26 to remove
the effect of the magnetic field from the sample processing chamber, when
desired. Thus, the
magnet is located on a movable magnet actuation means which can be moved to at
least two
positions: (1) an "on" position in which the magnet is adjacent to chamber C26
and sufficiently close
to apply a particle-retaining magnetic field to the chamber and its contents,
and (2) an "off' position
in which the magnet is positioned sufficiently distant from chamber C26 such
that no magnetic field
of substantial strength is applied to the chamber or its contents and any
magnetic particles present in
the chamber are not appreciably affected thereby.
[000337] Alternative means for applying the magnetic field to the desired
location may include
selective activation of an electromagnet, which is either located adjacent to
chamber C26 during an
assay or process by an automated instrument or which is moved into such
position prior to activation.
Still further means for selectively applying a magnetic field include a
permanent magnet mounted on
a platen that is movable transversely with respect to the plane of the
receptacle 10 into and out of
magnetically affecting proximity to chamber C26. Any suitable actuating
mechanism can be used to
move the platen, such as a threaded rod operatively coupled to a suitable
motor, an electronic linear
actuator, or a solenoid. Such magnetic separation means are known per se in
the art and can easily
be modified by the skilled artisan to any conformation or orientation of
receptacle chambers.
[000338] As mentioned above, opening the seals blocking passages between
chambers and then
transferring substances between adjacently connected chambers can be effected
by pressure
application mechanisms. The pressure application mechanisms of the automated
instrument deliver
a physical force to the outside of the receptacle at select locations and, in
particular, to the outside of
individual chambers of the receptacle at predefined instances as governed by a
computer controller.
In the context of the present invention, the term pressure application
mechanism refers to any means
for delivering a physical pressing force to the external surface(s) of the
receptacle. Preferably each
pressure application mechanism comprises a compression pad with a receptacle-
contacting surface.
The compression pad is coupled to an actuator that moves the pad relative to
the receptacle, generally
perpendicularly with respect to the face of the receptacle, selectively into
and out ofpressing contact
with the receptacle. Alternatively, roller bars or wheels may provide the
physical force. The
89
CA 02743477 2011-05-12
pressure application mechanisms may also have an additional function, such as
to provide a thermal
change to the area adjacent to the actuator. When an actuator comprises a
compression pad, the pad
can be made of any material suitable for exerting an appropriate force to the
surface of the receptacle
without damaging the receptacle. Typically, the compression pad applies a
pressure to either side of
the receptacle, while the opposite side of the receptacle, when in the
automated instrument, is
supported against a wall. Thus, application of external force by the
compression pad of the pressure
application mechanism pinches the receptacle at a selected location to
compress the receptacle at that
location and force fluid movement in a chamber and/or effect a temporary
separation of one chamber
from another (or one portion of a single chamber from another portion) by
bringing the two sides of
the receptacle into fluid sealing contact with each other. Alternatively, a
pair of compression pads
placed on opposing sides of the receptacle and both moveable toward and away
from the receptacle
can pinch the receptacle and its contents between them.
[000339] The functional architecture 700 of a system embodying aspects of the
present
invention is shown schematically in Figure 2. The system operates on a
reaction receptacle, or
container, 10 (300, described below, See Figures 12A and 1213) which is
schematically represented in
Figure 2 as a series of interconnected rectangles (i.e., chambers) as if the
container were shown in a
transverse cross-section. Operation of the system is controlled by a computer
or other
microprocessor, represented in Figure 2 as the control and processing computer
730, which is
programmed to control operation of the system and processing of data. The
system shown is a
schematic representation; overall system operation may be controlled by more
than one computer.
The control and processing computer 730 may reside within an instrument for
processing the
receptacle, or it may be a separate, stand-alone computer operably connected -
e.g., by serial cable,
by network connection, or wirelessly - to the instrument.
[000340] A first element of the system 700 is the substance movement control
system 701.
Substance movement control system 701 both causes and controls movements of
substances from
chamber to chamber within the system. More specifically, the substance
movement control system
701 may include substance moving members 710, which apply a substance moving
force to
individual chambers or to the contents of the chamber, passage blocking
members 708, which
selectively block and unblock portals or passageways between individual
chambers, and chamber
CA 02743477 2011-05-12
partitioning members 706 which selectively divide individual chambers into two
or more sub-
chambers by, for example, pressing against a flexible chamber with a narrow-
edged partitioning
member to collapse a narrow portion of the chamber thereby forming two sub-
chambers on opposite
sides of the narrow collapsed portion. Substance moving member 710, passage
blocking member
708, and chamber partitioning member 706 are moved, or actuated, by an
actuator drive mechanism
704 which may comprise pneumatics, pneumatic pistons, hydraulics, motors,
solenoids, etc. The
actuator drive mechanism 704 is controlled by an actuator controller 702,
comprising a computer or
other microprocessor device programmed to control operation of the actuator
drive mechanism 704
to regulate movement, sequence, and timing of substance moving member 710,
passage blocking
member 708, and chamber partitioning member 706. The actuator controller 702,
in combination
with the control and processing computer 730, selectively activates the
substance moving members
710, passage blocking member 708, and chamber partitioning members 706 in
selected sequences to
control movement of fluids throughout the receptacle during the performance of
an assay or other
process performed in the receptacle. In an alternate configuration, control of
the actuator drive
mechanism 704 may reside in the control and processing computer 730.
[000341] The architecture 700 may further include a temperature control system
720, which
may include heaters 724 and coolers 726 for selectively heating and/or cooling
the contents of one or
more chambers of the receptacle that are in proximity to the heaters or
coolers. It should be
understood that the heaters 724 and coolers 726 may comprise a single thermal
element, such as a
Peltier' chip. Operation of the heaters 724 and coolers 726 is controlled by a
temperature controller
722, which may comprise a computer or other microprocessor device programmed
to control
operation (temperature, timing, and sequence) of the heaters 724 and coolers
726, e.g_, by regulating
power to the heaters 724 and coolers 726. Temperature sensors 728 detect the
temperature of the
heaters 724 and coolers 726 and feed the temperature data to the temperature
controller 722 to
control operation of the heaters 724 and coolers 726 to achieve the desired
temperatures. The
temperature controller 722, in combination with the control and processing
computer 730, control
operation of the heaters 724 and coolers 726 to provide the desired
temperatures and sequences of
temperature variations (e.g., thermal cycling) to perform an assay or other
process within the
receptacle. In an alternate configuration, control of the heaters 724 and
coolers 726 may reside in the
control and processing computer 730.
91
CA 02743477 2011-05-12
[000342] A detector system 712 is provided to detect an output signal from the
contents of one
or more chambers, which signal may be indicative of the presence and/or
quantity of an analyte of
interest. The detector system 712 may comprise a fluorometric detector, or
fluorometer, comprising
an excitation source 714 for generating an excitation signal. The excitation
signal passes through
optics and filters 718, and a resulting excitation signal having a prescribed
wavelength or other optic
characteristic is directed at one or more of the chambers. Emissions from the
contents of the
chamber pass through the optics and filters 718 (which are not necessarily the
same optic elements
through which the excitation signal passes) to a detector element 716, wherein
the optics and filters
718 may pass only an emission signal of a prescribed wavelength to be detected
by the detector
element 716. Control of the operation of the detector system 712, as well as
processing of the data
collected by the detector system, may be performed by the control and
processing computer 730.
[000343] An automated instrument for performing a procedure on a sample in
cooperation with
a multi-chambered receptacle, such as receptacle 10, and which embodies
aspects of the present
invention, is designated by reference number 100 in Figure 3. The automated
instrument 100 can be
used to perform all or a portion of the steps of a process in a single, multi-
chambered receptacle,
without the need for interaction by a technician during the operation of the
process or steps of the
process. The instrument shown in Figure 3 includes a processing unit 102 and a
door assembly 200.
Certain components and surface panels are omitted from the processing unit
102, and a covering
shroud is omitted from the door assembly 200 in the illustrated embodiment so
that the underlying
components and features can be more readily observed.
[000344] Processing unit 102 includes a housing 104. It is noted that the top
panel of the
housing is omitted in the figure. Housing 104 contains electronics, circuitry,
and pneumatics for the
operation of the instrument 100. Barcode reader brackets 106 and 108 hold a
barcode reader (not
shown). In one embodiment, a barcode label is placed on the receptacle 10, and
the barcode reader
situated on the instrument will read this barcode and provide information such
as process
instructions, expiration information, calibration information, and sample
identification. Brackets 106
and 108 hold the barcode reader for hand-held reading of the receptacle
barcode label.
[000345] A display panel .110 projects upwardly from the housing 104 and is
positioned and
oriented to permit a user ready access to any control switches mounted on the
panel and to readily
92
CA 02743477 2011-05-12
view any displays mounted on the panel.
[000346] The housing 104 further includes a front portion 120 which carries
many of the
functional components of the processing unit 102. The front portion 120 of the
housing 104 includes
a pressure mechanism cluster 180 (described in more detail below) mounted with
an actuator plate
124, which may be formed (e.g., machined) from Delrin or aluminum, which may
be coated with
Teflon (PTFE). A recess 130 formed in the actuator plate 124 forms an opening
for receiving and
holding a receptacle 10 placed in the instrument unit 100 prior to closing the
door assembly 200.
The door assembly is hinged or otherwise mounted with respect to the front
portion 120 of the
housing so as to permit movement of the door assembly 200 between a receptacle-
receiving, open
position and a closed position. Latches or other similar mechanism (not shown)
maybe provided to
releasably hold the door assembly 200 in the closed position with respect to
the housing 104. More
specifically, the latch or other mechanism may be provided to hold the door
assembly 200 in the
closed position but be adapted to release the door and permit its movement to
the open position upon
application of a moderate amount of door opening force.
[000347] To begin a process, the receptacle 10 is placed in the instrument
100, and the door
assembly 200 is then closed. The receptacle 10 may include one or more
registration features, such
as alignment holes 74 and 75, which cooperate with mating features within the
instrument, such as
hooks or alignment pins (not shown) provided within the instrument 100 for
properly positioning and
orienting the receptacle with the receptacle-receiving opening of the
instrument. As an alternative to
the exemplary embodiment shown, an instrument incorporating aspects of the
present invention may
include a slot or other opening into which the receptacle is operatively
placed, and a pivoting door
assembly may be omitted. Sample material is preferably transferred to the
receptacle prior to its
placement in the instrument. Adding sample material to the receptacle before
positioning the
receptacle in the instrument minimizes opportunities for the instrument to be
contaminated with
spilled sample material.
[000348] Details of the door assembly 200 are shown in Figures 3 and 5. In the
figures, a
shroud, or housing, preferably covering portions of the door assembly is not
shown so as to permit
the underlying components of the door assembly to be seen.
93
CA 02743477 2011-05-12
[000349] Figure 5 shows the front side of the door assembly 200, i.e., the
side of the door
assembly 200 that faces the processing unit 1 02 and the receptacle when the
door assembly is closed.
The door assembly 200 may include one or more thermal zones for heating and/or
cooling regions of
the receptacle that are in proximity to the thermal zones. The exemplary door
assembly 200 shown
in Figure 4 includes five thermal zones 260, 262, 264, 266, and 268. The
thermal zones are
specifically located to provide heating and/or cooling to one or more specific
chambers of the
receptacle. In the illustrated embodiment, thermal zone 260 covers chamber Cl
6 and neck portion
51. Thermal zone 262 covers chambers C18 and C20. Thermal zone 264 covers
chambers C34,
C32, C30, and most of C36. Thermal zone 266 covers chamber C28. Thermal zone
268 is located
on the magnet translation mechanism 208 (described in more detail below) and
covers chamber C26,
and parts of chambers C22 and C24.
[0003 50] One or more thermal zones maybe used to provide localized heating
and/or cooling to
one or more specific chambers of the receptacle or to provide controlled and
stable ambient
temperatures within the instrument. The ambient temperature may be any
convenient temperature
for optimal performance of a process or particular steps of a process, as
described above. For
example, the ambient temperature may be in the range of about 20 C to about 40
or in the range of
about 25 C to about 37 C_
[000351) The thermal zones are preferably designed to rapidly heat (and/or
optionally rapidly
cool) an area of the receptacle and its contents to any desired temperature.
Rapid temperature
changes may be needed for processes requiring thermal cycling, such as PCR
amplification reactions.
Ideally, the thermal zones will have a high temperature range to accommodate
variations between
processes to be performed. Therefore, the temperature range of the thermal
zones is preferably from
about 5 C to about 95 C for water-based fluids, and may be much greater for
non-aqueous fluids,
such as those containing oil.
[000352] The portions of thermal zones 260, 262, 264, 266 and 268 visible in
Figure 5 are
conductor plates made from a thermally conductive material, such as copper or
aluminum, for
conducting thermal energy (heating and/or cooling) from a thermal energy
source, such as a Peltier
thermoelectric device, to the receptacle 10. The exposed surface of each
conductor plate, as shown
in Figure 5, has a size and shape conforming to the area of the receptacle
intended to be affected by
94
CA 02743477 2011-05-12
the thermal zone. Each conductor plate is mounted within a conforming opening
formed in blocks of
non-conductive material, which provide thermal separation between the
conductor plates.
Preferably, the exposed surfaces of each of the conductor plates for thermal
zones 260, 262, 264,
266, and 268 and the exposed surfaces of the separating blocks are coplanar,
together forming a flat
surface that contacts the side of the receptacle 10 when the door assembly 200
is in the closed
position.
[000353] The conductor plate of each thermal zone is in thermal contact with a
source of
thermal energy for conducting heating or cooling energy from the source to the
exposed surface of
the plate and then to the receptacle. In one embodiment, the source is a
thermoelectric module,
otherwise known as a Peltier device. In a preferred embodiment, thermoelectric
units are mounted
within the door assembly 200 in thermal contact with the thermal zones.
Suitable Peltier devices
include TEC1-12708T125 for thermal zone 264, TEC1-12705T125 for thermal zones
260 and 262,
and TESI-127047125 for thermal zones 266 and 268, all available from Pacific
Supercool Ltd.,
Bangkok, Thailand.
[000354] Thermal insulation, such as foam insulation, may be provided around
the
thermoelectric modules and between portions of the conductor plates. As is
generally known by
persons of ordinary skill in the art, means may be provided within the door
assembly 200 for
dissipating excess heat away from the source of thermal energy, such as one or
more thermally-
conductive heat sinks which may be combined with one or more fan mechanisms
for generating a
convective airflow with respect to the heat sink(s).
[000355] The thermal zones 260, 262, 264, 266, and 268 are under
microprocessor control for
controlling the magnitude and duration of the thermal conditions, including
thermal cycling where
indicated, affected by the thermal zones. And one or more of the thermal zones
can be deactivated
during a test in which heating and/or cooling in the area(s) of the inactive
thermal zone(s) is not
required- Accordingly, control of the thermal zones can be configured to
accommodate a variety of
different process requirements.
[000356] To improve heat transfer to particular chambers, it was found that
the use of oil or
other inert substance can reduce the volume of air (a very poor thermal
conductor) in a chamber and,
CA 02743477 2011-05-12
simultaneously, increase chamber pressure. Increased chamber pressure can
facilitate greater contact
between chambers and corresponding conductor plates, so that the contents of
the chambers are more
completely and rapidly heated.
[000357] The magnet translation mechanism 208 is constructed and arranged to
move a magnet
- including a single permanent magnet, a cluster of permanent magnets, and/or
one or more
electromagnets - relative to a chamber in which a magnetic separation
procedure is being performed
(e.g., chamber C26), referred to, for the purposes of this explanation, as the
magnetic separation
chamber. More specifically, the magnet translation assembly 208 is constructed
and arranged to
move the magnet with respect to the magnetic separation chamber between: (1)
an "on" position in
which the magnet is sufficiently close to the magnetic separation chamber so
that the magnetic field
generated by the magnet will have a sufficient effect on the contents of the
magnetic separation
chamber to substantially immobilize any magnetically-responsive materials
within the magnetic
separation chamber; and (2) an "off' position in which the magnet is
sufficiently removed from the
magnetic separation chamber so that the magnetic field generated by the magnet
will have an
insufficient effect on the contents of the magnetic separation chamber to
substantially immobilize
any magnetically-responsive materials within the magnetic separation chamber.
[000358] In the embodiment shown in Figure 5, the magnet translation mechanism
208 includes
a magnet carrier which supports a magnet or cluster of magnets and an actuator
coupled to the carrier
for moving the carrier up and down relative to the door assembly 200 between
the on and off
positions. In the illustrated embodiment, the magnet translation mechanism 208
caries a cluster of
three magnets 210, with a magnet being omitted from the top or "12 o'clock"
position on the
mechanism 208. The 12 o'clock position is closest to the portal 70 connecting
the magnetic
separation chamber 210 with the inlet 48 of the waste chamber C36. By omitting
a magnet from this
position, an accumulation of magnetic particles at this position is avoided.
This helps minimize the
number of magnetic particles inadvertently carried into the waste chamber C36
during the rinse and
wash steps of the magnetic separation procedure.
[000359] Referring to Figure 4, the compression pads of pressure mechanism
cluster 180 are
positioned in a pattern conforming to the location of the chambers and fluid
pathways of the
exemplary receptacle 10 shown in Figure I and are shaped to perform various of
the process-related
96
CA 02743477 2011-05-12
functions described herein. The automated instrument activates appropriate
pressure mechanisms,
magnets, and/or thermal zones in appropriate sequences, as controlled by an
internal microprocessor
controller.
[000360] The pressure mechanism cluster 180 is installed within the actuator
plate 124 and is
shown schematically in Figure 4. The cluster 180 includes a plurality of
individual compression
pads. constructed and arranged for reciprocal movement transversely to the
outer surface actuator
plate 124 to selectively apply pressure to selected portions of the receptacle
10. The pressure
mechanism cluster 180 includes a plurality of compression pads sized and
arranged to align with
various chambers and portals of the receptacle 10. Each compression pad
includes a head
operatively attached to a reciprocating pneumatic actuator, a magnetic
actuator, solenoid, or other
suitable mechanical, electro-mechanical or other actuator (not shown) for
moving the pad out into
compressing engagement with a corresponding portion of the receptacle 10 and
back into its stowed
position.
[000361] Compression pad P51-1 is positioned so as to align with a top portion
of the neck 51
of the receptacle 10. Compression pad P51-2 is positioned so as to align with
a lower portion of the
neck 51 of the receptacle 10 where the neck 51 enters chamber C 16 Compression
pads P 16-1, P 16-
2, P 16-3 and P 16-4 are all positioned so as to align with different portions
of chamber C 16 of the
receptacle 10. Compression pad P16-1 is the bottom compression pad for chamber
C 16,
compression pad P16-2 is the top compression pad for chamber C 16, compression
pad P 16-3 is the
divider for chamber C 16, and compression pad P 16-4 is the front compression
pad for chamber C 16.
[000362] Having multiple pads P 16-1, P 16-2, P1 6-3 and P 16-4 combined with
a large chamber
C16, which may be employed as the sample chamber, allows flexibility in the
size of the sample to
be assayed. Divider pad P 16-3 can be used to partition the chamber C 16 into
two smaller chambers.
Note that chambers C26 and C28 are much smaller than chamber C16 and, thus, it
is self-evident
that the entire contents of chamber C16, if filled substantially to capacity,
would not fit within
chamber C26 and/or chamber C28. For some applications, a relatively large
volume of sample
material may be required to ensure that there is a detectable amount of an
analyte, if present in the
sample, but subsequent chambers, such as chambers C26 and C28, for processing
the sample cannot
accommodate such a large volume of sample material. The multiple pads adapted
to compress
97
CA 02743477 2011-05-12
different portions of chamber C 16 allows the sample to be moved from chamber
C 16 to chamber
C26 one portion, or aliquot, at a time.
[000363] Compression pads P 18-1 and P18-2 are positioned so as to align with
the chamber
C18. Compression pad P18-1 is the rear compression pad for chamber C18 and
compression pad
P 18-2 is the front compression pad for chamber C 18.
[000364] Compression pad P20 is positioned so as to align with chamber C20.
Compression
pad P22 is positioned so as to align with chamber C22. Compression pad P24 is
positioned so as to
align with chamber C24. Compression pad P30 is positioned so as to align with
chamber C30.
Compression pad P32 is positioned so as to align with chamber C32.
[000365] Note that in the illustrated embodiment, there are no compression
pads associated
with chamber C28 or with region 50 of chamber C36 or region 38 of chamber C34.
The instrument
may, however, include other mechanisms for imparting forces onto the chambers,
or portions thereof,
as described in more detail below. Moreover, the illustrated embodiment is
exemplary, and other
embodiments encompassing aspects of the present invention may provide
compression pads for
chamber C28 as well as regions 50 and/or 38 or may omit compression pads for
other chambers
and/or regions thereof.
[000366] Compression pads P34-1, P34-2, and P34-3 align with the lateral
section 44 of
chamber C34. Compression pad P34-1 is the #1 wash compression pad, compression
pad P34-2 is
the #2 wash compression pad, and compression pad P34-3 is the #3 wash
compression pad.
[000367] Compression pad P34-4 is the #4 wash compression pad and aligns with
the vertical
section 42 of the wash reagent chamber C34.
[000368] Compression pad P34-5 is the #5 wash compression pad and aligns with
the lower
neck 40 of the wash reagent chamber C34. The #5 wash pad head P34-5 further
includes parallel
raised ribs P34-5a extending across the compression pad. Ribs P34-5a provide a
tight compressive
seal for closing off the neck portion 40 of receptacle 10 to prevent fluid
flow from the upper portion
38 of the wash reagent chamber 34 into the vertical section 42 and lateral
section 44 of chamber 34.
Compression pads P36-1, P36-2 and P36-3 are aligned with the vertical inlet 48
of chamber C36.
98
CA 02743477 2011-05-12
Note that compression pad P36-3 is wider than the compression pad P36-1 and
P36-2 so that a
portion of the compression pad P36-3 covers the neck 46 of chamber C36.
Compression pad P36-1
is the #1 waste compression pad, compression pad P36-2 is the #2 waste
compression pad, and pad
P36-3 is the #3 waste compression pad.
[000369] Compression pad P72 is a clamp aligned with portal 72. Similarly,
compression pad
P70 is a clamp that aligns with portal 70, compression pad P62 is a clamp that
aligns with portal 62,
compression pad P58 is a clamp that aligns with portal 58, compression pad P60
is a clamp that
aligns with portal 60, compression pad P66 is a clamp that aligns with portal
66, compression pad
P68 is a clamp that aligns with portal 68, compression pad P56 is a clamp that
aligns with portal 56,
and compression pad P54 is a clamp that aligns with portal 54. Compression pad
P64 is a clamp
aligned with portal 64.
[000370] Compression pad heads may be formed from a black acetal resin sold
under the brand
name Deirin by DuPont of Wilmington, DE.
[000371] Compression pad P26 is positioned so as to align with chamber C26. In
a preferred
embodiment, pad P26 is coupled to a screw actuator or other relatively slow-
moving actuator. A
screw actuator provides slow and steady compression, rather than the abruptly-
applied compressive
forces generated by pneumatically actuated compression pads. This controlled
motion can provide
several advantages. For example, a screw actuator allows the user to control
the rate and extent to
which the compression pad P26 moves, thereby making it possible to limit or
prevent turbulence
within a chamber being compressed. Avoiding turbulence is desirable when, for
example, using
detergent-based reagents that are prone to bubble under turbulent conditions
or when removing wash
reagent from a chamber during a magnetic separation wash procedure. While the
wash reagent is
being removed from a chamber containing immobilized, magnetically-responsive
particles,
turbulence within the chamber can cause the particles to become dislodged and,
thus, to be washed
away into a waste chamber. The controlled movement of the compression pad
P26can also help to
prevent over-compressing a chamber, which can result in peeling apart or
rupturing a wall of a
chamber.
[000372] Figures 12A and 12B show another receptacle 300 in accordance with
the present
99
CA 02743477 2011-05-12
invention. Like receptacle 10 described above, receptacle 300 includes a
generally planar vessel
having flexible top and bottom sheets formed from thin flexible materials,
such as foils and/or
plastics, and defining a pouch-like vessel. The receptacle 300 has an upper
edge 302 and a lower
edge 304 that indicate the preferred orientation of the receptacle during use
and define an upper
direction and a lower direction. An exemplary receptacle of the type shown in
Figure 12A has
dimensions of about 5.5 inches by about 3.4 - 4.0 inches and is about 0.4
inches thick (when filled
with sample and process materials), but may be of any dimensions suitable for
manual manipulation
or for use with an automated system, similar to the one described herein.
Preferred materials for
constructing receptacle 300 are the same as those described above for
receptacle 10. Receptacle 300
includes an inlet port 306 for loading a sample material, or other substance,
into the receptacle 300.
Receptacle 300 includes nine chambers C320, C322, C324, C328, C332, C334,
C336, C338, and
C340.
[000373] As shown in Figure 12A, the receptacle 300 may include a rigid frame
380
comprising vertical portions 381 and 383, a top horizontal portion 384, and a
bottom horizontal
portion 385. A panel 382 may receive an identifying label, such as a bar code
or other human or
machine readable indicia. The information carried on such label may include
lot number, serial
number, assay type, expiration date, etc.
[000374] A projecting tab 386 projects above the top portion 384 and provides
an appendage
for grasping the pouch 300 and inserting it into an instrument and removing it
from the instrument.
A port cover 388 (e.g., a one-way valve) is provided for introducing sample
into the sample chamber
C328A through inlet channel C328C. Frame 380, including the projecting tab 386
and sample cover
388, are preferably formed from a suitably rigid material such as plastic.
[000375] Further details of the receptacle 300 are shown in Figure 12B, which
is an exploded
view of the receptacle. Frame 380 comprises rear frame component 380a and
front frame component
380b between which is sandwiched a flexible pouch 301. Rear frame component
380a includes
vertical portions 381 a, 383a, a top horizontal portion 384a, a bottom
horizontal portion 385, and
projecting tab 386a. Front frame component 380b includes vertical portions
381b, 383b, top
horizontal portion 384b, and projecting tab 386b, but does not include a
bottom horizontal portion.
100
CA 02743477 2011-05-12
[000376] Each frame component 380a and 380b may be injection molded, and the
two
components may be connected to one another in a frame assembly by ultrasonic
welding. The
flexible pouch portion 301 of the receptacle 300 positioned and secured in the
frame 380 by pins on
the frame components 380a, 380b extending through holes formed in the
periphery of the pouch.
[000377] In an exemplary use of the receptacle 300, the chambers can be filled
with substances
needed to perform a binding reaction. For example, sample material may be
loaded into chamber
C328 through inlet port 306. Chamber C328 consists of an upper region C328A
and a lower region
C328B connected by a restricted section 364 that can be closed by a pressure
application mechanism
so that the lower region is segregated from the upper region. Chamber C332 may
be loaded with a
sample processing reagent for binding and immobilizing an analyte present in
the sample material on
a solid support, the lower region C328B of chamber C328 may, in addition to
receiving sample
material, function as a sample processing region of chamber C328 for
separating the immobilized
analyte from other components of the sample material, chamber C334 maybe
loaded with a dried,
first process material, chamber C340 may be loaded with a reagent for
reconstituting the first process
material, chamber C322 may be loaded with a dried, second process material,
chamber C324 maybe
loaded with a reagent for reconstituting the second process material, chamber
C336 may be loaded
with a wash reagent, chamber C338 may be loaded with a rinse reagent for
removing inhibitory
components of the wash reagent, and chamber C320 may function as a waste
chamber for receiving
and storing waste substances in relative isolation from other aspects of the
reaction. In addition to
containing the second process material, a lower region C322B ofchamber C322
may also function as
a detection region of chamber C322 for detecting a signal or change in a
reaction mixture that is
indicative of the presence of at least one analyte of interest in the sample
material.
[000378] Chamber C320 is configured to receive waste materials from chamber
C328 and
includes an initial, generally vertical inlet 370 extending from chamber C328,
an upper neck 372,
and a collection region 374. Vertical inlet 370 is positioned generally above
the lower region C328B
of chamber C328 and is connected to chamber C328 by means of portal 360
positioned near the top
of the lower region C328B of chamber C328. The arrangement of chamber C320
relative to the
chamber C328 allows for bubbles contained in chamber C328 to be transferred
directly into chamber
C320 when waste materials are moved from chamber C328 to chamber C320.
Furthermore, because
101
CA 02743477 2011-05-12
upper neck 372 is positioned above the collection region 374 of chamber C320,
waste material can
be retained within collection region 374 by force of gravity without the
application of a clamp or
other means for sealing the upper neck 372.
[000379] As illustrated in Figure 12, in the interconnected chamber system of
the receptacle
300, chamber C324 is connected to the lower region C322B of chamber C322 by
portal 350, the
lower region C322B of chamber C322 is connected to chamber C328 by portal 356,
chamber C340 is
connected to chamber C334 by portal 344, chamber C334 is connected to the
lower region C328B of
chamber C328 by portal 346, chamber C332 is connected to the lower region
C328B of chamber
C328 by portal 348, chamber C338 is connected to the lower region C328B of
chamber C328 by
portal 362, and chamber C336 is connected to the lower region C328B of chamber
C328 by portal
342. A wall 376 projects obliquely into chamber C336 for preventing air
bubbles that have collected
in an upper portion of chamber C336 from being moved through portal 342 and
into chamber C328
during a wash procedure. In one embodiment, each of the portals 342, 344, 346,
348,.350, 356, 360,
and 362 is temporarily closed by an openable seal or other barrier to prevent
fluid flow therethrough.
Like receptacle 10, receptacle 300 defines a non-linear arrangement of
chambers useful for
performing complex procedures requiring or benefiting from non-sequential
processing of samples.
[000380] Chamber C322 includes the lower region C322B discussed above and an
upper region
C322A which are connected by a restricted section 358. In the illustrated
arrangement, the combined
substances of chambers C324 and C322 (before or after being combined with
substances from
chamber C328) can be mixed in chamber C322 by moving the combined substances
back-and-forth
between the upper and lower regions C322A, C322B of chamber C322, while each
of portals 350
and 356 is clamped by a pressure application mechanism to prevent substances
from moving into
chambers C324 and C328. Due to the relative orientations of the upper and
lower regions C322A
and C322B of chamber C322, gravity assists in moving the combined substances
from the upper
region C322A into the lower region C322B as pressure being applied to the
lower region C322B is
removed. Thus, in the embodiment shown, there is no need for an external
pressure to move
substances from the upper region C322A to the lower region C322B of chamber
C322.
[000381] Receptacle 300 is processed in an instrument (not shown) having an
arrangement of
pressure application mechanisms - such as compression pads - and thermal zones
sized, shaped, and
102
CA 02743477 2011-05-12
positioned to conform to the chambers of the receptacle 300 for selectively
moving substances
between chambers and for selectively providing heating and/or cooling to one
or more selected
chambers.
[000382] A second embodiment of an instrument embodying aspects of the
invention, and
configured to process a receptacle 300, such as shown in Figures 12A and 12B,
is designated by
reference number 1000 in Figure 13. Instrument 1000 includes a housing 1002
having a top portion
1002 and a bottom 1002b. Housing 1002 further includes a handle 1004. Handle
1004 includes
opposed slots 1009 for holding a receptacle 300 during preparation. Instrument
1000 further
includes an air intake 1008, preferably covered by a suitable filter material,
and an exhaust vent
1010. A status screen 1012 displays status and other information useful to the
operator, and
operation buttons may be provided, for example below screen 1012, as shown. A
receptacle insert
slot 1014 in the top portion 1002a of the housing is configured to receive a
receptacle 300, as shown
in Figure 13. Receptacle insert slot 1014 is preferably convex so that spilled
liquid will run off the
housing 1002, rather than into the slot. A slot cover slide 1016 can be
manually manipulated after
the receptacle 300 is inserted into the slot 1014 to provide a closure over
the slot 1014 and may again
be opened to permit removal of the receptacle 300. Fan 1011 provides cooling
air for electronics and
other components internal to the housing 1002.
[000383] Looking into the interior of the housing 1002 in Figures 14 and 15,
the instrument
1000 includes an air compressor 1020 and an air reservoir 1024. The instrument
further includes a
detector, such as fluorometer 500 (described in more detail below), and a
magnet actuator 1090 for
selectively moving magnets into and out of operative position with respect to
the receptacle.
Instrument 1000 further includes an air manifold 1082 and an actuator plate
1080. Instrument 1000
may further include a coalescing air filter 1022. Aspects of the temperature
control system are also
shown and include a thermal isolating frame 1048 and a heat dissipating
system, including a fan
1070, a shroud 1068, and heat sink 1064. Fan 1070 draws air into the
instrument through the air
intake 1008, and the heated air, after flowing over the heat sinks 1064, exits
the housing 1002
through the exhaust vents 1010.
[000384] A pressure mechanism cluster of the instrument 1000 for applying
selective pressure
to the receptacle 300 shown in Figure 9 is shown in Figure 16. The cluster is
installed within the
103
CA 02743477 2011-05-12
actuator plate 1080, which may be formed (e.g., machined) from Delrin or
aluminum, which may
be coated with Teflon (PTFE). As with cluster 180 described above, the
cluster of Figure 16
includes a plurality of individual compression pads constructed and arranged
for reciprocal
movement transversely to the actuator plate 1080 to selectively apply pressure
to selected portions of
the receptacle 300. The pressure mechanism cluster includes a plurality of
compression pads sized
and arranged to align with various chambers and portals of the receptacle 300.
Each compression
pad includes a head operatively attached to a reciprocating pneumatic
actuator, a magnetic actuator,
solenoid, or other suitable mechanical, electro-mechanical or other actuator
(not shown) for moving
the pad out into compressing engagement with a corresponding portion of the
receptacle 300 and
then back into its stowed position.
[000385] In one embodiment, each compression pad is coupled to an air conduit
formed in the
manifold 1082, which directs pressurized air to the pad to move the pad to an
extended position.
More specifically, each compression member is controlled by a solenoid valve
which, when engaged,
connects pressurized system air to a pathway that goes to a portal where a
pressure regulator (not
shown) is installed, and the output of this regulator is connected back into
another portal on the
manifold 1082, which feeds the now-regulated & pressurized air to the
compression member.
Pressure sensors may be provided for monitoring pressure within the system.
[000386] Compression pads P328-1 and P328-2 are aligned with lower and upper
portions,
respectively, of the sample chamber C328A of the receptacle 300. Compression
pad P328-3 is
aligned with an upper neck C328C. Compression pad P364 is aligned with the
restricted area P364
between chamber C328A and chamber C328B and provides a means for selectively
opening or
closing the restriction 364 for controlling fluid flow between chambers C328A
and C328B.
[000387] Compression pad P338-2 is a circular pad aligned with an upper
portion of the rinse
chamber C338, and compression pad P338-1 aligns with a lower portion of the
chamber C338.
Compression pad P362 aligns with the portal 362 connecting the rinse chamber
with C338 with the
magnetic separation chamber C328B and provides a means for selectively opening
or closing the
portal 362 (after an initially-closed burstable seal has been opened) for
controlling fluid flow
between the chambers.
104
CA 02743477 2011-05-12
[000388] Compression pads P320-1, P320-2, and P320-3 align with different
portions of the
waste chamber C320. Compression pads P320-1 and P320-2 align with lower and
upper portions,
respectively, of an inlet passage connecting chamber C328B to the waste
chamber C320 and are
adapted for moving fluid up the passage into the upper neck 372 of the chamber
C320. Compression
pad P320-3 controls movement of fluid through the upper neck 372 and, in
particular, prevents fluid
from flowing back from the waste chamber C320 toward the chamber C328B.
Compression pad 360
is aligned with the portal 360 connecting the waste chamber C320 with chamber
C328B and
provides a means for selectively opening or closing the portal 360 to control
fluid flow between the
chambers.
[000389] Compression pad P356 is aligned with the portal 356 connecting
chamber C322B and
chamber C328B and provides a means for opening or closing the portal to
control fluid flow between
the chambers. Compression pad P322-1 is aligned with chamber C322A, and
compression pad P358
is aligned with the restricted section 358 connecting regions C322A and C322B
of chamber C322.
Compression pad P358 provides a means for opening or closing the restricted
section 358 for
controlling fluid flow between the chambers.
[000390] Compression P322-2 is aligned with the chamber C322B. In one
embodiment, lower
region C322B of chamber C322 functions as a detection chamber for detecting a
signal or change in
a reaction mixture that is indicative of the presence of at least one analyte
of interest in the sample
material. Accordingly, in some embodiments, compression pad P322 is configured
to allow optical
transmission through the compression pad, thereby permitting the detection of
an optical signal
emitted by the contents of chamber region C322B.
[000391] Compression pads P324-1 and P324-2 are aligned with upper and lower
portions,
respectively of the chamber C324. Compression pad P350 is aligned with the
portal 350 connecting
chamber C324 and C322B and provides a means for opening or closing the portal
to control fluid
flow between the chambers.
[000392] Compression pads P332-1 and P332-2 are aligned with the upper end
lower portions,
respectively of the chamber C332. Compression P348 is aligned with the portal
348 connecting
chamber C332 and chamber C328B and provides a means for selectively opening
and closing the
105
CA 02743477 2011-05-12
portal 348 to control fluid flow between the chambers.
[000393] Compression pad P340 is aligned with chamber C340, and compression
pad P334 is
aligned with chamber C334. Compression pad P344 is aligned with portal 344
connecting chambers
C340 and C334 and provides a means for selectively opening or closing the
portal 344 for
controlling fluid flow between the chambers. Compression P346 is aligned with
portal 346
connecting chamber C334 and C328B and provides a means for opening or closing
portal 346 for
controlling fluid flow between the chambers.
[000394] Compression pad P336-1 is aligned with a portion of the wash chamber
C336 and
compression pad P336-2 is aligned with another portion of the wash chamber
P336. Compression
pad P336-2 aligns with a portion of the wash chamber P336 extending from an
end of the oblique
wall 376 and a peripheral side wall of the chamber C336 and provides a means
for dividing chamber
C336 into two sub-chambers. Compression pad P342 aligns with the portal 342
connecting chamber
C336 and chamber C328B and provides a means for selectively opening or closing
the portal 342 to
control fluid flow between the chambers.
[000395] Compression pad P336-3 is aligned with a portion of wash chamber C336
and is a
bladder formed by a sheet of flexible, relatively non-porous material, secured
at its edges to a recess
formed in the actuator plate 1080. The bladder P336-3 is in communication with
an air conduit
formed in the manifold 1082 and may be controlled by a regulator. When filled
with air, the bladder
P336-3 inflates (expands) to apply pressure to a portion of chamber C336 to
displace wash fluid
from the chamber. The bladder P336-3 is preferred over a reciprocating
compression pad for this
location because inflation of the bladder can be controlled to provide a slow,
steady displacement of
chamber C336.
[0003961 The function of the bladder P336-3 is to gently pressurize the wash
chamber C336
enough to move an aliquot of wash buffer reagent into the channel area
adjacent portal 342 The
compress pad P336-2 has a single raised thin-surface to hold the wash aliquot
in place until it is
moved to the chamber C328B. The bladder P336-3 is actuated much like any of
the other
compression members, controlled by a solenoid valve which, when engaged,
connects the
pressurized system air to a pathway that goes to a portal where a pressure
regulator is installed, and
106
CA 02743477 2011-05-12
the output of this regulator is connected back into another portal on the
manifold 1082 which feeds
the now-regulated & pressurized air to the bladder. A suitable operating
pressure for the bladder
P336-3 is approximately 10 psi.
[000397] The pressure mechanism cluster may be covered by an elastomeric
shield (See
reference number 1081 in Figures 18 and 19) which stretches to permit the
compression pads to
operate and which covers and protects the compression pads, for example, from
spilled fluids. The
shield may include one opening through which a detector (e.g., fluorometer
500) may detect optical
singles emitted from the contents of a chamber located adjacent the opening.
The shield may be
provided with non-stick properties (e.g, a non-stick coating) to facilitate
insertion and removal of the
receptacle 300 from the instrument 1000 after processing.
[000398] As shown in Figure 17A, the pneumatic manifold 1082 is attached to
the actuator
plate 1080. The air reservoir 1024 is connected to the manifold 1082 as is the
magnet actuator 1090
and the detector 500. A window 1086 permits viewing of a bar code or other
label provided on the
panel 382 of the receptacle 300 (see Figure 12A), and bar code reader 1088 is
constructed and
arranged to read a bar code on the receptacle. Valves 1084 (e.g., solenoid
valves) control the
pressure distribution to the various conduits of the manifold, each of which
is connected to one of the
pneumatic compression pads shown in Figure 16.
[000399] More specifically, Figure 17B shows a circuit diagram of the
pneumatic system of the
instrument 1000. The system shown in Figure 17B differs somewhat from the
structure shown in
Figure 17A. For example, the system shown in Figure 17B lacks an air
reservoir. The pneumatic
system includes pump 1020 connected to a check valve 1030, a water trap 1032,
and an air dryer
1034 (e.g., a desiccant device for removing moisture from pressurized air).. A
valve 1028 (e.g., a
solenoid valve) is constructed and arranged for selectively disconnecting the
pump from the
pneumatic system by venting the pump to atmosphere "ATM". A pressure sensor
1036 detects the
pressure in the system and may communicate with the control and processing
computer 730 (See
Figure 2). The system may also include an accumulator 1038. The system next
includes the valves
1084 and the compression members (e.g., as shown in Figure 16). In Figure 17B,
only three valves
1084a, 1084b, 1084c and three associated compression members P332-2, P332-1,
and P348 are
shown. As can be seen in the Figure, each valve 1084a, 1084b, I084c can
selectively connect the
107
CA 02743477 2011-05-12
associated compression pad to the pressure source (pump 1020 or accumulator
1038), connect the
associated compression pad atmosphere "ATM" to vent the compression pad (to
remove pressure
that might inhibit complete retraction of the compression pad), or block the
compression member
branch from the rest of the pneumatic circuit.
[000400] The magnet actuator 1090, shown in cross-section in Figure 19,
includes a motor
1092 which moves a magnet holder 1136 holding magnets 1132, 1134 via a shaft
1094 coupled to a
lead screw 1096. Magnet actuator 1090 further includes cylinders 1 110, 1112
which are coupled to
an actuator fitting 1140 for reciprocally moving a compression cup 1130. Motor
1092 and cylinders
1110, 1112 are mounted atop a mounting block 1 120 attached to the manifold
1082.
[000401] More specifically, motor 1092 includes a shaft 1094 coupled to lead
screw 1096 by a
coupler 1098 that is rotatably mounted within bearings 1100, 1102. Lead screw
1096 is able to slide
axially within coupler 1098 and is threadably coupled to the magnet holder
1136 within which are
held a number of magnets, including magnets 1132, 1134 shown in the figure. In
a preferred
embodiment, the magnet holder 1136 holds three such magnets. The magnet holder
1136 is disposed
within a hollow portion of the compression cup 1130, which is disposed within
a circular hole
formed transversely through the actuator plate 1080. An end of the lead screw
1096 inserted into the
coupler 1098 has a square or other shaped cross-section that will prevent the
lead screw 1096 from
rotating with respect to the coupler 1098. Furthermore, the magnet holder 1136
has projecting ridges
or a non-circular peripheral shape that conforms to the inner wall of the
hollow compression cup
1130 to prevent the magnet holder 1136 from rotating within the compression
cup 1130. Thus
rotation of the lead screw 1096 by the motor 1092 causes corresponding
translation of the magnet
holder 1136.
[000402] In addition simply moving the magnet holder 1136 between on and off
positions, the
motor 1092 can be controlled to vary the speed with which the magnet holder
1136 is moved from
the on position to the off position and vice versa as well as to vary
positions between on and off.
Those skilled in the art could imagine how, using a combination of speed and
position, the strength
and rate of change of magnetic field strength can be optimized to maximize
magnetic particle
retention.
108
CA 02743477 2011-05-12
[000403] Each of the cylinders 1110, 1112 includes a cylinder housing 1114
within which is
disposed a reciprocating piston 1116. (Note: Cylinders 1110 and 1112 are
identical; accordingly,
only the features of cylinder 1110 are numbered in the figure). A pneumatic
port 1118 is provided
for coupling the cylinder 1110 to a source of air pressure. The pistons of
each of the cylinders 1110,
1112 are coupled to the actuator fitting 1140.
[000404] The actuator fitting 1140 includes a circular center portion which
fits into aportion of
the same hole into which the compression cup 1130 fits and two radial
projections 1142, 1144 which
fit into openings 1146, 1148, respectively, formed into the actuator plate
1080 adjacent the circular
opening that receives the compression cup 1130. The pistons 1116 are attached
to the radial
projections 1142, 1144.
[000405] As shown in the figure, the magnets 1132,1134 carried in the magnet
holder 1136 are
in an "on" position. That is, the magnets are in close proximity to the
elastomeric shield 1081
covering the actuator plate 1080, and thus are in close proximity to the
chamber of the receptacle
within which a magnetic separation procedure is being performed. The magnets
can be moved to an
"off' position by rotating the lead screw 1096 via the motor 1092 to translate
the magnet holder 1136
away from the shield 1081 (i.e., to the right in the figure) within the hollow
portion of the
compression cup 1130. Reversing the rotation of the motor 1092 and the lead
screw 1096 extends
the magnet holder 1136 back to the "on" position at the end of the compression
cup 1130.
Continued rotation of the lead screw 1096 will push the magnet holder 1136 and
compression cup
1130 out (to the left in the figure) against elastomeric shield 1081 to apply
a compressive force to a
chamber adjacent the compression cup 1130. Thus, the chamber is compressed to
displace liquid
from the chamber while the magnets are in the "on" position to hold and retain
magnetic particles
within the chamber, for example during a rinse step of the magnetic separation
procedure.
[000406] When the compression cup 1130 is extended by the lead screw 1096 and
the magnet
holder 1136, the actuator fitting 1140, which is rigidly attached to the
compression cup (e.g., the two
components are threaded together), also moves to an extended position (to the
right in the figure).
The pistons 1116 of the cylinders 1110, 1112 move passively along with the
actuator fitting 1140.
When the magnet holder 1136 is retracted by the lead screw 1096, springs (not
shown) within the
cylinders 1110, 1112 cause the actuator fitting 1140 and the compression cup
1130 to return to the
109
CA 02743477 2011-05-12
retracted position shown in the figure.
[000407] The magnet actuator 1090 also functions as a compression pad for
applying a
compressive force to a chamber of the receptacle to force the fluid contents
from the chamber when
the magnets are in the "off" position. This is accomplished by turning the
lead screw 1096 to
withdraw the magnet holder 1136 to the "off' position (to the right in the
figure) and then
pressurizing the pistons 1116 of the cylinders 1110, 1112 to extend the
pistons and thus extend the
actuator fitting 1140 and the compression cup 1130 (to the left in the figure)
against the shield 1081,
which will stretch and deflect in response to the reciprocating projection of
the cup 1130, to
compress a chamber adjacent the compression cup 1130. As the actuator fitting
1140 is extended,
the magnet holder 1136, which has been retracted back (to the right) into
contact with the actuator
fitting 1140, will also be moved in the direction of extension. To accommodate
this movement of
the magnet holder 1136, the end of the lead screw 1096 is able to slide with
the coupler 1098 (i.e.,
the lead screw 1096 "floats" within coupler 1098). Springs within the cylinder
1110, 1112 retract the
actuator fitting 1140 and compression cup 1130 when pressure is removed from
the pistons 1116.
[000408] The temperature control system of instrument 1000 is shown in Figure
20 and
includes thermal conductive elements 1040, 1042, 1044 disposed within a
thermal isolating frame
1048. The thermal conductive elements are preferably made from a thermally
conductive material,
such copper or aluminum, and the isolating frame 1048 is preferably formed
from a thermal
insulating material, such as Ultem or Delrin . In the illustrated embodiment,
ach of the
conductive elements 1040, 1042, 1044 is sized and shaped so as to be in
thermal communication
with a region of the receptacle 300 encompassing more than one chamber.
Furthermore, at least a
portion of each conductive elements 1040,1042, 1044 is in close proximity to
at least a portion of an
adjacent conductive element such that a chamber encompassed by one conductive
element is closely
adjacent to a chamber encompassed by the adjacent conductive element, and the
two chambers are
connected by a portal with no passageway extending between the two chambers.
Such close
proximity between adjacent conductive elements without thermal crosstalk
between the adjacent
conductive elements is facilitated by the insulation provided by the isolating
frame 1048.
[000409] The receptacle 300 is held in an operative position within the
instrument 1000
between the actuator plate 1080 and the isolating frame 1048 (See Figures 14
and 15). The
110
CA 02743477 2011-05-12
arrangement of the isolating frame 1048 and the actuator plate 1080 within the
instrument 1000
results in a receptacle-receiving gap therebetween, and that gap is
dimensioned with respect to the
receptacle 300 such that when a chamber of the receptacle that is adjacent one
of the conductive
elements 1040, 1042, 1044 is filled with fluid, the chamber expands to
increase the thermal contact
between the surface of the chamber and the adjacent conductive element.
[000410] Peltier' devices 1050, 1052, 1054 are positioned in thermal contact
with conductive
elements 1040, 1044, 1042, respectively. Temperature sensors 1056, 1058, 1060
are positioned in
thermal contact with the conductive elements 1040, 1042, 1044, respectively,
for sensing the
temperature of the respective thermal conductive element. Sensors 1056, 1058,
1060 may comprise
RTD sensors, and are coupled to a controller (e.g., temperature controller
722) for controlling
operation of the Peltier devices. Heating elements other than Peltier devices,
such as resistive foil
heaters, can be used as well.
[000411] The Peltier' devices 1050, 1052, 1054 (or other heating or cooling
elements), are
preferably mounted onto a heat sink 1062 which may comprise an aluminum block
having a first
portion 1064 with a first planar side on which the Peltier' devices are
mounted and heat dissipating
fins 1066 projecting from the opposite side. A shroud 1068 partially covers
the dissipating fins 1066
of the heat sink 1062, and a cooling fan mounted within a fan housing 1070 is
positioned for drawing
air into shroud 1068 and past the heat dissipating fins 1066.
[000412] The Peltier' devices 1050, 1052, 1054 can be selectively operated to
heat or cool the
conductive elements 1040, 1044, 1042 and thereby heat or cool the contents of
any chambers and
portions of chambers of the receptacle 300 that are in proximity to the
respective conductive
elements. The conductive elements 1040, 1042, 1044, as well as the isolating
frame 1048, are in a
fixed position with respect to the pouch 300. When the pouch 300 is inserted
into the slot 1014 of
the instrument 1000, the pouch 300 is disposed in close proximity to the
conductive elements 1040,
1042, and 1044. When a chamber is filled with a substance, the chamber of a
flexible pouch will
expand into the conductive element, thereby providing more complete physical,
as well as thermal,
contact with the conducting element positioned adjacent that chamber.
[000413] The results of an analytical procedure performed with the receptacle
10 or 300 and
111
CA 02743477 2011-05-12
instrument 100 or 1000 are determined by measuring an optical output of the
sample, such as
fluorescence or luminescence. Accordingly, an optical detector is provided
with a lens projecting
through opening 176 formed in the front portion 120 of the processing unit
102. In the illustrated
embodiment, the optical detector is a fluorometer 500. Alternative detectors
could be readily
adapted for use with the illustrated instrument, including detectors that
sense electrical changes or
changes in physical characteristics, such as mass, color or turbidity.
[000414] Details of a fluorometer embodying aspects of the present invention
are shown in
Figures 6, 7, 8a-c, and 9a-c. The fluorometer 500 includes a front housing 502
and a rear housing
520 together mounted to a base 580.
[000415] Front housing 502 partially encloses an interior lens chamber 506 and
includes an
upper barrel 504 having a generally cylindrical shape. The upper barrel 504
extends into opening
176 formed in an actuator plate 124 (see Figure 3) within the instrument 100,
or onto an opening
formed in the manifold 1082 of the instrument 3000 (see Figure 14, 18). Front
housing 502 further
includes three mounting legs 510 for securing the fluorometer 500 to the
actuator plate 124 within
the instrument 100 or to the manifold 1082 of the instrument 1000.
[000416] The rear housing 520 is mounted, beneath the front housing 502, to
the base 580 by
mechanical fasteners or the like. In the illustrated embodiment, the rear
housing 520 includes four
light conduits extending from one end thereof to the opposite end thereof. In
particular, the rear
housing includes a first emission conduit 522, a second emission conduit 524,
a first excitation
conduit 526, and a second excitation conduit 528. Housing 520 is exemplary;
the fluorometer 500
may include one or more emission conduits and one or more excitation conduits.
[000417] Further details of the rear housing 520 are shown in Figures 6 and 7a-
c. Within
housing 520, the two excitation conduits 526 and 528 are identical and the two
emission conduits
522 and 524 are identical.
[000418] Each excitation conduit 526, 528 includes a first portion 532 having
a cross-section in
the general shape of a right triangle with rounded corners and a convexly
rounded hypotenuse. The
purpose of this shape is to limit the weight of the rear housing 520. The
shape is merely preferred;
other cross-sectional shapes can be used for the conduits - including circular
or rectangular- so long
112
CA 02743477 2011-05-12
as the features of the conduit do not interfere with the passage of light. The
excitation conduits 526,
528 further include a second portion 534 that is generally cylindrical in
shape. A circular passage
536 connects the first portion 532 with the second portion 534, and the
diameter of passage 536 is
smaller than that of second portion 534, thereby forming an annular lens shelf
540 within second
portion 534. Finally, excitation conduits 526, 528 include an O-ring seat 538
formed at the end of
second portion 534.
[000419] Each emission conduit 522, 524 includes a first portion 552 having a
cross-section in
the general shape of a right triangle with rounded corners and a convexly
rounded hypotenuse. The
emission conduits 522, 524 further include a second portion 554 that is
generally cylindrical in
shape. A circular passage 556 connects the first portion 552 with the second
portion 554, and the
diameter of passage 556 is smaller than that of second portion 554, thereby
forming an annular lens
shelf 560 within second portion 554. Emission conduits 522, 524 also include
an O-ring seat 558
formed at the end of second portion 554. Finally, a circular photodiode seat
562 is superimposed
within an end of the first portion 552 of each of the emission conduits 522,
524.
[000420] Front housing 502 and rear housing 520 are preferably machined from
6061 T6
aluminum and have a black anodized finish. Alternatively, the front and rear
housings could be
molded or cast - either separately or as a single, integrated unit, from
anymaterial that can withstand
the temperature environment within the instrument and will provide
uninterrupted light conduits.
[000421) Base 580 includes a front printed circuit board ("PCB") 582 and a
rear printer circuit
board ("PCB") 586. Front PCB 582 and rear PCB 586 are held together in a
fixed, spaced-apart
relation by mechanical fasteners (e.g., bolts, screws) extending through
cylindrical spacer elements
584. Details of exemplary circuits for the PCB's 582, 584 are described below.
[000422] With reference to Figure 9b, installed within the first excitation
conduit 526 of the
rear housing 520 are first excitation optic elements. The first excitation
optic elements include a first
light emitting diode ("LED") 616 disposed at one end of the first portion 532
of the first excitation
conduit 526 and mounted to the front PCB 582 of the base 580. A first
excitation lens 618, which
may be a collimating lens, is seated on the lens seat 540 within the second
portion 534 of the first
excitation conduit 526. A first excitation filter 622 is positioned at the end
of the first excitation
113
CA 02743477 2011-05-12
conduit 526 and is aligned in series with (i.e., along the optic axis of) the
first excitation lens 618.
The first excitation filter 622 and the first excitation lens 618 are
separated from one another by a
spacer 620, preferably made from aluminum with a black anodized finish. The
first excitation
conduit 526, along with the associated optics elements, are referred to
collectively as the first
excitation channel.
[000423] Similarly, second excitation optic elements are installed within the
second excitation
conduit 528 of the rear housing 520. The second excitation optic elements
include a second LED
632 disposed at one end of the first portion 532 of the second excitation
conduit 528 and mounted to
the front PCB 582 of the base 580. A second excitation lens 634, which may be
a collimating lens, is
seated on the lens seat 540 of the second portion 534 of the second excitation
conduit 528. A second
excitation filter 638 is positioned at the end of the second excitation
conduit 528 and is aligned in
series with (i.e. along the optic axis of) the second excitation lens 634. The
second excitation filter
638 and the second excitation lens 634 are separated from one another by a
spacer 636, preferably
made from aluminum with a black anodized finish. The second excitation conduit
528, along with
the associated optics elements, are referred to collectively as the second
excitation channel.
[000424] With reference to Figure 9c, first emission optic elements are
installed within the first
emission conduit 522 of the rear housing 520. The first emission optic
elements include a first
photodiode 660 mounted within a photodiode mount 648 disposed in the
photodiode seat 562 within
the first portion 552 of the first emission conduit 522 and mounted to the
front PCB 582 of the base
580. A first emission lens 650, which may be a collimating lens, is seated on
the lens seat 560 of the
second portion 554 of the first emission conduit 522. A first emission filter
654 is positioned near
the end of the first emission conduit 522 and is aligned in series with (i.e.
along the optic axis of) the
first emission lens 650. The first emission filter 654 and the first emission
lens 650 are separated
from one another by a spacer 652, preferably made from aluminum with a black
anodized finish.
Furthermore, the first emission filter 654 is separated from the end of the
first emission conduit 522
by an additional spacer 656, also preferably made from aluminum with a black
anodized finish.
The first emission conduit 522, along with the associated optics elements, are
referred to collectively
as the first emission channel.
[000425] Similarly, second emission optic elements are- installed within the
second emission
114
CA 02743477 2011-05-12
conduit 524 of the rear housing 520. The second emission optic elements
include a second
photodiode 662 mounted within a photodiode mount 668 disposed in the
photodiode seat 562 within
the first portion 552 of the second emission conduit 524 and mounted to the
front PCB 582 of the
base 580. A second emission lens 670, which may be a collimating lens, is
seated on the lens seat
560 of the second portion 554 of the second emission conduit 524. A second
emission filter 674 is
positioned near the end of the first emission conduit 524 and is aligned in
series with (i.e. along the
optic axis of) the second emission lens 670. The second emission filter 674
and the second emission
lens 670 are separated from one another by a spacer 672, preferably made from
aluminum with a
black anodized finish. Furthermore, the second emission filter 674 is
separated from the end of the
second emission conduit 524 by an additional spacer 676, also preferably made
from aluminum with
a black anodized finish. The second emission conduit 524, along with the
associated optics
elements, are refered to collectively as the second emission channel.
[000426] A front housing cover disc 600 is disposed between the front housing
502 and the rear
housing 520 (see Figure 6). The front housing cover disc 600 includes a raised
circular ridge 602
projecting slightly within the lens chamber 506 of the upper housing 502 (see
Figures 8b, 8c). The
front housing cover disc 600 further includes four circular light openings
606, each being aligned
with one of the light conduits 522, 524, 526, and 528 formed in the rear
housing 520 when the cover
disc 600 is installed.
[000427] A common lens 680 is housed within the lens chamber 506 of the front
housing 502.
In one embodiment, the fluorometer 500 includes only a single, undivided
common lens 680 which
comprises the only optic element of the fluorometer outside the excitation and
emission conduits. In
this context, "undivided" means the common lens is made exclusively from
optically transmissive
material (e.g., glass) and includes no structure for redirecting or impeding
light, such as optically
opaque structure embedded in and/or applied to the surface of the lens to
physically and optically
divide the lens into two or more sub-parts.
[000428] An O-ring 682 is seated at an end of the lens chamber 506, and the
raised circular
ridge 602 of the front housing cover disc 600 projects into the lens chamber
506, pressing against the
O-ring 682 to ensure a light-tight connection between the front housing 502
and the front housing
cover disc 600. An O-ring 624 is provided within the O-ring seat 538 at an end
of the first excitation
115
CA 02743477 2011-05-12
conduit 526 and compresses against a rear side of the front housing cover disc
600 to provide a light-
tight connection. Similarly, an O-ring 640 is provided in the O-ring seat 538
of the second excitation
conduit, an O-ring 658 is provided in the O-ring seat 558 of the first
emission conduit 522, and an 0-
ring 678 is provided in the O-ring seat 558 of the second emission conduit
524. O-rings 624, 640,
658, 678 prevent light infiltration into the light conduits 526, 528, 522,
524, respectively. The 0-
rings prevent light infiltration by compensating for the dimensional
variations of the machined parts
within the specified tolerance and also by compensating for the deformations
induced by thermal
factors.
[000429] In operation, excitation light signals are emitted by the light-
emitting diodes 616 and
632. The signals are preferably of a prescribed wavelength corresponding to a
dye to be detected.
Light from the diode 616 is transmitted through the first excitation conduit
526 and impinges upon
lens 618 which focuses at least a portion of the light to the first excitation
filter 622. The first
excitation filter 622 passes light of only a prescribed wavelength (or a
prescribed range of
wavelengths) and removes undesirable wavelengths from the transmitted light.
The filtered light
progresses through the common lens 680, which focuses at least a portion of
the light out through the
upper barrel 504 of the front housing 502, where the excitation light impinges
upon a chamber (for
example, chamber C28) of a receptacle 10 within the instrument 100. Assuming
the presence of a
first analyte or group of analytes within that chamber, the dye of a first
binding agent or group of
binding agents mixed with the sample and adapted to detect the presence of the
first analyte(s) will
fluoresce. A portion of the fluorescent emission enters the upper barrel 504
and then travels through
the common lens 680 which directs at least a portion of the fluorescent
emission into first emission
conduit 522. Light entering first emission conduit 522 passes through the
first emission filter 654,
which will filter undesired wavelengths of emission light. The filtered
emission light then travels
through the lens 650 and finally onto the photodiode 660, which will detect
the presence of light at
the prescribed wavelength.
[000430] Similarly, excitation light signals emitted by diode 632 are
transmitted through the
second excitation conduit 528 and impinges upon lens 634 which focuses at
least a portion of the
light to the second excitation filter 638. The second excitation filter 638
passes light of only a
prescribed wavelength (or a prescribed range of wavelengths) and removes
undesirable wavelengths
116
CA 02743477 2011-05-12
from the transmitted light. The filtered light progresses through the common
lens 680, which
focuses at least a portion of the light out through the upper barrel 504 of
the front housing 502, where
the excitation light impinges upon a chamber (for example, chamber C28) of a
receptacle 10 within
the instrument 100. Assuming the presence of a second analyte or group of
analytes within that
chamber, the dye of a second binding agent or group of binding agents mixed
with the sample and
adapted to detect the presence of the analyte(s) will fluoresce. A portion of
the fluorescent emission
enters the upper barrel 504 and then travels through the common lens 680 which
directs at least a
portion of the fluorescent emission into second emission conduit 524. Light
entering second
emission conduit 524 passes through the second emission filter 674, which will
filter undesired
wavelengths of emission light. The filtered emission light then travels
through the lens 670 and
finally onto the photodiode 662, which will detect the presence of light at
the prescribed wavelength.
[000431] Light emissions detected by the photodiodes are converted to signals
that can provide
qualitative or quantitative information about the presence or amount of an
analyte or analytes in a
sample using known algorithms. Examples of quantitation algorithms are
identified in the "Uses"
section hereinabove.
[000432] In the illustrated embodiment, the excitation conduits 526, 528 are
located opposite
each other, and the emission conduits 522, 524 are located opposite each other
within the rear
housing 520 to minimize background from excitation light passing through the
emission filter.
However, the excitation conduits 526, 528 and the emission conduits 522, 524
could be located next
to each other.
[000433] Fluorometer 500 includes two excitation channels and two emission
channels, which
permit the fluorometer to differentially detect two dyes or reporter moieties
that are excited at
different wavelengths. Light emissions from these different dyes are generally
quenched in the
absence of target (e.g., an analyte, a control, or amplification product that
is representative of the
presence of either). Such dyes may include, for example, N,N,NN'-tetramethyl-6-
carboxyrhodamine
("TAMRA") and 6-carboxyfluorescein ("FAM") or 6-carboxy-X-rhodamine ("ROX")
and 2'7'-
dimethoxy-4'5-dichloro-6-carboxyfluorescein ("JOE"). DABCYL is useful quencher
moiety for
quenching light emissions from any of these dyes in the absence of target.
Thus, the instrument is
117
CA 02743477 2011-05-12
capable of distinguishing between two different analytes or groups of analytes
or of distinguishing an
analyte or group of analytes from an internal control. It is contemplated,
however, that a fluorometer
embodying aspects of the present invention may include more or less than two
excitation and
emission channels, with the number of excitation channels and the number of
emission channels
being equal.
[000434) The specific optical components selected for the excitation and
emission channel(s)
will depend on the wavelength of the dye fluorescence to be detected. For the
dye FAM, for
example, a suitable LED for the excitation channel is available from
Kingbright Corporation of Brea,
California as Part No. L71 I3PBCH, and a suitable excitation filter for the
same dye is-available
from Semrock of Rochester, New York as Part No. FF01-485/20-9.0-D. For the
same dye, a suitable
photo-detector for the emission channel is available from OSI Optoelectronics,
Inc. of Hawthorne,
California as Model No. PIN-44131, and a suitable emission filter is available
from Semrock as Part
No. FFO1-531/22-9.0-D. For the dye TAMRA, for example, a suitable LED for the
excitation
channel available from Nichia America Corporation of Torrance, California as
Model No.
NSPG500S, and a suitable excitation filter is available from Semrock as Part
No. FF01-543/22-9.0-
D. For the same dye, a suitable photo-detector for the emission channel is
available from OSI
Optoelectronics as Model No. PIN-44D1, and a suitable emission filter is
available from Semrock as
Part No. FF01-587/ 11-9.0-D.
[000435] Accordingly, a fluorometer embodying aspects of the present invention
is able to
excite and detect multiple, different signals (such as different wavelengths)
without moving with
respect to the sample or without the different excitation and emission
channels moving with respect
to each other. Moreover, the arrangement of the fluorometer with respect to
the actuator plate and
the detection chamber of the receptacle carried within the instrument, enables
the fluorometer to
direct excitation signals at the detection chamber and detect emissions from
the detection chamber
without the use of fiber optics. Moreover, the optic channels (excitation and
emission) defined by
the fluorometer are parallel throughout there extents, and thus excitation
light can be transmitted
toward the sample and emissions from the sample can be detected without the
use of reflective
elements (e.g., mirrors) that redirect substantially all the light impinging
on the element or light
characteristic separating elements that redirect a portion of a light signal
having a first optical
118
CA 02743477 2011-05-12
characteristic (e.g., wavelength) and transmit another portion of the light
signal having a second
optical characteristic, such as a dichroic beam splitter.
[000436] Figures 21-26 illustrate one embodiment of suitable circuitry. This
circuitry provides
for local control of the fluorometer 500 with operating modes selected,
measurements made, and
results reported in response to macro commands communicated remotely via a
serial interface.
[000437] Figure 21 represents circuitry comprising interconnection means and a
number of
power supply circuits. Figure 22 represents circuitry comprising control,
processing and
communication means, circuitry comprising means to program and debug the
processor, and a circuit
that provides a stable voltage reference. Figure 23 represents circuitry
comprising a voltage
measurement circuit and a means to provide processor control of LED intensity
and modulation.
Figure 24 represents circuitry comprising the excitation means (LEDs), an RF
shield, and
supplemental power filtering for sensitive preamplifier circuits (described in
the next figures). Figure
25A and Figure 25B represent two similar embodiments of a front-end amplifier
circuit (the
differences of which are explained later) which convert the modulated optical
signal from the
contents of a chamber into a modulated electrical voltage. Figure 26
represents a demodulation
circuit that converts the modulated and amplified signal into an analog
voltage proportional to the
amplitude of the modulated optical signal.
.[000438] This embodiment incorporates a modulation/demodulation scheme that
allows the
circuit to reject the effects of varying background ambient light whose
wavelength falls within the
band-pass range of the optical filters physically placed between the
photodiodes Dl (660) (Figure
25A) and D2 (662) (Figure 25B) and the chamber being interrogated.
Microprocessor U4 (Figure 22)
generates a clock (set at 275Hz in this embodiment) that is used to
alternately modulate LEDI (616)
and LED2 (632) while controlling the polarity of the analog switch U7 (Figure
26). By alternating
the polarity of the analog switch U7 at the same frequency and in phase with
the modulation of the
LED (either LED 1 (616) or LED2 (632)), a matched transmitter/receiver pair is
created. Only those
optical signals arriving at the same frequency and in phase with this clock
will be amplified at full
gain; all ambient light and other light signals modulated at a different
frequency are suppressed.
[000439] In Figure 21, J I provides the main interconnection means to the
circuit. Devices D 1
119
CA 02743477 2011-05-12
and D6 protect the circuit by absorbing transient voltages that are applied to
the circuit via this
connection to external circuits. Integrated circuit UI and associated
components (C1, C3-C5, and
R4-R7) form an adjustable voltage regulator that provides the positive analog
power supply for this
circuit. Integrated circuit U16 and associated components (C2, C51-C52, and
R58-R59) form an
adjustable voltage regulator that provides the negative analog power supply
for this circuit. A
separate +5V supply (U2 and associated components C7 and CI0-C11) forms the
digital power
supply. Lastly, several resistor-divider pairs are provided (R4/R5, R8/R9, R I
O/R 11, and R56/R57) to
translate the voltage level out of each supply to a voltage within the
conversion range of the A/D
converter found in the microprocessor (U4).
[000440] In Figure 22, microprocessor U4 provides the primary control and
processing means
for the circuit. A number of capacitors (C15-C19) provide power supply
bypassing for this device.
Power-on reset of the circuit is accomplished by circuitry incorporated within
the microprocessor,
however, the microprocessor can be manually reset by use of the pushbutton
switch SW I (in
combination with pull-up resistor R 15). A diode D3 is provided to protect the
circuit from potential
static discharge associated with ungrounded contact with the reset switch.
Crystal Yl and associated
components (C20 and C21) provide a stable timebase and clock for the circuit.
Communication
between the microprocessor (U4) and external circuits is accomplished by use
of integrated circuit
U5 and associated components (C23-C25), converting TTL level serial signals in
and out of the
microprocessor to signals in compliance with the RS-232 standard. Programming
and debugging of
the circuit is accomplished by use of the PROGRAMMING INTERFACE (components
J2, C22, and
D4-D5). Visual indicators are provided to indicate "Power On" (components LEDI
and RI 3) and
"Status" (components LED2 and R14 with on/off control provided by the
microprocessor U4).
Lastly, a precision voltage reference circuit (components U3, C9, and C12-C13)
is provided to
establish a stable reference for the A/D converter (which is incorporated
within the microprocessor
U4) and to the external A/D converter U I I and D/A converter U 12.
[000441] In Figure 23, integrated circuits U I I and U 12 provide an interface
between the analog
circuits of the device and the microprocessor U4. Both of these devices are
controlled by and
communicate with the microprocessor U4 via its Serial Peripheral Interface
(SPI). A/D
CONVERTER U 1 I converts the differential analog signal out of the DEMODULATOR
FILTER
120
CA 02743477 2011-05-12
(Figure 26) into a digital result with 24-bit resolution (signed, with
approximately 0.51iA of
resolution per bit). D/A CONVERTER U 12 receives a digital setting from the
microprocessor and
sets a corresponding analog voltage on its output, a voltage which is then
used to regulate LED
current. The DAC output voltage is connected to a resistor divider with a low-
pass filter
(components C45 and R37-R38) which lowers this output control voltage,
resulting in the circuit
being capable of controlling LED current over the range of 0-80mA with 20iA
/bit resolution. Two
identical circuits follow, one for FAM LED DRIVE and the other for TAM LED
DRIVE.
[000442] In Figures 23 and 24, circuits are provided that directly regulate
and modulate the
flow of current through the excitation LEDs (LED1 (616) and LED2 (632)). Power
to the LEDs
originates from the +12V supply. LED current flows first through resistor R3
that reduces the voltage
potential of the supply and, in conjunction with capacitor C3, provides
filtering of the switched
current load to the modulated LED. Current then flows through the LED (LED 1
(616) or LED2
(632)) and then through the drain source channel of a FET transistor (Q3 or
Q4). Finally, the LED
current passes through a feedback resistor (R53 or R54) that generates a
voltage proportional to the
current through the LED.
[000443] Referring to Figure 23, control of electrical current through the LED
is achieved by
use of a traditional feedback circuit comprised of an operational amplifier
(U13 or U14), FET
transistor (Q3 or Q4), and feedback resistor (R53 or R54). Control is achieved
when the voltage
potential is equal at both inputs of the operational amplifier. Should the
voltage at the inverting input
of the operational amplifier drop below the voltage at the non-inverting
input, the voltage at the
output of the operational amplifier increases. This increase in output voltage
is incident on the gate
of the FET transistor, which then starts to conduct more electrical current.
An increase in electrical
current through the feedback resistor results in an increase in voltage across
that resistor and a
corresponding increase in voltage on the inverting input of the operational
amplifier, completing the
feedback loop.
[000444] Additionally, to control switching (modulation or power on/off) of
the LED, circuits
are provided (Q 1 or Q2 and associated components) that force the operational
amplifier into and out
of saturation, thereby switching the controlling FET transistors (Q3 or Q4)
off and on, respectively.
In order to "inhibit" LED current, the voltage potential at the gate of the
FET transistor (Q I or Q2) is
121
CA 02743477 2011-05-12
taken several volts below the voltage at the source terminal of the FET. This
in turn allows current to
flow through the FET and into the circuit node formed at the inverting input
of the operational
amplifier, thereby causing the voltage to rise to approximately 2.5V at that
node. The output voltage
of the operational amplifier subsequently drops to zero volts and the FET
transistor (Q3 or Q4) is
turned off, along with the respective LED. To "enable" LED current, the
voltage potential at the gate
of the FET transistor (Q1 or Q2) is kept at a voltage equal to or slightly
below the voltage at the
source of the transistor. This keeps the transistor turned "off", with no
current flowing through the
FET into the circuit node at the inverting input of the amplifier. Voltage at
the inverting input of the
operational amplifier is now equivalent to the voltage across the feedback
resistor (R53 or R54), and
this voltage now tracks the voltage applied by the DAC circuit to the non-
inverting input of the
operational amplifier. LED current is controlled proportionally to this
applied voltage.
[000445] In Figure 23, LED health is monitored by the use of circuitry to
probe the voltage
across and current passing through the LED. Resistors R51 and R52 provide a
low impedance path
between the A/D converter of the microprocessor U4 (see Figure 22) and the
respective feedback
resistors (R53 and R54); voltage measured by the A/D circuit across these
resistors is proportional to
LED current. In addition, LED voltage can be determined by use of the voltage
buffering circuit
formed by operational amplifier U 15 and associated components. A resistor
divider circuit (R47/R49
or R48/R50) follows the buffering amplifier to bring the voltage down to a
level within the
conversion range of the A/D converter. LED voltage is calculated by taking the
reduced voltage out
of the buffer amplifier plus the voltage measured across the respective
feedback resistor (which
equates to the voltage across the dropping resistor R3) and subtracting these
from the measured value
of the +12V supply (with specific weighting for each measurement).
[000446] In Figure 23, additional features are illustrated that improve
circuit rejection of
transient stimuli (both internally and externally generated). An RF shield El
is provided to protect
the circuitry found in Figures 25A and 25B from electromagnetic and radio
frequency interference.
Two guard circuits (E8 and E9) are provided to prevent electrical leakage
between the current
carrying conductors to the LEDs (LED 1 (616) and LED2 (632)) and the sensitive
circuits found in
Figures 25A and 25B. These guard circuits are comprised of exposed ground
traces on the outside
layers of the board, adjacent to and encircling exposed pads and traces
connected to the LEDs.
122
CA 02743477 2011-05-12
Lastly, four low-pass filters (R 1 /C l , R2/C2, R26/C20, and R27/C21) are
utilized to provide
additional attenuation of power supply noise on the supplies used for the
preamplifiers (U 1 and U2,
Figures 25A and 25B).
[000447] Referring now to Figures 25A and 25B, a photodiode (D 1 (660) or D2
(662)) converts
incident light (both background illumination and the modulated fluorescent
signal from the contents
of the chamber being interrogated) into an electrical current which is
supplied into the circuit node
connected to the inverting input of the operational amplifier (U 1 or U2).
Components D3 and R4-R6
(Figure 25A; and D4 and R7-R9 in Figure 25B) create a bias voltage on the
anode of this
photodiode; a higher bias voltage increases dark current through the diode
while decreasing
photodiode noise. Next, a compensation circuit is provided (U3A and U4A and
associated
components) that generates an offsetting current equivalent to the current out
of the photodiode
attributable to ambient light. This compensation is vital as ambient light can
create current out of the
photodiode that is many orders of magnitude greater than the electrical
current attributable to the
modulated fluorescent signal. Without correction, any variation in the level
of ambient light can
result in offsets to the measurement. In addition, without compensation, the
current out of the
photodiode associated with ambient light is likely to saturate the output of
the preamplifier circuit.
Lastly, a trans-impedance amplifier circuit is provided (U 1 or U2 and
associated components) that
generates a voltage sufficient to source/sink any additional current out of
the photodiode. The voltage
out of this amplifier (U 1 or U2) is proportional to current out of the
photodiode that is attributable to
the modulated optical signal from the contents of the chamber being
interrogated. Additionally, there
is a small amount of signal associated with change in ambient background
lighting (which is filtered
out by the subsequent demodulator circuit).
[0004481 Due to the high gain of the trans-impedance preamplifier and the
small signal being
measured, the circuit described in the preceding paragraph can be highly
susceptible to drift as a
result of changes in temperature and humidity. To minimize these effects, a
number of design and
process provisions are implemented in preparing circuit boards 582, 586.
First, all circuit traces and
components are located as far as possible from other circuits. Second, the
printed circuit solder mask
has been eliminated from underneath components R12-R15 and Cl6-C17. This
provides a greater
clearance between the circuit board and the components, enabling wash and
rinse reagents to pass
123
CA 02743477 2011-05-12
through during sample processing procedures. Third, cylindrical resistors
(MELF type) are selected
for use at R I2-R 15 (again, maximizing clearance between components and the
circuit board). Lastly,
to minimize the amount of contaminants and residual flux remaining on the
circuit board after
assembly, the board is first washed with saponifiers appropriate for the
solder/flux used in the
soldering process, followed by a rinse with de-ionized water. Photodiodes Dl
(660) and D2 (662)
are preferably soldered to the circuit board (after the above assembly process
is completed) with a
"no-wash flux" core solder. Residual flux remaining on the circuit board after
this last soldering
process provides a protective barrier and, therefore, is preferably not
removed.
[000449] Referring again to Figures 25A and 25B, amplifiers U3B and U4B (and
associated
components) provide additional amplification of the signal. In addition,
feedback components R22
and CI8 form a simple low-pass filter within that amplifier circuit
(attenuating signals above
34KHz). Concerning the compensation feedback circuit (identified in Figures
25A and 25B as
"SERVO FEEDBACK"), operational amplifiers U3A and U3B (and associated
components) are
configured as integration amplifiers with a cut-off frequency of approximately
5Hz. The output
voltage of these amplifiers create a DC bias current that negates that portion
of the electrical current
from the photodiode (D 1 (660) or D2 (662)) that is attributed to background
ambient light and other
natural DC offsets in the circuit. This results in an output signal (at U3B or
U4B) that has a zero DC
voltage component, i.e., the signal is centered around OV.
[000450] In Figure 25A, components R28 and C22 (in combination with digital
control signal
"SERVO ENABLE" from the microprocessor) are used to disable the compensation
amplifier (U3A
only) from integrating at times when the TAM circuit (Figure 25B) is being
utilized. This is because
the output spectra of LED2 (632) (TAM) overlaps the band-pass of the optical
filters in front of
photodiode D1 (660). Without this disable feature, the FAM detector/amplifier
circuit (Figure 25A)
would integrate the excitation.signal used for the TAM circuit (Figure 25B),
resulting in longer
circuit settling times when the circuit is switched back to measure FAM
response. Disabling of the
integrating function in the FAM compensation circuit (U3A and associated
components) is
accomplished by setting the "SERVO ENABLE" output from the microprocessor U4
to active
ground. At other times, this digital signal from the microprocessor is tri-
stated and components R28
and C22 have no effect on the operation of the circuit.
124
CA 02743477 2011-05-12
[000451] Referring to Figure 26, the entire circuit takes an incoming AC
signal, modulated at
the same frequency as the respective LED, and converts this signal to a DC
voltage (referenced to
circuit ground) with amplitude six times that of the peak-to-peak AC voltage.
The two signals
"PREAMP_A" and "PREAMP_B" (the outputs from the circuits in Figures 25A and
25B,
respectively) are connected to the two contacts of a solid-state, single-pole,
double-throw analog
switch (U6). "FE_SEL" is the logic signal that determines whether the signal
"PREAMP_A" or
"PREAMP_B" will be connected through switch U6 to the subsequent analog switch
U7. Analog
switch U7 acts as a sort of buffer/inverter, alternately switching its two
inputs (the selected
preamplifier output and circuit ground) to either of the positive and negative
inputs of the
demodulator filter (operational amplifiers U8, U9, and associated components).
In this
implementation, the DEMODULATOR SWITCH (U7) is switched in phase with the LED,
such that
when the LED is powered, the more positive signal out of the preamplifier is
switched to the positive
input of the demodulator filter (through R20) and the more negative signal is
switched to the
negative input of the demodulator filter (through R21). Connections to the
DEMODULATOR
FILTER are reversed when the LED is turned off. In this manner, the maximum
positive gain is
attained from the demodulator and filter circuit.
[000452] Continuing with Figure 26, the output of the analog switch U7 is
connected to a low-
pass filter (comprised of components R20, R21, and C30) with a cut-off
frequency of approximately
9Hz. Capacitor C30 of this filter is charged to a DC voltage of amplitude
approximately one-half that
of the peak-to-peak amplitude of the modulated signal coming out of the
preamplifier as a result of
the switching of the preamplifier signal through analog switch U7. The
subsequent active filter
(operational amplifiers U8AB and U9AB and associated components) comprises a
multi-pole, low-
pass filter with gain of approximately 12. In the first part of this filter,
operational amplifier U8A/B
(and associated components) form a differential amplifier with a DC gain of 4.
The output of this
filter is passed through a low-pass filter (components R24, R25, and C35) with
a cut-off frequency of
approximately 3Hz. The second active stage of this filter is comprised of
operational amplifier
U9A/B (and associated components) which forms a differential amplifier with a
DC gain of 3.
Components R32 and C36 form a feed-forward compensation path for the positive
side of this filter
while components R33 and C37 form a feed-forward compensation path for the
negative side of this
filter. This filter is used to attenuate any and all signals from the pre-
amplifier that fall outside of a
125
CA 02743477 2011-05-12
10Hz range around the operating frequency of the LEDs (275Hz in this example).
[000453] Finally, in Figure 26, the outputs of the DEMODULATOR FILTER are fed
into a
differential amplifier circuit (U 10) with unity gain. Its function is to
convert the voltage differential
between the two signals out of the differential filter into a positive voltage
referenced to circuit
ground. The two output signals "FLUORO+" and "FLUORO-" connect to the A/D
converter
discussed above.
[000454] In certain circumstances, it is necessary or desirable to move
substance out of a
detection chamber of a receptacle. Under such circumstances, it may be
necessary to incorporate a
chamber compression member with a detector, such as the fluorometer 500
described above. One
such embodiment of a compression member for incorporation with a detector is
shown in Figures
I OA and I OB. The compression mechanism, or detector actuator, is designated
by reference number
1150 and includes a bracket 1152 connected to and projecting from the actuator
plate 124. A
compression tube 1154 is positioned in front of the lens of the detector 500
and extends through an
opening in the actuator plate. Compression tube 1154 may have a transparent
window 1 164 mounted
at its distal end relative to the detector 500. An actuating bar 1156 extends
transversely in opposite
directions from the compression tube 1154. An actuating mechanism, for
example, a pneumatic
piston represented by rod 1162 connected to a pressure source by pneumatic
line 1160, is carried on
the bracket 1152 and engages the actuating bar 1156 to move the compression
tube 1154. A guide
rod 1158 extends from the actuator plate 124 through an opening in a bottom
end of the actuating bar
1156.
[000455] Extending the actuating mechanism 1162 against the actuating bar 1156
moves the
compression tube 1154 town extended position (to the left as shown in Figure I
Ob) to compress the
chamber C against the door assembly 200. The guide rod 1158 extending through
the actuating bar
1156 helps keep the compression tube 1154 in a straight orientation and helps
prevent skewing of the
compression tube 1154 during movement of the tube by the actuating mechanism
1162. Guide rod
1158 may be omitted if skewing of the compression tube 1154 is not a concern.
[000456] An alternative mechanism for incorporating a compression member with
a detector is
shown in Figure 11. In Figure 11, detector 500 is mounted on a translating
mounting platform or
126
CA 02743477 2011-05-12
sled 1172 having a base 1174 with guide pins 1176 and 1178 extending through
longitudinal slots
1184 and 1182, respectively. A piston (for example, a pneumatic piston) 1180
causes reciprocal
movement of the sled 1172 and the detector 500 and thus provides a means for
moving the entire
detector into and out of engagement with a receptacle chamber.
[000457] Figure 18 shows a transverse cross section of an alternative
embodiment of a
compression pad 1180 integrated with the signal detector 500. The compression
pad 1180 comprises
an actuator cup 1182 disposed within an opening 1083 formed through the
actuator plate 1080. A
transparent window, or detection lens, 1184 is positioned in front of the
actuator 1182 within an
opening formed in the elastomeric shield 1081. A generally circular through
bore 1186 is formed
through the cup 1182 at an off center position with respect to an axis of
symmetry of the actuator cup
1182, thus forming an upper portion 1188 of the actuator cup 1182 that is
thicker than a lower
portion 1190 of the cup 1182. A reason for the off-center location is that
there may be extra air in
the detection chamber during the detection process to help ensure better
thermal contact between the
chamber and a heater element disposed adjacent the chamber and also helps with
the fluidics transfer
from the chamber of lower volumes of liquid. Because the extra air will cause
the liquid to pool
down towards the bottom of the "actuator area" (air rises to the top), the off-
center detection lens and
focal point of the fluorometer 500 are configured to read fluorescence in the
lower portion of the
detection area, where there will be more liquid (and therefore more
fluorescence). In an alternate
implementation, for example, when the detection chamber if fully full of
liquid, the bore 1186 may
be formed through the center of the actuator cup 1182.
[000458] The actuator cup 1182 further includes a first radial lug 1192 and a
second radial lug
1194 extending from diametrically opposed positions on the actuator cup 1182.
Radial lug 1192
resides within a radial opening 1196 extending from the opening 1083, and
radial lug 1194 resides
within a radial opening 1198 extending from the opening 1083. A circular blind
hole 1200 extends
from the radial opening 1196, and a circular blind hole 1202 extends from the
radial opening 1198_
Blind holes 1200 and 1202 hold coil compression springs (not shown) which
press against the radial
lugs 1 192 and 1194 to bias the cup 1182 in the retracted position, as shown.
[000459] Detector 500 is mounted to the manifold 1082 over an opening 1085
formed through
the manifold. First and second cylindrical projections 1204 and 1206 extend
from the manifold 1082
127
CA 02743477 2011-05-12
on opposite sides of the opening 1085. An 0 ring 1208 is positioned on the
cylindrical projection
1204, and an 0 ring 1210 is positioned on the cylindrical projection 1206.
Cylindrical projection
1204 extends in to a cup-like blind hole formed in the radial lug 1192, and
the cylindrical projection
1206 extends into a cup-like blind hole formed in the radial lug 1194. An air
pressure conduit 1212
extends into the cylindrical projection 1204 and exits at the top of the
projection. Similarly, an air
conduit 1214 extends into the cylindrical projection 1206 and exits from the
top of the projection.
The actuator cup 1 182 is moved from the retracted position shown in Figure 18
to an extended
position (to the left as shown in the figure), by applying pressure at the
conduits 1212 and 1214, thus
pushing the radial projections 1192 and 1194 up into the radial openings 1196
and 1198,
respectively, and moving the actuator 1182 to the left. The depth of the
circular blind holes 1200 and
1202 accommodate the length of the compressed springs (not shown) thereby
permitting the radial
lugs 1192, 1194 to move completely to the ends of the radial openings 1196,
1198.
EXAMPLES
[000460) Examples are provided below illustrating some of the uses of the
receptacles and
systems provided herein. Skilled artisans will appreciate that these examples
are not intended to limit
the invention to the particular uses described therein. Additionally, those
skilled in the art could
readily adapt the receptacles and systems provided herein for use in
performing other kinds of
reactions, processes or tests.
[000461] The following examples provides a number of experiments that were
conducted to
compare the sensitivity of a manual, real-time transcription-mediated
amplification ("TMA")
reaction with that of automated real-time TMA reactions using either liquid or
dried amplification
and enzyme reagents. TMA reactions are two enzyme, transcription-based
amplification reactions
that rely upon a reverse transcriptase to provide an RNase H activity for
digesting the RNA template
after producing a complementary DNA extension product with an antisense primer
or promoter-
primer. Examples of TMA reactions are disclosed in McDonough et al., U.S.
Patent No. 5,766,849;
Kacian et al., U.S. Patent No. 5,824,518; and Becker et al., U.S. Patent No.
7,374,885. The target for
this experiment was Chlamydia trachomatis 23S ribosomal RNA, referred to
hereinafter as "the
target nucleic acid.'
128
CA 02743477 2011-05-12
[000462] In each experiment, a "wobble" capture probe was used to non-
specifically bind the
target nucleic acid in the test samples. The wobble capture probe consisted of
a 3' region having a
random arrangement of 18 2'-methoxyguanine and 2'-methoxyuridine residues
(poly(K)18) joined to a
5' tail having 30 deoxyadenine residues (poly(dA)30). Complexes comprising the
wobble capture
probe and bound target nucleic acid were immobilized on magnetically-
responsive particles having
oligonucleotide tails consisting of 14 deoxythymine residues (poly(dT)14)
derivatized thereon and
then subjected to a wash procedure to remove interfering substances from the
test samples.
[000463] After the wash procedure, the target nucleic acid was exposed to TMA
reagents and
conditions and the resulting amplification product was detected in real-time
using a fluorescently
labeled, molecular beacon probe. See Kacian et al., U.S. Patent No. 5,824,518;
see also Tyagi et al.,
U.S. Patent No. 5,925,517. The primers used for amplification included an
antisense promoter-
primer having a 3' target binding sequence and a 5' T7 promoter sequencer and
a sense primer. The
molecular beacon probe was comprised of T-0-methyl ribonucleotides and had an
internal sequence
for binding to the target nucleic acid sequence. The molecular beacon probe
was synthesized to
include interacting FAM and DABCYL reporter and quencher moieties using
fluorescein
phosphoramidite (BioGenex, San Ramon, CA; Cat. No. BTX-3008) and 3'-DABCYL CPG
(Prime
Synthesis, Inc., Aston, PA; Cat. No. CPG 100 2N 12DABXS). The probes and
primers of this
experiment were synthesized using standard phosphoramidite chemistry, various
methods of which
are well known in the art, using an ExpediteTM 8909 DNA Synthesizer
(PerSeptive Biosystems,
Framingham, MA). See, e.g., Carruthers, et al., 154 Methods in Enzymology, 287
(1987).
[000464] Example 1: Manual Amplification Reactions
[000465] For this experiment, manual TMA reactions were set up in 12 x 75 mm
polypropylene
reaction tubes (Gen-Probe Incorporated, San Diego, CA; Cat. No. 2440), and
each reaction tube was
provided with 125 L of a Target Capture Reagent containing 160 .tg/mL I
micron magnetic
particles Sera-MagTM MG-CM Carboxylate Modified (Seradyn, Inc.; Indianapolis,
IN; Cat. No.
24152105-050450) derivatized with poly(dT)14 and suspended in a solution
containing 250 mM
HEPES, 310 mM LiOH, 1.88 M LiCI, 100 mM EDTA,adjusted to pH 7.5, and 10
pmol/reaction of
the wobble capture probe. Each reaction tube was then provided with 500 FL of
a mixture
containing a Sample Transport Medium (150 mM HEPES, 294 mM lithium lauryl
sulfate (LLS) and
129
CA 02743477 2011-05-12
100 mM ammonium sulfate,adjusted to pH 7.5) and water in a 1-to-1 ratio. The
mixtures contained
eitherl O5 copies of the target nucleic acid (test samples) or no target
nucleic acid (negative control
samples). The reaction tubes were covered with a sealing card and their
contents mixed by vortexing
for 15 seconds, and then incubated at 25 C for 5 minutes in a water bath to
facilitate binding of the
wobble capture probes to the target nucleic acid. (The wobble capture probes
bind to the derivatized
poly(dT)14 when the Target Capture Reagent is prepared.)
[000466] To purify bound target nucleic acid, a DTS 400 Target Capture System
(Gen-Probe;
Cat. No. 5210) was used to isolate and wash the magnetic particles. The DTS
400 Target Capture
System has a test tube bay for positioning the reaction tubes and applying a
magnetic field thereto.
The reaction tubes were placed in the test tube bay for about 3 minutes in the
presence of the
magnetic field to isolate the magnetic particles within the reaction tubes,
after which the supernatants
were aspirated. Each reaction tube was then provided with 1 mL of a Wash
Buffer (10 mM HEPES,
6.5 mM NaOH, 1 mM EDTA, 0.3% (v/v) ethanol, 0.02% (w/v) methylparaben, 0.01%
(w/v)
propylparaben, 150 mM NaCl, and 0.1% (w/v) sodium lauryl sulfate,adjusted to
pH 7.5), covered
with a sealing card and vortexed for 15 seconds to resuspend the magnetic
particles. The reaction
tubes were returned to the test tube bay and allowed to stand at room
temperature for 3 minutes
before the Wash Buffer was aspirated. The wash steps were repeated once.
[000467] Following purification of the target nucleic acid, 75 FL of an
Amplification/Detection
Reagent (44.1 mM HEPES, 2.82% (w/v) trehalose, 33 mM KCI, 0.01% (v/v) TRITON
X-100
detergent, 30.6 mM MgC12,- 0.3% (v/v) ethanol, 0.1 % methylparaben, 0.02%
(w/v) propylparaben,
0.47 mM each of dATP, dCTP, dGTP and dTTP, 1.76 mM each of rCTP and UTP, 9.41
mM rATP
and 11.76 mM rGTP,adjusted to pH 7.7 at 23 C) containing 11.9 pmollreaction
of the T7 promoter-
primer, 9.35 pmol/reaction of the non-T7 primer, and 10 pmol/reaction of the
molecular beacon
probe was added to each reaction tube. The reaction tubes were covered with a
sealing card and
mixed by vortexing for 15 seconds. After mixing, the contents of the reaction
tubes were transferred
to separate reaction wells of a white, 96-well microplate (Thermo Electron
Corporation, Waltham,
MA; Product No. 9502887), each reaction well containing 75 L of an Oil
Reagent (silicone oil
(United Chemical Technologies, Inc., Bristol, PA; Cat. No. PS038)). The
microplate was covered
with a ThermalSeal film (Sigma-Aldrich Co., St. Louis, MO; Cat. No. Z369675)
and incubated in a
130
CA 02743477 2011-05-12
Solo HT Microplate Incubator (Thermo Electron; Cat. No. 5161580) at 60 C for 5
minutes, and then
in a Solo Microplate Incubator (Thermo Electron; Cat. No. W1036) at 42 C for 5
minutes. While in
the second incubator, the sealing card was removed from the microtiter plate
and 25 L of an
Enzyme Reagent (58 mM HEPES, 50 mM N-acetyl-L-cysteine, 1.0 mM EDTA, 10% (v/v)
TRITON X-100 detergent, 3% (w/v) trehalose, 120 mM KCI, 20% (w/v) glycerol,
120 RTU/ L
Moloney murine leukemia virus reverse transcriptase ("MMLV-RT"), and 80 U/ L
T7 RNA
polymerase,adjusted to pH 7.0) was added to each reaction well. (One reverse
transcriptase unit
("RTU") of activity for MMLV-RT is defined as the incorporation of I nmol dTMP
into DE81
filter-bound product in 20 minutes at 37 C using (poly(rA)-p(dT)12_18) as the
substrate; and for T7
RNA polymerase, one unit ("U") of activity is defined as the production of 5.0
fmol RNA transcript
in 20 minutes at 37"C.) Immediately following addition of the Enzyme Reagent,
the contents of the
reaction wells were mixed by stirring with standard 200 gL pipette tips
engaged by an 8-channel
multi-pipettor and used to transfer the Enzyme Reagent to the microtiter
plates. The microtiter plate
was then re-sealed with a clear sealing card.
[000468] To detect the presence of amplification product in the reaction
wells, the sealed plate
was placed in a Fluoroskan Ascent 100 Microplate Fluorometer (Thermo
Electron; Product No.
5210480) pre-warmed to 42 C and fluorescent readings were taken at 30-second
intervals over a 50
minute period. Detection depended upon a conformational change in the
molecular beacon probes as
they hybridized to amplification products, thereby resulting in the emission
of detectable fluorescent
signals. As long as the molecular beacon probes maintained a hairpin
configuration, i.e., they were
not hybridized to an amplification product of the target nucleic acid,
fluorescent emissions from the
fluorescein reporter moieties were generally quenched by the DABCYL quencher
moieties. But as
more of the molecular beacon probes hybridized to amplicon in the reaction
wells, there was increase
in detectable fluorescent signals. Thus, fluorescent emissions that increased
over time provided an
indication of active amplification of the target region of the target nucleic
acid. The results of this
experiment are shown in Figure 27, where raw data from the fluorometer are
plotted as fluorescent
units (y-axis) versus time in minutes (x-axis) for each reaction well. Samples
containing the target
nucleic acid yielded strong fluorescent signals that emerged from background
approximately 15
minutes into the reaction, while control samples yielded no significant signal
above background.
131
CA 02743477 2011-05-12
[000469] Example 2: Automated Amplification Reactions in a Multi-Chambered,
Flexible Receptacle Using Liquid Reagents
[000470] In this experiment, the TMA reaction of Section 1 of this example was
performed
using the receptacle 10 and instrument 100 illustrated in Figures IA and 3.
The receptacle 10
illustrated in Figure IB was pre-loaded with reagents in the following manner:
(i) 125 pL of the
Target Capture Reagent was added to chamber C18; (ii) 3 mL of the Wash Buffer
was added to
chamber C34; (iii) 25 L of the Oil Reagent, followed by 85 L of the
Amplification/Detection
Reagent, was added to chamber C20; and (iv) 35 L of the Oil Reagent, followed
by 25 L of the
Enzyme Reagent, was added to chamber C32. After reagent loading, all of the
chambers of the
receptacle 10 except chamber C 16 were closed by heat sealing. A 500 gL test
sample having 105
copies of the target nucleic acid, as described in Example I above, was then
pipetted into chamber
C16, the sample chamber, which was then closed by heat sealing.
[000471) For the initial set-up, the sealed receptacle 10 was mounted on the
front portion 120
of the housing 104 and the door assembly 200 was closed, sandwiching the
chambers of the
receptacle 10 between the pressure mechanism cluster 180 and the thermal zones
260,262,264,266,
and 268 in the door assembly 200. The thermal zones were set to heat adjacent
chambers at 30 C.
The movement of materials between chambers of the receptacle 10 was controlled
by the
compression pads making up the pressure mechanism cluster 180 described infra.
Prior to starting
the test, compression pads P72, P70, P62, P51-1, P56, P58, P60, P64, P66 and
P68 were all activated
to clamp and protect corresponding seals associated with portals (or neck) 72,
70, 62, 51, 56, 58, 60,
64, 66 and 68, respectively, from prematurely opening or leaking. Wash Buffer
and air bubbles were
removed from the vertical and lateral sections 42, 44 of chamber C34, the wash
buffer chamber, and
the vertical inlet 48 of chamber C36, the waster chamber, was concurrently
closed by engaging the
following compression pads in the indicated order: (i) compression pads P34-1
and P36-1; (ii)
compression pads P34-2 and P36-2; (iii) compression pads P34-3 and P36-3; (iv)
compression pad
P34-4; and (v) compression pad P34-S.
[000472] After the initial set-up, compression pad P68 was retracted and
compression pads P32
and P68 were sequentially activated to press chamber C32 and portal 68,
thereby forcing open sealed
132
CA 02743477 2011-05-12
portal 68 and moving the Enzyme and Oil Reagents from chamber C32 to chamber
C30. At the
same time, compression pad P56 was retracted and compression pads P20 and P56
were sequentially
activated to press chamber C20 and portal 56, thereby forcing open sealed
portal 56 and moving the
Amplification/Detection and Oil Reagents from chamber C20 to chamber C22.
Compression pads
P68 and P56 remained activated to clamp portals 68 and 56, respectively,
thereby preventing a
backflow of the Enzyme and Amplification/Detection Reagents into chambers C32
and C20.
[000473] After moving the Enzyme and Amplification/Detection Reagents,
compression pads
P 18-1, P 18-2 and P54 were sequentially activated to press chamber C 18 and
portal 54, thereby
forcing open sealed portal 54 and moving the Target Capture Reagent ("TCR")
from chamber C 18 to
chamber C 16. The TCR and sample were mixed by twice moving the combined
contents back-and-
forth between chambers C 16 and C 18 using compression pads associated with
these chambers. Once
mixing was completed, compression pad P54 was activated to clamp portal 54,
thereby maintaining
the TCR/sample mixture in chamber C16, where it was incubated by heating
thermal zone 260 at
30 C for 5 minutes. This incubation step was carried out to facilitate non-
specific binding of the
target nucleic acid to the wobble capture probes and immobilization of the
wobble capture probes on
the magnetically-responsive particles present in the TCR.
[000474] To separate the target nucleic acid from other material in the test
sample, the magnet
translation mechanism 208 was activated to move the magnet into position
adjacent chamber C26
(referred to herein as the "on" position), the magnetic separation chamber 102
during the initial set-
up. Compression pad P62 was retracted and compression pads P16-3, P16-4 and
P62 were
sequentially activated to press a portion of chamber C16, thereby forcing open
sealed portal 62 and
moving a first aliquot of the TCR/sample mixture from chamber CI6 to chamber
C26. After moving
the first aliquot of the TCR/sample mixture to chamber C26, compression pad
P62 remained
activated to clamp portal 62, thereby preventing the movement of material
between chambers C16
and C26, and compression pads P 16-3 and P 16-4 were sequentially retracted.
In chamber C26, the
magnetically-responsive particles were subjected to the magnetic fields of the
magnet for I minute at
a temperature of 30 C provided by thermal zone 268. While the magnet remained
in the "on"
position, compression pads P26, P70, P36-1, P36-2 and P36-3 were sequentially
activated to press
chamber C26, portal 70 and the vertical inlet 48 of chamber C36, the waste
chamber, and to move
133
CA 02743477 2011-05-12
liquid from chamber C26 into chamber C36.
[000475] By activating different arrangements of the compression pads
associated with
chambers C 16 and C 18, three additional aliquots of the TCR/sample mixture
were moved from
chambers C16 and C18 to chamber C26. For the second aliquot of TCR/sample
mixture moved to
chamber C26, the sequential operation of the compression pads was as follows:
P 18-1 (+), P 18-2
(+), P 18-2 (-), P62 (-), P 16-3 (+), P 16-4 (+), P62 (+), P 16-3 (-) and P 16-
4 (-). For the third aliquot of
TCR/sample mixture moved to chamber C26, the sequential operation of the
compression pads was
as follows: P51-2 (+), P 18-1 (-), P 16-4 (+), P 16-3 (+), P 16-2 (+), P 16-1
(+), P 16-1 (-), P 16-2 (-),
P 16-3 (-), P 16-4 (-), P 18-1 (+), P 18-2 (+), P54 (+), P62 (-), P 16-3 (+),
P 16-4 (+), P62 (+), P 16-3 (-)
and P16-4 (-). And for the fourth aliquot of TCR/sample mixture moved to
chamber C26, the
sequential operation of the compression pads was as follows: P16-2 (+), P16-1
(+), P62 (-), P 16-3
(+), P16-4 (+), P16-3 (-) and P16-4 (-). The (+) designation indicates that
the referred to
compression pad was activated to press a corresponding portal or portion of a
chamber, and the (-)
designation indicates that the referred to compression pad was retracted from
corresponding portal or
portion of a chamber. The immobilization and liquid waste removal steps were
repeated for each
additional aliquot until all of the TCR/sample mixture had been processed and
the sample reduced to
a manageable size for further processing in the receptacle 10.
[000476] A wash procedure was then initiated to remove unwanted and
potentially interfering
material from the immobilized nucleic acids, during which the magnet remained
in the OonE
position. At the start of the wash procedure, compression pads P34-5, P34-4,
P34-3, P34-2 and P34-
1 were operated to prime the vertical and lateral sections 42, 44 (the "neck
region") of chamber C34
and, after retracting compression pad P72, to press on the neck region of
chamber 34, thereby
opening sealed portal 72 and moving about 200 L of Wash Buffer from chamber
C34 to chamber
C26. Compression pad P72 then clamped portal 72 and the Wash Buffer was moved
back-and-forth
three times between chamber C26 and the area covered by compression pads P62
and P164 of
chamber C.16 by the action of compression pads P62, P16-4 and P26 to remove
any residual
TCR/sample mixture material lodged in opened portal 62 and to purify bound
nucleic acids. In this
step, P26 was only partially activated to prevent overfilling the areas of
chamber C16 covered by
compression pads P62 and P 16-4 and to minimize foaming. All of the liquid was
finally collected in
134
CA 02743477 2011-05-12
chamber C26 and exposed to the magnetic fields of the magnet for 1 minute at a
temperature of
about 30 C to immobilize any magnetically-responsive particles. The Wash
Buffer was then moved
from chamber C26 into chamber C36 by the action of compression pads P26, P70,
P36-1, P36-2 and
P36-3. Another aliquot of about 200 pL of Wash Buffer was then moved from
chamber C34 to
chamber C26 by the operation of compression pads associated with the neck
region of chamber C34,
compression pad P72 was activated to clamp opened portal 72, and the washing
process was
repeated, except that the Wash Buffer was only moved into that portion of
chamber C 16 covered by
compression pad P62, and the movement between chamber C26 and chamber C16 was
only
performed twice. Finally, a third aliquot of about 200 L of Wash Buffer was
moved from chamber
C34 to chamber C26 by the operation of compression pads associated with the
neck region of
chamber C34, compression pad P72 was activated to clamp opened portal 72, and
the magnetically-
responsive particles were exposed to the magnetic fields of the magnet for 1
minute at a temperature
of 30 C to immobilize any dislodged magnetically-responsive particles.
Afterwards, the liquid was
moved from chamber C26 to chamber C34 by the action of compression pads P70,
P36-1, P36-2 and
P36-3. After the wash procedure was completed, the magnet was moved out of
alignment with
chamber C26 (the "off' position) and thermal zone 268 was moved into alignment
with chamber
C26.
[000477] Following separation of the target nucleic acid, compression pads P22
and P58 were
sequentially activated to press on chamber C22 and portal 58, thereby forcing
open sealed portal 58
and moving the Amplification/Detection Reagent from chamber C22 to chamber
C26. To ensure
that the magnetic particles were fully suspended in the
Amplification/Detection Reagent, the
Amplification/Detection Reagent was moved between chambers C22 and C26 two
times by
operation of compression pads P26, P58 and P22. In chamber C26, the
Amplification/Detection
Reagent was incubated with thermal zone 268 at 62 C (with a tolerance of,
e.g., 1.5 C) for 5
minutes to facilitate binding of the promoter-primer to target nucleic acids.
At the same time,
thermal zones 264 and 266 brought the temperature of chambers C28 and C30 to
42 C (with a
tolerance of, e.g., 0.45 'Q, an optimal temperature for TMA. Following the
62'C incubation, the
contents of chamber C26 were incubated at 42 C for another 5 minutes, after
which compression
pads P26 and P64 were sequentially activated to press on chamber C26 and
portal 64, thereby forcing
open sealed portal 64 and moving the heated Amp] ificati on/Detection Reagent
and magnetic particle
135
CA 02743477 2011-05-12
mixture from chamber C26 to chamber C28. Once in chamber C28, compression pads
P30 and P66
were sequentially activated to press on chamber C30 and portal 66, thereby
forcing open sealed
portal 66 and moving the heated Enzyme Reagent from chamber C30 to chamber
C28. After the
Enzyme Reagent was moved to chamber C28, the temperature of thermal zone 266
was adjusted to
38 C I C. To mix the Amplification/Detection Reagent, Enzyme Reagent and
magnetic particles,
gravity assisted in draining the contents of chamber C28 into chamber C30
through opened portal 66
and then moved back into chamber C28 by sequentially pressing on chamber C30
and portal 66 with
compression pads P30 and P66. This process was repeated three times to ensure
adequate mixing of
the reagents for amplification of the target sequence, after which mixing
compression pad P66 was
activated to clamp opened portal 66 and to move any residual reagents from
opened portal 66 to
chamber C28.
[000478] To detect amplification products generated in this mixture, the
fluorometer 500
positioned adjacent a transparent window of chamber C28 took fluorescent
readings at 5-second
intervals, each reading averaging just over 4 seconds, during a 27 minute
period. The results of this
experiment are represented in Figure 28, which is a graph showing fluorescence
units detected from
chamber C28 on the y-axis versus the time in minutes on the x-axis. These
results demonstrate that
the real-time TMA reaction performed using the instrument 100 and receptacle
10 described herein
gave equivalent results to the manually formatted real-time TMA reaction of
Example 1 above.
[000479] Example 3: Automated Amplification Reactions in a Multi-Chambered,
Flexible Receptacle Using Liquid Reagents and a Urine Samples
[000480] This experiment was designed to evaluate a real-time TMA reaction
using a urine
sample spiked with 105 copies of target nucleic acid. The materials and
methods of this experiment
were the same as those of the real-time TMA reaction described in Example 2,
with the following
exceptions: (i) chamber C18 was loaded with 150 L of the Target Capture
Reagent containing 10
pmol of the wobble capture probe in combination with 100 L of water, (ii)
chamber C34 was
loaded with 2 mL of the Wash Buffer; (iii) the Oil Reagent was not loaded into
chamber C20 or
chamber C32 prior to loading the Amplification/Detection and Enzyme Reagents
into these
chambers; (iv) the test sample included 250 L of urine from a healthy donor
and 250 L of the
Sample Transport Medium; and (v) the steps of moving the TCR/sample mixture
from chamber C 16
136
CA 02743477 2011-05-12
to chamber C26, subjecting the magnetic particles contained within chamber C26
to the magnetic
fields of the magnet, and moving liquid from chamber C26 to chamber C36 while
the magnetic
particles were immobilized was repeated only two times. The results of this
experiment are
illustrated in Figure 29, which is a graph showing fluorescence units detected
from chamber C28 on
the y-axis versus time in minutes on the x-axis. These results of this
experiment demonstrate that the
real-time TMA reaction detected the target nucleic acid provided in the urine
sample using the
instrument 100 and receptacle 10 described herein.
[000481] Example 4: Automated Amplifications Reaction in a Multi-Chambered,
Flexible Receptacle Using Dried Reagents
[000482] The purpose of this experiment was to evaluate the automated, real-
time TMA
reaction of Example 2 using dried forms of the Amplification and Enzyme/Probe
Reagents. The
receptacle 10 was pre-loaded with the following reagents: (i) 125 L of the
Target Capture Reagent
was added to chamber C18; (ii) 3 mL of the Wash Buffer was added to chamber
C34; (iii) 25 L of
the Oil Reagent, followed by 85 gL of an Amplification Reconstitution Reagent
(0.4% (v/v) ethyl
alcohol (absolute), 0.10% (w/v) methyl paraben, 0.02% (w/v) propyl paraben, 33
mM KCI, 30.6 mM
MgCl2, and 0.003% phenol red), was added to C-20; (iv) an Amplification
Reagent Pellet (formed
from a 14 L droplet containing 250 mM HEPES, 16% (w/v) trehalose, 53.4 mM
ATP, 10 mm CTP,
66.6 mM GTP, 10 mM UTP, 2.66 mM of each of dATP, dCTP, dGTP and dTTP, adjusted
to pH 7.0,
0.6 nmol/L of the antisense T7 promoter-primer and 0.47 nmol/L of the sense
non-T7 primer, where
the droplet was dispensed into liquid nitrogen and the resulting frozen pellet
was lyophilized) was
added to chamber C22; (v) 35 L of the Oil Reagent, followed by 25 L of an
Enzyme/Probe
Reconstitution Reagent (50 mM HEPES, 1 mM EDTA, 10% (v/v) TRITON X-100
detergent, and
120 mM KCI, adjusted to pH 7.0), was added to chamber C32; and (vi) an
Enzyme/Probe Reagent
Pellet (formed from a 7.28 L droplet containing 20 mM HEPES, 125 mM N-acetyl-
L-cysteine, 0.1
mM EDTA, 0.01 % (v/v) TRITON X- 100 detergent, 20% (w/v) Trehalose, 412 MR/L
MMLV-RT
(dialyzed), 687 MU/L T7 RNA polymerase (dialyzed), where "M" represents one
million, and 2.20
nmol/L of the molecular beacon probe) was added to chamber C30. After reagent
loading, all of the
chambers of the receptacle 10 except chamber C16 were closed by heat sealing.
A 500 L test
sample having 105 copies of the target nucleic acid, as described in Example I
above, was then
137
CA 02743477 2011-05-12
pipetted into chamber C16, which was then closed by heat sealing. The initial
set-up was the same
as Example 2 above.
[000483] Following the initial set-up, compression pads P32 and P68 were
sequentially
activated to press on chamber C32 and portal 68, thereby forcing open sealed
portal 68 and moving
the Enzyme/Probe Reconstitution Reagent and Oil Reagent combination from
chamber C32 to
chamber C30, where the Enzyme/Probe Reagent Pellet was allowed to dissolve in
the Enzyme/Probe
Reconstitution Reagent for two minutes. Compression pads P20 and P56 were then
sequentially
activated to press on chamber C20 and portal P20, thereby forcing open sealed
portal 56 and moving
the Amplification Reconstitution Reagent and Oil Reagent combination from
chamber C20 to
chamber C22, and compression pads P20 and P22 were activated to move the
contents of chamber
C22 back-and-forth four times between chambers C20 and C22 to fully
reconstitute the
Amplification Reagent, after which compression pad P56 was activated to clamp
portal 56.
Following a two minute dwell period in chamber C30, the compression pads P30
and P32 were
activated to move the contents of chamber C30 back-and-forth two times between
the chambers C30
and C32 to fully reconstitute the Enzyme/Probe Reagent, after which
compression pad P68 was
activated to clamp portal 68. The remainder of the steps were the same as
those Example 2.
[000484] The results of this experiment are illustrated in Figure 30, which is
a graph showing
fluorescence units detected from chamber C28 on the y-axis versus the number
of time in minutes on
the x-axis. These results show that this real-time TMA reaction, using
pelleted amplification and
enzyme/probe reagents that are reconstituted on-board, detected the targeted
transcript using the
instrument 100 and receptacle 10 described herein.
[000485] Example 5: Automated Amplification Reactions Using Liquid Reagents in
the Presence or Absence of an Oil Reagent
[000486] This experiment was designed to evaluate the benefits of providing an
immiscible
liquid to open chambers of a multi-chambered receptacle prior to loading
liquid reagents for
performing TMA reactions. For this experiment, receptacles of the type
illustrated in Figure 4 were
prepared in replicates of two as follows: (i) in the controls, no Oil Reagent
was added to chambers
containing the Amplification/Detection Reagent (chamber C20) or the Enzyme
Reagent (chamber
138
CA 02743477 2011-11-09
C32); (ii) 25 gL Oil Reagent was added to chamber C32 prior to adding the
Enzyme Reagent
and no oil was added to chamber C20; and (iii) 25 L Oil Reagent was added to
each of
chambers C20 and C32 prior to adding the Amplification/Detection and Enzyme
Reagents.
Chamber C18 was loaded with 250 gL of the Target Capture Reagent containing 10
pmol of
the wobble capture probe and 100 L of water. The materials and methods of
this
experiment were otherwise substantially the same as those of the reactions
described in
Example 2, except that the amplification reaction was conducted at about 40 C
for 40
minutes. Figure 31 is a graph illustrating the results of this experiment,
showing the
fluorescence units detected from chamber C28 on the y-axis versus time in
minutes on the x-
axis. The results show that the real-time TMA reactions of this experiment
performed
noticeably better when at least one of the Amplification/Detection and Enzyme
Reagents was
combined with the Oil Reagent.
[000487] While the present invention has been described and shown in
considerable
detail with reference to certain illustrative embodiments, those skilled in
the art will readily
appreciate other embodiments of the present invention. Accordingly, the
present invention is
deemed to include all modifications and variations encompassed within the
scope of the
following appended claims.
139
