Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
Diagnostic Devices and Apparatus for the Controlled Movement
of Reagents Without Membranes
This is a divisional application of Canadian
Patent Application No. 2,284,801 filed March 24, 1998.
Field of the Invention
This invention relates to devices for conducting
assays, including qualitative, semi-quantitative and
quantitative determinations of one or more analytes in a
single test format.
Claimed in this divisional application are an
assay device and a method for detecting an analyte of
interest in a fluid sample.
In an embodiment of the invention, there is an
assay device comprising:
a housing comprising married top and bottom
members or a round tube;
a sample addition zone as a first device region
formed by or within the housing for receiving a fluid
sample; and
a second device region fluidly connected to the
first device region, the second device region comprising one
or more capture zones on a surface within the housing, each
capture zone comprising a first receptor reactive with an
analyte of interest and at least one analyte sensor
configured and arranged to detect an electrochemical signal
related to a reaction of a detectable amount of the analyte
of interest with the first receptor and produce a measurable
signal in response,
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wherein the assay device is configured to control
a volume of the fluid sample flowing from the first device
region to the second device region for determining the
presence or amount of the analyte of interest therein.
In a further embodiment of the invention, there is
an assay device comprising:
a housing comprising married top and bottom
members or a round tube;
a sample additional zone as a first device region
formed by or within the housing for receiving a fluid
sample;
a second device region fluidly connected to the
first device region, the second device region comprising one
or more capture zones on a surface within the housing, each
capture zone comprising a first antibody or binding fragment
thereof reactive with an analyte of interest and at least
one analyte sensor configured and arranged to detect an
electrochemical signal related to a reaction of a detectable
amount of the analyte of interest with the first antibody or
binding fragment thereof and produce a measurable signal in
response; and
a labeled reagent species dissolvably disposed on
the surface within the housing that is in fluid
communication with the second device region, wherein the
labeled reagent species comprise an enzyme conjugated to a
second antibody or binding fragment thereof reactive with
the analyte of interest, wherein molecules of the labeled
reagent species form sandwich complexes with molecules of
the analyte of interest and with molecules of the first
antibody or binding fragment thereof.
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The parent application is directed to an
analytical device comprising a reaction chamber having a
surface comprising an array of texture structures wherein
numerous small menisci are formed when a fluid is placed in
contact with the surface, at least one capillary channel and
a diagnostic element.
It should be understood that the expression
"present invention" encompasses the subject matters of both
this and the parent application.
Background of the Invention
Over the years, numerous simplified test systems
have been designed to rapidly detect the presence of a
target ligand of interest in biological, environmental and
industrial fluids. A synonym for target ligand is analyte
or target analyte. In one of their simplest forms, these
assay systems and devices usually involve the combination of
a test reagent which is capable of reacting with the target
ligand to give a visual response and an absorbent paper or
membrane through which the test reagents flow. Paper
products, glass fibers and nylon are commonly used for the
absorbent materials of the devices. In certain cases, the
portion of the absorbent member containing the test reagents
is brought into contact, either physically or through
capillarity, with the sample containing the target ligand.
The contact may be accomplished in a variety of ways. Most
commonly, an aqueous sample is allowed to traverse a
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porous or absorbent member, such as porous polyethylene or
polypropylene or membranes by capillarity through the portion
of the porous or absorbent member containing the test reagents.
In other cases, the test reagents are pre-mixed outside the
test device and then added to the absorbent member of the
device to ultimately generate a signal.
Commercially available diagnostic products employ a
concentrating zone methodology. In these products, such as
ICONR (Hybritech Incorporated), TESTPACKTM (Abbott Laboratories)
or ACCULEVELR (Syva Corporation), the device contains an
immunosorbing or capture zone within a porous member to which a
member of a specific binding pair is immobilized. The surface
of the porous member also may be treated to contain one or more
elements of a signal development system. In these devices,
there is a liquid absorbing zone which serves to draw liquid
through the immunosorbing zone, to absorb liquid sample and
reagents and to control the rate at which the liquid is drawn
through the immunosorbing zone. The liquid absorbing zone is
either an additional volume of the porous member outside of the
immunosorbing zone or an absorbent material in capillary
communication with the immunosorbing zone. Many commercially
available devices and assay systems also involve a wash step in
which the immunosorbing zone is washed free of nonspecifically
bound signal generator so that the presence or amount of target
ligand in the sample can be determined by examining the porous
member for a signal at the appropriate zone.
In addition to the limitations of the assay devices and
systems of the prior art, including the limitations of using
absorbent membranes as carriers for sample and reagents, assay
devices generally involve numerous steps, including critical
pipetting steps which must be performed by relatively skilled
users in laboratory settings. Accordingly, there is a need for
one step assay devices and systems, which, in addition to
controlling the flow of reagents in the device, control the
timing of the flow of reagents at specific areas in the device.
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In addition, there is a need for assay devices which do not
require critical pipetting steps but still perform semi-
quantitative and quantitative determinations.
Description of the Drawings
Figure 1 is a partially schematic, top perspective view of
a device in accordance with the present invention.
Figure 1A is a partially schematic, perspective exploded
view of the device showing the detail in the area of the sample
addition reservoir, the sample-reaction barrier, the reaction
chamber, the time gate and the beginning of the diagnostic
element.
Figure 1B is a partially schematic, perspective exploded
view of the device showing the detail in the area of the
optional reagent reservoir, the sample addition reservoir, the
sample-reaction barrier, the reaction chamber, the time gate
and the beginning of the diagnostic element.
Figure 1C is a partially schematic, perspective exploded
view of the device showing the detail in the area of the
optional reagent reservoir in fluid contact with the sample
addition reservoir and the reaction chamber.
Figure 1D is a partially schematic, perspective cutaway
view of the flow control means.
Figure 2 is a partially schematic, perspective view of a
second device in accordance with this present invention, which
may be used to add pre-mixed reaction mixtures.
Figure 3 is a partially schematic top view of the
diagnostic element showing some potential placements of capture
zones.
Figure -4 is a partially schematic, perspective view of a
used reagent reservoir.
Figure 5 is a partially schematic view of embodiments of
these devices which are columnar or have curved opposing
surfaces.
Figure 6 is a top view of time gates.
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Figure 7 shows typical dimensions for a preferred time
gate.
Figure 8 is a top view of sequential time gates.
Figure 9A-D are views of preferred textured surfaces; as
illustrated a textured surface can comprise texture structures
which have curved or linear surfaces; the surfaces can be
smooth or uneven. Exemplary texture structures are conical
(Fig. 9B-C), hexagons (Fig. 9D) or mounds (Fig. 9A). The
structures depicted in Figure 9 are broadly considered posts.
Figure 10 depicts convex and concave flow fronts.
Figure 11 depicts a preferred embodiment of a device in
accordance with the invention.
Figure 12A-F, respectively depict various embodiments of
stops and energy directors in accordance with the invention;
Figures 12A, 12C and 12E depict various embodiments without a
lid attached; Figure 12B depicts an embodiment of Figure 12A
with a lid attached, Figure 12D depicts an embodiment of Figure
12C with a lid attached, Figure 12F depicts an embodiment of
Figure 12E with a lid attached. The energy directors and stops
in Figure 12 can be configured as posts or ridges.
Figure 13 depicts a electron micrograph of an embodiment
of the invention illustrating a sample addition reservoir 1, a
textured sample reaction barrier 3, a textured reaction chamber
4, a textured used reagent reservoir 7, a stop 60, a point 70,
and energy directors 62.
Figure 14 is an enlarged view of a portion of Fig. 13,
illustrating textured sample reaction barrier 3, textured
reaction chamber 4, an energy director 62, and stop 60.
Fig. 15 depicts an electron micrograph of an embodiment of
the invention illustrating a time gate 5, a textured diagnostic
lane 6, and an energy director 62.
Figure 16 A-B depict electron micrographs of two views of
a textured surface adjacent an energy director 62. The energy
director depicted in this embodiment has the form of a ridge.
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Summary of the Invention
The devices and methods of this invention
overcome the problems f ound in the prior art providing devices
and methods which do not require precise pipetting of sample,
5 which do not use absorbent members, which include novel
textures and elements for the controlled movement of reagents
in the device and which are capable of providing quantitative
assays.
The devices described herein do not use bibulous or porous
materials, such as membranes and the like as substrates for the
immobilization of reagents or to control the flow' of the
reagents through the device. A disadvantage of, for example,
membranes in diagnostic devices is that on both microscopic and
macroscopic scales the production of membranes is not easily
reproducible. This can result in diagnostic devices which have
differential properties of non-specific binding and flow
characteristics. Membranes are very susceptible to non-
specific binding which can raise the sensitivity limit of the
assay. In one embodiment, the time gates of this invention
can, however, be embedded in membranes or used in devices with
membranes.
In the case of immunochromatographic assay formats such as
those described in U.S. Pat. Nos. 4,879,215, 4,945,205 and
4,960,691, the use of membranes as the diagnostic element
requires an even flow of reagents through the membrane. The
problem of uneven flow of assay reagents in
imrnunochromatographic assays has been addressed in U.S. Patents
4,756,828, 4,757,004 and 4,883,688. These patents teach
that modifying the longitudinal edge of the bibulous
material cbntrols the shape of the advancing front.
The devices of the present invention circumvent these
membrane associated problems by. the use of defined surfaces,
including grooved surfaces, capillarity, time gates, novel
capillary means, including channels and novel fluid flow
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control means alone or in various combinations, all of which
are constructed from non-absorbent materials. In a preferred
mode of this invention, the capillary channel of the diagnostic
element is composed of grooves which are perpendicular to the
flow of the assay reagents. The manufacture of grooved
surfaces can be accomplished by injection molding and can be
sufficiently reproducible to provide control of the flow of
reagents through the device.
The assay devices, assay systems and device components of
this invention can comprise two opposing surfaces disposed a
capillary distance apart; at least one of the surfaces
comprises the ability to detect at least one target ligand or a
conjugate in an amount related to the presence or amount of
target ligand in a sample. The inventive device components may
be incorporated into conventional assay devices with membranes
or may be used in the inventive membrane-less devices herein
described and claimed. Components of the invention comprise
flow control elements, measurement elements, time gates,
elements for the elimination of pipetting steps, and generally,
elements for the controlled flow, timing, delivery, incubation,
separation, washing and other steps of the assay process.
Unlike assay devices of the prior art, the inventive assay
devices described herein do not require the use of bibulous
materials, such as papers or membranes. The inventive devices
of the present invention rely on the use of defined surfaces,
including grooved and textured surfaces, and capillarity alone
or in various combinations to move the test reagents. The
inventive devices described herein provide means for the
controlled, timed movement of reagents within the device and do
not require precise pipetting steps. The concepts and devices
of the present invention are especially useful in the
performance of immunoassays and nucleic acid assays of
environmental and industrial fluids, such as water, and
biological fluids and products, such as urine., blood, serum,
plasma, spinal and amniotic fluids and the like.
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According to one aspect of the present invention,
there is provided an analytical device for determining the
presence or amount of a target ligand in a test sample, the
device comprising: a reaction chamber having at least one
reconstitutable reagent on a surface thereof, the surface
comprising a first array of texture structures wherein
numerous small menisci are formed when a fluid is placed in
contact with the surface to facilitate preparation of a
uniform layer of the reagent in a dry form; at least one
capillary channel; and a diagnostic element comprising a
second array of texture structures in fluid communication
with the capillary channel, each texture structure of the
second array comprises a surface having an immobilized
ligand receptor covalently or non-covalently attached
thereto, the immobilized ligand receptor capable of binding
the target ligand such that when the test sample containing
the target ligand flows through the capillary channel, the
target ligand diffuses across a width of the capillary
channel and binds to the immobilized ligand receptor,
wherein the capillary channel extends through the reaction
chamber and the diagnostic element.
The device may further comprise an inlet port and
a vent; an array of structures, where each structure has a
surface providing an immobilized receptor covalently or non-
covalently attached to the surface of the structure, and
capable of binding a ligand; a plurality of channels wherein
the test sample containing a ligand, flows through the
channels, the ligand diffuses across the width of the
channels to bind the immobilized receptor; and, a labeled
reagent comprising a specific binding member conjugated to a
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detectable label, where the detectable label is capable of
producing a signal at the immobilized receptor which
indicates the presence or amount of a ligand in a test
sample.
Disclosed is an assay device comprising: a sample
addition reservoir; a sample reaction barrier fluidly
connected to the sample addition reservoir; a reaction
chamber fluidly connected to the sample reaction barrier,
the chamber having at least two fingers in the walls
thereof, wherein the barrier has a higher capillarity than
the reaction chamber; a time gate fluidly connected to the
reaction chamber, the time gate capable of permitting fluid
to pass therethrough at a desired flow rate; a diagnostic
element fluidly connected to the time gate, the diagnostic
element capable of immobilizing at least
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one conjugate in at least one zone; and, a used reagent
reservoir fluidly connected to said diagnostic element, whereby
fluid can flow in sequence from said reservoir, to said
barrier, to said reaction chamber, to said time gate, to said
diagnostic element then to said reservoir.
Disclosed is a device capable of performing an assay, said
device comprising two or more surfaces that are in contact by
fluid during performance of the assay, wherein a first device
surface comprises a first immunoassay reagent immobilized
thereon and a second capillary space surface comprises a second
immunoassay reagent immobilized thereon.
Disclosed is a device capable of performing an assay, said
device comprising a stop and an energy director. Accordingly
during manufacture of this device, the energy director serves
to seal a first device piece to a second device piece and to
define a capillary space in the device, and the stop serves to
allow preparation of a device chamber with uniform dimensions.
Disclosed is a zone comprising a region capable of having
a fluid placed thereon, and a hydrophobic region adjacent to
the region capable of containing a fluid placed thereon,
whereby the hydrophobic region impedes the flow of fluid into
that hydrophobic region. Also disclosed is an assay device
comprising this zone. Also disclosed is a method to facilitate
uniform drying of a liquid, where the method comprises:
providing the zone; introducing liquid into the zone region
capable of having a fluid placed thereon; and, and drying said
liquid.
Devices in accordance with the invention were used to
conduct assays on liquid samples suspected of containing an
analyte of interest.
Disclosed is a surface configured to facilitate placement
of a uniform layer of dried reagent thereon, said surface
comprising a plurality of texture structures, whereby a
plurality of menisci are formed when a fluid is placed in
contact with the surface. Surfaces in accordance with the
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invention, and devices comprising such surfaces, were used to
facilitate preparation of a uniform layer of a dried reagent on
said surface.
Disclosed is a method of manufacturing analytical devices
from a master. A master is provided that comprises device
features in accordance with the invention, e.g., a master
having an array of structures which have one or more channels
therebetween. Thereafter, in accordance with manufacturing
techniques known to those of ordinary skill in the art, copies
of the master are made.
Disclosed is a method for manufacturing a capillary space
comprising a hydrophobic surface and a hydrophilic surface.
The method comprises applying a hydrophobic material to a
hydrophilic surface that is capable of forming a lumenal
surface of a capillary space; or, masking a region of a
hydrophobic surface; applying a means for producing a
hydrophilic surface of the surface whereby areas of the surface
which are nonmasked become hydrophilic, and removing the
masking to expose a hydrophobic region of a surface that is
capable of forming a lumenal surface of a capillary space.
Disclosed is a capillary space that comprises a lumen
comprising at least one rectilinear angle when viewed in a
cross section, where the capillary also comprises a hydrophobic
zone on a lumenal surface thereof. Also disclosed is a
material configured to fit into a capillary space, said
material comprising a hydrophobic zone on a surface thereof.
Also disclosed is a material comprising a hydrophobic zone;
where the zone, upon addition of liquid to the material, is
capable of delimiting a discrete area of liquid on a suxface on
or within the material.
Definitions
In interpreting the claims and specification, the
following terms shall have the meanings set forth below.
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Target ligand - The binding partner to one or more
receptors. Synonyms for target ligand are analyte, ligand or
target analyte.
Ligand - Binding partner to one or more ligand
5 receptor(s). A synonym for ligand is analyte. For example, a
ligand can comprise an antigen, a nucleotide sequence, lectin
or avidin.
Ligand Analogue - A chemical derivative of the target
ligand which may be attached either covalently or noncovalently
10 to other species, for example, to the signal development
element. Ligand analogue and target ligand may be the same and
both generally are capable of binding to the ligand receptor.
Synonyms for ligand analogue are analyte analogue or target
analyte analogue.
Ligand Analogue Conjugate - A conjugate of a ligand
analogue and a signal development element. A ligand analogue
conjugate can be referred to as a labeled ligand analogue.
Signal Development Phase - The phase containing the
materials involving the signal development element to develop
signal, e.g., an enzyme substrate solution.
Receptor - Chemical or biochemical species capable of
reacting with or binding to target ligand, typically an
antibody, a binding fragment, a complementary nucleotide
sequence, carbohydrate, biotin or a chelate, but which may be a
ligand if the assay is designed to detect a target ligand which
is a receptor. Receptors may also include enzymes or chemical
reagents that specifically react with the target ligand. A
receptor can be referred to as a reagent or a binding mernber.
A receptor which is neither a labeled receptor nor an
immobilized receptor can be referred to as an ancillary
receptor or an ancillary binding member. For example, a
receptor can comprise an antibody.
Ligand Receptor Conjugate - A conjugate of a ligand
receptor and a signal development element; synonyms for this
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term include binding member conjugate, reagent conjugate,
labeled reagent or labeled binding member.
Signal Development Element - The element which directly or
indirectly causes a visually or instrumentally detectable
signal as a result of the assay process. Receptors and ligand
analogues may be bound, either covalently or noncovalently to
the signal development element to form a conjugate; when so
bound these substances can be referred to as labeled. The
element of the ligand analogue conjugate or the receptor
conjugate which, in conjunction with the signal development
phase, develops the detectable signal, e.g., an enzyme.
Reaction Mixture - The mixture of sample suspected of
containing target ligand and the reagents for determining the
presence or amount of target ligand in the sample, for example,
the ligand analogue conjugate or the receptor conjugate. As
used herein the reaction mixture may comprise a proteinaceous
component which may be the target, a component of the sample or
additive (e.g., serum albumin, gelatin, milk proteins).
Ligand Complement - A specialized ligand used in labeling
ligand analogue conjugates, receptors, ligand analogue
constructs or signal development elements.
Ligand Complement Receptor - A receptor for ligand
complement.
Ligand Analogue-Ligand Complement Conjugate - A conjugate
composed of a ligand analogue, a ligand complement and a signal
development element.
Capture Efficiency - The binding efficiency of the
component or components in the reaction mixture, such as the
ligand analogue conjugate or the receptor conjugate, to the
capture zone of the diagnostic element.
Capture Zone - The area on the diagnostic element which
binds at least one component of the reaction mixture, such as
the ligand analogue conjugate or the receptor conjugate.
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Capillarity - The force induced by a capillary space, or
the exhibition of capillary action. Capillarity can be
affected by the solid surface or the liquid surface or both.
Biosensor - Any electrochemical, optical, electro-optical
or acoustic/mechanical device which is used to measure the
presence or amount of target ligands. For example,
electrochemical biosensors utilize potentiometric and
amperometric measurements, optical biosensors utilize
absorbance, fluorescence, luminescence and evanescent waves.
Acoustic/mechanical biosensors utilize piezoelectric crystal
resonance, surface acoustic waves, field-effect transistors,
chemical field-effect transistors and enzyme field-effect
transistors. Biosensors can also detect changes in the
physical properties of solutions in which receptor binding
events take place. For example, biosensors may detect changes
in the degree of agglutination of latex particles upon binding
antigen or they may detect changes in the viscosity of
solutions in response to receptor binding events.
Detailed Description of the Invention
The present invention is directed to diagnostic testing
devices for determining the presence or amount of at least one
target ligand. Figure 1 shows a preferred embodiment of a
device 10 according to the invention. Generally, the devices
of the invention have thicknesses of about 2 mm to 15 mm,
lengths of about 3 cm to 10 cm and widths of about 1 cm to 4
cm. The dimensions may be adjusted depending on the particular
purpose of the assay.
One device of this invention, as depicted in Fig. 1,
generally illustrates some features of the inventive devices
and portions of devices herein disclosed and claimed. The
device 10 comprises various elements, a sample addition zone 1,
a sample addition reservoir 2, a sample reaction barrier 3, a
reaction chamber 4, a time gate 5, a diagnostic element 6, and
a used reagent reservoir 7. The devices are comprised of
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capillary channels which are formed when a top member 8 is
placed on the bottom member 9 a capillary distance apart and
which move the reagents and sample throughout the device.
The top and bottom members may be married to form a housing,
the various chambers sealed and the capillaries formed by a
number of techniques, including but not limited to, gluing,
welding by ultrasound, riveting and the like. The elements
of the device can be used in various combinations with the
diagnostic element 6 to achieve a variety of desired
functions. As one skilled in the art will recognize these
elements may be combined to perform one-step or multistep
assays. The devices 10 may also be used in the formation of
reaction mixtures for the assay process. The device 20 in
Fig. 2 may be used to add pre-mixed reaction mixtures for
the generation of signal which relates to the presence or
amount of the target ligand.
An optional reagent chamber 17 may be incorporated
into device 10 or 20 as depicted in Fig. 1B and Fig. 1C.
The devices 10 and 20 may also be used with an optional
fluid control means 18 as shown in Fig. 1D.
Features include, but are not limited to: 1)
diagnostic elements which are not comprised of bibulous
materials, such as membranes, 2) means to control the volume
of sample or reaction mixture, 3) time gates, 4) novel
capillary means, termed fingers herein and 5) novel flow
control means, sometimes referred to as a "gap" herein and
6) used reagent reservoir which prevents backward flow of
reagents. Those of skill in the art will appreciate that
these elements are separately novel and nonobvious, and may
be incorporated into diagnostic devices in various
combinations and may be used with other elements known to
those skilled in the art to achieve novel and nonobvious
diagnostic test devices and heretofore unrealized benefits.
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Components of the device (i.e., a physical
structure of the device whether or not a discrete piece from
other parts of the device) can be prepared from copolymers,
blends, laminates, metallized foils, metallized films or
metals. Alternatively, device components can be prepared
from copolymers, blends, laminates, metallized foils,
metallized films or metals deposited one of the following
materials: polyolefins, polyesters, styrene containing
polymers, polycarbonate, acrylic polymers, chlorine
containing polymers, acetal homopolymers and copolymers,
cellulosics and their esters, cellulose nitrate, fluorine
containing polymers, polyamides, polyimides,
polymethylmethacrylates, sulfur containing polymers,
polyurethanes, silicon containing polymers, glass, and
ceramic materials.
Alternatively, components of the device are made
with a plastic, elastomer, latex, silicon chip, or metal;
the elastomer can comprise polyethylene, polypropylene,
polystyrene, polyacrylates, silicon elastomers, or latex.
Alternatively, components of the device can be
prepared from latex, polystyrene latex or hydrophobic
polymers; the hydrophobic polymer can comprise
polypropylene, polyethylene, or polyester.
Alternatively, components of the device can
comprise TEFLONO, polystyrene, polyacrylate, or
polycarbonate.
Alternatively, device components are made from
plastics which are capable of being milled or injection
molded or from surfaces of copper, silver and gold films
upon which are adsorbed various long chain alkanethiols.
The structures of plastic which are capable of being milled
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or injection molded can comprise a polystyrene, a
polycarbonate, or a polyacrylate.
Each of the elements of devices 10 and 20 will be
described separately, then representative descriptions of
the devices of this invention will follow.
Sample Addition Zone
Referring to Figs. 1 and 2, the sample addition
zone 1 of the devices 10 and 20 is the area which is formed
by or within the housing and in which sample is introduced
to
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the device. The sample addition zone 1 can be a port of
various configurations, that is, round, oblong, square and the
like or the zone can be a trough in the device.
Sample Addition Reservoir
5 Referring to Figs. 1 and 2, the sample addition reservoir
2 is an element of the device which receives the sample.
Referring now to Fig. 1, the volume of the sample addition
reservoir 2 should be at least the volume of the reaction
chamber 4 or greater. The sample addition reservoir 2 can be a
10 capillary space or it can be an open trough. In addition, a
filter element can be placed in or on the sample addition
reservoir 2 to filter particulates from the sample or to filter
blood cells from blood so that plasma can further travel
through the device. The sample addition reservoir can comprise
15 a vent (not illustrated) to facilitate escape of gas and liquid
filling of the reservoir.
In a preferred embodiment, the volume or capacity of the
sample addition reservoir 2 is 1 to 5 times the volume of the
reaction chamber 4. In general, one selects a volume or
capacity of this reservoir 2 such that if the excess sample is
used to wash the diagnostic element 6 then enough volume of
sample is needed to thoroughly remove any unbound reagents from
the diagnostic element 6 arising from the assay process.
Reservoir 2 may also contain certain dried reagents which
are used in the assay process. For example, a surfactant can
be dried in this reservoir 2 which dissolves when sample is
added. The surfactant in the sample would aid in the movement
of the sample and reaction mixture through the device by
lowering the surface tension of the liquid. The sample
addition reservoir 2 is generally in direct fluid contact with
the sample-reaction barrier 3 (Fig. 1) or the diagnostic
element 6 (Fig. 2).
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Sample-Reaction Barrier
As depicted in Fig. 1, the sample-reaction barrier 3
separates the sample in the sample addition reservoir 2 from
the reaction mixture in the reaction chamber 4. The sample-
reaction barrier is desired because it provides the device with
the capability of forming a precise reaction mixture volume. A
precise volume of the reaction mixture is generally necessary
for assays in which semi-quantitative or quantitative results
are desired. Thus, a precise pipetting step of the sample to
the device is not required because the sample reaction barrier
forms a reaction chamber of precise volume into which the
sample is capable of flowing. The sample reaction barrier 3 is
desired because the reactions which take place in the reaction
chamber 4 should preferably be separated from the excess sample
in the sample addition reservoir 2.
The sample reaction barrier 3 comprises a narrow
capillary, generally ranging from about 0.01 mm to 0.2 mm and
the surfaces of the capillary can be smooth or have a single
groove or a series of grooves which are parallel or
perpendicular to the flow of sample. In a preferred embodiment
of the sample reaction barrier 3, now referring to Fig. 1A,
grooves 12, parallel to the flow of sample, are incorporated
onto one surface of the device a capillary distance, for
example, 0.02 mm to 0.1 mm, from the other surface. The volume
of sample which fills the sample-reaction barrier 3 (Fig. 1A)
should be kept to a minimum, from about 0.01% to 10% of the
reaction chamber 4 volume so that the reagents of the reaction
chamber 4 do not significantly diffuse back into the sample in
the sample addition reservoir 2. That is, the diffusion of the
reaction mixture back into the excess sample should be kept to
a minimum so that the chemical or biochemical reactions
occurring in the reaction mixture are not substantially
influenced by the excess sample in the sample addition
reservoir 2. Groove depths can range from about 0.01 mm to 0.5
mm and preferably from about 0.05 mm to 0.2 mm. When more than
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17
one groove is used for this element, the number of grooves in
this element is typically between 10 and 500 grooves per cm and
preferably from about 20 to 200 grooves per cm. Sample from
the sample addition reservoir 2 flows over the grooves 12 by
capillary action and then into the reaction chamber 4. In a
further preferred embodiment, grooves, hereafter termed
"fingers" 16, are situated in the wall of the reaction chamber
4 in fluid contact with the grooves 12 or capillary space of
the sample-reaction barrier 3. These fingers 16 are typically
0.5 mm to 2 mm wide, preferably 1 mm to 1.5 mm wide and
typically 0.1 mm to 1.5 mm in depth, preferably about 0.2 to 1
mm in depth. The fingers 16 in the wall of the reaction
chamber 4 aid in the capillary flow of the sample into the
reaction chamber 4. That is, the fingers allow fluid to move
from a capillary where the capillarity is relatively high to a
capillary where the capillarity is lower. Thus, the capillary
at the sample-reaction barrier is generally more narrow and has
a greater capillarity than the capillary or space of the
reaction chamber. This difference in capillarity can cause the
flow of sample or fluid in the device to stop in the sample-
reaction barrier capillary. Presumably, the fingers break the
surface tension of the fluid at the interface of the two
capillaries or spaces and thereby cause the fluid to move into
a capillary or space of lower capillarity. One can appreciate
that the utility of fingers can be extended to any part of the
device where fluid must flow from high capillarity to low
capillarity. In practice, this is usually when the direction
of fluid flow is from a narrow capillary (higher capillarity)to
a wider capillary (lower capillarity).
The top surface of the sample reaction barrier may also be
used to immobilize reagents used in the assay process such that
the sample flows over the sample reaction barrier, dissolves
the reagents and moves into the reaction chamber. The movement
of the sample and reagents into the reaction chamber may act as
a mixing means.
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18
Reaction Chamber
Referring to Fig. 1, the sample moves into the reaction
chamber 4 from the sample-reaction barrier 3. .The reagents of
the device 10 are preferably placed in the reaction chamber 4,
for example, as dried or lyophilized powders, such that when
the sample enters the reaction chamber 4 the reagents quickly
reconstitute. The volume of the reaction chamber 4 is the
volume of sample which defines the reaction mixture. The
reaction chamber may be sealed on two sides, for example, by
ultrasonic welding of the top and bottom members. Thus,
delivery of the sample to the device 10 at the sample addition
zone 1 does not require a precise pipetting step to define the
volume of the reaction mixture. Mixing features which mix the
reaction mixture can also be incorporated in conjunction with
the reaction chamber element 4. The sample fills the
reaction chamber 4 because of capillary forces and also,
potentially, because of the hydrostatic pressure exerted
by the sample in the sample addition reservoir 2.
A surface of reaction chamber 4 may be smooth or comprised
of texture structures such as posts or grooves. Texture on a
device surface can facilitate drying of reagents on the surface
during preparation of the device, and can facilitate movement
of sample into the reaction chamber 4. Texture on a device
surface facilitates uniform placement of dried reagents on the
surface as follows: A liquid reagent-containing fluid is placed
in contact with the textured surface, and small reagent fluid
menisci form adjacent each texture structure. Absent the
presence of texture, the fluid would tend to form larger
menisci at corners of the entire chamber, which when dried
would produce a non-uniform layer of dried reagent. When
texture structures are designed into the device, the presence
of numerous small menisci leads to a more uniform layer of
reagent that is dried throughout the chamber.
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The volume of the reaction chamber 4, and thereby the
reaction mixture, may be any volume which accommodates the
reagents and which provides the desired sensitivity of the
assay. The shape of the reaction chamber 4 should be such that
the movement of the reaction mixture from the reaction chamber
4 is not turbulent and eddies are not formed as a result of the
movement out of the reaction chamber 4. A preferred shape of
the reaction chamber 4 is shown in Fig. 1. The depth of the
reaction chamber 4 should be commensurate with the width of the
chamber to accommodate the desired reaction mixture volume.
The depth of the reaction chamber can range from about 0.05 mm
to 10 mm and preferably from 0.1 mm to 0.6 mm. To accommodate
a particular volume of the reaction chamber, the length and
width of the reaction chamber should be adjusted and the depth
maintained as narrow as is practical. The reaction chamber 4
is in direct fluid contact with the sample-reaction barrier 3
and the diagnostic element 6 or time gate 5. In addition, the
reaction chamber 4 may also be in direct fluid contact with an
optional reagent reservoir 17 as shown in Figs,. 1B and 1C.
A preferred embodiment of the reaction chamber utilizes a
ramp which extends from the bottom of the reaction chamber to
the surface of the diagnostic element. The ramp minimizes or
prevents mixing and eddy formation of the reaction mixture with
the sample at the interface of the reaction chamber and the
diagnostic element as the fluid moves through the device.
Thus, the ramp allows a smooth transition of the fluid out of
the reaction chamber and onto the diagnostic element. The
length of the ramp should be optimized for each depth of the
reaction chamber, but generally, the ramp is at an angle of
between 25 and 45 degrees relative to the floor of the reaction
chamber.
Time Gate
Referring to Fig. 1A, the time gate 5 holds the reaction
mixture in the reaction chamber 4 for a given period of time.
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The concept of the time gate is that a predominantly aqueous
solution cannot pass through a sufficiently hydrophobic zone
until the hydrophobic zone is made sufficiently hydrophilic.
Furthermore, the hydrophobic zone is made hydrophilic through
5 the binding of a component in the aqueous solution to the
hydrophobic zone. The sufficiently hydrophobic zone is
generally in a capillary space. The driving force for fluid
movement over or through the time gate may be either the
capillarity of the space or hydrostatic pressure exerted by the
10 sample or a combination of both of these forces. The amount of
time which is required to hold the reaction mixture in the
reaction chamber 4 is relative to the assay process such that
the reactions which occur in the reaction chamber 4 as a result
of the assay process will reflect the presence or amount of
15 target ligand in the sample. Thus, the time gate 5 delays the
flow of the reaction mixture onto the diagnostic element 6.
The time gate 5 delays the flow of the reaction mixture by the
principle that a hydrophilic liquid, such as an aqueous
solution or one which has a dielectric constant of at least 40,
20 cannot move past a sufficiently hydrophobic barrier in a
capillary channel. In designing and building azime gate, one
can begin with a hydrophobic surface, such as are found on
native plastics and elastomers (polyethylene, polypropylene,
polystyrene, polyacrylates, silicon elastomers and the like) or
silicon chip surfaces or metal surfaces, either smooth, grooved
or textured and a capillary is formed by an opposing surface
which can be hydrophobic or hydrophilic in nature and smooth,
grooved or textured. The hydrophobic surface(s) in the
capillary have a microscopic surface area onto which can bind
components which are generally soluble in a predominantly
aqueous solution. The hydrophilic character and the
concentration of the component(s) in the reaction mixture and
the overall surface area of the time gate affects the mechanics
of the time gate. The amount of time for which the time gate 5
holds the reaction mixture is related to the rate of binding of
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21
a component(s) from the reaction mixture to the hydrophobic
barrier. The binding of the component(s) from the reaction
mixture changes the hydrophobic barrier to a zone which is
sufficiently hydrophilic over which or through which the
reaction mixture can flow. Creating the sufficiently
hydrophilic surface then allows the fluid to flow as if the
time gate had not been in the device. Thus, fluid flow through
the remainder of the device is not affected once the time gate
has been made hydrophilic. Other devices described which
incorporate fluid delay means,.for example, in U.S Patent
Nos. 4,426,451 and 4,963,498, only require an
external manipulation of the device to start fluid
flow or utilize.capillary constrictions to slow fluid flow. In
this latter case, the capilla,ry const-riction used to delay
'15 fluid flow will affect the fluid =flow through the remainder of
the device.
In a preferred embodiment, for example, the time gate 5
can be composed of latex particles 15 (Fig. 1A, not drawn to
scale), such as polystyrene latexes with diameters of between
about 0.01 m and 10 m or hydrophobic polymers, such as
polypropylene, polyethylene, polyesters and the like, which are
introduced onto the device in the appropriate zone where the
reaction mixture must travel. In another preferred embodiment,
.the time gate can be created by application of a hydrophobic
chemical, such as an ink or a long chain fatty acid, or a
hydrophobic decal to the desired zone. The hydrophobic
chemical or decal is generally not soluble or is poorly soluble
in the reaction mixture. In yet another preferred embodiment,
the time gate can also be formed by changing a hydrophilic
surface to a hydrophobic surface. For example, hydrophobic
surfaces made hydrophilic by plasma treatment can be converted
back to a hydrophobic surface by the application of solvents,
ultraviolet light or heat and the like. These treatments can
act to change the molecular structure of the hydrophilic,
plasma modified surface back to a hydrophobic form.
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The component(s) in the reaction mixture which bind to the
hydrophobic zone may be various proteins, polypeptides,
polymers or detergents. A preferred protein is bovine serum
albumin. The time delay provided by the time gate 5 depends on
the concentration of the component(s) in the reaction mixture,
for example, bovine serum albumin, which binds to the
hydrophobic zone, for example, the surface area provided by the
latex particles 15. Another preferred embodiment of the time
gate 5 utilizes polyelectrolytes which are hydrophobic and
which become hydrophilic by exposure to the buffering capacity
of the reaction mixture. The time gate 5 would be comprised
of, for example, polyacrylic acid, which in its protonated form
it is hydrophobic. The reaction mixture, if buffered above the
pKa of the polyacrylic acid, would deprotonate the acid groups
and form the hydrophilic salt of the polymer. In this case,
the time delay is related to the mass of polyelectrolyte and
the pH and the buffering capacity of the reaction mixture.
The geometry or shape of the time gate can influence the
area of the time gate that the fluid will pass over or through.
That is, the time gate can be designed to direct the flow of
liquid through a specific area of the time gate. By directing
the fluid to flow through a defined area of the time gate the
reproducibility of the time delay is improved. Figure 6 shows
representative geometries of time gates. For example, as shown
in figure 6, time gates a-d, the time gates have V-shapes
incorporated into their design, and more specifically, the
length of the time gate (defined as the distance the fluid must
cross over or through in order to pass the time gate) is less
at the tip of the V than in the body of the time gate. Thus,
in a preferred mode, the fluid will cross over or pass through
the time gate where the length is shortest thereby directing
fluid flow through the time gate in a consistent manner. In
general, the directionality of fluid flow over or through the
time gates is represented by opposing arrows in Figure 6. In a
preferred embodiment, the orientation of the time gates b, c
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23
and d of figure 6 are such that the fluid touches the flat
portion of the time gate first rather than the V shape. In
other words, the preferred direction of flow for the time gates
b, c and d of figure 6 is represented by the up arrow. In
cases where the time gate is simply a line, for example as seen
in figure 6, time gate e and f, the path of fluid flow over or
through the time gate can occur at any point on the time gate.
Thus, the time gates which have geometries directing the fluid
flow over or through a consistent area of the time gate are
preferred. For example, time.gates with lengths ranging from
about 1.3 mm to 0.13 mm achieve delay times of approximately
0.3 min to 5.5 min, respectively, when the distance between
surfaces is about 0.018 mm. When the time gate is V-shaped,
the length of the time gate 5 at the tip of. the V has
dimensions smaller than the length of the time gate at the
remaining portion of the V; that is, the arms of the V should
have a length roughly 2 to 5 times the length of the V tip, as
for example, figure 7, time gate a, illustrates. Figure 7,
time gate b, shows -that only a small area of the time gate is
crossed over or through at the tip of the V as compared with
the remainder of the time gate. The time gate should span the
width of the capillary or space so that the entire fluid front
comes in contact with the time gate. If the time gate was not
as wide as, for example, the diagnostic element, then the fluid
front would go around the time gate. Thus, the time gate
should "seal" the fluid in the space during the delay period.
Referring to Fig. 1, one skilled in the art can recognize
that each device 10 could incorporate one or more time gates to
achieve the desired function of the device. Figure 8 shows
some examples of the sequential placement ofseveral time gates
of figure 6. For example, as discussed in the next section,
Optional Reagent Chambers, if a sequential addition immunoassay
was to be performed by the device then two time gates would
allow two sequential incubation steps to be performed by the
35, device prior to the movement of the reaction mixture to the
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24
diagnostic element. In another example, if an incubation of
the reaction mixture on the capture zone or zones of the
diagnostic element(s) 6 was required then a time gate(s) would
be placed immediately behind the capture zone or zones. This
use of the time gate may arise in cases where poor efficiency
of binding of the component in the reaction mixture to the
capture zone of the diagnostic element would prevail.
Another application of the time gate involves the
placement of a time gate on a surface which is not part of a
capillary space. For example, the time gate can be placed on a
hydrophilic surface, which alone without a capillary space will
allow liquids to move. This is generally the case when a
substantial volume of liquid is placed on a surface and it
spreads because of surface tension and because of the
hydrostatic pressure of the liquid pushing the meniscus
outwardly. The time gate then would function to delay the
advance of the fluid front because the hydrostatic nature of
the surface of the time gate would stop the movement of liquid.
As the meniscus of the advancing liquid touches the time gate,
the component or components in the liquid binds to the time
gate to create a sufficiently hydrophilic surface for a
continued advance of the liquid on the surface.
Yet another embodiment of the time gate involves the
positioning of a time gate prior to a membrane which is used to
capture a conjugate or receptor. In yet another embodiment of
the time gate, the time gate can be composed of hydrophobic
surfaces in a membrane. In those cases, the hydrophobic
membrane is positioned prior to the portion of membrane which
captures the conjugate or receptor and may be positioned after
a reaction chamber or a portion of membrane where reagents of
the assay are placed or embedded and where the reagents
incubate for a defined period of time. The time gate in the
membrane can be formed by application of raw latex particles in
the membrane at an appropriate solids concentration ranging
from about 0.01% to 10%. The size of the latex particles
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should be slightly less than the pore size of the membrane so
that the latex becomes imbedded within the membrane. The
density of latex within the membrane at the time gate should be
uniform so that the reaction mixture does not circumvent the
5 time gate. For example, the latex size used to create a time
gate for a membrane with a pore size of 1 m can range between
0.05 and 0.2 m. Since the distribution of pore sizes in
membranes varies widely, the actual size of latex used must be
arrived at by experimentation. The hydrophobic nature of the
10 membrane used for the time gate can also be formed by plasma
treatment or by treatment of the membrane with hydrophobic
chemicals or polymers that adsorb to the membrane. One skilled
in the art can appreciate that the teachings described herein
of the inventive features of the time gate can be utilized to
15 design time gates in a variety of diagnostic devices which
utilize membranes. That is, devices described, for example, in
U.S. Patents 4,435,504, 4,727,019, 4,857,453, 4,877,586 and
4,916,056, can incorporate a time gate, for example,
prior to the membrane or.in the membrane which captures
20 the conjugate or receptor.
Optional Reagent Chambers
Referring to Figs. 1B and 1C, the optional reagent chamber
17 is useful for the introduction of reagents into the assay
process. In general, the optional reagent chamber 17 may be in
25 direct fluid contact with the sample addition reservoir 2 via a
sample reaction barrier 3 or a port the reaction chamber 4 or
the diagnostic element 6, via a sample reaction barrier 3 or a
port. For example, Fig. 1B shows the optional reagent chamber
17 in direct fluid contact with the reaction chamber 4. The
flow of the introduced reagent may be controlled by a time gate
5a and fingers 16 can aid in the movement of reagents into the
reaction chamber 4. Referring now to Fig. 1C, for example, if
a sequential addition immunoassay was to be performed by the
device then 2 time gates 5 and 5a would and fingers 16 can aid
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in the movement of reagents into the reaction chamber 4.
Referring now to Fig. 1C, for example, if a sequential addition
immunoassay was to be performed by the device then 2 time gates
and 5a would allow 2 sequential incubation steps to be
5 performed in the optional reagent chamber 17 and then in the
reaction chamber 4 by the device prior to the movement of the
reaction mixture onto the diagnostic element 6. That is,
sample would be applied to the sample addition reservoir 2
through the sample addition zone 1 and the sample flows over
the sample reaction barrier 3 and into the optional reagent
chamber 17 by the aid of fingers 16 where the first set of
reactions would occur. The time gate 5a, after the appropriate
amount of time, would allow the reagents to flow over the
sample reaction barrier 3a and into the reaction chamber 4 by
the aid of fingers 16a where the next set of reactions would
take place. After the appropriate amount of time, the time
gate 5 allows the flow of reaction mixture onto the diagnostic
element 6.
Fluid Control Means
Referring to Fig. 1D, the optional fluid control means 18
is designed to control the flow of the reaction mixture in the
device. More specifically, the optional fluid control means 18
causes the volume of the reaction mixture to flow over the
capture zone of the diagnostic element 6 at a rate which allows
for an optimum capture of reagents onto the capture zone.
After the volume of the reaction mixture flows over the capture
zone the rate of flow of the excess reagents may be increased.
The differential rate of flow of the reagents in the device is
achieved by designing a gap 18 between the surfaces of the
capillary space 19 of the diagnostic element 6. The size of
the gap 18 is larger than the capillary space 19 of the
diagnostic element 6. The gap 18 generally follows the capture
zone or the zone where the rate of flow is required to be
decreased. The gap 18 in the diagnostic element 6 thus has an
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associated volume. The volume of the gap 18 is filled with the
reaction mixture by capillary action as it moves through the
device. Since the gap 18 after the capture zone is greater
than the capillary space 19 of the diagnostic element 6 a drop
in capillary pressure at the beginning of the gap 18 results in
a decrease in the rate of flow of the reaction mixture into the
gap 18 and therefore a decrease in the rate of flow of the
reaction mixture over the capture zone. Varying the size of
the gap 18 changes the capillarity in the gap and thus the flow
of the reaction mixture over the capture zone. In the case of
immunoassays requiring a wash step to remove unbound reagents
from the diagnostic element 6, it is generally desired that the
rate of flow of the wash solution over the diagnostic element 6
is faster than the rate of flow of the reaction mixture over
the diagnostic element 6 because this decreases the time of the
assay. The shape of the gap can take many forms. As shown in
Fig. 1D, the gap has square corners, however, the gap can be
shaped as a trapezoid or triangle which would change the rate
of flow of the reaction mixture while flowing into the gap.
One skilled in the art can also appreciate that for certain
immunoassays a wash step is not required.
The control of the rate of flow of the reagents in the
device can also be used to allow chemical reactions to take
place in one zone of the device before the reagents move to
another area of the device where the extent of reaction of the
reagents is monitored or where further reaction may take place.
For example, several fluid control means could be incorporated
into a device for use in immunoassays where a sequential
addition and incubation of reagents is necessary. That is, the
sample comes in contact with the first reagents and the time
for the reaction of the sample and first reagents is controlled
by a first gap. When the first gap is filled with fluid, the
reaction mixture continues to the second reagents at which time
an additional chemical reaction can subsequently take place.
The time required for completion of this second reaction can
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also be controlled by a second gap before further flow of the
reaction mixture along the diagnostic element. Chemical and
biochemical reactions also take place in the volume of the gap,
for example, by immobilizing reagents in the gap.
Diagnostic Element
Referring to Figs. 1 and 2, the diagnostic element 6 is
formed by opposing surfaces which are a capillary distance
apart through which the reaction mixture flows and on which are
placed one or more capture zones. The capture zones are
comprised of reagents, such as receptors, or devices, such as
biosensors which bind or react with one or more components from
the reaction mixture. The binding of the reagents from the
reaction mixture to the capture zones of the diagnostic element
6 is related to the presence or amount of target ligand in the
sample. One or more receptors or biosensors can be placed on
the diagnostic element 6 to measure the presence or amount of
one or more target ligands. The receptors or biosensors can be
placed in discrete zones on the diagnostic element 6 or they
can be distributed homogeneously or heterogeneously over the
surface. Receptors or other chemical reagents, For example, a
receptor against the signal generator can also be immobilized
on the diagnostic element 6 to verify to the user that the
reagents of the reaction mixture are viable and that the
reaction mixture passed through the zones of the receptors or
biosensors. A single receptor or biosensor can be placed over
the majority of the diagnostic element 6 such that as the
reaction mixture flows through the diagnostic element 6 the
components from the reaction mixture bind to the surface of the
diagnostic element 6 in a chromatographic fashion. Thus, the
distance which the component of the reaction mixture binds
would be related to the concentration of the target ligand in
the sample. The reagents, such as receptors, are immobilized
on the surface of the diagnostic element 6 through covalent
bonds or through adsorption. A preferred embodiment is to
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immobilize receptor coated latex particles, for example of
diameters ranging from about 0.1 m to 5 m. In addition,
particles termed "nanoparticles" can also be coated with
receptor and the resulting nanoparticles can be immobilized to
the diagnostic element through adsorption or covalent bonds.
Nanoparticles are generally composed of silica, zirconia,
alumina, titania, ceria, metal sols, and polystyrene and the
like and the particle sizes range from about 1 nm to 100 nm.
The benefit of using nanoparticles is that the surface area of
the protein coating the nanoparticle as a function of the
solids content is dramatically enhanced relative to larger
latex particles.
The surfaces of the diagnostic element 6 would allow the
receptor coated nanoparticles or latex particles to bind to the
diagnostic element 6. In a preferred embodiment, the receptors
bind to the surface of the diagnostic element through
electrostatic, hydrogen bonding and/or hydrophobic
interactions. Electrostatic, hydrogen bonding and hydrophobic
interactions are discussed, for example, in Biochemistry 20,
3096 (1981) and Biochemistry 29, 7133 (1990). For example, the
diagnostic element 6 can be treated with a plasma to generate
carboxylic acid groups on the surface. The receptor coated
latex particles are preferably applied to the diagnostic
element 6 in a low salt solution, for example, 1-20 mM, and at
a pH which is below the isoelectric point of the receptor.
Thus, the negative character of the carboxylic acid groups on
the diagnostic element 6 and the positive charge character of
the receptor latex will result in enhanced electrostatic
stabilization of the latex on the diagnostic element 6.
Hydrogen bonding and hydrophobic interactions would also
presumably contribute to the stabilization and binding of the
receptor latex to the diagnostic element 6. Magnetic fields
may also be used to immobilize particles which are attracted by
the magnetic field.
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In an additional embodiment of the diagnostic
element, now referring to Fig. 5, the diagnostic element 6
is a cylindrical surface which may be composed of grooves.
When the diagnostic element is composed of grooves, the
5 grooves generally run perpendicular to the flow of the
reaction mixture. A capillary space is formed around the
diagnostic element by a round tube which is generally clear;
thus, the surface of the diagnostic element and the opposing
surface of the tube are a capillary distance apart. The
10 round tube acts as a housing in this embodiment. The
capillary formed allows the flow of the reaction mixture
over the round diagnostic element 6. Generally, the
reaction mixture would travel up against gravity or down
with gravity through the cylindrical capillary space. The
15 capture zones of the round diagnostic element 6 can be
placed in discrete zones or over the entire length of the
diagnostic element 6. The capture zones may also circle the
diameter of the diagnostic element 6 or may be applied to
only a radius of the diagnostic element 6. The reaction
20 mixture may be delivered to the diagnostic element 6 through
the tube 58. Furthermore, the cylindrical volume of the
tube 58 may be used as a reaction chamber 4 and a disc
shaped sample reaction barrier 3 with grooves on its
perimeter may also be inserted to form the reaction chamber
25 4 and the sample addition reservoir 2. From this discussion,
now referring to Fig. 1 and 2, one skilled in the art can
also appreciate that the flat diagnostic element 6 may also
be curved such that the curvature is a radius of a circle.
One skilled in the art can appreciate that various
30 means can be used for the detection of signal at the capture
zone of the diagnostic element. in the case of the use of
biosensors, such as, for example, a piezoelectric crystal,
the piezoelectric crystal onto which would be immobilized a
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30a
receptor, would be the capture zone and the response
generated by binding target ligand would be generally
reflected by an electrical signal. Other types of detection
means include, but are not limited to visual and
instrumental means, such as
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spectrophotometric and reflectance methods. The inventive
features of the diagnostic element described herein allows for
improved capture efficiencies on surfaces over which a reaction
mixture flows and that various means for detection may be used
by one skilled in the art.
The surfaces of the capillaries in the device are
generally hydrophilic to allow flow of the sample and reaction
mixture through the device. In a preferred embodiment the
surface opposing the diagnostic element 6 is hydrophobic such
that the reaction mixture repels this surface. The repulsion
of reaction mixture to the surface opposing the diagnostic
element 6 forces the reaction mixture, and particularly the
protein conjugates, to the surface where capture occurs, thus
improving the capture efficiency of the components of the
reaction mixture to the capture zone. The hydrophobic surfaces
opposing the diagnostic element can have a tendency to become
hydrophilic as the reaction mixture progresses through the
diagnostic element because various components which may be
present endogenously or exogenously in the sample or reaction
mixture, such as, for example, proteins or polymers, bind to
the hydrophobic surface. A preferred hydrophobic surface
opposing the diagnostic element can be composed of TEFLON . It
is well known to those skilled in the art that TEFLON surfaces
bind proteins poorly. Thus, the TEFLON surface opposing the
diagnostic element would not become as hydrophilic as would
surfaces composed of, for example, polystyrene, polyacrylate,
polycarbonate and the like, when the reaction mixture flows
through the diagnostic element.
In another preferred embodiment, the diagnostic element 6
is hydrophilic but the areas adjacent to the diagnostic element
6 are hydrophobic, such that the reagents of the assay are
directed through only the hydrophilic regions of the diagnostic
element. One skilled in the art will recognize that various
techniques may be used to define a hydrophilic diagnostic
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element or zone, such as plasma treatment of hydrophobic
surfaces using masks which shield the surfaces, except for the
diagnostic element, from the treatment or by application of
hydrophobic adhesives to hydrophilic surfaces to define a
diagnostic element or by the use of viscous hydrophobic
compounds, such as an oil or a grease. In another preferred
embodiment, the capillary of the diagnostic element can be
formed by ultrasonic welding. The boundaries of the diagnostic
element are dictated by the energy directors which are used to
form the sonicated weld.
The surfaces of the diagnostic element 6 or of the other
components of the device may be smooth, grooved, or grooved and
smooth. Various textured surfaces may. also be employed, alone
or in combination with smooth or grooved surfaces. For
example, surfaces composed of posts, grooves, pyramids, and the
like referred to as protrusions; or holes, slots, waffled
patterns and the like, referred to as depressions may be
utilized. Referring now to Fig. 9, the surface can comprise
texture structures that comprise the form of diamonds,
hexagons, octagons, rectangles, squares, circles, semicircles,
triangles or ellipses. The textured surface can comprise
texture structures in geometries ordered in rows, staggered or
totally random; different geometries can be combined to yield
the desired surface characteristics. Typically, the
depressions or protrusions of the textured surface can range
from about 1 nm to 0.5 mm and preferably from about 10 nm to
0.3 mm; the distance between the various depressions or
protrusions can range from about 1 nm to 0.5 mm, and preferably
from about 2 nm to 0.3 mm.
A surface of diagnostic element 6 may be smooth or
comprised of texture structures such as posts or grooves.
Texture on a device surface can facilitate drying of reagents
on the surface during preparation of the device, and can
facilitate movement of sample in the diagnostic element.
Texture on a device surface facilitates uniform placement of
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dried reagents on the surface as follows: A liquid reagent-
containing fluid is placed in contact with the textured
surface, and small reagent fluid menisci form adjacent each
texture structure. Absent the presence of texture, the fluid
would tend to form larger menisci at corners of the entire
surface or chamber, which when dried would cause a non-uniform
layer of dried reagent. When texture structures are designed
into the device, the presence of numerous small menisci leads
to a more uniform layer of reagent that is dried throughout the
surface or chamber.
In a preferred mode as shown in Figs. 1 and 2, one surface
of the diagnostic element 6 is grooved and the grooves are
perpendicular to the flow of the reaction mixture and the
opposing surface is smooth. In another embodiment, one surface
of the diagnostic element 6 is grooved at the capture zone and
the areas adjacent to the capture zone are smooth. The
opposing surface of the diagnostic element 6 may be smooth or
may be grooved, for example, the grooves of each surface
intermesh. The positioning of the grooves of the diagnostic
element perpendicular to the flow of the reaction mixture is
beneficial in that the flow of the reaction mixture through the
diagnostic element 6 occurs in an organized manner with a
distinct, straight front dictated by the grooves in the
capillary space.
In addition, when one surface is in close proximity, for
example 1 .m to 100 m, to the peaks of the grooves then the
capture efficiency of the components from the reaction mixture
can be enhanced. The enhancement of capture efficiency at the
capture zones in grooved diagnostic elements as compared to
smooth surface elements may be related to the movement of the
reaction mixture in the capillary space; that is, in the case
of the grooved surface the reaction mixture is forced to move
over the peak of the groove and into the trough of the next
groove. Thus, a finer grooved surface, that is, more grooves
per cm, would provide a better capture efficiency than a
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coarser grooved surface. The reaction mixture is thus driven
closer to the surface of the grooved diagnostic element than it
would be if both surfaces were smooth.
Also, the close proximity of the surfaces decreases the
volume of the bulk reaction mixture above the surface of the
diagnostic element and therefore decreases the diffusion
distance of the components which bind to the diagnostic
element. The proximity of the surfaces of the diagnostic
element should minimize the volume of reaction mixture in the
diagnostic element at the capture zone without blocking the
capillary flow through the element. In addition, in
embodiments where a reagent is dried on a device surface and
another reagent is dried on a separate surface of the device,
these reagents can diffuse from their respective surfaces upon
introduction of fluid to those surfaces. The surfaces having
reagent immobilized thereon can be surfaces in a particular
chamber of the device or can be surfaces in different regions
of the device. The regions can be separate chambers or can be
device surfaces that do not delimit a chamber.
The capture of, for example, the complex of target ligand:
Ligand receptor conjugate at the capture zone can approach 100%
efficiency if the proximity of the surfaces is optimized. The
capture of nearly all of the ligand receptor conjugate which is
bound by target ligand is most desired because a greater
sensitivity of the assay as a function of sample volume can be
achieved. Other advantages of improved capture efficiency are
that less reagents are used because the sample volume is
decreased, the assay device can be miniaturized because of the
smaller sample volume and the reproducibility of the assay
result will be improved because changes in the rate of flow of
the reaction mixture through the capture zones will have less
or no effect on the capture of the labeled conjugates.
The capillary space can be defined by a variety of ways,
for example, machining the surfaces to the appropriate
tolerances or using shims between the surfaces. In a preferred
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embodiment, ultrasonic welding of the surfaces defines the
capillary. In this case, the capillary space is defined by the
energy directors and the distance between the surfaces is a
function of the size of the energy director, the welding
5 energy, the time of energy application and the pressure applied
during welding. The surfaces of the diagnostic element can be
parallel or non-parallel. In the latter case, the flow rate of
the reagents through the diagnostic element will not be uniform
throughout the length. A preferred embodiment is to maintain
10 the surfaces of the diagnostic. element approximately parallel.
The surfaces of the diagnostic element can be made from
materials, such as plastics which are capable of being milled
or injection molded, for example, polystyrene, polycarbonate,
polyacrylate and the like or from surfaces of copper, silver
15 and gold films upon which are adsorbed various long chain
alkanethiols as described in J. Am. Chem. Soc. 1992, 114, 1990-
1995 and the references therein. In this latter example, the
thiol groups which are oriented outward can be used to
covalently immobilize proteins, receptors or various molecules
20 or biomolecules which have attached maleimide or alkyl halide
groups and which are used to bind components from the reaction
mixture for determining the presence or amount of the target
ligand.
Referring to Figs. 3A and 3B, the zones of immobilization
25 of one or more receptors or the placement of biosensors at the
capture zone 37 on the diagnostic element 6 can take many
forms. For example, if the target ligand is very low in
concentration in the sample then one would desire that all of
the reaction mixture pass over the zone of immobilized receptor
30 or biosensor to obtain the best signal from the given volume of
reaction mixture. In this case, the placement of the reagents
or biosensors on the diagnostic element 6 at the capture zones
37 could, for example, resemble that shown in Fig. 3A. If the
target ligand in the sample is high in concentration and the
35 sensitivity of the analytical method is not an issue then the
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placement of the receptors or biosensors at the capture zones
17 could, for example, resemble that in Fig. 3B. One skilled
in the art can appreciate that the placement of receptors or
biosensors on the diagnostic element is a function of the
sensitivity requirements of the analytical method.
One or more diagnostic elements can be comprised in a
device. The reaction mixture may be applied to a device with
multiple diagnostic elements. In addition, the sample may be
applied to the device and then separated into different
reaction chambers, each with separate diagnostic elements. The
capture zone can be various geometrical symbols or letters to
denote a code when the sample is positive or negative for the
target ligand. One skilled in the art will recognize the
useful combinations of the elements of this invention.
The diagnostic element can also be configured to perform a
semi-quantitative or quantitative assay, as for example, is
described in Clinical Chemistry (1993) 39, 619-624, herein
referred to by reference only. This format utilizes a
competitive binding of antigen and antigen label along a solid
phase membrane. The improvement is that the use of the
diagnostic element described herein for the above cited method
would require a smaller sample volume and improved binding
efficiency to the solid phase surface.
Diagnostic Elements Other Than Capillaries
The inventive teachings described herein of the adsorption
of proteins, particularly receptors to plastic surfaces, can be
utilized for adsorption of receptors to many plastic surfaces
which are not a part of a capillary. Nanoparticles and latex
particles coated with receptors can also be applied to surfaces
of many types of immunoassay devices, such as, to "dipsticks"
or lidless devices. For example, dipsticks are generally a
solid phase onto which are bound, as a result of the assay
process, for example, the ligand receptor conjugate. Dipsticks
generally incorporate membranes; however, a disadvantage in the
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use of membranes in dipsticks is the difficulty in washing the
unbound ligand receptor from the membrane. Thus, an
improvement in the use of dipsticks is to immobilize receptor
coated latex or nanoparticles directly onto a plastic surface
of the dipstick. The removal of unbound ligand conjugate from
the plastic surface is thus more efficient than removal from a
membrane.
Textured surfaces such as disclosed herein can be used in
diagnostic elements other than capillaries. In such
embodiments, a textured surface can serve to provide additional
surface area which allows for a higher density of assay
reagents to be immobilized thereon. Furthermore, a textured
surface, or other surface modifications, can be provided to
affect the flow characteristics of a fluid on or within the
surface. For example, as disclosed herein a surface can be
provided with hydrophobic regions to diminish the extent of
fluid flow in the hydrophobic region, textures can be used that
provide for a more uniform distribution of dried reagents on
the surface, textures can be provided to modify the
configuration of the meniscus at the fluid flow front, or
textures can be used that provide the capillary driving force
for movement of fluid within the surface.
Used Reagent Reservoir
Referring to Figs. 1 and 2, the used reagent reservoir 7
receives the reaction mixture, other reagents and excess sample
from the diagnostic element 6. The volume of the used reagent
reservoir 7 is at least the volume of the sample and extra
reagents which are added to or are in the device. The used
reagent reservoir 7 can take many forms using an absorbent,
such as a bibulous material of nitrocellulose, porous
polyethylene or polypropylene and the like or the used reagent
reservoir can be comprised of a series of capillary grooves.
In the case of grooves in the used reagent reservoir 7, the
capillary grooves can be designed to have different capillary
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38
pressures to pull the reagents through the device or to allow
the reagents to be received without a capillary pull and
prevent the reagents from flowing backwards through the device.
The size and quantity of the grooved capillaries determine the
volume and capillarity of the used reagent reservoir 7. In a
preferred embodiment, as shown in Fig. 4, the fingers 52 at the
end of the diagnostic element 6 are in fluid contact with a
capillary space 55 and the capillary space 55 is in fluid
contact with a grooved or textured capillary space 56. The
depth of the grooves or textured surface can be, for example,
about 0.1 mm to 0.6 mm, preferably about 0.3 mm to 0.5 mm and
the density can range from about 5 to 75 grooves per cm and
preferably about 10 to 50 grooves per cm.
Referring to Fig. 4, the reagents of the device move to
the fingers 52 at the end of the diagnostic element ,6' and into
the capillary channel 55. The reagents either partially or
completely fill the capillary space 55 and then come in contact
with the grooved or textured surface 56. The width of the
capillary space 55 is generally about 1 mm to. 3 mm and the
20. depth is generally about 0.1 mm to 2 mm. The length of the
capillary space 55 should be sufficient to be in fluid contact
with the grooved or textured surface 56. The grooved or
textured surface 56 partially or completely pulls the reagents
fr.om the capillary channel 55 depending on the rate of delivery
of the reagents into the capillary space 55 from the diagnostic
element 6. When the flow of reagents is complete in the
device, the grooved or textured surface 56 has greater
capillarity than the capillary channel 55 and the reagents are
removed from the capillary channel 55 by the grooved or
textured surface 56. In addition, the reverse flow of the
reagents from the grooved or textured surface is not preferred
because the capillarity in the grooved or textured surface 56
holds the reagents and prevents their backward flow. One
skilled in the art can recognize from these inventive features
that the arrangement of grooves or a used reagent reservoir
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within the device can be adapted to a variety of desired
objectives.
The Description of the One-Step Assay Device
The elements of the device which. have been described
individually can be assembled in various ways to achieve the
desired function. The term "one-step" implies that one manual
action is required to achieve the assay result, for example,
adding sample to the device is one step. In the case of the
device performing a one-step assay which involves both a timed
incubation of reagents and a wash step, the wash solution is
excess sample and the assay device is built with the elements
in fluid communication using the sample addition reservoir, the
sample-reaction barrier, the reaction chamber, the time gate,
the diagnostic element and the used reagent reservoir as
depicted in Fig. 1. The devices are generally about 3 cm to 10
cm in length, 1 cm to 4 cm in width and about 2 mm to 15 mm
thick. Typically, a top member with smooth surfaces is placed
onto a bottom member which has a surface onto which are built
the elements stated above. The relationship of the elements
are as depicted in Fig. 1. The reagents required for
performing the assay are immobilized or placed in the
respective elements. The surfaces are brought together, a
capillary distance apart, and in doing so, the regions of the
sample addition reservoir, the sample reaction barrier, the
reaction chamber, the time gate, the diagnostic element, the
gap and the used reagent reservoir are all formed and are
capable of functioning together. Also, the surfaces are
brought together such that the opposing surfaces touch to form
and seal the sample addition reservoir, the reaction chamber,
and the used reagent reservoir.
When performing a qualitative, non-competitive assay on
one or more target ligands, the signal producing reagents,
which could include, for example, a receptor specific for the
target ligand adsorbed to a colloidal metal, such as a gold or
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selenium sol, are placed on the sample reaction barrier or in
the reaction chamber in dried or lyophilized form. Another
receptor for each target ligand is immobilized onto the surface
of the diagnostic element at the capture zone. The time gate
5 is positioned generally on the diagnostic element between the
reaction chamber and the capture zones by the placement of, for
example, a surfactant-free polystyrene suspension onto the
device in an amount which dictates the desired incubation time.
The incubation time is usually the amount of time for the
10 reactions to come to substantial equilibrium binding. The
assay is then performed by addition of sample to the sample
addition reservoir of the device. The sample moves over the
sample-reaction barrier, into the reaction chamber by the aid
of the fingers and dissolves the reagents in the reaction
15 chamber to form the reaction mixture. The reaction mixture
incubates for the amount of time dictated by the time gate.
The excess sample remaining in the sample addition reservoir
and reaction mixture in the reaction chamber are in fluid
communication but are not in substantial chemical communication
20 because of the sample-reaction barrier. Thus, the reaction
chamber defines the volume of the reaction mixture. The
reaction mixture then moves past the time gate and onto the
diagnostic element and over the capture zones. The complex of
receptor conjugate and target ligand formed in the reaction
25 mixture binds to the respective receptor at the capture zone as
the reaction mixture flows over the capture zones. The
reaction mixture may also flow over a positive control zone,
which can be for example, an immobilized receptor to the signal
development element. As the reaction mixture flows through the
30 diagnostic element and into the used reagent reservoir by the
aid of the fingers, the excess sample flows behind the reaction
mixture and generally does not substantially mix with the
reaction mixture. The excess sample moves onto the diagnostic
element and removes the receptor conjugate which did not bind
35 to the capture zone. When sufficient excess sample washes the
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diagnostic element, the signal at the capture zones can be
interpreted visually or instrumentally. Referring to Fig. 1D,
in a preferred mode of the above description, the reaction
mixture moves onto the diagnostic element 6, over the capture
zone or zones and then the reaction mixture proceeds into a
capillary gap 18. The capillary gap 18 generally has less
capillarity than that of the diagnostic element 6. The
capillary space 19 of the diagnostic element 6 is generally
smaller than the capillary space of the gap 18. The volume of
the capillary gap 18 generally approximates the volume of the
reaction mixture such that the capillary gap 18 fills slowly
with the reaction mixture and once filled, the capillarity of
the remaining portion of the diagnostic element 6 or used
reagent reservoir is greater than the capillarity of the gap 18
resulting in an increased rate of flow to wash the diagnostic
element 6. As one skilled in the art can appreciate, the gap
18 can be formed in the top member 8 or in the bottom member 9
or a combination of both members 8 and 9.
In the case of the device performing a one-step assay
which does not involve a timed incubation step but does involve
a wash step in which the wash solution is excess sample, the
assay device is built with the elements in fluid communication
using the sample addition reservoir, the sample-reaction
barrier, the reaction chamber, the diagnostic element and the
used reagent reservoir. The assay reagents are used as
described above for the non-competitive qualitative assay. The
assay device without the time gate allows the reaction mixture
to flow onto the diagnostic element without an extended
incubation time. The capillary flow of the reaction mixture
and the excess sample are as described above.
The optional reagent chamber is incorporated into the
device in the case of the device performing a one-step assay
with the introduction of an additional assay reagent into or
after the reaction mixture or the introduction of a wash
solution which flows behind the reaction mixture through the
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device. The optional reagent chamber may be in fluid contact
with any element of the device and is generally in fluid
contact with the reaction chamber. When in fluid contact with,
for example, the reaction chamber, the optional reagent chamber
and the reaction chamber may be separated by a time gate.
Various reagents may be dried or lyophilized in the optional
reagent chamber, such as detergents for a washing step or
reagents which are sequentially provided to the diagnostic
element after the reaction mixture.
In the case of performing one-step, non-competitive,
quantitative assays the reagents as described above for the
non-competitive, qualitative assay may apply. The device is
comprised of the elements, sample addition reservoir, sample-
addition barrier, reaction chamber, time gate, diagnostic
element and used reagent reservoir. In this case, the capture
zone of the diagnostic element is generally the entire
diagnostic element. That is, the capture zone is a length of
the diagnostic element onto which the receptor conjugate binds.
The receptor conjugate binds along the length of the capture
zone in proportion to the amount of target ligand in the
sample. Alternatively, one or more capture zones 37 can be
placed on the diagnostic lane (Fig. 3A-B), and signals from the
capture zone(s) can be read by an instrument such as a CCD
camera, a fluorometer or a spectrophotometer.
The device of the present invention is preferred for this
quantitative assay because of the high efficiency of capture of
the reagents, for example, the binding of a complex of target
ligand and receptor conjugate to an immobilized receptor to the
target -ligand on the capture zone, and because the movement of
the reaction mixture over the diagnostic element proceeds with
a sharp front. The receptors on the capture zone sequentially
become saturated with the complex of target ligand and receptor
conjugate as the reaction mixture moves over the length of the
capture zone. The length of the diagnostic element containing
bound conjugate then determines the concentration of the target
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ligand. Those skilled in the art will recogr.ize the format of
this type of immunoassay as a quantitative
immunochromatographi.c assay as discussed in U.S. Pat.
Nos. 4,883,688 and 4,945,205.
In the case of the device performing a one-step,
quantitative or qualitative competitive assay which involves
both a timed incubation of reagents and a wash step and the
wash solution is excess sample, the assay device is built with
the elements in fluid commur_ication us.ing the sample addition
reservoir, the sample-reaction barrier, the reaction chamber,
the time gate, the diagnostic element and the used reagent
reservoir. When performing a qualitative competitive assay an
one or more target ligands, the conjugate is composed of, for
example, a ligand analogue coupled to signal development
.15 element, such as a gold or selenium so1. The conjugate and
receptor for each target ligand are placed in the reaction
chamber in dried or lyophilized form, for example, in amounts
which are taught by U.S. Pat. Nos. 5,026,535 and 5,089,391.
Another receptor for each
target ligand is immobilized onto the surface of the diagnostic
element at the capture zone.. The time gate is positioned
generally on the diagnostic element between the reaction
chamber and the capture zones as described previously. The
incubation time is usually the amount of time for the reactions
to come to substantial equilibrium binding.
The assay is then performed by addition of sample to the
device. The sample moves over the sample-reaction barrier and
into the reaction chamber, dissolves the reagents to form the
reaction mixture and incubates for the time dictated by the
time gate. The excess sample and reaction mixture are in fluid
communication but not in substantial chemical communication
because of the sample-reaction barrier. The reaction mixture
then moves onto the diagnostic element and over the capture
zones. The ligand anal.ogue_-conju.gate binds to the respective
receptor or receptors at the capture zone or zones. As the
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reaction mixture flows over the diagnostic element and into the
used reagent reservoir, the excess sample flows behind the
reaction mixture and generally does not substantially mix with
the reaction mixture. The excess sample moves onto the
diagnostic element and removes conjugates which do not bind to
the capture zone or zones. When sufficient excess sample
washes the diagnostic element the results at the capture zones
can be interpreted visually or instrumentally.
In a preferred mode of the present invention, the reaction
mixture moves onto the diagnostic element, over the capture
zone or zones and then the reaction mixture proceeds into a
capillary gap. The capillary gap has less capillarity than
that of the diagnostic element. The volume of the capillary
gap generally approximates the volume of the reaction mixture
such that the capillary gap fills slowly with the reaction
mixture and once filled, the capillarity of the remaining
portion of the diagnostic element or used reagent reservoir is
greater resulting in an increased rate of flow of excess sample
to wash the diagnostic element.
In another aspect of the one-step, competitive assay, the
reaction mixture is composed of ligand analogue-ligand
complement conjugate to each target ligand and receptors
adsorbed to latex particles with diameters of, for example, 0.1
m to 5 m to each target ligand, in appropriate amounts, for
example, as taught by U.S. Pat. Nos. 5,028,535 and 5,089,391.
The ligand complement on the conjugate can be any chemical or
biochemical which does not bind to the receptors for the target
ligands. The assay is begun by addition of sample to the
device. Sample fills the reaction chamber and is incubated for
a time which allows the reagents to come to substantial
equilibrium binding. The reaction mixture flows over the time
gate and onto or into a filter element to prevent ligand
analogue-ligand complement conjugates which have bound to their
respective receptor latexes from passing onto the diagnostic
element. Typical filter elements can be composed of
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nitrocellulose, cellulose, nylon, and porous polypropylene and
polyethylene and the like. Thus, only the ligand analogue-
ligand complements conjugate which were not bound by the
receptor latex will pass onto the diagnostic element. The
5 receptor to the ligand complement of the conjugate is
immobilized on the diagnostic element at the capture zone and
binds the conjugate. A wash step may not be required because
the filter removes the conjugate bound to latex; however, the
excess sample or a wash solution from the optional reagent
10 chamber may be used to wash the diagnostic element.
In the case of a one-step quantitative, competitive assay,
the receptor to the ligand analogue conjugate or the ligand
complement of the conjugate is immobilized onto the diagnostic
element as described previously for the one-step quantitative,
15 non-competitive assay. Thus, the concentration of the target
ligand in the sample is visualized by the distance of migration
on the diagnostic element of the conjugate. In another mode, a
quantitative assay could be performed by the binding of the
labeled conjugate, for example, the ligand analogue-ligand
20 complement conjugate, to sequential, discrete capture zones of
receptor on the diagnostic element. The quantitative result is
achieved by the depletion of the conjugate as the reaction
mixture flows through the capture zones of the diagnostic
element. Signal related to analyte concentration is measured,
25 e.g., by a CCD camera, a fluorometer or a spectrophotometer.
The Device as a Diagnostic Element
The diagnostic element of the device can be utilized with
a sample addition means to perform a separation step of bound
and unbound conjugates. An example of this type of device
30 which has a sample addition means, a diagnostic element and a
used reagent reservoir is depicted in Fig 2. For exaniple, in
the case of a non-competitive assay, at least one receptor
conjugate is incubated with sample which is suspected of
containing at least one target ligand in a suitable vessel and
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46
this reaction mixture is applied to the sample addition zone of
the device. The reaction mixture then flows onto the
diagnostic element and over the capture zone of, for example,
immobilized receptor to the target ligand. When target ligand
is present in the sample, the target ligand-receptor conjugate
complex binds to the receptor on the capture zone. If the
signal development element is an enzyme, then either a
substrate for the enzyme which produces a visual color or a
wash solution followed by a substrate is next added to the
device. Excess reagents flow to the used reagent reservoir.
The presence or amount of each target ligand in the sample is
then determined either visually or instrumentally.
In the case of a competitive immunoassay, for example as
taught by U.S. Pat. Nos. 5,028,535 and 5,089,391,
1~ the diagnostic element may be used to separate
bound and unbound ligand analogue conjugates such
that the unbound ligand analogue conjugates bind to the
receptors of the diagnostic element in proportion to the
presence or amount of target ligand in the sample.
One skilled in the art can appreciate that all formats of
immunoassays or nucleic acid assays which require a separation
step of free and bound conjugates or the separation of free of
bound reagents which subsequently leads to the ability to
detect a signal can utilize the inventive features of the
diagnostic element. One skilled in the art can also recognize
that the inventive elements of this invention, namely, the
fingers, the sample reaction barrier, the reaction chamber, the
time gate, the diagnostic element, the fluid control means and
the used reagent reservoir can be used separately or in various
combinations and in conjunction with other devices not
described here. Furthermore, textured surfaces, such as
described herein, can be utilized in one or more regions of the
device to facilitate placement of a uniform layer of dried
reagent in the area, or to modify fluid flow characteristics
through the region. In addition, hydrophobic zones can be
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placed in a region of the device to modify fluid flow
characteristics in the region. As appreciated by one of
ordinary skill in the art, features disclosed herein can be
utilized in various combinations in the preparation and use of
assay devices.
For example, the sample reaction barrier with fingers and
the reaction chamber can be used in conjunction with devices
incorporating porous members, such as membranes to deliver
precise volumes of reagents to the porous member. The time
gate can also be incorporated into the aforementioned devices
or the time gate may be used alone in conjunction with devices
incorporating porous members. The fluid control means can also
be used in devices incorporating porous members to control the
rate of flow of reagents through the porous member. In the
context of performance of assays in accordance with the
invention, channels can exist such as' the distance between
opposing walls of a particular region, e.g., between the lid
and the base; or the distance between adjacent texture
structures. Accordingly, when a ligand receptor is immobilized
on a device surface, a ligand of interest in a sample can
diffuse across the width of a channel to bind with its
receptor.
EXAMPLES
Example 1
Preparation of anti-DhCG Antibody-Collodial Gold Conjugate
Colloidal gold with an average diameter of 45 nm was
prepared according to the method of Frens, Nature, Physical
Sciences, 241, 20 (1973). The colloidal gold conjugate was
prepared by first adding 5.6 ml of 0.1 M potassium phosphate,
pH 7.58, dropwise with rapid stirring to 50 ml of colloidal
gold. Anti B-subunit monoclonal antibody to hCG (Applied
Biotech, San Diego, CA; 1 ml of 4.79 mg/ml in phosphate
buffered saline, 0.02% sodium azide, pH 7) was added in a bolus
to the colloidal gold with rapid stirring. After complete
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mixing the stirring was stopped and the solution was incubated
at room temperature for 1 h. Polyethylene glycol (average
molecular weight=20,000) was added (0.58 ml) as a 1% solution
to the colloidal gold solution and the solution was mixed. The
colloidal gold solution was subjected to centrifugation at
27,000 g and 5 C for 20 min. The supernatant was removed and
each pellet was washed twice bv resuspension and centrifugation
with 35 ml of 10 mM potassium phosphate, 2 mM potassium borate,
0.01% polyethylene glycol (average molecular weight=20,000), pH
7. After the final centrifugation, the pellet was resuspended
in 0.5 ml of the wash buffer. The gold conjugate was diluted
for the assay of hCG into a buffered solution containing 10
mg/ml bovine serum albumin at pH 8.
Example 2
Preparation of Anti-ahCG Antibody Latex
Surfactant-free polystyrene particles (Interfacial
Dynamics Corp., Portland, OR; 0.106 ml of 9.4% solids, 0.4 m)
was added while vortexing to anti a-subunit hCG monoclonal
antibody (Applied Biotech, San Diego, CA; 0.89 ml of 6.3 mg/ml
in 0.1 M 2-(N-morpholino) ethane sulfonic acid, (MES), pH 5.5)
and the suspension was incubated at room temperature for 15
min. The suspension was subjected to centrifugation to pellet
the latex particles. The pellet was washed three times by
centrifugation and resuspension of the pellet with 10 mM MES,
0.1 mg/ml trehalose, pH 5.5. The final pellet was resuspended
in the wash buffer at a solids concentration of 1%.
Example 3
Preparation of Goat Anti-Mouse Latex
Surfactant-free polystyrene particles (Interfacial
Dynamics Corp., Portland, OR; 0.11 ml of 9.4% solids, 0.6 m)
were added while vortexing to goat IgG antibody against mouse
IgG (Jackson ImmunoResearch Laboratories, Inc.; 0.89 ml of 0.34
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mg/ml in 0.1 M MES, pH 5) and the suspension was incubated at
45 C for 2 h. The suspension was subjected to centrifugation
to pellet the latex particles. The pellet was washed three
times by centrifugation and resuspension of the pellet with 10
mM MES, 0.2 mg/mi trehalose, pH 5.5. The final pellet was
resuspended in the wash buffer at a solids concentration of 1%.
Example 4
Preparation of the One-Step Device for a Qualitative or
Quantitative hCG Assay
A one-step device made of plastic was built having an 80
to 100 l sample addition reservoir, a 20 l reaction chamber
and a 40 l used reagent reservoir. This device is designed
for applying samples of about 20 l to 100 l, but the reaction
chamber is fixed at 20 l. In cases where a larger reaction
mixture volume is required for the desired assay, then the
reaction chamber would be increased to that volume and the
sample addition reservoir would be about 2 to 4 times the
volume of the reaction chamber volume.
The devices were plasma treated to graft functional groups
which create a hydrophilic surface. Those skilled in the art
will recognize that the plasma treatment of plastic is
performed in a controlled atmosphere of a specific gas in a
high frequency field. The gas ionizes, generating free
radicals which react with the surface.
The sample addition reservoir was shaped as a trapezoid
with dimensions of 14 mm and 7 mm for the parallel sides and 7
mm for the other sides with a depth of 0.49 mm. The sample
addition reservoir was adjacent to the sample reaction barrier.
The sample-reaction barrier was 1.5 mm long and 7 mm wide
including grooves running parallel to the flow of the sample at
a density of 50 grooves per cm and a depth of 0.1 mm. In the
case of sample volumes larger than 20 to 80 l, the width of
the reaction barrier and thereby the reaction chamber could be
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increased to accommodate the desired flow rate but the groove
size or density could remain as indicated.
The fingers in the walls of the reaction chamber and the
used reagent reservoir were 1 mm wide and 0.4 mm deep with 7
5 fingers in each wall of the reaction chamber and the used
reagent reservoir. The reaction chamber volume was 20 l. The
reaction chamber was shaped as a trapezoid with dimensions of 7
mm and 3.5 mm for the parallel sides and 7.1 mm for the other
sides with depths of 0.56 mm for 20 l reaction chambers.
10 The diagnostic element was about 2.5 cm long, 2 mm wide
and 1 mm from the base of the device including grooves running
perpendicular to the flow of reaction mixture at a density of
100 grooves per cm and a depth of 0.05 mm. In the case of a
time gate on the diagnostic element, the time gate was
15 positioned on the diagnostic elementimmediately adjacent to
the reaction chamber. The width of the diagnostic element
could be increased to increase the flow of the reaction mixture
to the desired rate past the capture zones.
The anti-ahCG antibody latex (1 l) and the goat anti-
20 mouse latex (1 l) were applied to the diagnostic element of
the devices approximately 1.5 cm apart. The anti-13hCG antibody
colloidal gold conjugate (10 l) was pipetted into the trough
of the reaction chamber.
The devices were placed under vacuum for about 15 min. to
25 dry the reagents. The used reagent reservoir had the shape of
a trapezoid with dimensions of 7 mm and 15 mm for the parallel
sides and 8 mm for the other sides with a depth of 0.5 mm.
Referring to Fig. 4, in a preferred embodiment of the used
reagent reservoir, the reaction mixture moved to a capillary
30 space 55 (1.25 mm long, 27.5 mm wide and 0.48 mm deep) from the
diagnostic element 6, aided by fingers 52 (1 mm wide and 0.4 mm
deep with 7 fingers), and then into a grooved capillary
structure (13.6 mm long, 25.4 mm wide, 0.61 mm deep with a
density of 16 grooves per cm). The outer walls and the top
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surface of the walls of the sample addition reservoir and the
reaction chamber had applied a thin coating of silicon grease
to prevent the leakage of the reagents from the reservoir and
chamber of the assembled device. The capillary spaces in the
~ devices were then formed by placing a clear plastic
polycarbonate sheet on top of the device. The plastic sheet
was held to the opposing surface with binder clips. The clear
plastic sheet had a sample port above the sample addi:ion
reservoir for the introduction of sample.
Example 5
'rlvdror)hobic Borders to Fluid-Containing Areas
Fluid flow on a surface or in a capillary is affected by
the surface tension of the fluid. For example, i.n a capillary
channel that is formed by essentially planar wall's that
intersect along corners, fluid flow preferentially precedes
along the corners. The predisposition for fluid flow to
proceed at corners occurs because the corners of a capillary
create the lowest surface tension for the fluid.
However, when a uniform flow front is required within a
capillary, the reduced surface tension at corners of the
capillary can cause a non-uniform flow front. Non-uniform flow
fronts can result in the creation of air pockets within the
capillary. If air pockets occur, wetting of the. capillary
surfaces within the air pocket is impaired or prevented.
Consequently, when surfaces of capillaries are used, for
reactions such as binding of antibodies or antigens, for
chemical reactions, or for nucleic acid hybridization
reactions, the creation of air pockets decreases the efficiency
of the reaction. Furthermore, the creation of air pockets
within analogous capillaries of individual devices of the same
design is not predictable, consequently the consistency of
binding or chemical reactions between the individual devices
will be poor. Thus, air pockets within capillaries can alter
fluid flow or even prevent it in the capillary.
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Embodiments of this invention. which comprise hydrophobic
areas on a lumenal surface of a capillary space act to control
fluld flow within capillaries, and more specifically to
minimize fluid flow at the corners of capillaries so that the
fluid flow front is convex rather than concave.
The inventive teachings herein show that hydrophobic
borders lining capillary channels, preferably along edges or at
corners, slow.s.fluid flow at these locations, thereby creating
convex flow fronts instead of the native concave flow front.
Concave flow fronts are disadvantageous in capillary channels
because air can be trapped as the concave flow front proceeds
through the capillary, since a concave flow front increases the
propensity of the advancing fluid to form air pockets in the
capillary. Hydrophobic borders facilitate the escape of air
i5 from the advancing fluid flow front because the likelihood is
substantially reduced that fluid can be held within the
capillary in the hydrophobic zones.
?n a preferred embodiment, hydrophobic zones are applied
to a surface. Specifically, a hydrophobic zone can be located
on at least one surface of a capillary, each hydrophobic zone
bordering the edge or corner of the capillary, being located
adjacent a hydrophilic surface in which fluid is intended to
flow. On at least one surface of the capillary, the
hydrophobic zones or borders occupy between 1% to. 90% .of.-the
surface, each zone being adjacent to a hydrophilic surface and
the edge or corner of the capillary.
Hydrophobic zones delimit the edges of a surface or occupy
the edges of materials placed in capillary spaces. In another
preferred embodiment, hydrophobic zones delimit the edges of
materials placed in capillary spaces, for example, materials
such as filters, membranes and polymeric meshes. The
hydrophobic zone can cover from 1% to 90% of the surface of the
material to be placed in the capillary space. For additional
disclosure concerning the use of materials within capillary
spaces, see e.g., copending U.S. Patent No. 6,391,265.
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Accordingly, fluid flow in the capillary is delayed
at the edges of the material in contact with the
corners of the capil-lary relative to the fluid flow within the
hydrophilic zones of the capillary. The hydrophobic zones of
the material in the capillary occupy between 1% and 90% of the
surface of the material, each zone being adjacent to the
hydrophilic surface and the edge of the capillary.
In addition, embodiments of the invention allow for the
application of fluids to discrete zones on surfaces or within
surfaces or membranes. Thus, in another preferred embodiment
hydrophobic zones of surfaces prevent the movement of reagents
on or within a surface or within a membrane. In this
embodiment, these zones act as corrals to hold fluid within an
area of the surface. These embodiments overcome a difficulty
of applying reagents to discrete zones on or within surfaces or
in membranes, such that a volume of applied reagent will move
on a surface or within a membrane because of the hydrostatic
pressure cf the reagent. This problem is especially prevalent
in the case of surfaces which comprise a texture to facilitate
movement of fluids by capillary action along the surface such
as during the performance of an assay on a fluid sample.
However, when manufacturing such assay devices, it can be
desirable to place reagents on such surfaces, application of
discrete zones of reagents is especially difficult because the
surface is designed to facilitate fluid movement by capillary
action, and the effects of hydrostatic forces exacerbate the
difficulties produced by capillary action. These factors
create an unpredictable area of reagent, which may not be
discrete. Consequently, if the volume of the applied reagent
relative to the surface is too great, adjacent reagent areas
may run together to create one undesired, commingled zone.
This situation is particularly problematic when the surface
comprises grooves or texture that cause fluids to flow by
capillarity on or within the surface. Generally, such surfaces
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are substantially fluid impermeable. Accordingly, the creation
of hydrophobic borders on or within a surface or in a membrane
to encompass or retain the applied reagent allows the
appiication of reagents in discrete areas.
In another preferred embodiment, hydrophobic zones are
applied to surfaces to control the overall movement of the
fluid on or within a surface or in a capillary. For example,
hydrophobic zones can be utilized to direct or prevent fluid
flow to various areas of a device.
In an alternative preferred embodiment, hydrophobic
borders are placed at the edge(s) of a surface such as adjacent
to corners of a chamber, to facilitate uniform drying of
liquids on the surface. This embodiment is useful in the
drying of liquid on a surface, wherein, in the absence of
hydrophobic borders, liquid which is added to the surface or
chamber accumulates at the edge or corners of the surface or
chamber as evaporation occurs. This latter situation occurs
because the corners of a chamber create a meniscus and it is
energetically favorabie for a fluid to move to a corner
meniscus as i: evaporates. Therefore, the outcome of
eva:::,ration is a disproportionately larger amount of dried
reagent in the corners of the chamber than in the surface of
the chamber. The novel use of hydrophobic borders adjacent to
corners of chambers prevents the fluid meniscus from forming at
the corner, since the fluid will not accumulate at the
hydrophobic border. Thus, by use of this embodiment, the
resultant dried liquid is more uniformly dispersed on the floor
of the chamber.
Hydrophobic zones can also be created for surfaces which
had previously been made hydrophilic. Several techniques are
known to those skilled in the art for use to make a surface
hydrophilic, for example, corona discharge, treating with a gas
plasma, treating with detergents or proteins and the like. For
surfaces made hydrophilic by the aforementioned techniques,
hydrophobic zones can be created by application of organic
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solvents that destroy the plasma treatment or denature the
proteins, to recreate a native hydrophobic plastic surface or
to create a hydrophobic surface by the denatured proteins, or
by local heating of the surface using focused laser beams to
5 destroy the hydrophilic nature of the surface. Alternatively,
one can mask hydrophobic areas before creating a hydrophilic
area by any of the foregoing methods. The areas can be masked
by objects such as a template or can be masked by materials
that are applied to the surface and then are subsequently
10 removed.
Hydrophobic compounds, such as aliphatic and/or aromatic
compounds and various inks and polymers and the like can be
used for the creation of hydrophobic zones in accordance with
the invention. The compounds are generally dissolved in
15 organic solvents or mixtures of aqueous and organic solvents.
One skilled in the art will recognize that a variety of
techniques known in the art (such as ink jet printing,
spraying, silk screening, drawing, embossing and the like) are
techniques that permit the application of hydrophobic zones on
20 or within surfaces.
Example 6
Textured Surfaces to Facilitate Uniform Drying of Reagents
In an additional embodiment, texture structures such as
posts are positioned, generally in an ordered array on a
25 surface. When fluid is placed in contact with the structures,
small menisci are formed at each structure. When the reagent
fluid dries, these menisci thereby provide a very uniform
distribution of dried reagent on the surface. Generally, the
structures are posts which are substantially perpendicular to a
30 surface such as a floor of a chamber. A rectilinear angle is
defi-ned between a surface and a wall of a post located thereon.
The density and size and shape of posts on the surface can
vary, depending on the degree of uniformity desired for the
dried reagents. Post height can also vary, and generally the
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height of the posts should be about 1% to more than 100% of the
height of the fluid in the chamber; that is, the posts can
protrude from the fluid or the fluid can cover the posts after
applying the fluid to the surface. In all cases, the posts
will act as zones to form menisci as liquid evaporates from the
surface or chamber.
Example 7
Qualitative or Quantitative One-Step Assay for hCG
The devices described in Example 4 were used for the
qualitative or quantitative one-step assay for hCG. The assay
times for the devices without the time gates were about 5 to 10
min. A urine solution (60 1) containing 0, 50, 200 and 500
mIU hCG/ml was added to the sample reservoir of the devices.
The sample moved into the reaction chamber,' dissolved the
colloidal gold conjugate and the reaction mixture moved onto
the diagnostic ei.ement over the anti-hCG latex and goat anti-
mouse IgG latex capture zones. The reaction mixture moved into
the used reagent reservoir and the excess sample washed the
diagnostic element. The color density of the capture zones for
hCG was measured instrumentally using a MINOLTA CHROMA METER CR
241 at.540 nm. A red color was visible for samples containing
hCG and not visible for the sample without hCG at the capture
zones for hCG. The AE* values for the 0, 50, 200 and 500 mIU/ml
were 0, 7.78, 12.95 and 20.96, respectively, and for the
positive control (goat anti-mouse IgG) zones a distinctive red
bar was observed with a AE' of about 35.
Example 8
Qualitative or Quantitative One-Step Assay for hCG Using a Time
.Gate
Devices as described in Example 4 were prepared with the
addition of the time gate. The time gate was formed on the
diagnostic element which is in contact with the reaction
mixture in the reaction chamber.
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The time gate was prepared by adding 1 l of 2% solids of
surfactant-free, sulfated latex, 1.0 m, (Interfacial Dynamics
Corp., Portland, OR)._ The other reagent latexes and gold
conjugate were also added to the devices and dried as described
in Exampie 7. Clear plastic sheets were placed on the devices
and various aiiquots of sample (about 60 41) containing 0, 50,
200 and 500 mIU hCG/ml, respectively, was added to the devices.
The sample moved into the reaction chamber, dissolved the
colloidal gold con;ugate and the reaction mixture remained in
the reaction chamber for about 8 to 10 min, whereas in devices
without time gates the reaction mixture remained in the
reaction chamber for 5 sec to 15 sec. The proteinaceous
components of the reaction mixture, which may be present in the
sample and which was added as a component of the reaction
mixture, namely, bovine serum albumin, bound to the latex
particles of the time gate and changed the hydrophobic surface
of the time gate into a hydrophilic surface. Other proteins,
such as aelatin, serum albumins, immunoglobulins, enzymes and
the like and polypeptides and hydrophilic polymers will aiso
function to bind to the hydrophobic zone.
The gradual transformation of the hydrophobic surface to a
hydrophilic surface, which resulted through binding -of the
proteinaceous components of the reaction mixture to the latex
particles allowed the reaction mixture to flow over the area of
the time gate.
In control experiments in which protein, namely bovine
serum albumin, was not added to the reaction mixture, flow of
the reaction mixture over the time gate and onto the diagnostic
element did not occur during the time (5 h) of the experiment.
This control experiment showed that the urine sample alone did
not contain sufficient protein or components which bind to the
applied latex of the time gate to allow a change in the
hydrophobic character of the time gate. In the event that the
components in the sample should only be used to cause the
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transformation of the hydrophobic time gate to a hydrophilic
one for the reaction mixture to flow, then one would be
required to lower the mass and total surface area of the latex
applied to the time gate to an extent which would allow flow of
the reaction mixture over the time gate in an appropriate
amount of time.
The reaction mixture then moved onto the diagnostic
element over the anti-hCG latex and goat anti-mouse IgG latex
capture zones. The reaction mixture moved into the used
reagent reservoir and the excess sample washed the diagnostic
eiement. The color density of the capture zones for hCG was
measured instrumentally using a MINOLTA CHROMA METER CR 241. A
red color was vi.sible, for samples containing hCG and not
visible for the sample without hCG at the capture zones for
hCG. The AE* values for the 0, 50, 200 and 500 mIU/ml were 0,
6.51, 13.14 and 18.19, respectively. A red color bar was
visible at the goat anti-mouse IgG capture zones of each
device.
Example 9
Qualitative or Quantitative One-Step Assay for hCG Using a Flow
Control Means
Devices as described in Example 4 were prepared with the
addition of the optional flow control means.
The optiohal flow control means or "gap" was placed behind
the capture zone for hCG gold conjugate on the diagnostic
element. The gap between the two surfaces was 0.38 mm, the
length of the gap was 13.2 mm and the width of the gap on the
top member was 9 mm; however, the effective width of the gap
was the width of the diagnostic element (2 mm). This gap
volume above tkie diagnostic element was about 10 l which was,
in this case, half the volume of the reaction chamber.
The anti-hCG and the goat anti-mouse latexes and gold
c,onjugate were added to the device and dried as described in
Example 7. Clear plastic sheets of polycarbonate having a gap
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in one surface were placed on the devices with the gap fac:.r.g
the diagnostic element. Sample (about 60 l) contain;ng 0 and
200 mIU hCG/ml was added to the devices. The sample moved into
the reaction chamber, dissolved the colloidal gold conjugate
and the reaction mixture then moved onto the diagnostic element
over the anti-hCG latex. The reaction mixture then entered the
gap which was immediately behind the capture zone o_' ant-,-hCG
iatex. The flow rate over the capture zone slowed while :he
react_on mixture moved over the capture zone and filled the
gap. The time for the 10 l reaction mixture to fill the gap
was aiDout 12 min to 16 min, whereas with devices without the
optional flow control means, the times were about 1 min to 3
min. for the reaction mixture to pass over the capture zone.
When the reaction mixture filled the gap, the reaction mixture
then moved into the narrow capillary of the diagnostic element
and cver the goat anti-mouse capture zone. The reaction
mixture moved into the used reagent reservoir and the excess
sample washed the diagnostic element.
The color density of the capture zones for hCG was
measured instrumentally using a MINOLTA CHROMA METER CR 241. A
red color was visible for samples containing hCG and not
visible for the sample without hCG at the capture zones for
hCG. The DE' values for the 0 and 200 mIU/ml were 0 and 16.12.
The SE* value of the hCG capture zone for the device without the
flow control means for the 200 mIU/mi sample was 16.32. A red
color bar was visible at the goat anti-mouse IgG capture zones
of each device.
Example 10
Preoaration of the Diagnostic Element for Multi-step Assays
A device was built comprising a sample addition reservoir
and a diagnostic element. The devices were plasma treated to
graft functional groups which create a hydrophilic surface.
The sample addition reservoir had dimensions of 12 mm long, 6
mm wide and 0.05 mm deep. The diagnostic element was about 5.5
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cm long, 1.3 mm wide and 1 mm from the base of the device and
included grooves running perpendicular to the flow of reaction
mixture at a density of 100 grocves per cm and a depth of 0.05
mm. In the case of qualitative assays, the antibody latex (1
5 l) was applied to the diagnostic element, covering the entire
width and 1 cm length of the diagnostic element. In the case
of an immunochromatographic assay, the antibody latex (6 l)
was applied to the entire width and length of the diagnostic
element. The devices were placed under vacuum f.or a=bout 1 h to
~0 dry the reagents. The capillary spaces in the device were then
formed by placing a clear plastic polystyrene sheet on top of
the device. The plastic sheet was held to the opposing surface
with binder clips.
Example 11
15 Assay for hCG Using the Diagnostic Element
The diagnostic element described in Example 10 was used
fo-- the assay of hCG. Urine samples (20 41) containing 0, 50,
200 and 500 mIU/ml hCG were added to tubes containing anti-f3hCG
antibody colloidal gold conjugate (2 l). The tubes were
20 vortexed and the reaction mixtures were incubated for 5 min at
room temperature. The reaction mixtures (20 l) were applied
in 10 l aliquots to the sample addition reservoir of the
device. The reaction mixture flowed onto the diagnostic
element from the sample reservoir and over the capture zone.
25 An absorbent at the end of the capture zone removed the used
reagent from the diagnostic element. The color density of the
capture zones for hCG was measured instrumentally using a
MINOLTA CHROMA METER CR 241. A red color was visible for
samples containing hCG and not visible for the sample without
30 hCG at the capture zones for hCG. The DE* values for the 0, 50,
250 and 500 mIU/ml were 0.00, 1.24, 3.16 and 5.56,
respectively.
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0'1
Examole i2
S=/nthesis cf meta-Nitrophencvclidine
o an ice cooled solution of ohencyclidine hydrochloride
(5g, 1.8 X 10"2 mcl) ir. concentrated sulfuric acid (9ml) was
~ addea-4 dropwise, and with stirring, fuming nitric acid (2mi).
':'he reaccicr. mixture was stirred In an ice-water bat:n for 1
hour and then poured onto crushed ice/water. The mixture was
made basic wit!-i lON sodium hydroxide (50 ml) to pH 12 and
extracted with diethyl ether (2 X 100 ml). The combined
organic layers were washed with water (2 X 100 ml) , dried over
anhyarous magnesium sulfate, filtered and evaporated under
vacuum. The residue was treated with methy'- alcohol (20 ml)
and heated on a hoi: water bath (80 C) until solute dissolved.
The flask was covered with aluminum foil (product is light
13 sensitive) and the solution was allowed to stir at room
temperature overnight when a yellow solid precipitated. The
solid was collected by fi'_tration and dried under vacuum to
afford 3.0 g(58 s) of m-nitrophencyclidine as fine yellow
crystals waich were protected from light: mp 81-82 C.
Examole 13
Syr.,:hesis of ineta-Aminophencyclidine
To a stirring solution of m-nitrophencyclidine (3.0 g,
10.4 X 10-3 mol) in methyl alcohol (150 ml) was added, under a
flow of argori, 10% pallddium-carbon (0.5 g) followed by
ammonium formate ( 4. 0 g, 6.3 X 10-2 mol ). The reaction mixture
was stirred at room temperature for 2 hours after which time
the catalyst was removed by filtration and the solvent was
evaporated under vacuum. The residue was treated with 1 N
potassium hydroxide solution (30 ml) and extracted with diethyl
ether (2 X 50 ml) . The combined organic extracts were washed
with water (50 ml), dried over anhydrous magnesium sulfate,
filtered and evaporated under vacuum. The residue was
dissolved in hexane (20 ml) and the solution was stirred at
room temperature overnight when a white solid precipitated.
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The solid was collected by fi'_trazion and dried under vacuum to
afford 1.4 g(52a) of m-aminophencyclidine: mp 121-122 C.
Example 14
Synthesis of Acetylthiopropionic Acid
To a stirred solution of 3-mercaptoproprionic acid (7 mi,
0.08 moles) and imidazole (5.4 g, 0.08 moles) in
tetrahydrofuran (THF, 700 ml) was added dropwise over 15
minutes, under argon, a solution of 1-acetyl imidazole (9.6 g,
0.087 moles) in THF (100 ml). The solution was allowed to stir
a further 3 hours at room temperature after which time the THF
was removed in vacuo. The residue was treated with ice-cold
water (18 ml) and the resulting solution acidified with ice-
cold concentrated HC1 (14.5 ml) to pH 1.5-2. The mixture was
extracted with water (2 X 50 ml), dried over magnesium sulfate
and evaporated. The residual crude yellow oily solid product
(10.5 g) was recrystallized from chloroform-hexane to afford
4.8 g (41% yield) acetylthiopropionic acid as a white solid
with a melting point of 44-45 C.
Examole 15
Synthesis of ineta-Acetylthiopropionamide Phencyclidine
To a stirring solution of m-aminophencyclidine (1.4 g, 5.4
X 10-3 mol)'and acetylthiopropionic acid (0.87 g, 5.8 X 10-3 mol)
in anhydrous tetrahydrofuran (7 ml) was added
dicyclohexylcarbodiimide (1.19 g, 5.8 X 10"3 mol) . The flask
was purged with argon and the solution stirred at room
temperature for- 2 hours. The mixture was filtered from
insoluble dicyclohexylurea and evaporated under vacuum. The
residual solid was recrystallized from chloroform/hexane to
afford 1.5 g (71%) of m-acetylthiopropionamide phencyclidine as
a white crystalline solid: mp 152-4 C.
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ExamDle 16
Synthesis of meta-3-Mercaptoproprionamide Phencyclidine
meta-Acetylthiopropionamide phencyclidine (0.01 g, 2.57 X
10-' mol) was dissolved in 1.29 ml 0.12 M potassium carbonate in
80% methanol/20% water (v/v). The solution sat at room
temperature for 5 min and then 0.2 ml 0.5 M potassium
phosphate, pH 7, was immediately added and the solution was
adjusted to pH 7-7.5 with hydrochloric acid (1 N) . The title
compound in solution was used as is to react with BSA-SMCC.
Examole 17
preparation of Phencyclidine Analogue Attached to Bovine Serum
Albumin (BSA-?CP)
3ovine serum albumin (BSA, 3.5 ml of 20 mg/ml) was reacted
with succinimidyl 4-(Iv-maleimidomethyl)-cyclohexane-l-
carboxylate (SMCC, Pierce Chemical Co.) by adding a solution of
6.7 mg SMCC in 0.3 ml acetoni*_rile and stirring the solution at
room temperature for 1 h while maintaining the pH between 7 and
7.5 with 1 N potassium hvdroxide. The protein was separated
from unreacted compounds by gel filtration chromatography in
0.1 M potassium phosphate, 0.02 M potassium borate, 0.15 M
sodium chloride, pH 7Ø The meta-3-mercaptoproprionamide
phencyclidine (0.2 ml of 13 mM) was added to the BSA-maleimide
(2 ml at 8.2 mg/ml) and the solution was stirred at room
temperature for 4 h. The solution was then dialyzed 3 times
against 1000 ml of 10 mM MES, pH 5.5. Recover 1.8 ml BSA-PCP
at 8 mg/ml.
Example 18
Precaration of Phencyclidine Analogue Colloidal Gold Conjugate
A solution (4.7 ml) containing BSA (22 mg) and BSA-PCP
(5.6 mg) in 10 mM MES, pH 5.5 was added in a bolus to colloidal
gold (105 ml) in 10 mM MES, pH 5.5 with rapid stirring. After
complete mixing the stirring was stopped and the solution was
incubated at room temperature for 1 h. The colloidal gold
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conjugate was subjected to diafiltration against 50 mM
potassium phosphate, 10 mM potassium borate, pH 7, using a
tangential flow device (Sartorius Easy Flow, mclecular weight
cutoff was 100,000) to remove BSA and BSA-PCP which was not
bound to colloidal gold. The gold conjugate was diluted for
the assay of PCP into a buffered solution containing 10 mg/ml
bovine serum albumin at pH 7.5.
Example 19
Preparation of anti-Phencyclidine Antibody Latex
=0 Surfactant-free polystyrene particles (Interfacial
Dynamics Corp., Portland, OR; 0.074 ml of 9.4% solids, 0.4 m)
was added while vortexing to anti-phencyclidine monoclonal
antibody (0.926 ml of 5.86 mg/ml in 0.1 M MES, pH 5) and the
suspension was incubated at 45 C for 2 h. The suspension was
subjected to centrifugation to pellet the latex particles. The
pellet was washed three times by centrifugation and
resuspension of the pellet with 10 mM MES, 0.1 mg/ml trehalose,
pH 5.5. The final pellet was resuspended in the wash buffer at
a solids concentration of 1%.
Example 20
Preparation of Latex-Immobilized Affinity-Purified Goat IgG
Antibody Against the Fc Fragment of Mouse IgG (Goat anti-mouse
Fc latex)
Affinity-purified goat anti-mouse (Fc (Immunosearch) and
polystyrene' latex particles (sulfated, 1.07 m) (Interfacial
Dynamics) were incubated separately at 45 C for one hour, the
antibody solution being buffered with 0.1 M 2-(N-morpholino)
ethane sulfonic acid at pH 5.5. While vortexing the antibody
solution, the suspension of latex particles was added to the
antibody solution such that the final concentration of antibody
was 0.3 mg/ml and the solution contained 1% latex solids. The
suspension was incubated for 2 hours at 45 C prior to
centrifugation of the suspension to pell.et the.latex particles.
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The latex pellet was resuspended in 1% bovine serum albumin in
phosphate-buffered-saline (PBS) and incubated for one hour at
room temperature. Following centrifugation to pellet the
latex, the pellet was washed three times by resuspension in PBS
5 and centrifugation. The final pellet was resuspended in PBS
containing 0.1% sodium azide at pH 7.0 at a latex concentration
of 1% solids.
Example 21
Assay for Phencyclidine Using the DiaQnostic Element
10 The diaanostic element described in Example 10 was used
for the assay of phencyclidine (PCP). Urine samples (133 l)
containing 0, 100, 200 and 300 ng/ml PCP were added to tubes
conta'-r.ing a lyoph'_lized buffer formulation (containing 10 mM
potassium phosphate, 150 mM sodium chloride and 10 mg/ml BSA,
15 pH 8) and phencyclidine analogue colloidal gold conjugate (4
41) was added and the solution was vortexed. Anti-PCP antibody
(2.8 i of 0.1 mg/mi) was added to each tube and the solutions
were vortexed and incubated at room temperature for 5 min.
Goat anti-mouse Fc latex (50 ml of a 1% suspension) was added
20 to the tubes, the tubes were vortexed and incubated at room
temperature for 10 min. The solutions were then filtered to
remove the complex of the PCP analogue gold conjugate:anti-PCP
antibody:goat anti-mouse latex from the reaction mixture using
a GELMAN ACRODISC 3 syringe filter (0.45 m). The filtrates
25 of the reaction mixtures (20 l) were applied to the diagnostic
elements described in example 10. The reaction mixture flowed
onto the diagnostic element from the sample reservoir and over
the capture zone. An absorbent tissue placed 1 cm after the
capture zone removed the used reagent from the diagnostic
30 element. The color density of the capture zones was measured
instrumentally using a MINOLTA CHROMA METER CR 241. The AE*
values for the 0, 100, 200 and 300 ng/mi samples were 0.69,
9.28, 14.04 and 21.6, respectively.
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Example 22
Exemplary Device Configuration
A presently preferred mode of the invention utilizes a
device embodiment capable of performing one-step immunoassays.
The device preferably comprises a sample addition reservoir 1,
a sample reaction barrier 3, a reaction chamber 4, a time gate
5, a diagnostic lane 6, a used reagent reservoir 7, and a lid
64. Figure 11 depicts a preferred embodiment of.the device
where the lid is removed to permit illustration of various
portions of the device. A..lid_is__not illustraed in Fig. 11, as
appreciated by one of ordinary skill in the art, the lid has an
access port so that fluid can be introduced into the sample
addition reservoir; the lid can also have a vent to facilitate
escape of gas as the device fills with liquid. In one
embodiment, a vent is located in the lid at an area of the used
reagent reservoir.
The sample addition reservoir can comprise a filter (not
illustrated) for the separation of plasma from red blood cells
or for the separation of debris in the sample from the sample
to be assayed and a reservoir for the storage of sample used in
the assay device. For additional_ disclosure conc.exning
filters, see e.g., copending U.S. Patent No. 6,391,265.
The sample addition reservoir is in fluid communication
with the sample reaction barrier.
The sample reaction barrier can comprise a texture
composed of texture structures on a surface thereof. Preferred
texture height is about 0.01 to 0.02 mm and width of each
texture structure is about 0.09 to 0.20 mm. The distance
between adjacent texture structures is about 0.080 to 0.100 mm.
The height of the capillary space in the sample reaction
barrier is about 0.02 to 0.08 mm. Preferably, the surface of
the sample reaction barrier at both edges of the capillary.is
made hydrophobic to prevent fluid from preferentially flowing
at the edges of the capillary. Since the hydrophobic surfaces
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minimize the flow along the edges of the reaction chamber,
these surfaces also direct fluid flow into , -the r=eaction
chamber, the access to wL:ich occurs toward the -center of the
sample reaction barrier. The sample .reaction barrier
preferably comprises ten vertical grooves 116 that are in fluid
communication with the capillary spaces of the sample reaction
barrier and the reaction chamber. The grooves are
approximately 0.02 to 0.03 mm high and are spaced about 0.5 to
1.5 mm apart.
The reaction chamber is comprised of a capillary about
0.03 to 1.0 mm high and contains a volume of about 0.2 to 6 i.
Preferably, both inner lid and base surfaces of the reaction
chamber capillary space comprise a texture of small texture
structures, about 0.015 to 0.03 mm high, with a diameter of
0.05 to 0.1 mm, at a spacing of about 0.1 to 0.3 mm. The
reaction chamber is in fluid communication with the time gate.
One surface of the reaction chamber adjacent to the time gate
comprises grooves to define a flow front perpendicular to the
direction of fluid flow. The grooves are oriented
substantially perpendicular to the direction of fluid flow.
The grooves are generally 0.03 to Ø07 mm trigh and are spaced
0.08 to 0.12 mm apart. The surface at an edge of the reaction
chamber, such as at a corner, is made hydrophobic. As
disclosed herein, a hydrophobic region slows flow at the edges
of the capillary and prevents fluid from preferentially flowing
at the edges.
The time gate is comprised of a capillary about 0.02 to
0.12 mm high. One surface of the time gate is comprised of
grooves about 0.03 to 0.07 mm high and spaced about 0.08 to
0.12 mm apart; these grooves are contiguous with similar
grooves in the. reaction chamber. The grooves are =oriented
substantially perpendicular to the predominant direction of
fluid flow through the device. As disclosed herein, a surface
of the time gate is made hydrophobic to delay fluid flow out of
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the reaction chamber. The time gate is in fluid communication
with the diagnostic lane.
The d:agnostic lane/element comprises a capillary
oreferably about 0.01 to 0.05 mm high and comprises a texture
composed of texture structures about 0.01 to 0.02 mm high, 0.03
to 0.07 mm in diameter/width, and spaced about 0.04 to 0.09 mm
apart. The volume of the diagnostic lane is about 0.5 to 3 l.
The edges of the capillary in the diagnostic lane are made
hydrophobic to slow fluid flow at the edges of the capillary,
and to prevent fluid from preferentially flowing at the edges
of the capillary. The diagnostic lane is in fluid
communication with the used reagent reservoir. As shown in
Figure 15, diagnostic lane 6 preferably begins at a point 70.
When the diagnostic lane begins at a point, this allows fluid
to enter the lane at a more predictable location, generally the
proximal-most location of the diagnostic lane. When fluid
enters the lane in a predictable location, this in turn leads
to increased predictability of fluid flow in the diagnostic
lane itself which allows for uniformity of performance for
2~ devices of the same configuration.
The used reagent reservoir preferably comprises a
capillary space similar in dimension to the capillary of the
diagnostic lane, and is generally of equal or greater volume.
It is comprised of a texture comprising texture structurres that
have a height of about 0.01 to 0.02 mm, widths/diameters of
about 0.03 to 0.07 mm which are spaced 0.04 to 0.09 mm apart.
The used reagent reservoir can comprise zones that exhibit a
color change upon addition of fluid, to visibly indicate to the
user that fluid has flowed past that particular zone. For
example, the reservoir can contain colored zones that be.come
colorless when fluid comes into contact with them. These
colored zones can be made of water soluble dyes, such as green
food coloring, that wash away through dissolution by the
advancing fluid. Alternatively, the region can contain a zone
that develops a color change when fluid has flowed in the
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=egion. These zones can consist of receptors that bind a dye
label in the sample or bind an enzyme that generates color at
the zone. These novel color change features have application
in indicating to the user of the test device the extent of
completion of the procedure.
Referring now to Figure 12, a device oreferably comprises,
generally at the outer edge and in areas where capillary spaces
of particular dimension are important, structures referred to
as stops 60. The stops serve to establish a capillary space of
1.0 uniform height, e.g., among various devices manufactured in the
same way. A device also preferably comprises energy directors
n2. The energy directors also serve to eStablish a capillary
space of uniform height, e.g., among various devices
manufactured in the same way, where an energy source such as
ultrasonic welding is used to join two parts by melting them
together. The energy directors also function to join two
pieces of a device, such as joining a lid and a base. As
depicted in Figure 12, an energy director has a height greater
zhan that of a stop. When stops and energy directors are used
together, the portion of the energy director that is higher
than a stop is induced to melt by an externally applied energy
source. As such melting occurs, the two parts being joined
come closer together. The closeness of the approximation of
the two parts is limited by the stops which, when preferably
are used with-energy directors, do not melt and thus serve to
define a uniform separation of two joined parts. The uniform
separation is a capillary space in preferred embodiments of a
device in accordance with the invention.
Accordingly, the stops and energy directors are designed
to'define a capillary space and to maintain a stable union of a
lid 64 to the base. Thus, adjacent to the sample addition
reservoir, the sample reaction barrier, the reaction chamber,
the time gate and the diagnostic lane are energy directors that
adjoin the lid to the base, to complete the formation of
capillary spaces within the device, and to seal fluid in the
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capillary spaces. Typically, the lid is ultrasonically welded
to the base. Adjacent to the energy directors are stops that
are about 0.02 to 0.06 ;nm high. The stops act to prevent the
iid from being attached to the base in a manner that prevents
5 formation of a capillary space; the stops thus serve to define
a reproducible capillary space between many devices. The stops
are bordered by energy directors so that fluid does not enter
the area of the stops.
Furthermore, as depicted in Figure 11, the device
10 preferably comprises one or more regions of dead space 66
adjacent sides of the diagnostic lane. The dead space(s) allow
for detection of a sensible signal, e.g., a color change in the
fluorescent or visible spectra, without interference from any
signal contained in reactants that are located in a used
15 reagent reservoir or other device region.
The novel use cf stops, as described herein, serve to
define capillaries or uniform height in devices. As
appreciated by one of ordinary skiil in the art, the stop
height can be varied to establish a variety of capillary spaces
20 in a device. Furthermore, as illustrated in Figure 12, various
stop 60 and energy director 62 embodiments can be prepared in
accordance with the present invention. In general, the
dimensions of a capillary space designed into a device is
determined based on the nature of the sample to be assayed.
25 For example, whole blood or lysed blood has a higher viscosity
than plasma or serum. Accordingly, devices were designed with
higher capillary gaps to assay whole or lysed blood, these
devices had higher gaps than devices for serum or plasma. When
whole, or lysed blood was used in assays in the devices with the
30 higher capillary gaps, these devices achieved similar assay
times and assay characteristics relative to devices configured
for use with plasma or serum samples. The devices requiring
higher capillary gaps have had correspondingly higher stops.
The stops can be formulated using shims, layers of glue or
35 hardening agents, or they can be molded directly into the part,
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=ssing injection molding or other conventional molding or
fabrication processes. In the case of using silicon chips,
stoos can be incoroorated into devices utilizing
photolizhography or micromachining techniques.
~ Figsre 13 depicts a electron micrograph of a~ embodiment
of the invention illustrating a sample addition reservoir 1, a
textured sample reaction barrier 3, a textured reaction chamber
4, a textured used reagent reservoir 7, a stop 60, and energy
direc=ors 62. In this embodiment energy directors 62 and stop
60 collectively constitute a dead space. Figure 14 is an
enlarged view of a portion of Fig. 13, illustrating textured
sample reactior, barrier 3, textured reaction chamber 4, an
energy director 62, and stop 60. Fig. 15 depicts an electron
micrograph of an embodiment of the invention illustrating a
time gate 5, a textured diagnostic lane 6, and an energy
director 62. Figure lo' A-B depict two views of a textured
surface in a capiLlary space adjacent an energy director 62.
ln a further preferred aspect of the invention, depicted
in Figure 11, the device is fabricated to have positioners 68,
the lid (not shown) is fabricated to have aositioning elements
that mate with the asymmetrically placed positioners 68, to
ensure that the lid is placed on the device with the correct
orientation, so that the surface facing the facing into the
device has the appropriate texture. In addition, the lid has a
hole at the region of sample addition chamber 1, to permit
introduction of fluid into the device.
In a preferred embodiment of an immunoassay device in
accordance with the invention, immunoassay reagents are placed
on separate surfaces located in a given region of the device.
For example, an immunoassay reagent can be immobilized on the
lid in the area of the sample reservoir, sample reaction
barrier, reaction chamber or diagnostic lane; and a separate
immunoassay reagent can be immobilized on the base in an area
of the sample reservoir, sample reaction barrier, reaction
chamber or diagnostic lane. It is particularly advantageous to
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place one reagent on a lid and another reagent on a base of the
device when the lid and base initially constituted separate
pieces that are subsequently attached together in the
manufacture of the device. One or more of such immobilized
reagents can be diffusible when contacted by fluid.
In accordance with this embodiment of the invention,
reagents that could not otherwise be packaged together in a
capillary space of a device without the occurrence of adverse
cross reactions, can be placed in a device in a single
capillary space. For example, if a labeled antibody and a
capture antibody were placed together in a capillary space,
non-specific interactions can occur in the absence of any
target material. Such non-specific interactions lesson the
sensitivity of an assay. Fundamental assay types that can
ut'-'_ize reagents localized on separate surfaces in a capillary
space include, but are not limited to, competitive
imrnunoassays, sandwich imrnunoassays and nucleic acid probe
assays. Thus, in embodiments where a reagent is dried on a
device surface and another reagent is dried on a separate
surface of the device, these reagents can diffuse from their
respective surfaces upon introduction of= fluid to those
surfaces. The surfaces having reagent immobilized thereon can
be surfaces in a particular chamber of the device or can be
surfaces in different regions of the device. The regions can
be separate chambers or can be device surfaces that do not
delimit a chamber.
Additionally, immunoassay reagents can be immobilized on
particles or nanoparticles (collectively referred to herein as
particles). Such particles can be placed on a surface, such as
a surface delimiting a capillary space, in a device in
accordance with the invention. By use of such particles
comprising reagents immobilized thereon, one can provide a
zone, comprising particles and a surface, where the zone
comprises reagents that could not otherwise be provided
together. For example, particles comprising immdbilized
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reagents can be placed on a surface where the surface itself
comprises a- reagent; when the surface is a surface cf a
capillary space, one or more capillary space surfaces can have
a reagent immobilized thereon. Different reagents can be
~ placed on different surfaces. A reagent immobilized by the
particles (or on a surface) can be diffusible or nor.-diffusible
when placed in contact with a liquid.
Accordingly, use of a device with the preferred
configuration has allowed the performance of one-step
immunoassaysthat simultaneously measure multiple analytes from
a'biological fluid in an assay time of about 10 minutes.
Ciosing
Although the foregoir.g invention has been described in
some detail by way of illustration and example, it will be
obvious that certain changes or modifications may be practiced
within the scope of the appended claims.
It must be noted that as used herein and in the appended
claims, the singular forms "a," "and," and "the" include plural
referents unless the context clearly dictates otherwise. Thus,
for example, reference to "a iormulation" includes mixtures of
different formulations and reference to "the method of
treatment" includes reference to equivalent steps and methods
known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific
terms used herein have the same meaning as commonly "understood
by one of ordinary skill in the art to which this invention
belongs. Although any methods and materials similar to
equivalent to those described herein can be used in the
practice or testing of the invention, the preferred methods and
materials are now described.
