Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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RETAINING MICROFLUIDIC MICROCAVITY AND OTHER MICROFLUIDIC
STRUCTURES
TECHNICAL FIELD
The present invention concerns a microfluidic device in which there is a
microchannel
structure which comprises (a) one or more inlet ports, (b) one or more outlet
ports, and (c)
a structural unit which comprises a fluidic function and is located between
one of the inlet
ports and one of the outlet ports. The structural unit (c) may include an
inlet or an outlet
port.
According to the invention the structural unit is selected by certain
innovative structures
permitting a) retaining nl-aliquots of liquids in which the constituents have
been defined
by mixing of aliquots within the microfluidic device (unit A), b) mixing of
aliquots of
liquids (unit B), c) partition of larger aliquots of liquids into smaller
aliquots of liquids
and distributing the latter individually and in parallel to different
microchannel structure
of the same microfluidic device (unit C), d) quick penetration into a
microchannel
structure of an aliquot of a liquid dispensed to an inlet port of a
microchannel structures
(unit D), and e) volume definition integrated within a microchannel structure
(unit E).
There may in addition also be other structural units and/or microfluidic
functionalities
included.
The microchannel structures are intended for transport and processing of one
or more
aliquots of liquids (thus the device is named microfluidic). In preferred
variants capillary
force and centrifugal force are used for the transport of the aliquots.
The term "aliquot" will refer to an aliquot of a liquid if not otherwise
specified.
The invention also concerns various methods in which the microfluidic
device/microchannel structures is/are used.
Patent applications and issued patents that are referenced are incorporated by
reference.
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Z
DRAwINGs
The structural units are viewed from above. The cross-sectional areas of the
microconduits and microcavities are typically rectangular. The depths of the
microchannel
structures shown are typically constant and within the interval 100-150 Vim.
The widths
for liquid transport microconduits are typically within the interval 100-300
pin and for air
microconduits within the interval of 40-100 Vim. Also compare the figures in
PCT/SE02/531, PCT/SE02/537, PCT/SE02/00538 and PCT/SE02/539 (all of Gyros AB),
which disclose other structures of comparable dimensions. Figures 2d-a
specifically
include certain dimensions in pin (italics). Circles represent openings to
ambient
atmospheres (inlet port, outlet ports, vents etc).
Figure 1 illustrates the definitions of "edge" and "circumferential zone".
Figures 2a-a illustrate innovative variants of units A and B.
Figures 3a-c illustrate innovative variants of unit C.
1 S Figures 4a-b illustrate innovative variants of unit D.
Figure 5 illustrates an innovative variant of unit E.
BACKGROUND TECHNOLOGY
Microfluidic structures have been considered promising for assays, chemical
synthesis etc
which are to be performed with a high degree of parallelity. A generally
expressed desire
has been to run the complete sequence of steps of test protocols, including
sample
treatment within microfluidic devices. This has lead to a desire to dense-pack
microchannel structures on planar substrates (chips) and to integrate valve
functions,
separation functions, means for moving liquids etc within microfluidic
devices. In the
macroscopic world these kinds of functionalities can easily be integrated into
various
kinds of liquid transportation systems, but in the microscopic world it has
become
expensive, unreliable etc to miniaturize the macroscopic designs. The
situation becomes
still worse when moving from p1- to nl-aliquots or from microchannel
dimensions of
above 100 pin down to those less than 100 pin. One of the main reasons for
this is that the
surface forces of liquids are more influential on liquid behavior when going
down in
volume from the pl-volumes to the n1 volumes and smaller, for instance when
going
below S p1. A typically example is that wicking/imbibing will promote quick
liquid
RECTIFIED SHEET (RULE 91)
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transport from a nl-vessel making it difficult to retain a specified liquid
volume in such a
vessel. See below.
Background publications: Centrifugal force for moving liquids in micofluidic
devices
S The use of centrifugal force for moving liquids within microfluidic systems
has been
described for instance by Abaxis Inc (WO 9533986, WO 9506870, US 5,472,603);
Molecular devices (US 5,160,702); Gamera Biosciences/Tecan (WO 9721090, WO
9807019, WO 9853311), WO 01877486, WO 0187487; Gyros AB/Amersham Pharmacia
Biotech (WO 9955827, WO 9958245, WO 0025921, WO 0040750, WO 0056808, WO
0062042, WO 0102737, WO 0146465, WO 0147637, WO 0147638, WO 0154810, WO
0241997, WO 0241998, PCT/SE02/00531, PCT/SE02/00537, PCT/SE02/00538,
PCT/SE02/00539).
See also presentations made by Gyros AB at various scientific meetings:
(1) High-through put screening SNP scoring in microfabricated device. Nigel
Tooke
(September 99);
(2) Microfluidics in a rotating CD (Ekstrand et al) MicroTAS 2000, Enschede,
The
Netherlands, May 14-18, 2000.
(3) (a) SNP scoring in a disposable microfabricated CD device (Eckersten et
al) and
(b) SNP scoring in a disposable microfabricated CD device combined with solid
phase
PyrosequencingTM (Tooke et al) Human Genome Meeting, HGM 2000, Vancouver,
Canada, April 9-12, 2000,
(4) Integrated sample preparation and MALDI MS on a microfluidic compact disc
(CD
with improved sensitivity (Magnus Gustavsson et al) ASMS 2001 (spring 2001 ).
Documentation on Gyros' presentations can be found on www. lyros.com.
Background publications: Unit A (retaining microcavity for nl-aliquots).
The proprietor of the present invention has during the last year developed
microfluidic
systems comprising structural units comprising microcavities intended for nl-
volumes of
liquids. See for instance WO 9955827, WO 9958245; WO 0040750, WO 0146465, WO
0147638, WO 0241997, and WO 0241997 and scientific presentations made by Gyros
AB
(see above). Hydrophobic surface breaks for preventing undesired creeping of
liquid
around corners or as valves have in particular been emphasized in WO 9958245.
See also
PCT/SE02/00531, PCT/SE02/00537, PCT/SE02/00538 and PCT/SE02/00539.
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Background publications: Unit B (mixing unit)
Units for mixing aliquots within microfluidic devices have previously been
described.
These units have been based on
S (a) mechanical mixers in mixing microcavities or microconduits including
creation of
turbulence by fixed streric hinders (e.g. WO 9721090 and US 4279862
(Bretaudiere et
al)),
(b) creation of turbulent flow in a microcavity by two incoming liquid flows
(e.g. WO
9853311),
(c) creation of a laminar flow in the inlet end of a mixing microconduit and
achieving
mixing by diffusion during the transport through the microconduit (e.g. US
5,637,469,
(Wilding & Kricka).
(d) mixing by pumping layered aliquots back and forth in a mixing microcavity
or
microconduit. This can be accomplished by applying pulsed centrifugal force by
spin
1 S pulses that drive the liquid in one direction and a higher spin pulse and
in the reverse
direction at a lower spin pulse utilizing energy built up in the system during
a high
pulse for driving the liquid in the reverse direction during a lower spin
pulses. This
can be accomplished by utilizing enclosed air ballast chambers and/or
hydrophobic/hydrophilic as outlined in WO 0187487. The principle of back and
forth
transport is also described in PCT/SE02/00531 (unit S) and WO 9958245.
Background publications: Unit C (distribution manifold).
According to the inventors knowledge publications related to this topic are
rare. US
6,117,396 (Orchid) gives a non-centrifugal gravity based microfluidic device
in which a
common reagent channel is used both as an overflow channel and as a reagent
fill
channel. A plurality of parallel volume metering capillaries is connected at
different
positions to the reagent fill channel from below. A centrifugally based
distribution
manifold for microfluidic systems has been given in WO 9958245 and WO 0187486.
This
latter variant is based on an annular distribution microconduit and comprises
at least one
waste/overflow microconduit per aliquot to be dispensed.
Microfluidic devices with a microchannel structures that comprises a part that
bents
towards a lower level (downward bent) and/or a part that bents towards a
higher level
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(upward bent) have been described previously. Downward and upward bents have
been
linked to each other in short series. Bent structures for centrifugal based
system have been
used for metering liquids, process chambers etc.
5 Downward bents have been combined with centrifugal force and used for
retaining liquid
(valve function) that is to be subjected to distinct process steps in the
bent, e.g. chemical
or biochemical reactions, affinity reactions, measurement operations, volume
metering
etc. By including an outlet microconduit with a valve function, for instance a
passive
valve, in the lower part of the bent, processed aliquots can been transported
further
downstream in the structure in a controlled manner.
Further details about previously known bent structure are given in: WO
9958245; WO
0147638; WO 0146465; WO 0040750; PCT/SE02/00531, PCTlSE02/00537,
PCT/SE02/00538 and PCT/SE02/00539; WO 0241997 and WO 0241998. Bent structures
have also been indicated in scientific presentations made by Gyros AB and
given
elsewhere in this specification.
Background publications: Unit D (inlet port).
Imbibing has been utilized to promote liquid penetration into microchannel
structures by
including edge/corner structures associated with inlet ports. See US 4,233,029
(Eastman
Kodak) and US 4,254,083 (Eastman Kodak).
Background publications: Unit E (integrated volume-defining unit).
Integrated volume defining units in microfluidic systems are previously known.
US
6,117,396 (Orchid), for instance, gives a non-centrifugal gravity based system
in which a
common reagent channel may act as an overflow/filling channel along which
there is
spaced a plurality of volume metering capillaries for pl-volumes. Integrated
units for
metering volumes in centrifugal based system by the use of an overflow channel
have
been described in WO 9853311, WO 0146465 and WO 0040750.
OBJECTS
Major object: The present invention provides novel fluidic functionalities
that can be
used when transporting and processing nl-volumes of liquids in microchannel
systems of
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the kind defined under the heading "Technical Field". A particular intention
is to create
functionalities that do not require movable mechanical parts, e.g. to
accomplish valuing,
pumping; mixing etc, and can be integrated into the microchannels and/or the
substrates.
The various novel functionalities are based on local surface characteristics
of the inner
walls of the microchannels and/or on properties of the liquids, such as
surface tension and
wetting ability.
Other objects
~ A first object is to provide a structural unit with a functionality that
permits retaining
of a defined nl-aliquot for prolonged period of times in a predetermined
microcavity
(retaining microcavity) of a microchannel structure of a microfluidic device.
The nl-
aliquots concerned are obtained by mixing nl-aliquots within the microchannel
structure. The term "prolonged" typically means that the liquid is retained in
the
retaining microcavity under static conditions, i.e. non-flow conditions. The
period of
time concerned is typically >_ 15 seconds, such as 30 seconds or >_1 minute,
such as >_
5 minutes or >_ 10 minutes, for instance >_ 1 hours or >_ 10 hours. The
contemplated
periods last typically <_ 24 hours such as <_ 12 hours. This object primarily
aims at
minimizing liquid losses due to wicking and/or evaporation from retaining
microcavities with volumes in the nl-range during incubations for performing
reactions and measurements in the mixed nl-aliquot, for storage purposes etc.
Acceptable losses are typically <_ 20%, such as <_ 10% or <_ 5%.
~ A second object is to provide a structural unit with a functionality that
permits simple,
quick, safe, reproducible and reliable mixing of two aliquots that are
miscible with
each other within a microchannel structure of a microfluidic device.
~ A third object is to provide a structural unit with a functionality that
permits
simplified and reliable distribution in parallel to separate substructures of
a plurality of
microchannel structures in a microfluidic device.
~ A fourth object is to provide a structural unit with a functionality that
facilitates rapid
introduction of an aliquot into a microchannel structure of a microfluidic
device.
~ A fifth object is to provide a structural unit with a functionality that
enables
reproducibly metering of an aliquot within a microchannel structure before the
aliquot
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is transported further downstream in a microchannel structure of a
microfluidic
device.
Subobjects related to the above-mentioned objects correspond to methods and
uses of the
microfluidic devices/structural units for transporting and processing the
aliquots of
liquids. A particular subobject to the first object is a method for reducing
evaporation
caused by wicking from the type of nl-aliquot referred to.
THE INVENTION
We have now found that these objectives can be at least partially met in a
microfluidic
device of the type defined under the heading "Technical Field".
The invention is among others based on the recognition that the appropriate
surface
tension of a liquid is important for controlling a liquid flow in a
microsystem. This in
particular applies when dealing with aliquots in the nano-litre range and/or
if the control is
exerted without mechanical valves and pumps, i.e. by driving the transport of
aliquots
through a functional unit of the invention by capillary force and/or inertia
force etc.
Typical examples of inertia force are gravitational force and centrifugal
force. See also
under the heading "Means for driving the liquid flow"
2O SUMMARY OF THE FIRST MAIN ASPECT OF THE INVENTION
In the first main aspect, the invention relates to the microfluidic device
generally defined
under the heading "Technical Field". The main characteristic feature of this
aspect of the
invention is that at least one of the structural units is selected amongst the
innovative units
A-E described below. Units that combine the functionality and/or structure of
two or more
of the units A-E may be included. Other units that are known or will be known
in the
future may also be included as long as at least one of the innovative units A-
E is present.
For additional units see also PCT/SE02/00531.
In preferred variants of this aspect at least one of the aliquots referred to
in the description
of a structural unit should have a surface tension, which is >_ 5 mN/m, such
as >_ 10 mN/m
or >_ 20 mN/m.
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Summary of the second main aspect of the invention.
In the second main aspect, the invention is a method for transporting one, two
or more
aliquots through a microchannel structure of the microfluidic device, which is
generally
defined under the heading "Technical Field". The method comprises the steps of
(i) providing the microfluidic device,
(ii) providing said one, two or more aliquots,
(iii) introducing each of said aliquots through an inlet port of one, two or
more
microchannel structures of the device,
(iv) transporting the aliquots through at least one of the structural units
which is
present between an inlet port and an outlet port without utilizing valves and
pumps
containing movable mechanical parts, and
(v) possibly collecting the aliquots in treated form in one or more of the
outlet ports of
the microchannel structure.
One main characteristic feature of the second aspect is that one, two, three
or more of the
aliquots that are to be introduced through an inlet port of the microchannel
structures have
a surface tension which is > 5 mN/m, such as >_ 10 mN/m or >_ 20 mN/m.
The microfluidic device provided in step (i) is according to the first main
aspect.
In step (ii), at least one of the aliquots has a volume in the nano-litre
range.
In step (iii) two or more of the aliquots may be introduced via the same or
different inlet
ports.
In step (iv) the driving force utilized for transport of the aliquots
typically is capillary
force and/or inertia force without excluding other kinds of forces as
discussed elsewhere
in this specification.
Steps (iii) and (iv) include that the various aliquots are processed according
to an intended
protocol, i.e. the transport step (step (iv)) includes that an aliquot
introduced into a
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microchannel structure may be transported to a certain position (structural
unit) and/or
processed in a predetermined manner before the next aliquot is introduced. The
part
sequence that comprises steps (iii) and (iv) may thus be interrupted for
dispensation steps,
process steps etc to take place, be divided in substeps. For instance, The two
reactants
may be dispensed separately in sequence to the same or to different inlet
ports and then
mixed in a separate mixing unit as discussed elsewhere in this specification.
Subsequent
to the mixing the reaction mixture is transported to a reaction microcavity
and retained
therein while the reaction is allowed to proceed according to the desired
protocol, after
which the result of the reaction is analysed in the same microcavity or
further downstream
or outside the microchannel structure. The analysis may involve
determination/detection
of products and/or the disappearance of one or more of the reactants. By
properly
designing the system the proceeding of the reactions may be followed through
the wall of
the microcavity, i.e. there may also be substeps run in parallel (measuring
and
incubation).
In step (v) the term "treated form" contemplates that the aliquots have passed
the structure
and been subjected to one or more predetermined treatments. The chemical
composition
may have changed and/or aliquots may have been mixed.
At least one of the aliquots is typically aqueous and/or may contain one or
more surface-
active agents that increase or decrease the surface tension of a liquid, such
as water.
Typical agents that reduce surface tension are detergents that may be
cationic, anionic,
amphoteric or non-ionisable. Surface-active agents include organic solvents,
preferably
miscible with water. Examples are methanol, ethanol, isopropanol, formamide,
acetonitrile etc. Charged or chargeable polymers, biomolecules such as
proteins, certain
sugars etc may also act as surface-active agents.
MICROFLUIDIC DEVICES.
The microfluidic device of the present invention typically comprises one, two,
three, four
or more sets of microchannel structures in which aliquots are transported or
processed for
various purposes, for instance analytical or synthetic purposes. The prefix
"micro"
contemplates that an individual microchannel structure comprises one or more
cavities
and/or channels that have a depth and/or a width that is 5 103 pm, such as 5
102 pm. The
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lower limit for the width/breadth is typically significantly larger than the
size of the
largest reagents and constituents of aliquots that are to pass through a
microchannel. The
volumes of microcavities and thus also of aliquots to be transported and
processed are
typically <_ 1000 n1, such as <_ 500 n1 or _< 100 n1 or < 50 n1. The nl-range
comprises, if not
5 otherwise specified, volumes < 5000 n1, e.g. within the ranges specified in
the preceding
sentence. There may also be larger cavities, e.g. directly connected to inlet
ports, with a
volume within intervals, such as 1-10 p1, 1-100 p.1, and 1-1000 p.1 (pl-
range). These
cavities are typically used for the introduction of samples that are to be
concentrated
within a microchannel structure, or of washing liquids and the like.
The term "microconduit" means a part of a microchannel structure.
A microconduit may be intended for transport of liquids (liquid flow
microconduits) or
for transport into or out of the microchannel structure (air microconduits).
The dimensions
of the two types may be different, for instance an air microconduit may have a
smaller
cross-sectional area and/or a higher aspect ratio (depth:width) compared to a
liquid flow
microconduit or vice versa. The liquid flow microconduit thus may have an
aspect ratio <_
1 while an air microconduit may have an aspect ratio >_ 1 or vice versa, or
the aspect ratio
may be equal for the microconduit irrespective of their use. A liquid flow
channel
typically has hydrophilic inner surfaces as discussed elsewhere in this
specification while
an air channel typically has hydrophobic inner surfaces. Liquid flow
microconduits may
also be used for venting air into or out of a microchannel structure.
The terms "inlet port" and "outlet port" contemplate port for air and ports
for liquids.
A microchannel structure may comprise a number of functional units that are
necessary to
carry out a predetermined protocol within the structure. A microchannel
structure thus
may comprise one, two, three or more units selected amongst inlet ports,
outlet ports,
units for distributing samples, liquids and/or reagents to individual
microchannel
structures, microconduits for liquid transport, units for defining liquid
volumes, valuing
units, units venting to ambient atmosphere, units for mixing liquids, units
for performing
chemical reactions or bioreactions, units for separating soluble constituents
or particulate
materials from a liquid phase, waste liquid units including waste cavities and
overflow
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channels, detection units, units for collecting an aliquot processed in the
structure and to
be transferred to another device e.g. for analysis, branching units for
merging or dividing
a liquid flow, etc. In one and the same microchannel structure there may be
several inlet
ports and/or several outlet ports that are connected to a main flow path via
microconduits
at a different or at the same downstream position. These microconduits may
also contain
functional units of the type discussed above.
Typically a microfluidic device comprises in total >_ 50, such as >_ 100 or >_
200,
microchannel structures per microfluidic device. The microchannel structures
of a set are
essentially identical and may or may not extend in a common plane of a
substrate. There
may be channels providing liquid communication between individual microchannel
structures of a set and/or to one or more other sets that may be present in
the same device.
The microchannels are typically covered, i.e. surrounded by walls or other
means for
directing the flow and to lower evaporation. Openings such as in inlet ports,
outlet ports,
1 S vents etc are typically present where appropriate.
The cross-section of a microchannel may have rounded forms all around, i.e. be
circular,
ellipsoid etc. A microchannel may also have inner edges, i.e. have cross-
sections that are
triangular, squaric, rectangular, partly rounded, planar etc. Microcavities or
microchambers may have the same or a different cross-sectional geometry
compared to
surrounding microconduits.
If not otherwise indicated the term "edge" of a microconduit will refer to the
intersection
of two inner walls of the microconduit. This kind of edges is typically
extending more or
less in parallel along the flow-direction (length-going edges). See figure 1
that shows a
microchannel having a rectangular cross-section (101), four inner walls (102)
with four
wall intersections or edges (103). The arrow (105) gives the flow direction.
A circumferential zone of a microchannel is also illustrated in figure 1. It
is an inner
surface zone (104) in the inner wall of a microchannel and extends in a sleeve-
like
manner around the flow direction (105). The length of this kind of zone is at
least from
0.1-10, 0.1-100, 0.1-1000 or more times the breadth or depth of the
microchannel/microconduit at the upstream end of the zone.
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The microfluidic device may have an axis of symmetry that is n-numbered (Cn)
where n is
an integer between 2 and oo, preferably 6, 7, 8 and larger, for instance oo.
In preferred
cases the microfluidic device as such may have a cylindrical, spherical or
conical
symmetry (C~) and/or is disc-shaped. Axes of symmetry may be combined with
utilizing
centrifugal force created by spinning around the axis of symmetry for driving
a liquid
flow through a microchannel structure.
The microfluidic device is typically in the form of a disc with the
microchannel structures
extending in a plane parallel to the disc plane.
The devices can be manufactured as summarized in PCT/SE02/00531.
The devices are preferably of the same dimension as a conventional CD, but may
also be
smaller, for instance down to 10% of conventional CDs, or larger, for instance
up to more
than 200% or more than 400 % of a conventional CD. These percentage values
refer to
the radius.
In the preferred variants the microchannel structures comprises inner surfaces
that have
been hydrophilised, for instance as described in WO 0056808. If necessary the
inner
surfaces may be coated with a non-ionic hydrophilic polymer as described in WO
0056808 or and US 5,773,488 (Gyros AB), for instance. The preferred variants
are the
same as given in these publications, e.g. to a wettability allowing for
capillarity to draw a
liquid into a structural unit once having passed the inlet thereof. Where
appropriate
hydrophobic surface breaks are introduced as outlined in WO 9958245 and
PCT/SE02/00531. See also WO 0185602 (t~mic AB & Gyros AB)
The exact demand on liquid contact angles (hydrophilicity/hydrophobicity) of
inner
surfaces of the microchannel structure may vary between different functional
units.
Except for local hydrophobic surface breaks the liquid contact angel for at
Least two or
three inner walls of a microconduit at a particular location should be
wettable
(hydrophilic) for the liquid to be transported, with preference for liquid
contact angels that
are 5 60°, such as 5 50° or S. 40° or S 30° or S
20°. In the case one or more walls have
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higher liquid contact angles, for instance non-wettable (hydrophobic), this
can be
compensated by a lowered liquid contact angle on the remaining walls. This may
be
particular important if non-wettable lids are used to cover open hydrophilic
microchannel
structures. The values above apply for the liquid to be transported and to the
functional
units given above (except for local hydrophobic surface breaks) and at the
temperature of
use. Surfaces having water contact angles within the limits given above may
often be
used for other aqueous liquids.
The terms "wettable surface" and "hydrophilic surface" are mostly contemplated
a surface
that has a liquid contact angle of <_ 90° (in particular for water and
other aqueous media).
Surfaces that are "non-wettable" or "hydrophobic" thus typically have a liquid
contact
angle > 90°. The liquid contact angle in the normal case refers to
equilibrium contact
angles although it sometimes may refer to receding and/or advancing contact
angles
depending on the purpose of a measurement. In the context of the invention
equilibrium
contact angles are primarily contemplated.
Valve functions.
Three categories of valves that previously have been suggested for
microfluidic devices
are:
1. Mechanical valves.
2. Valves that comprise intersecting channels together with means that
determine
through which channel a liquid flow shall be created.
3. Inner valves, i.e. valves in which the passage or non-passage of a liquid
depends on
physical and/or chemical properties of the liquid and the material in the
surface of the
inner wall of a microconduit at the position of an inner valve.
Type 1 valves typically require physically closing of a microconduit are
therefore called
"closing valves". They often have movable mechanical parts for closing a
microconduit.
Type 2 valves function without closing and are therefore "non-closing". A
typical
example is directing an electrokinetic flow at the intersection of two
channels by
switching the electrodes. See for instance US 5,716,825 (Hewlett Packard) and
US
5,705,813 (Hewlett Packard).
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In type 3 valves, the non-passage or passage of a liquid may be based on:
(a) changing the cross-sectional area in a microconduit at the valve position
by changing
the energy input to the material of the wall in the microconduit (closing
valves),
S and/or
(b) locally changing the interaction energy between a through-flowing aliquot
and an
inner surface of a microconduit at the valve position (non-closing valves),
and/or
(c) a suitable curvature of the microconduit at the valve function (geometric
valves, non-
closing).
Type 3a valves are illustrated in WO 0102737 (Gyros AB) in which stimulus-
responsive
polymers (intelligent polymers) are suggested to create a valve function, and
in WO
9721090 in which relaxation of non-equilibrium polymeric structures and
meltable wax
plugs are suggested to create a valve function.
In type 3b valves, the microconduit at the position of the valve is open even
if the liquid is
stopped (inner valves including capillary valves, also called passive valves).
Through
flow in this kind of valves is accomplished simply by increasing the force
driving the
liquid. The use of hydrophobic surface breaks (changes in chemical surface
characteristics) as valves is described in for instance WO 9958245, WO
0146465, WO
0185602 (~mic AB & Gyros AB), WO 0187486 and PCT/SE02/00531. The use of
changes in geometric surface characteristics as valves is described in for
instance WO
9615576 (David Sarnoff Res. Inst.), EP 305210 (Biotrack), and WO 9807019. Type
3b
valves comprise an anti-wicking function if they utilize changes in chemical
and/or
geometrical surface characteristics in edges as described for anti-wicking
means.
Type 3c valves may be achieved by linking an upward bent of a microchannel
immediately downstream to a downward bent in centrifugal based systems. This
is
illustrated in WO 0146465 that suggests connecting an upward bent microconduit
downstream to a UlY-shaped microconduit, e.g.
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Anti-wicking means
Anti-wicking means are typically local surface modifications that counteract
wicking/
imbibing.
5 Imbibing (wicking) means that liquid transport is initiated in the edges of
micro channels.
See for instance Dong et al (J. Coll. Interface Science 172 (1995) 278-288)
and Kim et al
(J. Phys. Chem. B 101 (1997) 855-863). Imbibing renders it difficult to retain
a defined
volume of a liquid in a desired microcavity for a longer period of time in
case there is a
microconduit having a length-going edge directly connected to the microcavity.
This in
10 particular applies if the volume < S ~1, such as in the nl-range or less.
If the microconduit
is connected to ambient atmosphere, for instance via an inlet port, imbibing
will promote
evaporation and irreversible loss of a predispensed volume of a liquid.
Anti-wicking means typically comprises a change in surface characteristics,
such as in
15 geometric and/or chemical surface characteristics, in an inner edge of a
microconduit. The
edge typically starts in a microcavity and stretches into the microconduit.
Anti-wicking
means may be present upstream or downstream a microcavity intended to contain
a liquid.
An anti-wicking functionality may inherently also be present in inner valves
that are
based on the presence of a hydrophobic surface break in an inner edge.
The change in geometric surface characteristics is typical local and may be
selected from
indentations, protrusions (projections), and an increase in the angle between
the two inner
walls defining a length-going inner edge. In most cases the deformation will
also stretch
into and/or across a wall delineated by this kind of edge, for instance into
and/or across a
wall delineated by two edges comprising the deformation. For indentations and
protrusions this will mean valleys/grooves and ridges, respectively, across
the wall. An
increase in the angle between two intersecting walls means in its extreme that
the inner
edge can be rounded within a zone carrying the anti-wicking means but not
rounded
between this zone and the microcavity. The microconduit thus may locally be
cylindrical.
Also other physical deformations of the edges may result in anti-wicking.
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Deformations in the form of indentations, for instance, may be "ear-like" as
illustrated in
the figures (214, 406, 509) of the present specification or similar to a
triangular groove as
illustrateii in figure 13 (1312) of PCT/SE02/00531.
A change in chemical surface characteristics (surface break) in the context of
anti-wicking
primarily refers to a change in hydrophocity/hydrophilicity of the surface of
an inner wall
of a microchannel structure. Typically the inner surface of the microconduit
is hydrophilic
as discussed above with a change into hydrophobicity where anti-wicking is to
be
achieved.
In a similar manner as for changes in geometric surface characteristics a
change in
chemical surface characteristics typically may extend into and/or across the
inner surface
of a wall in a microconduit.
A change in geometric and chemical surface characteristics may fizlly or
partially coincide
in the inner surface of microconduit. An indentation and the like which
stretches across an
inner wall thus physically should comprise the change in chemical surface
characteristics
if the aim is to avoid valuing effects. Compare figure 4 (406 and 407).
The anti-wicking means in a circumferential zone that comprise inner edges
should be at
different positions (or be lacking) in at least one compared to the position
of the anti-
wicking means in the other edges of the circumferential zone. For instance, if
the
microconduit has a four-edged cross-section (rectangular) with all four edges
extending
into a microcavity, opposite inner walls typically may have the change in
surface
characteristics at different distances, e.g. pair-wise, from the microcavity.
Compare for
instance figure 4.
The anti-wicking means described herein is adapted to prevent wicking for
aliquots that
have a surface tension, which is >_ S mN/m, such as >_ 10 mN/m or >_ 20 mN/m.
The
importance of including anti-wicking means is primarily related to handling of
aliquots <_
S ~1, such as aliquots in the nl-range, in the microfluidic devices described
herein.
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Further information about various kinds of anti-wicking means possibly
combined with an
inner valve function is given in PCT/SE02/00531.
Means for driving the liquid flow
The liquid flow may be driven in the microfluidic device of the present
invention by
distinct means that either is present on a substrate comprising the
microchannel structures
or is external to the substrate. The former variants typically means liquid
flow created by
electroendosmosis, by micropumps that are present on the substrate, expanding
gas etc.
The latter variants typically mean external pressure-generating means that
create a liquid
flow that is in fluid communication with the microchannel structure. Another
alternative
is to use forces such as capillary forces and inertia force including
gravitational force and
centrifugal force. In this latter case no means for moving the liquids is
required in the
microchannel structures or in the substrates carrying the microchannel
structures.
Variants in which the microchannel structures are oriented from an inner
position to an
outer position in relation to a spinning axis, such as an axis of symmetry of
a substrate as
described above are typically combined with a spinner that is capable of
spinning the
substrate around the spinning axis that rnay coincide with the axis of
symmetry. Useful
spinners should be able to create the necessary centrifugal force for driving
the liquids
through at least a part of a microchannel structure. The centrifugal force may
be utilized
in combination with a second liquid aliquot to create a sufficient local
hydrostatic
pressure within a structure to drive a first aliquot through an outward
(downward) and/or
an inward (upward) bent of a microchannel structure. See for instance WO
0146465.
Typically spinning speeds are within the interval 50-25000 rpm, such as 50-
15000 rpm.
The spinning speed within a given protocol may vary and depends on the part
structure
that is to be passed by a liquid, for instance. In case the microfluidic
device contains a
plurality of microchannel structures that are to be run in parallel, it may be
beneficial to
start the passage of liquid through a particular structural unit with a short
pulse of
increased spinning followed by a slower spinning.
Orientations and positions in a microfluidic device
The present invention is primarily intended for geometric arrangements in
which the
microchannel structure is present in a substrate and arranged about an axis of
symmetry
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(spinning axis) that typically is going through substrate. The term "radial
distance" means
the shortest distance between an object and the axis of symmetry and/or a
spinning axis
The radial distance for an inlet port and a structural unit may be the same,
or the inlet port
may be at a shorter or longer radial distance compared to the structural unit.
In a typical
case there is also an outlet port for liquid downstream the structural unit,
which in most
cases is at a larger radial distance than the inlet port. The microchannel
structure may or
may not be oriented in a plane perpendicular to the axis of symmetry. The
terms "higher"
and "upper" for a level/position means that an object is at a shorter radial
distance (inner
position) compared to being at a "lower" level/position (outer position).
Similarly, the
terms "up", "upward", "inwards", and "down", "downwards", "outwards" etc will
mean
towards and from, respectively, the spinning axis. This terminology applies if
not
otherwise is specified. With respect to other arrangements/substrates and
conventional
driving forces, i.e. gravity force, externally applied pressure, electro-
osmotically
(electrokinetically, by electroendoosmosis etc) driven flows etc, these terms
have their
conventional meaning.
The terms "downstream" and "upstream" are related to the process protocols and
liquid
flow as such. The terms thus refer to the order in which a unit, a part, a
process step, etc is
utilized. A downstream position is coming after an upstream position.
DETAILED DESCRIPTION OF THE MAIN SUBASPECTS OF THE INVENTION (STRUCTURAL
UNITS A-E.
Inlet ports typically have hydrophobised areas to direct applied liquid into
the ports. Local
surface breaks that are hydrophobic for aqueous liquids are represented by
straight or bent
rectangles. They are primarily present for controlling liquid flow, e.g. in
valves (inner
valves), in anti-wicking means, in vents and for directing liquid inwards the
structures in
inlet ports.
UNIT A (RETAINING MICROCAVITY UNIT)
The first aspect of the invention is a microfluidic device comprising a
microchannel
structure in which there is a structural unit accomplishing retaining of a nl-
aliquot of
liquid in a microcavity (retaining microcavity) as discussed in the first
object. The n1-
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19
aliquot has been obtained by mixing two liquid aliquots within the microcannel
structure
and is henceforth named "mixed nl-aliquot" or "mixed aliquot".
The present inventors have recognized that a nl-aliquot placed in a
microcavity of a
microchannel structure under static non-flow conditions is quickly reduced in
volume and
may disappear from the microcavity in the case the microchannel structure is
openly
connected to other parts of the structure, e.g. to inlet ports or outlet ports
which directly
communicate with ambient atmosphere and to other microcavities not containing
liquid.
The present inventors have discovered that this effect is related to wicking
in inner edges,
and that the effect is enhanced if evaporation of a wicked liquid from the
outlet and/or
inlet ports is possible. The present inventors hereby present a solution to
this problem.
The solution for a mixed nl-aliquot is to a) place anti-wicking means in the
microconduits
directly connected to a microcavity intended to retain a well-defined aliquot
of liquid,
and/or b) secure that the distance within the microchannel structure between
the
microcavity and each of the outlet ports has a sufficient length.
The structural unit of the first aspect of the invention is illustrated in
figures 2a-b and d-e.
The unit is characterized in comprising
(a) a microcavity (retaining microcavity) (219) which is intended for
retaining a n1-
liquid aliquot (mixed aliquot) under static non-flow conditions and is located
between at least one (205,215,237) of said one or more inlet ports
(205,215,237) and
at least one (238,241) of said one or more outlet ports (207,238,239,240,241);
(b) a mixing unit (302+303+301) which is located upstream the retaining
microcavity
(219) and downstream said at least one inlet port (205,215,237) and in which
two or
more aliquots (aliquot 1, aliquot 2 etc) are to be mixed to form said mixed
aliquot;
(c) two or more microconduits (218,220,242) directly connected to the
retaining
microcavity (219) and communicating with one of said inlet or outlet ports
(205,207,215,237,238, 239,240,241).
Each of the microconduits (218,220,242) comprises anti-wicking means (221 a,e)
in
association with the joint between the retaining microcavity (219) and the
microconduit.
Alternatively, if a microconduit is directly attached to a retaining
microcavity (219) and
does not contain anti-wicking means, then the distance (dl) from the retaining
microcavity
(219) to an opening to ambient atmosphere in an inlet or outlet port
(205,207,215,237,
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The distance (d,) refers to the shortest distance, if alternatives are
available. The distance
is measured inside the microchannel structure and includes length of the
microconduit
concerned. The cross-sectional dimension refers to an inner dimension.
5 The effect of reducing evaporation to ambient atmosphere without anti-
wicking means
may be further enhanced if the distance (d~) is further increased, e.g. to >_
20 times or >_ 50
times or >_ 100 times or >_ 500 times or >_ 1000 times >_ 5000 times the
largest cross-
sectional dimension of this microconduit at its joint to the retaining
microcavity (219).
10 In principle the above-mentioned conditions for microconduits not
containing anti-
wicking means may be applied to microconduits containing anti-wicking means.
Accordingly any microconduit connected to a retaining microcavity (219) may
contain
both anti-wicking means and comply with the conditions for the distance (d~).
15 The microconduits connected to the retaining microcavity may be either an
air
microconduit (242, figure 2d) or a liquid flow microconduit (218,220, figures
2a-b and d-
e). The latter typically also functions as a microconduit for venting out air
displaced by an
incoming liquid. Further differences between the two kinds of microconduits
are
discussed under the heading "Microfluidic device".
One, two or more up to all of the microconduits (218,220,242) directly
connected to the
retaining microcavity (219) have one or more length-going edges extending
continuously
from said retaining microcavity. Each of these edges preferably has anti-
wicking means.
Air microconduits having hydrophobic inner surfaces at their joint to the
retaining
microcavity (219) will inherently provide anti-wicking means. See further
under the
heading "Anti-wicking means".
A liquid flow microconduit (218,220) directly connected to a retaining
microcavity (219)
typically also comprises a non-closing valve function in association with the
joint
between the microconduit (218,220) and the microcavity (219). This valve
function may
be based on a change in geometric and/or chemical surface characteristics
and/or on a
suitable curvature of these flow microconduits (upward bents) as illustrated
in figures ta-
b and a (microconduit 220). The anti-wicking means and the valve functions may
fully or
RECTIFIED SHEET (RULE 91 )
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be based on a change in geometric and/or chemical surface characteristics
and/or on a
suitable curvature of these flow microconduits (upward bents) as illustrated
in figures ta-
b and a (microconduit 220). The anti-Wicking means and the valve functions may
fully or
partially coincide in a liquid flow microconduit (218,220). See further under
the heading
"Valve functions".
The mixing unit of the microchannel structure may in principle be any kind of
mixing unit
that can be adapted to the instant kind of microfluidic structures. This
includes the kind of
mixing units discussed under the heading "Background publications mixing units
(unit
B)" and the innovative unit B discussed below. Thus the mixing unit may
comprise two
inlet microconduits (224 and 225) for the aliquots to be mixed (aliquot 1,
aliquot 2).
These inlet microconduits merge in the downstream direction into a common
microconduit (302) that communicates with the retaining microcavity (219) in
the
downstream direction. At the intersection of the two inlet microconduits (224
and 225),
1 S there may be a microcavity (303) with a total volume that is essentially
the same as or
larger than the total volume of the aliquots to be mixed and introduced via
the inlet
microconduits. There may also be further inlet microconduits merging at the
intersection
or elsewhere for mixing of additional aliquots with aliquot 1 and aliquot 2.
Mixing may
occur in the common microconduit (302) (mixing microconduit), or in the
microcavity
(303) (mixing microcavity). The preferred mixing unit is according to unit B
below.
As discussed above a plurality of microchannel structures may be arranged
around a
spinning axis combined with using centrifugal force created by spinning around
the
spinning axis for driving the liquid flow in parallel through the structures.
Centrifugal
force may be combined with capillary force. Other forces may also be used for
this and
other configuration. See for instance under the heading "Means for driving the
liquid
flow".
In particular a plurality of the microchannel structures may be present on a
microfluidic
device that has an axis of symmetry coinciding with a spinning axis. In this
variant the
microcahnnel structures are typically arranged to permit the use of
centrifugal force for
driving a liquid flow in parallel within individual microchannel structures.
See above
under the heading "Microfluidic device".
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The retaining microcavity (219) may have different forms as known in the
field. Preferred
variants often define or are part of a U/Y-shaped structures, possibly linked
to upwardly
bent microconduits at either one or both of the upwardly directed shanks of
the U/Y as
described previously for reaction microcavities (WO 0040750, WO 0146465). The
U-
shaped structure may also be as presented in figure 2e where the U is defined
by a
reaction microcavity which comprises two upwardly directed shanks, the upper
parts of
which are connected to microconduits (218,220) containing the anti-wicking
means/valves (221e,a). Microconduit (218) plus the most downstream part of the
retaining microcavity (219) define an upward bent that will provide a valve
function.
This latter variant may be advantageous in the case the mixed aliquot is to be
transported
further downstream in the structure. See figure 2a-b and e. Another variant is
that the
microcavity (219) is circle like with the down stream or upstream microconduit
attached
without formation of this kind of bent. See for instance figure 2e in which
one of the
microconduits (242) is a pure air channel which preferably has an hydrophobic
inner
surface that in fact creates an anti-wicking effects and renders passage of
liquids difficult.
The mixed aliquot may be retained in the microcavity (219) for different
purposes, such
as performing a chemical and/or biochemical reaction and/or a measurement of
one or
more chemical or physical parameters of the mixed aliquot under static non-
flow
conditions within the microcavity (219) with a high accuracy (that would have
suffered
from loss of liquid and changes in concentrations if wicking and evaporating
would have
been allowed to act). Typically the reaction and/or measurement are part of an
assay
procedure for determining/detecting a component present in the mixed aliquot
or in some
other aliquots dispensed to the microchannel structure. The reaction may also
be
performed for synthetic purposes. Biochemical reactions include bioaffinity
reactions (e.g.
reactions between an antibody and an antigen/hapten, an enzyme and its
substrate,
cofactor, cosubstrate etc, complementary nucleic acids, and lectin
carbohydrate) including
enzyme reactions, cell reactions, etc. The reactions may take place in a
homogeneous
liquid phase or involve reactions between solid phase bound reactants and
dissolved
reactants or reactants in suspended form (heterogeneous reactions). Retaining
may also be
for the storing of the mixed aliquot, for instance awaiting certain process
steps to take
place outside or inside the microfluidic device on other aliquots that are to
be used in the
microfludic device, possibly together with the mixed aliquot. The periods of
time for
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retaining are as outlined in the first object. After the retaining period has
lapsed, further
processing of the liquid aliquot may take place in the reaction microcavity
(219) or further
downstream in the microchannel structure.
The surface tension of the liquid, the liquid contact angles of the inner
surfaces of the
microchannel structures, kind of liquids etc are selected as described under
the headings
"Microfluidic device", "Valve functions" and "Anti-wicking means".
The use of unit A is defined by the method of the second main aspect of the
invention and
comprises in addition a mixing step utilizing the mixing subunit and a process
step of the
mixed aliquot that may be performed for any of the reasons discussed above.
UNIT B (MIXING UNIT)
The second subaspect of the invention is a microfluidic device as defined
under the
heading "Technical field" comprising a microchannel structure in which there
is a
structural unit accomplishing mixing of aliquots (unit B).
This subaspect is based on our recognition that quick, efficient and reliable
mixing of
aliquots that are miscible can take place by first collecting the aliquots in
a microcavity,
preferably under the formation of a phase system, and then permitting the
aliquots to pass
through a microchannel of sufficient length to permit homogeneous mixing.
Preferred variants of our mixing units are illustrated in figures 2a-c. The
variants shown
are arranged as discussed above on a spinnable substrate (compare the arc-like
arrangement). Figures 2a-b comprises four microchannel structures connected to
each
other by a common distribution channel.
In general terms unit B comprises an inlet arrangement (201) and a mixing
microconduit
(202) as described in prior publications. Between the inlet arrangement (201)
and the
mixing microconduit (202) we have introduced a microcavity (203) to precollect
the
aliquots to be mixed in the mixing microconduit (202). The precollecting
microcavity
(203) has an opening (223) in its lower part which opening is in register with
the mixing
microconduit (202). This precollecting microcavity may have various designs
with one
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feature being that it should enable formation of a liquid interface between
the two aliquots
to be mixed. The flow direction should be essentially perpendicular at the
interface, i.e.
90°~ 45°.
In addition to the mixing unit as such, figures 2a-b show:
(a) A common distribution channel (204) as described for unit C below with an
inlet port
(205) with ridges/projections (206,216) as described for unit D above, an
outlet port
(207), and inlet vents (208) to ambient atmosphere via a common venting
channel
(209) and an air inlet (237). When the distribution channel is filled with
liquid and a
downward driving force is applied, liquid will be forced out through the
microconduits connecting the distribution channel (204) with the microcavities
(203).
At the same time air will enter through the vents (208).
(b) A common waste channel (210) comprising outlet ports (238)
(c) Volume-defining units (213) as described for unit E and comprising anti-
wicking
means (214,221 g) as described above, an inlet port (21 S) with
ridges/projections
(206,216) as described for unit D, and an overflow channel (217) ending in an
outlet
(212) in the common waste channel (210); and
(d) A microcavity (219) in which various kinds of processes may be carried out
as
discussed elsewhere in this specification, and an enlarged waste outlet
conduit (220),
which merges into the common waste channel (210) via the outlet (211 ).
Surface breaks (non-wettable) are represented by straight or arc-formed
rectangles (e.g.
221 a,b,c etc and 222, respectively).
The mixing unit of the present invention is characterized by comprising
(a) the microcavity (203) with an outlet opening (223), typically in its lower
part;
(b) an inlet arrangement (201) linked to the microcavity (203), and
(c) a mixing microconduit (202) connected to the outlet opening (223).
The microcavity (203) shall have a volume sufficient to contain simultaneously
the
aliquots to be mixed.
The inlet arrangement is connected to the upper or lower part of the
microcavity (203).
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Preferably there is a valve associated with the mixing conduit (202),
preferably close to its
joint to microcavity (203). This valve function is preferably an inner valve
of the same
kind as discussed elsewhere in this specification, for instance in the form of
a surface
break (non-wettable) (221b). The valve may also be mechanical.
5
The inlet arrangement may comprise a common inlet microconduit (not shown) for
several aliquots and/or separate inlet microconduits (224 and 225) for
individual aliquots.
The joint between these microconduits and the inlet openings are preferably
located at the
upper part of precollecting microcavity (203). In the upstream direction each
of these inlet
10 microconduits (224 and 225) communicates with an inlet port (205 and 215).
Each inlet
microconduit (224 and 225) may comprise a submicrocavity permitting separate
predispensing of an aliquot to a microchannel structure before transport down
into the
microcavity (203). In figures 2a-b one of these submicrocavities is
microcavity (226) of
the volume-defining unit (213) and the other Y-shaped structure (227) a part
of which
15 belongs to the common distribution channel (204). Between each
submicrocavity
(226,227) and microcavity (203) there may be a valve function (221d,c,
respectively) that
enables for aliquots to be transported into the submicrocavities (226,227)
without leakage
into the microcavity (203). The valve function at these positions is
preferably an inner
valve of the same kind as discussed for the valve functions (221 a,b)
associated with the
20 mixing microconduit (202), e.g. a surface break (non-wettable) (221a,b).
As illustrated in figures 2a-b the mixing conduit (202) may have various
forms. It may be
a single channel that is meandering or coiled in order to save space as
suggested in figure
2a. It may also be built up of a chain of interlinked small microcavities
(228), each of
25 which has a smoothly increasing cross-sectional area from the inlet end and
a smoothly
decreasing cross-sectional area when approaching the outlet end as suggested
in figure 2b.
Figure 2b also illustrates that these small microcavities can be of
continuously increased
breadth from their inlet and outlet ends with the steepest increase from the
outlet end
(droplet-shaped breadth).
When the aliquots are introduced into microcavity (203) there should be formed
a phase
system in the microcavity. Each aliquot should be represented by a liquid
phase. The flow
direction out of the microcavity (203) should be essentially perpendicular to
the interface
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between the phases. During passage of the phase system into the mixing
microconduit
(202), the upper phase is typically entering in the center of the microconduit
and the lower
phase next to the inner wall. Mixing will occur during the transport in the
microconduit
(202) probably due to the fact that the center of the liquid flow will have a
higher flow
rate than the peripheral part next to the inner wall of the mixing conduit.
This means that
the two aliquots repeatedly will replace each other in the front position
while traveling
through the mixing microconduit. This may be the reason for the quick and
efficient
mixing that is accomplished in the inventive mixing structure. If the mixing
microconduit
(202) is of sufficient length in relation to the flow rate and the
constituents of the aliquots,
complete mixing will have occurred at the end of the mixing microconduit
(202).
Sufficient length typically means that the phase system should have a smaller
volume than
the volume of the mixing microcoduit (202).
Figure 2c shows a third variant of the inventive mixing unit. This variant has
a
microcavity (229) corresponding to microcavity (203) in figures 2a-b. The
microcavity
(229) comprises an upper downward bent (230) and a lower downward bent (231)
and a
channel part (232) going from the lower part of the upper bent (230) to the
lower part of
the lower bent (231). In the lower part of the lower bent (231) there is an
opening (233)
leading into a mixing microconduit (234). Preferably there is a valve (235) in
the mixing
microconduit (234), typically close to the opening (233). This valve
preferably is an inner
valve for instance comprising a change in surface characteristics (non-
wettable surface
break). Figure 2c in addition shows inlet vents to ambient atmosphere (236a-d)
at top
positions of the bents. When filling the downward bents with aliquot 1 and
aliquot 2,
respectively, a liquid interface can be formed in the communicating
microconduit (232).
By applying a downwardly directed driving force the two aliquots will be
forced into the
mixing microconduit in the same manner as for the variants described in
figures 2a-b.
In the variant of figure 2c, the inlet arrangement of figures 2a-b is fully
integrated with the
precollecting microcavity (203) and therefore more or less indistinguishable.
The microcavity (229) of figure 2c may be part of two aligned common
distribution
channels of the same kind as outlined in figures 2a-b.
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27
In preferred variants, a microchannel structure comprising unit B may be
oriented about a
spinning axis that in turn may coincide with an axis of symmetry of a
spinnable
substrate/device as discussed elsewhere in this specification. The flow
direction through
the outlet opening of microcavity (203) is typically oriented essentially
outward in
relation to the axis of symmetry (spinning axis).
The use of unit B comprises a method for mixing two or more aliquots within a
microfluidic device comprising a microchannel structure. The aliquots may have
the same
or different volumes and/or compositions. The method is characterized in
comprising the
steps of:
(i) providing a microchannel structure comprising unit B as defined above;
(ii) introducing the aliquots via the inlet arrangement of unit B into
microcavity (203),
preferably to form a phase system therein;
(iii) applying a driving force to transport the phase system through mixing
microconduit (202);
(iv) collecting the homogenously mixed aliquots at the end of the mixing
microconduit
(202) for further transport and/or treatment within the microchannel
structure.
If submicrocavities (226,222) are present in the inlet arrangement (201 ), the
aliquots to be
mixed may be individually predispensed to these submicrocavities before the
driving
force for transport into precollecting microcavity (203) is applied.
The rules for selecting driving force are the same as discussed as discussed
above. For
spinnable substrate centrifugal force is preferred.
At least one of the aliquots should have a surface tension, which is >_ 5
mN/m, such as >_
10 mN/m or >_ 20 mN/m.
Common waste channel: In figures 2a-b the common waste channel (210) have
supporting
means for minimizing the risk for collapse due to the breadth of the channel.
The surface
break (227) improves the emptying of the overflow channel (217) and facilitate
its
refilling.
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UNIT C (UNIT FOR FORMING A PLURALITY OF ALIQUOTS OF DEFINED VOLUMES WITHIN A
MICROFLUIDIC DEVICE, DISTRIBUTION MANIFOLD).
The third subaspect of the invention is a microfluidic device comprising a
microchannel
structure in which there is a structural unit (unit C) accomplishing metering
one, two,
three or more aliquots (two or more = plurality of aliquots).
This subaspect is based on our recognition that the relative loss of liquid by
evaporation
may be significant when dispensing small aliquots, in particular nl-volumes,
to individual
microchannel structures in a microfluidic device. We have also found that the
prior
systems utilizing a common reagent fill channel from which metering is done in
parallel
in a plurality of metering microcavities are insufficient when the cross-
sectional
dimensions of the channels are in the lower part of the ~tm-range and/or the
volumes are
decreased into the nl-range.
1 S Unit C presents a solution to these problems and makes it possible to
reproducibly meter a
number of smaller aliquots within the same microfluidic device and to
transport these
aliquots in parallel into separate microchannel structures of the microfluidic
device or into
separate parts of the same microchannel structure. The aliquots may be
identical or
different with respect to size, composition etc, and are typically in the nl-
range as defined
above
Unit C is represented in figures 3a-c that show variants that are arranged
about a spinning
axis that may coincide with an axis of symmetry as discussed above. In these
figures the
distribution unit as such is encircled (300).
Based on figures 3a-b, the unit comprises
(a) a continuous microconduit (301) containing an upper pant at each end (end
parts, 302,
303) and therebetween alternating lower and upper parts (304a-h/f and 305a-e,
respectively);
(b) the number of upper parts including the end parts is n and the number of
lower parts
is n-1 where n is an integer > 2, i.e. >_ 3;
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(c) each of the upper parts (302, 303, 305a-e/g) has means for venting (top
vent, inlet
vents) (306a-g/i) to ambient atmosphere and/or anti-wicking means (326a-i) in
length-
going edges delineating its lower wall(s);
(d) each of the lower parts (304a-f/h) has an emptying opening which in a
downstream
direction via a connecting microconduit (307a-f/h) communicates with a
substructure
of a microchannel structure and/or with a corresponding substructure of
another
microchannel structure;
(e) each of the connecting microconduits (307a-f/h) has a valve (308a-f/h),
i.e. a valve
function in close association with the joint between the connecting
microconduit and
the corresponding lower part;
(fj an inlet port (309) is connected to the continuous rnicroconduit (301)
directly or
indirectly at one of the upper parts (302, 303, 305a-e/g), preferably via one
of the end
parts (302 or 303);
(g) an outlet port (310) is connected to the continuous microconduit (301)
directly or
indirectly at another upper part (302, 303, 305a-e/g), preferably via one of
the end
parts (302 or 303) (which preferably is not connected to the inlet port, i.e.
an inlet port
and an outlet port should not connected at the same upper part).
In a lower part (304a-f/h), the continuous microconduit (301) is preferably
shaped as a
downward bent. This kind of bents includes that the microconduit in the bent
is enlarged
to a microcavity. Similarly an upper part is preferably in the form of an
upwardly bent
microconduit but without enlargement of the type that can be present in a
downward bent.
The smallest cross-sectional areas of the continuous microconduit (301)
between the ends
(302, 303) should be in the upper parts, with preference for in association
with the top
vents (306a-g/i) and/or the anti-wicking means (326x-i). The cross-sectional
area of the
continuous microconduit (301) may be of constant size and/or shape along the
length of
the continuous microconduit.
The inlet ports (309) and the outlet ports (310) are typically at a lower
level than the
extremes of the upward bents and may even be at a lower level than the
extremes of the
lower parts (304) and/or than a desired part of the individual microchannel
structures that
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are downstream the lower parts (304) (for instance at a lower level than a
waste outlet
port).
The liquid aliquot is preferably transported from an inlet port (309) to an
outlet port (310)
S of the continuous microconduit (301) by capillarity meaning that the liquid
contact angle
in this part of the microchannel structure continuously has to be well below
90°, i.e.
preferably S 40°, such as < 30° or 5 20°, and enabling
filling by capillarity of the
continuous microconduit (301) to valves (308a-f/h) by self suction from an
inlet port of
the microconduit (301).
In the preferred variants the continuous microconduit (301 ) has meander-form.
The integer n is preferably > 2, such as 3, 4, S, 7, 8, 9, 10, 11, 12 or more.
The joints between a connecting microconduit (307a-f/h) and a lower part (304a-
f/h) are
preferably located at the same level and/or at the lowest part of a downward
bent. The
valves (308a-f/h) in the connecting microconduit (307a-f/h) preferably are
inner valves
that may be closing or non-closing.
The top vents (306a-g/i) are preferably located at the same level on the
upward bents
(302, 303, 305a-e/g). Each top vent (306a-g/i) comprises an opening in an
upper part
(302, 303, 305a-e/g) of the continuous microconduit (301) and possibly also a
microconduit. Each top vent may have an inner valve and/or may be equipped
with anti-
wicking means in the case the top vent has a length-going edge that might
promote
imbibing and evaporation of liquid. The anti-wicking means are described
elsewhere in
this specification. The top vents may be connected via a common venting
channel (311)
and an inlet (325) to ambient atmosphere.
The openings associated with top vents in the upper part may be directed
upward as
illustrated in figures 3a-c but may also be directed in other directions, e.g.
as illustrated in
figure 2c (236x-d).
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As outlined for unit D preferred variants of unit C may have: A) inlet port
(309) designed
with a hydrophobic surface break at the rim of the inlet opening which directs
a dispensed
aliquot into the opening of the port, and B) an inner valve in the
microconduit connecting
an upper part to an inlet port (310). Compare also figure 7 and figure 8 of
PCT/SE02/00538 and PCT/SE02/00539, respectively.
One or both of the end parts (302,303) may directly or indirectly be connected
to another
distributing unit C according to the invention as illustrated in figure 2 of
PCT/SE02/00537.
Unit C is intended for distributing (n-1) aliquots to (n-1) microchannel
structures or (n-1)
part structures of a microchannel structure. The volume between two close top
vents
(306a-e/g) will in most variants define the volume of the aliquot to be
dispensed through
the connecting microconduit (307a-f/h) between these top vents (segment). By
varying
the depth and/or width of different segments, one can envisage that the
volumes dispensed
through different connecting microconduits (307a-f/h) can differ in a
controlled manner.
By first filling the continuous microconduit (301) with liquid between its end
parts (302
and 303), for instance by self suction, and then forcing liquid to pass
through the
connecting microconduits (307), the metered aliquots between close top vents
will pass
into separate connecting microconduits. Spillover between neighboring segments
of the
continuous microchannel (301) will be minimized due to the top vents and/or by
the
presence of anti-wicking means (326) in edges delineating lower walls in upper
parts.
By filling the segments with the same liquid, for instance in one step,
aliquots of the same
composition will be dispensed through all the emptying openings.
Figure 3b illustrates a non-meander form of unit C (straight form) in which
the lower
parts (304a-h) are in form of microcavities that are connected to each other
via upper parts
(305a-g). At the end of the continuous microconduit (30I) there are also upper
parts
(302,303) via which an inlet and an outlet port may be connected (309 and 310,
respectively). Means for venting (306a-i) the continuous microconduit (301)
may be
associated with upper parts of the continuous microconduit, for instance in
the conduit
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32
parts (305a-g) and/or in the end parts (302,303). The lower part of each
microcavity
(304a-h) has an outlet opening to which a connecting microconduit (307a-h)
with a valve
functiom(308a-h) is associated. There may also be anti-wicking means
(rectangles, 326a-
i) at both sides of each microcavy (304a-h) in edges that extend down into a
neighboring
S microcavity/lower part (304a-h). The anti-wicking means may be of the same
kind as
discussed elsewhere in this specification. A variant is shown in figure 7 and
figure 8 of
PCT/SE02/00538 and PCT/SE02/00539, respectively, and illustrates a
distribution
manifold with a centrally located inlet port and anti-wicking means in the
edges as
discussed above but without the top vents (306a-g).
Figure 3c represents a variant, which will enable distribution of aliquots of
different
compositions to individual microchannel substructures. The distribution unit
as such is
encircled (300). Upstream the distribution unit (300) there is a microchannel
substructure
(311), which will enable filling of segments between close top vents (306a-d)
of the
continuous microchannel (301) with aliquots of different compositions. In
order to
accomplish this, substructure (311) comprises a volume-defining unit (312),
which is
capable of metering a liquid volume that is equal to the volume of the segment
between
two close top vents (306a-d) in the continuous microchannel (301). If the
volumes of the
segments are different, subunits defining different volumes may be included in
substructure (311). In figure 3c, the substructure (311) upstream the
distribution unit
(300) may comprise further functionalities. Thus substructure (311) may
comprise a first
downward bent (313) which has one of its shanks (314) connected to the end
part (302) of
the continuous microchannel (301) and the other shank (315) connected to the
lower part
of a second downward bent (316) that in turn is connected to a metering part
of volume-
defining unit (312) at the upper part of one of its shanks (317). The other
shank (318) of
the second downward bent (316) may be venting to ambient atmosphere via an
inlet
(327). The illustrated metering part of volume-defining unit (312) is of the
same kind as
unit E and includes an overflow system and an inlet port (319) of the same
kind as unit D.
The volume of the metering microcavity (320) of the volume-defining unit (312)
is the
same as in a segment between two close top vents (306a-d). The substructure
(311) of
figure 3c also comprises (a) a large waste chamber (321) with a relatively
wide opening
(322) into the lowest part of the first downward bent (313), and (b) a valve
function (323)
associated with the connection between the first and second downward bent
(313,316).
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Due to the size of the waste chamber (321 ) there are supporting means in form
of pillars
(324) securing that its top and bottom are kept apart from each other.
The kind of design presented in figure 3c makes it possible to consecutively
fill the
segments between the top vents (306a-d) of the continuous microconduit (301)
with
aliquots of different compositions, and thus to distribute aliquots of
different composition
to the individual substructures connected to unit C via the connecting
microconduits
(308a-c). With reference to figure 3c this means (presuming waste chamber
(321) is
closed or absent):
Step 1: Aliquot 1 is metered in the volume-defining unit (312) and transported
to
downward bent (313), for instance by spinning if the unit is present on a
spinnable
substrate that may be a circular disc by the hydrostatic pressure created by
centrifugal force.
Step 2: Aliquot 2 is metered in the volume-defining unit (312) and transported
into the
downward bent (313). This will push aliquot 1 to segment 1 (between top vents
406a and b) of the continuous microconduit (301).
Step 3: Aliquot 3 is metered in the volume-defining unit (312) and transported
into the
downward bent (313). This will push aliquot 1 to the second (next) segment and
place aliquot 2 in the first segment.
When the desired number of segments has been filled a downwardly directed
driving
force is applied to pass the aliquots through their respective connecting
microconduit/valve (307a-c/308a-c).
A simplified variant of figure 3c means that the first downward bent (313) is
designed as a
volume-defining unit, for instance by placing an overflow system at the same
level as the
top vents (306a-d) of the continuous microconduit (301) in shank (315).
By introducing a chemical functionality, for instance in the form of
substructure
comprising an inlet port followed by a reaction zone in front of unit C, unit
C may be used
for collecting separate fractions between each pair of neighboring top vents
in the
continuous microconduit (301) from liquids that have passed through the
reaction zone.
Collected fractions can then be further processed, for instance analysed, by
taking them
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further down into the microchannel structure via the connecting microconduits
(307a-c).
With respect to figure 3c, such a zone suitably is positioned between the
first and second
downward bents (316 and 313, respectively), for instance combined with the
valve (323)
The reaction zone may for instance comprise an immobilized reactant selected
from (a) a
catalysts such as an enzyme, (b) a ligand capable of binding to a component of
a liquid
which is to pass through the zone, (c) an affinity complex between a ligand
and a binder
etc. Based on the presence of particular components in the fractions that are
collected one
can analyse for features related to the zone as such or to the liquids
applied, e.g. features
of compounds present in the zone and/or in a fraction.
Unit C is preferably present in a spinnable microfluidic device of the kind
discussed
elsewhere in this specification. The continuous microconduit (301) may then be
oriented
in an annular-like fashion around a spinning axis and may occupy at least a
sector of an
annular zone defined by the continuous microconduit. The sector typically
covers at least
0.5-10° and at most 360° relative a spinning axis and/or an axis
of symmetry. The lower
parts (304) of the unit are directed outwards from the spinning axis and the
upper parts
(302, 303, 305) inwards towards the spinning axis.
The driving force is selected according to the same principles as outlined for
the
microfluidic device above, with preference capillarity for filling the
continuous
microconduit (301) and centrifugal force or overcoming the valve functions
(308a-f/h) in
the connecting microconduits (307a-f/h).
The aliquot applied should have a surface tension, which is >_ 5 mN/m, such as
>_ 10
mN/m or >_ 20 mN/m.
UNIT D (INLET UNIT WITH MEANS SUPPORTING LIQUID ENTRANCE INTO A MICROCHANNNEL
STRUCTURE)
This subaspect of the invention refers to an improvement that lowers the time
for
undesired evaporation of an aliquot that has been dispensed to a microfluidic
device of the
same kind as the invention. The advantages are primarily related to dispensing
and/or
metering nl-aliquots within microfluidic devices.
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The fourth sub-aspect of the invention is a microfluidic device comprising a
microchannel
structure in which there is an inlet unit promoting liquid entrance into a
microchannel
structure.
5 The unit is illustrated in figures 4a-b. The unit comprises:
(a) an inlet port comprising a microcavity (401) and an inlet opening (402),
and
(b) an inlet conduit (403) which is positioned downstream said microcavity
(401) and
which communicates with the interior of the microchannel structure.
The inner wall of the microcavity (401) comprises one or more grooves and/or
10 projections (ridges/valleys) (404) directed towards the connection between
the inlet
conduit (403) and the microcavity (401). The microcavity (401) is typically
tapered
(narrowing) when approaching the inlet microconduit (403).
The main purpose of the grooves and/or the projections is to increase the
capillary suction
15 in the inlet port. This will speed up liquid penetration and lower the time
for undesired
evaporation and loss of liquid during the dispensing operation.
The narrowing design of microcavity (401) as such assist in promoting liquid
penetration
and of retaining a dispensed aliquot within the covered part of a microchannel
structure.
Figure 4b illustrates a variant comprising a non-wetting surface break (405)
in association
with the rim of the inlet opening (401), primarily at a side which is closest
to the spinning
axis if the inlet port is located on a spinning substrate. This figure also
illustrates a variant
of unit D that comprises anti-wicking means downstream the inlet opening
(401). These
means may comprise changes in geometric surface characteristics (406) and/or
in
chemical surface characteristics (407).
The projections may have a height that at maximum is equal to the depth of the
microcavity (401) but may be significantly lower as long as a sufficient
capillary action
(self suction) is maintained in the inlet port in order to draw a dispensed
aliquot
completely into the covered part of a microchannel structur.
The liquid to be introduced typically has a surface tension as discussed
above.
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The width of the inlet opening is typically smaller than the width of
microcavity (401 ) as
illustrated in figures 4a-b.
The inlet opening (402) may have one or more edges directed inwards the port,
preferably
with an n-numbered axis of symmetry perpendicular to the opening. n is
preferably an
integer < 7, such as 3, 4, 5 or 6. See for instance US 4,233,029 (Eastman
Kodak) and US
4,254,083 (Eastman Kodak).
Unit D is typically combined with a dispenser that is capable of dispensing an
aliquot in
the nl-range to the inlet port. The dispenser can be one of the dispensers
generally
described elsewhere in this specification.
Other forces than capillary force may be used for promoting penetration
through the inlet
port, for instance inertia force including centrifugal force.
Microchannel structures that comprise unit D are in a preferred variant placed
on a
spinnable substrate as discussed elsewhere in this specification.
This kind of inlet unit is particularly well adapted to receive aliquots that
are in the form
of particle suspensions.
UNIT E (DEFINITION OF THE VOLUME OF ALIQUOTS).
In spite of the previously known devices for metering aliquots in the pl-range
there is still
a need for improvements, in particular with respect to the nl-range. The
reason is that
uncontrolled evaporation has a stronger influence on a smaller aliquot more
compared to a
larger aliquot (respect relative loss in volume). This is further accentuated
when a large
number of aliquots are to be dispensed in sequence before the aliquots are
further
processed within a microfluidic device.
The present inventors have recognized these problems and designed a volume-
metering
unit (unit E) to meter primarily nl-volumes of liquids. The unit can be
integrated into
microchannel structures of microfluidic devices.
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The fifth subaspect of the invention thus is a microfluidic device that
comprises a
microchannel structure in which there is volume-defining unit enabling
accurate metering
of small volumes within a microfluidic device, primarily nl-volumes.
Unit E is illustrated in figure 5. Unit E comprises
(a) a volume-defining microcavity (501),
(b) an inlet microconduit (502) which is connected to the microcavity (501)
via an inlet
opening on the microcavity (501) (at the joint between said microcavity and
the inlet
microconduit),
(c) an outlet microconduit (503) which is connected to microcavity (501) via
an outlet
opening in microcavity (501) (at the joint between said microcavity and the
outlet
microconduit), and
(d) an overflow microconduit (504), which is connected to an overflow opening
on
microcavity (501) (at the joint between said microcavity and the overflow
microconduit).
The inlet opening and the overflow opening are typically at the same level on
the
microcavity (501). The overflow opening is at a higher level than the outlet
opening and
the volume between these two openings defines the volume to be metered in the
volume-
defining microcavity (501). The metered volume is typically in the nl-range as
defined
above, but may also be larger, such as _< 10 p1 or <_ 100 ~1 or <_ 1000 ~,1.
The liquid typically has a surface tension as discussed above.
The overflow microconduit (504) is typically communicating with ambient
atmosphere
via an enlargement at the end of the overflow microconduit (504) (typically a
waste
chamber or a waste conduit (511). The joint between the overflow microconduit
(504)
and the enlargement is at a lower level than both the connection between the
overflow
microconduit (504) and the lowest part of the volume-defining microcavity
(501) (in
reality the valve function (506) at the outlet opening of the volume-defining
microcavity).
The outlet microconduit (503) is used to transport a metered liquid aliquot
further into the
microchannel structure.
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The volume-defining microcavity (501) may have different forms, for instance
comprise
(a) one or more capillaries,
(b) a downward bent structure with one shank acting as the inlet and the other
shank
ending in an upward bent that can be used as the overflow microconduit, and
with the
outlet micrconduit (503) being joined at the lower part of the downward bent
and
intended for transporting a metered aliquot further downstream in the
microchannel
structure.
The cross-sectional area (al) in the volume-defining microcavity (501) at the
overflow
opening is in preferred variants smaller than the largest cross-sectional area
(a2) between
the overflow opening and the outlet opening. The ratio a~/az typically is S
1/3, such as S
1/10. This means a significant constriction of the microcavity (501) at the
joint between
the overflow microconduit (503) and the microcavity (501), i.e. at the joint
between inlet
microconduit (502) and volume-defining microcavity (5O1).
The inlet microconduit (502) upstream the overflow opening typically widens,
for
instance to an inlet port (505), such as unit D.
Between the volume-defining unit and a true inlet port there may other
structural/functional units, for instance a unit for sample treatment such as
for the removal
of particulate materials.
Unit E may have a valve function (506,507,508) associated with at least one of
(a) the outlet opening of microcavity (501),
(b) the inlet microconduit (502) closely upstream the overflow opening, and
(c) the overflow microconduit (504), preferably its lower part such as in
association with
its joint with the waste conduit/chamber (511).
These valves may be mechanical valve or of any of the other types discussed
above, but is
preferably an inner valve of the closing or non-closing type with emphasis of
the former.
At least one of the inlet microconduit (502), the outlet microconduit (503)
and the
overflow microconduit (504) may have anti-wicking means of the kinds defined
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elsewhere in this specification. The variant shown in figure 5 comprises anti-
wicking
means (509) in the inner edges of inlet microconduit (502). The anti-wicking
means
stretches across the corresponding inner walls as discussed above in general
terms.
A microchannel structure comprising unit E may in its preferred variants be
equipped
with valve functions (506, 508), preferable inner valves of the non-closing
type, and be
present on a spinnable substrate as discussed elsewhere in this specification.
If the
intention is to drive the liquid out of the overflow channel (504) before the
metered
aliquot is released via the outlet microconduit (503), it becomes important to
have a
sufficiently large difference in radial distance (r1) between the overflow
opening in the
volume-defining microcavity (501) and the ending (512) of the overflow
microconduit
(504) in a waste chamber (511) relative to the difference (r2) in radial
distance between
the overflow opening and the valve (506) in the outlet microcoduit (503). r~
shall be
essentially larger than r2. This particularly applies if the valve function
(506) in the outlet
microconduit (503) is an inner non-closing valve. By properly selecting r1 >
rz , e.g. r1 >
1.25r2, or r> > 1.5r2 , or r1 > 2r2 , or r> > Sr2, or r> > l Orz , it will be
possible for the liquid in
the over-flow microconduit to pass through the valve (508) at a lower driving
force (e.g.
lower spinning speed) than required for the liquid in the volume-defining
microcavity to
pass through the valve (506). The optimal relation between the two distances
depends on
various factors, such as width, breadth, wettability, roughness etc of the
microconduits
concerned as well as surface tension, density et of the liquid concerned.
A variant that may be adapted to spinnable substrates comprises a downward
bent with
the volume-defining microcavity being a part of the lower part of the bent.
The overflow
microconduit typically is connected to one of the shanks of the downward bent
and forms
together with this shank an upward bent. The upper part of the same shank
vents to
ambient atmosphere (inlet vent). An inlet port for sample (corresponds to 505)
may be
connected to the other shank of the same downward bent. The vent to ambient
atmosphere
may be designed with a sample/liquid inlet function. The outlet conduit with a
valve is
connected to the lower part of the downward bent (corresponds to 503 and 506,
respectively). The overflow microconduit (corresponds to 504) ends in a waste
channel or
waste chamber with a valve function (corresponds to 508).
CA 02456421 2004-02-03
WO 03/018198 PCT/SE02/01539
There are advantages with having the outlet opening connected to the outlet
microconduit
(503) on microcavity (501) somewhat higher than the lowest part of the volume-
defining
microcavity. In such variants there will be a small volume present below the
outlet
opening in which it will be possibly to sediment and collect particulate
materials and only
S flow the supernatant that corresponds to a metered volume through the outlet
microconduit (503). Sedimenting can be assisted by the use of centrifugal
force
(spinning).
The use of unit E defines a method for introducing metered aliquots into
microchannel
10 structures. The method comprises the steps of
(i) providing a microchannel structure comprising unit E and an aliquot having
a
larger volume than then the volume to be metered in the unit;
(ii) introducing the liquid of aliquot into the unit;
(iii) applying a first driving force to move excess liquid out through the
overflow
15 microconduit (504) and a second driving force to move the metered volume
through the outlet microconduit (503) into the remaining part of the
microchannel
structure.
The driving force is selected as discussed above for the other units with
preference for
inertia force including centrifugal force when the substrate is spinnable.
The invention is further defined in the appending claims that are part of the
specification.
