U.S. patent application number 11/268952 was filed with the patent office on 2006-03-23 for optimized high throughput analytical systems.
This patent application is currently assigned to Caliper Life Sciences, Inc.. Invention is credited to Andrea W. Chow, H. Garrett Wada.
Application Number | 20060062696 11/268952 |
Document ID | / |
Family ID | 36074212 |
Filed Date | 2006-03-23 |
United States Patent
Application |
20060062696 |
Kind Code |
A1 |
Chow; Andrea W. ; et
al. |
March 23, 2006 |
Optimized high throughput analytical systems
Abstract
The present invention provides novel microfluidic devices and
methods for controlling/manipulating fluidic materials in
microfluidic devices. In particular, the devices and methods of the
invention create and utilize differences between dispersion rates
and/or average velocity of fluidic materials in order to manipulate
fluidic materials.
Inventors: |
Chow; Andrea W.; (Los Altos,
CA) ; Wada; H. Garrett; (Atherton, CA) |
Correspondence
Address: |
CALIPER LIFE SCIENCES, INC.
605 FAIRCHILD DRIVE
MOUNTAIN VIEW
CA
94043-2234
US
|
Assignee: |
Caliper Life Sciences, Inc.
Mountain View
CA
|
Family ID: |
36074212 |
Appl. No.: |
11/268952 |
Filed: |
November 7, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10206787 |
Jul 26, 2002 |
|
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11268952 |
Nov 7, 2005 |
|
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60308368 |
Jul 27, 2001 |
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Current U.S.
Class: |
422/400 |
Current CPC
Class: |
B01L 2300/0816 20130101;
B01L 2400/0487 20130101; B01L 3/502746 20130101; B01L 2400/0406
20130101; B01L 3/502707 20130101; B01L 2400/084 20130101; B01L
2300/0877 20130101; Y10T 436/2575 20150115; G01N 27/44743 20130101;
B01L 2300/0858 20130101; Y10T 436/25 20150115 |
Class at
Publication: |
422/100 |
International
Class: |
B01L 3/00 20060101
B01L003/00 |
Claims
1. A microfluidic device comprising a body structure having a
microchannel disposed therein, wherein the microchannel includes a
region having a cross-sectional geometry that comprises a center
segment and two side segments, each of the two side segments having
a first maximum depth, the center segment having a second maximum
depth different from the first maximum depth.
2. The device of claim 1, wherein the second maximum depth is
greater than the first maximum depth.
3. The device of claim 2, wherein the body structure comprises a
first substrate and a second substrate, and wherein the
microchannel region is formed into a surface of at least one of the
two substrates, the first substrate and the second substrate being
joined such that the microchannel region is defined by the
interface of the two substrates.
4. The device of claim 3, wherein a first portion of the
microchannel region is formed into a surface of the first substrate
and a second portion of the microchannel region is formed into a
surface of the second substrate.
5. The device of claim 4, wherein the first portion is narrower
than the second portion, and wherein the substrates are joined such
that the first portion is substantially centered laterally over the
second portion.
6. The device of claim 4, wherein the width of the first portion is
substantially the same as the width of the second portion, and
wherein the substrates are joined such that the first portion is
offset laterally from the second portion, the first portion
partially overlapping the second portion, the overlap of the two
portions forming the center segment of the microchannel region
cross-sectional geometry.
7. The device of claim 4, wherein the cross-sectional geometry of
the first portion is substantially the same as the cross-sectional
geometry of the second portion, each portion comprising a center
section and two side sections, the center section having a maximum
depth greater than that of the side sections, and wherein the
substrates are joined such that the first portion is substantially
centered laterally over the second portion.
8. The device of claim 1, wherein the second maximum depth is less
than the first maximum depth.
9. The device of claim 8, wherein the body structure comprises a
first substrate and a second substrate, and wherein the
microchannel region is formed into a surface of at least one of the
two substrates, the first substrate and the second substrate being
joined such that the microchannel region is defined by the
interface of the two substrates.
10. The device of claim 8, wherein the body structure comprises a
first substrate and a second substrate, and wherein a first portion
of the microchannel region is formed into a surface of the first
substrate and a second portion of the microchannel region is formed
into a surface of the second substrate, the first substrate and the
second substrate being joined such that the microchannel region is
defined by the interface of the two substrates.
11. The device of claim 8, wherein the cross-sectional geometry of
the first portion is substantially the same as the cross-sectional
geometry of the second portion, each portion comprising a center
section and two side sections, the center section of each portion
having a maximum depth less than that of the side sections, and
wherein the substrates are joined such that the first portion is
substantially centered laterally over the second portion.
12. The device of claim 1, wherein the microchannel region is
formed by one or more of photolithographic etching, plasma etching,
wet chemical etching, laser drilling, micromilling, injection
molding, stamp molding, embossing, and ablation.
13. The device of claim 1, wherein the microchannel region is
formed using a double etching technique.
14. A microfluidic system, comprising: a body structure having a
microchannel disposed therein, wherein the microchannel includes a
region having a cross-sectional geometry that comprises a center
segment and two side segments, each of the two side segments having
a first maximum depth, the center segment having a second maximum
depth different from the first maximum depth; a source of a first
fluidic material, fluidly coupled to the at least one microchannel;
a source of a second fluidic material, fluidly coupled to the at
least one microchannel; and a fluidic direction system for
controllably moving the first fluidic material and the second
fluidic material into and through the microchannel region, which
fluid direction system does not comprise electrokinetic flow.
15. The system of claim 14, wherein the fluid direction system
comprises one or more of positive pressure, negative pressure,
hydrostatic pressure, and wicking forces.
16. The system of claim 14, wherein the body structure includes a
detection region, the system further comprising: a detection system
positioned proximal to the detection region.
17. The system of claim 16 further comprising: a computer operably
coupled to the detection system, wherein the computer comprises an
instruction set for acquiring data from the detection system and
for tracking one or both of the average velocity and dispersion
rate of the first fluidic material and one or both of the average
velocity and dispersion rate of the second fluidic material.
18. The system of claim 14, wherein the source of the first fluidic
material and the source of the second fluidic material comprise the
same source.
19. The system of claim 14, wherein the first fluidic material
comprises cells or beads.
20. The system of claim 14, wherein the second fluidic material
comprises a test compound.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/206,787, filed Jul. 26, 2002, which claims
the benefit of U.S. Provisional Patent Application No. 60/308,368,
filed Jul. 27, 2001, both of which are incorporated herein by
reference in their entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] When carrying out chemical or biochemical analyses, assays,
syntheses or preparations, a large number of separate manipulations
are performed on the material(s) or component(s) to be assayed,
including measuring, aliquotting, transferring, diluting, mixing,
separating, detecting, incubating, etc. Microfluidic technology
miniaturizes these manipulations and integrates them so that they
can be executed within one or a few microfluidic devices. For
example, pioneering microfluidic methods of performing biological
assays in microfluidic systems have been developed, such as those
described by Parce et al., "High Throughput Screening Assay Systems
in Microscale Fluidic Devices" U.S. Pat. No. 5,942,443 and Knapp et
al., "Closed Loop Biochemical Analyzers" (WO 98/45481).
[0003] Of particular interest in numerous applications utilizing
microfluidic devices is the movement/transport of, e.g., samples,
reagents, analytes, etc. often in discrete bands (or plugs). For
example, in many experimental/assay situations it is desirous to
keep plugs of different samples (e.g., a selection of possible
enzymatic inhibitors) from diffusing or dispersing into one another
as the samples are flowed through various regions of a microfluidic
device. This is especially true in high throughput systems where
muddying or intermingling of sample plugs can severely decrease
throughput efficiency.
[0004] Conversely, it is also of interest in the use of
microfluidic devices to move/transport fluidic materials (e.g.,
samples, reagents, analytes, etc.) in such a way as to, e.g.,
separate multiple materials from within a single plug into various
separate plugs and/or to "stretch" a particular sample plug into a
longer, and therefore, e.g., less concentrated, length.
[0005] The amount/degree of dispersion of samples, etc. in
microfluidic devices is influenced by how the samples, etc. are
transported through the microfluidic device. Fluidic materials
(e.g., in sample plugs) are transported through microfluidic
devices in numerous ways using, e.g., electrokinetic flows
(electrophoresis or electroosmosis), pressure (e.g., via
application of a positive force or via a vacuum), hydrostatic
forces, etc. However, various flow regimens used in microfluidic
devices can lead to dispersion of plugs of fluid material in the
microfluidic elements (e.g., microchannels). For example, pressure
driven flow can result in sometimes large amounts of Taylor
dispersion of a fluidic material. Additionally, even electroosmotic
flow and hydrostatic flow can cause small pressure gradients along
a microchannel due to, e.g., mismatch of electroosmotic flow rates,
etc. Such can lead to, e.g., dispersion even when fluidic materials
are transported via electrokinetic methods.
[0006] A welcome addition to the art would be the ability to
manipulate the length of sample plugs (e.g., to minimize
lengthening [i.e., to keep plugs intact] and/or to maximize
lengthening [i.e., to separate mixed samples or to dilute samples])
as the plugs are flowed through a microfluidic device. The current
invention describes and provides these and other features by
providing new methods, microchannels, and microfluidic devices that
meet these and other goals.
SUMMARY OF THE INVENTION
[0007] The present invention provides methods, systems,
microchannels, kits, and devices for controlling and manipulating
aliquots of fluidic materials in microfluidic devices. Fluidic
materials are flowed through microfluidic devices comprising
microchannels of "regular" cross-sectional geometry and/or
"specifically configured" cross-sectional geometry. The invention
utilizes differences in dispersion rates and/or average velocities
of the fluidic materials created by the particular cross-sectional
geometry of the channels in which the fluidic materials are
flowed.
[0008] In one aspect, the invention comprises an integrated system
or microfluidic device having a body structure with at least one
microchannel with a cross-sectional geometry configured to
manipulate a dispersion rate and/or an average velocity of at least
one fluidic material. Such integrated system or microfluidic device
further has one or more detection regions of the microchannel; a
source(s) of one or more fluidic materials coupled to the
microchannel; fluid direction systems and a detection system
proximal to the detection region. Such microchannel in the system
or device optionally has a cross-sectional geometry that
manipulates the dispersion rate and/or the average velocity of the
fluidic material relative to the dispersion rate and/or average
velocity of the same fluidic material in a microchannel having a
substantially rectangular cross-sectional geometry. In some
embodiments, at least 2 fluidic materials each having a dispersion
rate and/or each having an average velocity are flowed through the
system/device. The cross-sectional geometry of the microchannel
optionally manipulates the dispersion rates and/or average
velocities of such fluidic materials to either be the same and/or
to be different rates/velocities (e.g., 1.25, 1.5, 1.75, 2, 3, 4,
5, or 10 times different, i.e., the dispersion rate and/or average
velocity of one fluidic material is 1.25, etc. times greater than
the other).
[0009] In other aspects, the invention comprises a method of
manipulating a dispersion rate and/or average velocity of a fluidic
material in an integrated system or microfluidic device by flowing
the material through a microchannel whose cross-sectional geometry
is configured to manipulate the dispersion rate and/or average
velocity relative to the dispersion rate and/or average velocity of
the same fluidic material in a microchannel of substantially
rectangular cross-sectional geometry. In some embodiments, such
method includes flowing at least 2 fluidic materials through the
microchannel, each material having a dispersion rate and/or average
velocity. The cross-sectional geometry of the microchannel is
optionally specifically configured to manipulate the dispersion
rates and/or average velocities of the fluidic materials to either
be the same and/or to be different rates/velocities (e.g., one
rate/velocity being 1.25, 1.5, 1.75, 2, 3, 4, 5, or 10 times
greater than the other rate/velocity). In some embodiments, the
microchannels of such systems/devices change over the length of the
microchannels. Also such systems/devices optionally comprise fluid
direction systems using one or more of electrokinetic flow,
positive pressure, negative pressure, hydrostatic pressure, or
wicking forces (or a combination of such) as well as optionally
comprising an operably attached computer attached to the detection
system for acquiring data and tracking dispersion rates and/or
average velocities of the fluidic materials.
[0010] In other aspects, the invention comprises a microchannel
with one or more region whose cross-sectional geometry is
configured to manipulate the dispersion rate and/or average
velocity of at least one fluidic material relative to the
dispersion rate and/or average velocity of the same material in a
microchannel of substantially rectangular cross-sectional geometry.
Such manipulation can be to increase and/or to decrease the
dispersion rate and/or average velocity of the fluidic material. In
some embodiments, multiple fluidic materials are flowed through the
microchannel (e.g., a first fluidic material and at least a second
fluidic material) each of which has a dispersion rate and/or
average velocity (e.g., a first dispersion rate and/or average
velocity and a second dispersion rate and/or average velocity,
etc.). The cross-sectional geometry of the microchannel is
specifically configured to optionally manipulate the first
dispersion rate and/or average velocity to be the same as the
second dispersion rate and/or average velocity or, alternatively
and/or additionally, to manipulate the first dispersion rate and/or
average velocity to be different than the second dispersion rate
and/or average velocity. The cross-sectional geometry of the
microchannel can be configured so that the first dispersion rate
and/or average velocity is, e.g., 1.25, 1.5, 1.75, 2, 3, 4, 5, or
10 times greater than the second dispersion rate and/or average
velocity.
[0011] In other aspects, the invention comprises a method of
designing a microchannel (and/or a region(s) of a microchannel)
comprising one or more region by selecting a cross-sectional
geometry to manipulate the dispersion rate and/or average velocity
of at least one fluidic material. Such microchannel can be of
multiple regions (e.g., a first region and a second region) having
different cross-sectional geometries. Such dispersion rate and/or
average velocity is optionally manipulated relative to the
dispersion rate and/or average velocity of the same fluidic
material in a microchannel of substantially rectangular
cross-sectional geometry. In some embodiments such method involves
a first fluidic material (with a first dispersion rate and/or
average velocity) and at least a second fluidic material (with a
second dispersion rate and/or average velocity). Such method can
comprise selecting a specific cross-sectional geometry of a region
of the microchannel to either make the dispersion rates and/or
average velocities of the two fluidic materials be the same and/or
for the dispersion rates and/or average velocities to be different
(e.g., the first dispersion rate and/or average velocity can be
1.25, 1.5, 1.75, 2, 3, 4, 5, or 10 times greater than the
second).
[0012] In yet other aspects, the invention comprises an integrated
system or microfluidic device for separating at least two fluidic
materials based upon a difference in the dispersion rate and/or
average velocity of the fluidic materials. Such integrated system
or microfluidic device comprises a body structure with at least one
microchannel that causes a first fluidic material (with a first
dispersion rate and/or average velocity) to have a different
dispersion rate and/or average velocity than a second fluidic
material (with a second dispersion rate and/or average velocity).
Furthermore, such microchannel has a detection region and does not
have a separation matrix for separating the fluidic materials.
Additionally, the system or device has a source of the first and
second fluidic materials (both of which sources are coupled to the
at least one microchannel and which optionally are the same
source); a fluidic direction system to move the fluidic materials
without the use of electrokinetic flow; and a detection system
proximal to the detection region. In some embodiments of the
system/device, the dispersion rate and/or average velocity of the
first fluidic materials is 1.25, 1.5, 1.75, 2, 3, 4, 5, or 10 times
greater than the dispersion rate and/or average velocity of the
second fluidic material. Additionally, in some embodiments, the
microchannel in such system or device changes its cross-sectional
geometry over the length of the microchannel. Such system or device
also has a fluidic direction system that uses one or more (or a
combination of) positive pressure, negative pressure, hydrostatic
forces, or wicking forces. Such system or device, furthermore,
optionally has a computer operably coupled to the detector system
with instructions to acquire data, track the dispersion rates
and/or average velocities of the fluidic materials, etc.
[0013] In yet other aspects, the invention comprises a method of
separating at least two fluidic materials in an integrated system
or microfluidic device based upon differences in dispersion rate
and/or average velocity of the fluidic materials. Such method
comprises flowing a first fluidic material (with a corresponding
first dispersion rate and/or average velocity) and at least a
second fluidic material (with a corresponding second dispersion
rate and/or average velocity) through a microchannel that does not
have a separation matrix and wherein the flow is not via
electrokinetic force. In some embodiments, such method involves
flowing through microchannels whose cross-sectional geometry causes
the dispersion rate and/or average velocity of the first fluidic
materials to be 1.25, 1.5, 1.75, 2, 3, 4, 5, or 10 times greater
than the dispersion rate and/or average velocity of the second
fluidic material.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 is a schematic cross-view of a microchannel of
regular cross-sectional geometry.
[0015] FIG. 2, panels A through H, are schematic cross-views of
sample specifically configured microchannels.
[0016] FIG. 3, panels A, B, and C, are schematic views of optional
embodiments of the invention comprising microchannels of various
cross-sectional geometries.
[0017] FIG. 4 is a schematic view of an integrated system
comprising a microfluidic device incorporating the elements of the
invention.
[0018] FIG. 5 is a graph representing separation of fluidic
materials in a microchannel of regular cross-sectional
geometry.
[0019] FIG. 6, panels A through E, are graphs representing
separation of fluidic materials in a microchannel of regular
cross-sectional geometry.
[0020] FIG. 7 illustrates average particle velocity relative to the
average fluid velocity versus normalized particle size in a
two-dimensional channel with Poiseuille flow.
DETAILED DISCUSSION OF THE INVENTION
[0021] The methods and devices of the current invention directly
address and solve problems associated with control and manipulation
of aliquots of fluidic material in microfluidic devices. Briefly,
the invention provides devices and methods for altering
microchannel cross-sectional geometry in order to control
dispersion and/or average velocity of various fluidic materials
(e.g., in order to separate or not to separate the various fluidic
materials); and separating various fluidic materials based upon
their dispersion rates and/or average velocity in a "regular"
cross-sectional geometry microchannel under non-electrokinetic
flow.
[0022] The methods and devices of the current invention used to
control and manipulate aliquots of fluidic material are flexible
and can be utilized in many different embodiments of microfluidic
devices which perform myriad assays, tasks, etc. The methods and
devices herein can be utilized in microfluidic devices to, e.g.,
maximize throughput time, when such is applicable to the assay(s)
being performed. For example, the screening of large libraries (or
extremely large libraries, etc) such as combinatorial libraries can
be time consuming due to the aggregation of time requirements for
each individual assay. While microfluidic devices ease the process
of such large screenings, assays that have low throughput or
non-optimized throughput can still have substantial time
requirements. The combination of elements that constitute the
methods and devices of the current invention cleverly allow for
optimizing of throughput by decreasing or eliminating intermingling
and/or spreading of sample aliquots, thus substantially decreasing
time requirements for assays in microfluidic devices. Furthermore,
the methods and devices of the present invention allow aliquots of
mixed materials to be separated into their individual components in
situations which preclude use of, e.g., electrophoretic separation,
separation matrices (e.g., gel matrices, etc.), etc. due to the
parameters/characteristics of the particular usage.
[0023] The current invention differs from other, previous, methods
and devices in numerous ways. For example, the current invention
utilizes, e.g., specific alterations of microchannel
cross-sectional geometry to manipulate/control dispersion and
average velocity of fluidic aliquots. Additionally, the current
invention utilizes differences in the dispersion rates and/or
average velocity of fluidic materials in "regular" shaped
microchannels to separate such materials without the use of, e.g.,
electrophoresis, separation matrices, etc. The combination of these
elements allows for adjustment and modification of balances between
the several elements of the invention in order to, e.g., optimize
throughput for the specific assay(s) to be performed, allow for
specific needs of particular constituents (e.g., ones that cannot
be separated through separation matrices, etc.), etc.
[0024] The present invention also optionally includes various
elements involved in, e.g., transporting fluidic materials
involved, reconstitution of dried or immobilized samples,
temperature control, fluid transport mechanisms, detection and
quantification of molecular interactions (e.g., fluorescence
detectors), robotic devices for, e.g., positioning of components or
devices involved, etc.
METHODS AND DEVICES OF THE INVENTION
[0025] Manipulation/control of molecules, compounds, etc. in
microfluidic devices is often done within one or more microchannels
(sometimes referred to herein as microfluidic channels) or
microreservoirs, etc. The term "microfluidic," as used herein,
refers to a device component, e.g., chamber, channel, reservoir, or
the like, that includes at least one cross-sectional dimension,
such as depth, width, length, diameter, etc., of from about 0.1
micrometer to about 500 micrometer. Examples of microfluidic
devices are detailed in, e.g., U.S. Pat. No. 5,942,443 issued Aug.
24, 1999, entitled "High Throughput Screening Assay Systems in
Microscale Fluidic Devices" to J. Wallace Parce et al. and U.S.
Pat. No. 5,880,071 issued Mar. 9, 1999, entitled "Electropipettor
and Compensation Means for Electrophoretic Bias" to J. Wallace
Parce et al., both of which are incorporated herein by reference
for all purposes.
[0026] In general, microfluidic devices are planar in structure and
are constructed from an aggregation of planar substrate layers
wherein the fluidic elements, such as microchannels, etc., are
defined by the interface of the various substrate layers. The
microchannels, etc. are usually etched, embossed, molded, ablated,
or otherwise fabricated into a surface of a first substrate layer
as grooves, depressions, or the like. A second substrate layer is
subsequently overlaid on the first substrate layer and bonded to it
in order to cover the grooves, etc. in the first layer, thus
creating sealed fluidic components within the interior portion of
the device. Optionally, either one or both substrate layer has
microchannels devised within it. Such microchannels can be aligned
one on top of another when the substrate layers are joined
together. Such microchannels as thus constructed can be symmetrical
(i.e., the microchannel on the first substrate is the same shape as
that of the microchannel on the second substrate, thus forming a
symmetrical microchannel when the two substrate layers are joined,
see, e.g., FIGS. 2B, E, and F) or such microchannels can be
asymmetrical (i.e., the microchannel on the first substrate is a
different shape from that of the microchannel on the second
substrate, thus forming an asymmetrical channel when the two
substrate layers are joined, see, e.g., FIGS. 2A, D, G, and H).
Additionally, open-well elements can be formed by making
perforations in one or more substrate layers, which perforation
optionally can correspond to depressed microreservoir,
microchannel, etc. areas on the complementary layer.
[0027] Manufacturing of these microscale elements into the surface
of the substrates can be carried out through any number of
microfabrication techniques that are well known in the art. For
example, lithographic techniques are optionally employed in
fabricating, e.g., glass, quartz, or silicon substrates, using
methods well known in the semiconductor manufacturing industries,
such as photolithographic etching, plasma etching, or wet chemical
etching. Alternatively, micromachining methods such as laser
drilling, micromilling, and the like are optionally employed.
Similarly, for polymeric substrates, well known manufacturing
techniques may also be used. These techniques include injection
molding or stamp molding methods wherein large numbers of
substrates are optionally produced using, e.g., rolling stamps to
produce large sheets of microscale substrates, or polymer
microcasting techniques where the substrate is polymerized within a
micromachined mold. Furthermore, various combinations of such
techniques are optionally combined to produce the microelements
present in the current invention.
[0028] As stated above, the substrates used to construct the
microfluidic devices of the invention are typically fabricated from
any number of different materials, depending upon, e.g., the nature
of the samples to be assayed, the specific reactions and/or
interactions being assayed for, etc. For some applications, the
substrate can optionally comprise a solid non-porous material. For
example, the substrate layers can be composed of, e.g.,
silica-based materials (such as glass, quartz, silicon, fused
silica, or the like), polymeric materials or polymer coatings on
materials (such as polymethylmethacrylate, polycarbonate,
polytetrafluoroethylene, polyvinylchloride, polydimethylsiloxane,
polysulfone, polystyrene, polymethylpentene, polypropylene,
polyethylene, polyvinylidine fluoride,
acrylonitrile-butadiene-styrene copolymer, parylene or the like),
ceramic materials, metal materials, etc.
[0029] The surface of a substrate layer may be of the same material
as the non-surface areas of the substrate or, alternatively, the
surface may comprise a coating on the substrate base. Furthermore,
if the surface is coated, the coating optionally can cover either
the entire substrate base or can cover select subparts of the
substrate base. For example, in the case of glass substrates, the
surface of the glass of the base substrate may be treated to
provide surface properties that are compatible and/or beneficial to
one or more sample or reagent being used. Such treatments include
derivatization of the glass surface, e.g., through silanization or
the like, or through coating of the surface using, e.g., a thin
layer of other material such as a polymeric or metallic material.
Derivatization using silane chemistry is well known to those of
skill in the art and can be readily employed to add, e.g., amine,
aldehyde, or other functional groups to the surface of the glass
substrate, depending upon the desired surface properties. Further,
in the case of metal substrates, metals that are not easily
corroded under potentially high salt conditions, applied electric
fields, and the like are optionally preferred.
[0030] Although described in terms of a layered planar body
structure, it will be appreciated that microfluidic devices in
general and the present invention in particular can take a variety
of forms, including aggregations of various fluidic components such
as capillary tubes, individual chambers, arrangement of channel(s)
etc., that are pieced together to provide the integrated elements
of the complete device. For example, FIG. 3, panels A, B, and C,
illustrates one of many possible arrangements of the elements of
the present invention. In one such possible arrangement, as shown
in FIG. 3, body structure 302 has main channels 304 and 306
disposed therein, which are fluidly connected to various reservoirs
that can optionally contain, e.g., buffer, reagents, etc. Channel
304 as presented in FIG. 3 comprises a microchannel whose
cross-sectional geometry has been specifically configured to
manipulate the dispersion rate and/or average velocity of one or
more fluidic material flowed through the microchannel.
Alternatively, only a sub-portion or sub-region of channel 304 is
so configured. Channel 306 as presented in FIG. 3 comprises a
microchannel of "regular" shape, as described herein, whose effect
on the dispersion rate and/or average velocity of fluidic materials
flowed through the channel is used to separate such fluidic
materials without the use of, e.g., separation matrices,
electrophoresis, etc.
[0031] The microfluidic devices of the invention typically include
at least one main channel (herein, as termed a specifically
configured microchannel and/or a "regular" microchannel), where,
e.g., analysis, separations, etc. are performed, but may include
two or more main channels in order to multiplex the number of
analyses being carried out in the microfluidic device at any given
time. Typically, a single device will include from about 1 to about
100 or more separate main channels, which main channel(s) are often
ones specifically configured in cross-sectional areas and/or
"regular" cross-sectional channels for separation as well. In most
cases, the main channel is intersected by at least one other
microscale channel disposed within the body of the device.
Typically, the one or more additional channels are used, e.g., to
bring the samples, test compounds, assay reagents, etc. into the
main channel, in order to carry out the desired assay, separation,
etc. Additionally, the main channel can be intersected by one or
more shunt microchannels as well.
[0032] The reservoirs or wells of microfluidic devices
incorporating the methods and devices of the current invention are
locations at which samples, components, reagents, and the like are
added into the device for assays, etc. to take place. Introduction
of these elements into the system is carried out as described
herein. The reservoirs are typically placed so that the sample or
reagent is added into the system upstream from the location at
which it is used. For example, a dilution buffer is added upstream
from the source of a reagent if the sample is to be diluted before
reaction with the reagent. Alternatively, waste wells or reservoirs
are used to store samples after a reaction or assay has been
completed. The removal of the completed samples provides space in
the channels to load and incubate other samples. In this fashion,
the devices of the invention are optionally used in a high
throughput manner. The throughput is maintained by continuously
loading, incubating, and unloading samples into and from the
incubation channels of the device.
[0033] In the present invention, a dilution buffer is typically
added into a main channel upstream of a shunt channel, so that the
increase in flow rate due to the addition of buffer material
downstream of its entry point may be counteracted by the reduction
in pressure due to the shunt channel. Reagent materials, on the
other hand, are typically added downstream of a shunt channel so
that they are added after the downstream flow rate in the main
channel has been reduced, so that smaller quantities of reagent are
added.
[0034] In these systems, a "capillary element" (a channel in which
fluidic materials can be moved from a source to a microscale
element) or other similar pipettor element is temporarily or
permanently coupled to a source of fluidic material. The source of
the fluidic material can be internal or external to the
microfluidic device comprising the capillary element. Example
sources include microwell plates, membranes, or other solid
substrates comprising lyophilized components, wells, or reservoirs
in the body of the microscale device itself, etc.
[0035] For example, the source of a cell type, sample, or buffer
can be a microwell plate external to the body structure of the
microfluidic device, having at least one well with a sample of
interest, i.e., the sample plug(s) and/or buffer plug(s) to be
drawn into the device will be within the microwell plate.
Alternatively, the fluidic material source is a well or reservoir
disposed on the surface or within the body of the structure of the
microfluidic device comprising a selected cell type, component,
reagent, etc.; a container external to the body structure of the
microfluidic device comprising at least one compartment comprising
the selected particle type, component, reagent, etc.; or a solid
phase structure comprising the selected cell type, reagent, etc. in
lyophilized or otherwise dried form.
[0036] Manipulation/Control of Fluidic Material Within Microfluidic
Devices
[0037] The present invention provides methods and devices for
manipulating and controlling aliquots of fluidic materials in
microfluidic devices and systems by utilizing (and/or changing)
differences in dispersion rate and/or average velocity of different
fluidic materials as such materials pass through the channels of
the device or system. The present invention is applicable to both
homogeneous and non-homogeneous assays.
[0038] As used herein, the term "dispersion" refers to the
convection-induced, longitudinal dispersion of material within a
fluid medium due to velocity variations across streamlines, e.g.,
in pressure driven flow systems, electrokinetically driven flow
systems around curves and corners, and electrokinetically driven
flow systems having non-uniform buffer ionic concentrations, e.g.,
plugs of high and low salt solutions within the same channel
system. For the purposes of the present invention, dispersion is
generally defined as that due to the coupling between flow and
molecular diffusion, i.e., Taylor dispersion. In this regime, the
time-scale for dispersion due to convective transport is long or
comparable to the time scale for molecular diffusion in the
direction orthogonal to the flow direction. For discussions on
dispersion and Taylor dispersion in particular, see, e.g., Taylor
et al., Proc. Roy. Soc. London, (1953) 219A:186-203; Aris, Proc.
Roy. Soc. London (1956) A235:67-77; Chatwin et al., J. Fluid Mech.
(1982) 120:347-358; Doshi et al., Chem. Eng. Sci. (1978)
33:795-804; and Guell et al., Chem. Eng. Comm. (1987) 58:231-244,
each of which is incorporated herein by reference for all purposes.
Channel design optimization in light of dispersion and diffusion of
serially introduced reagents is described in "Methods and Software
for Designing Microfluidic Devices," U.S. Ser. No. 09/277,367 filed
Mar. 26, 1999, by Chow et al. and in "Optimized High-Throughput
Analytical System," U.S. Ser. No. 09/233,700 filed Jan. 19, 1999,
by Kopf-Sill et al., which are incorporated herein by reference for
all purposes. For more information on dispersion as it relates to
high throughput in microfluidic devices, see, e.g., U.S. Pat. No.
6,150,119 issued Nov. 21, 2000, entitled "Optimized High-Throughput
Analytical System" to A. Kopf-Sill et al., which is incorporated
herein by reference for all purposes.
[0039] In typical microfluidic devices fluid is moved through
micro-etched channels via electrokinetic flow (electrophoresis or
electroosmosis) or through the application of small pressure
differentials. In the absence of bends in the channels,
electrokinetically driven flows do not produce convective
dispersion. In electrokinetic flow, all solute molecules across the
microchannel travel with the same velocity, hence no shear results
from this motion, and therefore no Taylor dispersivity is produced.
However, pressure driven flows of small molecule fluidic material
i.e., colloidal material (less than 1 .mu.m) which may include
small molecular weight material, through a channel (e.g., a
microchannel as is used herein) can lead to large amounts of
dispersion. This is also true when a pressure gradient is produced
unintentionally, e.g., through the result of hydrostatic pressure
differentials or mismatches in electrokinetic flow rates along a
microchannel. The cause of such dispersion is that the convective
velocity of the fluidic material is lower near the walls than it is
in the center of the microchannel, and thus a plug of fluidic
material that starts out as discrete will spread out as convection
proceeds in the axial direction. Ultimately this convection
spreading process is cut off by diffusion across streamlines. This
results in an effective dispersivity "K." For example, in flow of a
fluidic material through a tube, the dispersivity in the axial
direction is given by K D = 1 + 1 48 .times. ( Ua D ) 2 ##EQU1##
where U is the average velocity in the tube, a is the tube radius,
and D is the molecular diffusivity. See, Taylor, Proc. Roy. Soc.
(1953) 219A:186-203.
[0040] In a rectangular or isotropically etched channel that is
substantially rectangular, the dispersivity is determined by K D =
1 + 1 210 .times. f ( d w ) .times. ( Ud D ) 2 ##EQU2## where d is
the channel depth and w is the channel width. The function f(d/w)
is dependent on the aspect ratio of the channel. For very small
(d/w) ratio, f approaches the value 8 in a rectangular channel. See
Doshi et al., Chem. Eng. Sci (1978) 3:795-804.
[0041] In the microchannels of the present invention, the
dispersion of small molecule fluidic material is dictated by the
Taylor-Aris dispersion mechanism, which is an interplay between
convection and molecular diffusion. For the majority of systems
involving small molecule fluidic materials, the Taylor dispersivity
(or Taylor-Aris dispersion) is much greater than the molecular
diffusivity, and thus the Taylor dispersivity predominantly
controls the spread of the plug or band (i.e., the aliquot) of the
fluidic material. The Taylor-Aris dispersion of a fluidic material
can be modified by modifying the cross-sectional geometry of the
conduit (e.g., microchannel) in which the material flows.
[0042] The dispersion of fluidic materials that are not small,
e.g., cells, beads, etc., is not controlled by Taylor-Aris
dispersion. Instead, because of their large size, thermal diffusion
is negligible and their dispersion is controlled by convection
only. Consequently, the dispersion rate of non-Brownian materials,
e.g., cells, beads, etc., is generally larger than that for
colloidal or Brownian fluidic materials in, e.g., microchannels.
Additionally, they can flow at an average velocity different than
that of the small molecule or Brownian fluidic materials. The
difference in velocity is determined by two factors. First, the
center of mass of larger non-Brownian fluidic materials (i.e.,
non-small molecules) is excluded from the region near the channel
wall (i.e., the slower flowing region) comparable to the radius of
the non-small molecule materials. This factor causes the non-small
molecular non-Brownian materials to flow faster than the small
molecule materials in the same channel. Second, hydrodynamic
interactions with the wall slow down the flow of the non-Brownian
materials compared to the Brownian or small molecular materials in
the same streamline. The importance of these two opposing factors
can be controlled by varying the ratio of the half channel depth
(h) to the radius of the non-Brownian materials (r) to the half
channel depth (h). At very small values of (r/h), the velocity
ratio of the non small to small molecule fluidic materials reaches
unity. As the ratio (r/h) increases, the velocity ratio increases
initially and then decreases as is shown by Staben et al. in
"Motion of a particle between two parallel plane walls in
low-Reynolds number Poiseuille Flow" Physics of Fluid. Staben et
al. show that particles that have diameters that are 42% of the
channel depth have a maximum average velocity that is greater than
the average fluid velocity; however, this is not the case for
particles that have diameters greater than 82% of the channel
depth. These large particles were found to have smaller average
velocities than the fluid. See FIG. 7 illustrating the average
particle velocity relative to the average fluid velocity in
relation to particle size in a two-dimensional channel with
Poiseuille flow. While the differences in dispersion rates and/or
average velocity between small molecule fluidic materials and
non-small molecule fluidic materials such as cells, as outlined
above, can be problematic in some assay situations, the current
invention utilizes such in the methods and devices herein to
produce desired manipulations/controls of the fluidic
materials.
[0043] The current invention cleverly takes advantage of the
varying dispersion rates and/or average velocity of small molecule
fluidic materials (which are governed by Taylor-Aris dispersion)
and non-small molecule fluidic materials, such as cells (which are
governed solely by convection). For example, the methods and
devices of the current invention utilize the above disparities to
perform separations between fluidic materials without having to
resort to use of, e.g., electrophoresis, gel matrices, etc.
Furthermore, again, as illustrated in more detail below, the
methods and devices of the current invention utilize the
differences in dispersion rates and/or average velocity produced by
changed cross-sectional geometries of microchannels to
manipulate/control aliquots of fluidic material (e.g., to keep
disparate fluidic materials such as cells and small molecule
compounds together in the same fluidic plug or, conversely, to
separate such fluidic materials into different fluidic plugs).
[0044] For example, a microfluidic device can be designed to
include at least one microchannel that includes at least one region
comprising a first cross-sectional geometry along its length which
is shaped and dimensioned to enhance a dispersion of at least one
of at least two differently sized particles flowing in a fluid
through the microchannel such that the at least two particles have
substantially the same average velocity in the at least one region
of the microchannel, as shown for example in FIGS. 2C, 2D, and 2E,
wherein the microchannel includes lateral wings that are shallower
than a central portion of the channel. Such a configuration with
shallower side wings can help enhance the dispersion of small
compounds such as test compounds, and thus further increase their
average velocity, relative to flowing cells in the microchannel in
order to substantially match the average velocity of the compounds
relative to the average velocity of the cells in the microchannel.
Such a cross-sectional geometry configuration may find particular
applicability in high-throughput screening applications as
described, for example, in U.S. Pat. No. 5,942,443, which is
incorporated by reference herein in its entirety. In
high-throughput screening of cell-based assays as described in the
'443 patent, the compounds (e.g., potential drug candidates) are
brought in as discrete bands in a serial manner, and the throughput
is dictated by how far apart the compound bands must be spaced. In
cell-based assays, cells typically move at an average velocity and
dispersion rate that is higher than those of the compounds. To
avoid dispersion between the compound bands, typically the bands
are increased in size to ensure that some of the cells are always
in contact with a given compound for a required incubation period.
However, the throughput of the system decreases with long compound
bands. In order to substantially match the velocity and/or
dispersion of the cells and compounds (or any other differently
sized/charged/mass species) in the microchannel, the
cross-sectional geometry of the microchannel can be configured as
described above with reference to FIGS. 2A and 2C-E to enhance the
dispersion of the small compounds relative to the cells so that the
average velocity of the two species is substantially equal.
[0045] On the other hand, in other applications such as
cell-washing, one may want to enhance the differential velocities
of particles or molecules (e.g., target drug compounds versus
cells) flowing down the microchannel. Again, the cross-sectional
geometry of the microchannel can be manipulated to accomplish that
goal. For example, a microfluidic device can be designed to include
at least one microchannel that has at least one (or more) region
along its length having a first cross-sectional geometry that is
shaped and dimensioned to enhance a differential velocity of at
least one of at least two differently sized particles flowing in a
fluid through the microchannel, such as shown in FIGS. 2F and 2H,
for example. Of course, the microchannel can further include at
least a second region having a second cross-sectional geometry that
is dimensioned and shaped to alter the dispersion of at least one
of the least two differently sized particles flowing through the
microchannel such that the particles have a different average
velocity as they flow through the microchannel, as discussed
above.
[0046] As used herein, the term "cross-sectional geometry,"
"channel geometry," "geometry," etc. is to be understood to
optionally include the dimension/size of the channel (i.e., of the
elements of the channel such as height, depth, wall curvature,
etc.) as well as the layout/pattern of the channel (i.e., the
arrangement of the elements of the channel such as wall height,
curvature, placement of any troughs/ridges/etc.). In other words,
either or both of the dimension/size of a microchannel (or its
elements) or the layout/pattern of a microchannel (i.e., of its
elements) are included within its "cross-sectional geometry" and
are manipulated herein in order to perform the separations, etc. of
the current invention.
[0047] As used herein, some microchannels are described as
"regular," "non-specifically configured," "rectangular,"
"substantially rectangular," or the like (e.g., the separation of
mixed fluidic materials in "regular" microchannels without the use
of electrophoresis or matrices, etc.). Such channels are typically
ones similar to that illustrated in cross-section in FIG. 1. As
shown in FIG. 1, the microchannel has basically vertical sides and
a horizontal bottom with rounded transitions between the side walls
and the bottom (i.e., depth d, width l, side transition regions of
radius r as used in FIG. 1). Such microchannels are typically
fabricated by isotropic etching processes as used to construct the
microfluidic devices of the current invention. It is to be
understood that other similarly shaped microchannels (i.e., ones of
slightly different character but basically the same shape) are also
included in the preceding terms (e.g., "regular," etc.). For
example, the bottom of a regular microchannel can comprise a
concave or "half-moon" shape, etc. The area of a regular
microchannel (e.g., one as is shown in FIG. 1) is given by the
equation: A = r 2 .function. ( .pi. 2 + l d ) ##EQU3##
[0048] Additionally, as used herein, some microchannels are
described as "configured," "specifically configured," etc. Such
channels can comprise a myriad of channel shapes depending upon the
specific end result desired. For example, non-limiting exemplars of
specifically configured microchannels are shown in FIGS. 2A, 2C-2F,
and 2H. Such microchannels are similar in some ways to a "regular"
microchannel as shown in FIG. 1, but contain further complexity in
their cross-sectional geometry. While the regular microchannel in
FIG. 1 can be produced by typical isotropic-etching techniques, the
more complex "specifically configured" microchannels in FIG. 2 are
optionally fabricated through, e.g., a double (or triple, etc.)
etching technique. Additionally, and/or alternatively, two
substrate layers can be etched (either isotropically or otherwise)
and then placed (e.g., joined, bonded, etc.) together to form the
microchannel. Each substrate layer can be etched in either the same
or different geometry, thus resulting in either a symmetrical
channel geometry (see, e.g., FIG. 2E) or an asymmetrical channel
geometry (see, e.g., FIG. 2D). Again, depending upon the specific
end result desired (e.g., minimizing Taylor-Aris dispersion;
separating, either quickly or slowly, a plug comprising a number of
mixed fluidic materials into separate bands of single fluidic
material; keeping disparate types of fluidic materials, such as,
e.g., cells and small molecule compounds, together in the same
plug; etc.), the cross-sectional geometry of specifically
configured microchannels varies in different embodiments of the
current invention, and FIG. 2 represents only several of the many
possible configurations of the invention.
[0049] The cross-sectional geometry of microchannels of the present
invention, be they regular or specifically configured, can change
over their length. In other words, a microchannel can change from,
e.g., a channel as shown in FIG. 1 to, e.g., one having a
perpendicular transition between the side walls and the bottom, or,
e.g., from one as shown in FIG. 1 to any one as shown in FIG. 2 (or
any other specifically configured microchannel) all depending upon
the particular needs of the assays/systems used.
[0050] In order to correctly manipulate/control the dispersion
rates and/or average velocity of the fluidic materials flowed
through the "regular" channels involved in the methods and devices
of the current invention, the dispersivity of materials flowing in
the microchannels used in the microfluidic devices of the current
invention is determined. This is done by using the method of
moments (see, e.g., Aris, Proc. Roy. Soc. (1956) 235A:67-77) to
calculate the dispersivity for a channel of particular
cross-section geometry. Such process involves first calculating the
velocity field within the channel, wherein u is the unidirectional
velocity in the direction z. The dimensionless velocity
distribution is controlled by the Poisson equation:
.gradient.*.sup.2u*=-1; u*|.sub..differential.D*=0 wherein the
unidirection velocity is rendered dimensionless with respect to the
characteristic velocity U.sub.c, and all lengths are held
dimensionless with respect to the channel depth d. Thus, u * = u U
c ; .times. U c = ( - .DELTA. .times. .times. p L ) .times. d 2
.mu. . ##EQU4## Because there is no variability in the direction z,
the Laplacian .gradient.*.sup.2 is the two-dimensional Laplacian.
Once the dimensionless velocity u* is determined, the average
velocity in channel U is determined by: U = U c .times. 1 A *
.times. .intg. D * .times. u * .times. .times. d A * ##EQU5##
wherein A*=A/d.sup.2. Once the above velocity is determined, it is
used to re-normalize velocity u* by: u ^ = u U . ##EQU6##
[0051] The determination for Taylor-Aris dispersivity thus reduces
to the simple integral: K D = 1 + ( Ud D ) 2 .times. 1 A * .times.
.intg. D * .times. u ^ .times. g ^ .times. .times. d A * ##EQU7##
wherein the function is the solution to the following problem:
.gradient. * 2 .times. g ^ = 1 - u ^ ; .times. .gradient. .fwdarw.
.times. * g ^ n .fwdarw. .times. .differential. D * = 0 ; .times.
.intg. D * .times. g ^ .times. .times. d A * = 0. ##EQU8##
[0052] While the above equations are used to determine dispersivity
in channels of regular cross-sectional geometry (e.g., ones such as
shown in FIG. 1 used to, e.g., separate mixed fluidic materials
without the use of electrophoresis, matrices or the like), by
accounting for the correct cross-sectional geometry, the equations
can be adapted for use with microchannels of specifically
configured cross-sectional geometry (e.g., ones used to keep
specific fluidic materials, e.g., in the same fluidic plug or to
separate such into separate plugs).
[0053] Illustrative Examples of Sample Microfluidic Device
Incorporating Manipulation/Control of Aliquots of Fluidic
Materials
[0054] As stated previously, the use of pressure driven flow in
microfluidic devices often leads to large amounts of Taylor
dispersion. Furthermore, even use of electroosmotic flow or
hydrostatic pressure driven flow can lead to occurrence of Taylor
dispersion due to small pressure gradients that may arise due to
mismatch of forces. The present invention utilizes the dependence
of Taylor-Aris dispersivity on a microchannel's cross-sectional
geometry to control (e.g., minimize) dispersion of materials in
"specifically configured" microfluidic channels. The present
invention also cleverly takes advantage of such differences in
dispersion and/or flow rate to manipulate and control aliquots of
fluidic materials in microfluidic devices. The present invention
uses the mismatch of dispersion rates and/or average velocity
between different fluidic materials in herein termed "regular"
channels to allow separation between such fluidic materials without
having to use, e.g., electrophoresis, separation matrices (e.g.,
gel matrices), etc. Additionally, the present invention
specifically configures the cross-sectional geometry of
microchannels in order to take advantage of, and to change,
differences in dispersion rate and/or average velocity between
different fluidic materials in specifically configured
microchannels (i.e., in microchannels whose cross-sectional
geometry has been specifically configured to achieve such desired
result).
[0055] Not only can configuration of microchannel cross-sectional
geometry, as described herein, allow for the separation of
different fluidic materials based upon varying dispersion rates
and/or average velocity, but such configuration can also allow
non-separation of different fluidic materials. In other words, the
specifically configured microchannels herein can allow fluidic
materials which would normally separate (i.e., in a "regular"
channel shape) in flow due to their different dispersion/average
velocity rates to NOT separate, and thus stay in the same "plug" or
aliquot in a microchannel.
[0056] By having the specific configuration of a microchannel
change over its course, myriad effects can be achieved. For
example, one fluidic material can be "washed" by another fluidic
material. In such a case, e.g., cells (containing a specific
receptor) and a ligand specific for such receptor can be mixed into
the same aliquot and flowed through a specifically configured
microchannel of the present invention. The specific configuration
of the microchannel thus keeps the, e.g., cells and ligand together
in the same plug where otherwise the cells and the ligand would
tend to separate into separate bands due to their different rates
of dispersion and/or average velocity. Once the cells and ligand
have been in each other's presence for the required period of time,
the mixed plug can flow through a different section of microchannel
which has been specifically configured to maximize the differences
between the dispersion rates and/or average velocity of the two
components and thus separate them into different bands.
Alternatively, the mixed plug could be flowed through a "regular"
cross-sectional geometry channel in order for the cells to be
washed free of the unbound ligand, again, due to differences
between the dispersion rates and/or average velocity of the two
components.
[0057] Sample plugs (i.e., discrete aliquots of sample) within
microfluidic elements of microfluidic devices, such as those using
the devices/methods of the current invention, often undergo a
blurring or smearing of their original boundaries. Such blurring is
typically caused by diffusion and/or dispersion of the sample
plug.
[0058] The methods and devices of the present invention are useful
in numerous situations, for example, they can be used to maximize
throughput of serially introduced samples by preventing or reducing
unwanted intermingling of fluidic materials in microfluidic devices
or systems (i.e., by reducing dispersion of fluidic plugs). The
"throughput" of a microfluidic device/system/channel is typically
defined as the number of different materials that can be serially
introduced into the device/system/channel per unit time. Compounds
that are screened at rates greater than one compound per minute
within a single channel are generally termed high throughput, while
screening of compounds at a rate greater than one compound per 10
seconds generally falls into the ultra-high throughput category.
Decreasing the amount of unwanted intermingling of sample plugs
allows for minimization of spacing between serially introduced
materials, e.g., samples, thus allowing a greater number of
different materials, e.g., samples, to be serially introduced into
the microfluidic device per unit time. The closer that plugs are
able to be loaded, the more plugs that can be analyzed per unit
time.
[0059] Spacers and/or buffers are optionally used to keep samples
separated and/or prevent mixing of samples. For example, a buffer
is optionally loaded into a channel after each sample plug in order
to separate samples from one another and prevent contamination
between samples. A sample plug includes an initial sample aliquot
and any products produced by incubation or reaction of the initial
sample aliquot. The buffer plugs optionally can comprise immiscible
fluids to decrease diffusion. Buffer plug lengths are calculated in
the same way as sample plug lengths, e.g., based on diffusivity
and/or dispersion of the material. For example a buffer plug is
typically 500 .mu.m to 5 mm, preferably 600 .mu.m to 3 mm or 850
.mu.m 1 mm. The last buffer plug loaded or added into a channel or
the device is optionally longer, e.g., 500 um to about 10 mm, to,
e.g., allow for flow pinching.
[0060] As stated previously, the elements (i.e., methods and
devices) of the current invention can be incorporated into numerous
microfluidic devices that perform any number of different assays,
tasks, etc. Whenever throughput needs to be optimized, the elements
of the current invention can be combined and interlaced to help
achieve proper (or more efficient) throughput. High throughput
assays are useful in, e.g., diagnostic assays, genomic assays, and,
in particularly preferred aspects, pharmaceutical screening assays.
The various types of assays that benefit from such systems and
methods as are found within the present invention are described
generally in Published International Patent Application Nos.
98/00231 and 98/00705, which are incorporated herein by reference
in their entirety for all purposes.
[0061] Additionally, the methods and devices of the current
invention are readily incorporated into numerous microfluidic
devices that require manipulation/control of fluidic materials to,
e.g., keep mixed fluidic materials in the same plug(s) and/or
separate plugs of mixed fluidic materials into separate plugs
(without having to resort to use of separating matrices and with or
without use of electrophoresis). These systems are described in
numerous publications by the inventors and their coworkers. These
include certain issued U.S. Patents, including U.S. Pat. Nos.
5,699,157 (J. Wallace Parce) issued Dec. 16, 1997, U.S. Pat. No.
5,779,868 (J. Wallace Parce et al.) issued Jul. 14, 1998, U.S. Pat.
No. 5,800,690 (Calvin Y. H. Chow et al.) issued Sep. 1, 1998, U.S.
Pat. No. 5,842,787 (Anne R. Kopf-Sill et al.) issued Dec. 1, 1998,
U.S. Pat. No. 5,852,495 (J. Wallace Parce) issued Dec. 22, 1998,
U.S. Pat. No. 5,869,004 (J. Wallace Parce et al.) issued Feb. 9,
1999, U.S. Pat. No. 5,876,675 (Colin B. Kennedy) issued Mar. 2,
1999, U.S. Pat. No. 5,880,071 (J. Wallace Parce et al.) issued Mar.
9, 1999, U.S. Pat. No. 5,882,465 (Richard J. McReynolds) issued
Mar. 16, 1999, U.S. Pat. No. 5,885,470 ( J. Wallace Parce et al.)
issued Mar. 23, 1999, U.S. Pat. No. 5,942,443 (J. Wallace Parce et
al.) issued Aug. 24, 1999, U.S. Pat. No. 5,948,227 (Robert S.
Dubrow) issued Sep. 7, 1999, U.S. Pat. No. 5,955,028 (Calvin Y. H.
Chow) issued Sep. 21, 1999, U.S. Pat. No. 5,957,579 (Anne R.
Kopf-Sill et al.) issued Sep. 28, 1999, U.S. Pat. No. 5,958,203 (J.
Wallace Parce et al.) issued Sep. 28, 1999, U.S. Pat. No. 5,958,694
(Theo T. Nikiforov) issued Sep. 28, 1999, U.S. Pat. No. 5,959,291
(Morten J. Jensen) issued Sep. 28, 1999, U.S. Pat. No. 5,964,995
(Theo T. Nikiforov et al.) issued Oct. 12, 1999, U.S. Pat. No.
5,965,001 (Calvin Y. H. Chow et al.) issued Oct. 12, 1999, U.S.
Pat. No. 5,965,410 (Calvin Y. H. Chow et al.) issued Oct. 12, 1999,
U.S. Pat. No. 5,972,187 (J. Wallace Parce et al.) issued Oct. 26,
1999, U.S. Pat. No. 5,976,336 (Robert S. Dubrow et al.) issued Nov.
2, 1999, U.S. Pat. No. 5,989,402 (Calvin Y. H. Chow et al.) issued
Nov. 23, 1999, U.S. Pat. No. 6,001,231 (Anne R. Kopf-Sill) issued
Dec. 14, 1999, U.S. Pat. No. 6,011,252 (Morten J. Jensen) issued
Jan. 4, 2000, U.S. Pat. No. 6,012,902 (J. Wallace Parce) issued
Jan. 11, 2000, U.S. Pat. No. 6,042,709 (J. Wallace Parce et al.)
issued Mar. 28, 2000, U.S. Pat. No. 6,042,710 (Robert S. Dubrow)
issued Mar. 28, 2000, U.S. Pat. No. 6,046,056 (J. Wallace Parce et
al.) issued Apr. 4, 2000, U.S. Pat. No. 6,048,498 (Colin B.
Kennedy) issued Apr. 11, 2000, U.S. Pat. No. 6,068,752 (Robert S.
Dubrow et al.) issued May 30, 2000, U.S. Pat. No. 6,071,478 (Calvin
Y. H. Chow) issued Jun. 6, 2000, U.S. Pat. No. 6,074,725 (Colin B.
Kennedy) issued Jun. 13, 2000, U.S. Pat. No. 6,080,295 (J. Wallace
Parce et al.) issued Jun. 27, 2000, U.S. Pat. No. 6,086,740 (Colin
B. Kennedy) issued Jul. 11, 2000, U.S. Pat. No. 6,086,825 (Steven
A. Sundberg et al.) issued Jul. 11, 2000, U.S. Pat. No. 6,090,251
(Steven A. Sundberg et al.) issued Jul. 18, 2000, U.S. Pat. No.
6,100,541 (Robert Nagle et al.) issued Aug. 8, 2000, U.S. Pat. No.
6,107,044 (Theo T. Nikiforov) issued Aug. 22, 2000, U.S. Pat. No.
6,123,798 (Khushroo Gandhi et al.) issued Sep. 26, 2000, U.S. Pat.
No. 6,129,826 (Theo T. Nikiforov et al.) issued Aug. 10, 2000, U.S.
Pat. No. 6,132,685 (Joseph E. Kersco et al.) issued Oct. 17, 2000,
U.S. Pat. No. 6,148,508 (Jeffrey A. Wolk) issued Nov. 21, 2000,
U.S. Pat. No. 6,149,787 (Andrea W. Chow et al.) issued Nov. 21,
2000, U.S. Pat. No. 6,149,870 (J. Wallace Parce et al.) issued Nov.
21, 2000, U.S. Pat. No. 6,150,119 (Anne R. Kopf-Sill et al.) issued
Nov. 21, 2000, U.S. Pat. No. 6,150,180 (J. Wallace Parce et al.)
issued Nov. 21, 2000, U.S. Pat. No. 6,153,073 (Robert S. Dubrow et
al.) issued Nov. 28, 2000, U.S. Pat. No. 6,156,181 (J. Wallace
Parce et al.) issued Dec 5, 2000, U.S. Pat. No. 6,167,910 (Calvin
Y. H. Chow) issued Jan. 2, 2001, U.S. Pat. No. 6,171,067 (J.
Wallace Parce) issued Jan. 9, 2001, U.S. Pat. No. 6,171,850 (Robert
Nagle et al.) issued Jan. 9, 2001, U.S. Pat. No. 6,172,353 (Morten
J. Jensen) issued Jan. 9, 2001, U.S. Pat. No. 6,174,675 (Calvin Y.
H. Chow et al.) issued Jan. 16, 2001, U.S. Pat. No. 6,182,733
(Richard J. McReynolds) issued Feb. 6, 2001, U.S. Pat. No.
6,186,660 (Anne R. Kopf-Sill et al.) issued Feb. 13, 2001, U.S.
Pat. No. 6,221,226 (Anne R. Kopf-Sill) issued Apr. 24, 2001, U.S.
Pat. No. 6,233,048 (J. Wallace Parce) issued May 15, 2001, U.S.
Pat. No. 6,235,175 (Robert S. Dubrow et al.) issued May 22, 2001,
U.S. Pat. No. 6,235,471 (Michael Knapp et al.) issued May 22, 2001,
and U.S. Pat. No. 6,238,538 (J. Wallace Parce et al.) issued May
29, 2001.
[0062] These systems are also described in various PCT applications
by the inventors including, e.g., WO 98/00231, WO 98/00705, WO
98/00707, WO 98/02728, WO 98/05424, WO 98/22811, WO 98/45481, WO
98/45929, WO 98/46438, and WO 98/49548, WO 98/55852, WO 98/56505,
WO 98/56956, WO 99/00649, WO 99/10735, WO 99/12016, WO 99/16162, WO
99/19056, WO 99/19516, WO 99/29497, WO 99/31495, WO 99/34205, WO
99/43432, WO 99/44217, WO 99/56954, WO 99/64836, WO 99/64840, WO
99/64848, WO 99/67639, WO 00/07026, WO 00/09753, WO 00/10015, WO
00/21666, WO 00/22424, WO 00/26657, WO 00/42212, WO 00/43766, WO
00/45172, WO 00/46594, WO 00/50172, WO 00/50642, WO 00/58719, WO
00/60108, WO 00/70080, WO 00/70353, WO 00/72016, WO 00/73799, WO
00/78454, WO 01/02850, WO 01/14865, WO 01/17797, and WO
01/27253.
[0063] FIG. 3 illustrates one non-limiting example of a
microfluidic device that incorporates methods and devices of the
present invention. The microfluidic device as shown in FIG. 3
comprises body structure 302, in which are disposed various
microchannels, reservoirs, etc. Specifically, channel 306 comprises
a regular cross-sectional geometry (as described herein). Channel
304 comprises a channel of specifically configured cross-sectional
geometry (as described herein), see, e.g., FIG. 2A-H for
non-limiting examples of specifically configured microchannels.
Both channel 304 and 306 are fluidly coupled to channel 324, which
is optionally connected to capillary element 320, which accesses
samples, etc. that are stored, e.g., outside of the device in,
e.g., a microwell plate or the like. For example, capillary element
320 can access a microwell plate (or even numerous microwell plates
provided in, e.g., a robotic armature) that contain a number of
putative pharmaceutical compounds to be screened within the
microfluidic device.
[0064] The fluidic material (or, more typically, the mixture of
fluidic materials) in channel 324 is next mixed with, e.g., a
buffer in order to, e.g., dilute the sample to a proper
concentration for the necessary assays/reactions to occur and/or to
help dilute unwanted sample storage materials such as DMSO. To
accomplish such, in FIG. 3, a quantity of buffer is flowed into
channel 324 from, e.g., buffer reservoir 310. Alternatively,
additional fluidic materials in place of, or in addition to,
buffers are optionally flowed into channel 324 from, e.g.,
reservoir(s) 308, 310, 312, or 314.
[0065] The fluidic material (or, again, more typically the mixture
of fluidic materials) then is flowed from channel 324 into either
(or both) channel 304 or 306. In channel 304 the fluidic materials
are manipulated/controlled by having their dispersion rates and/or
average velocity changed because of the specifically configured
cross-sectional geometry of the microchannel (see, supra). The
manipulation can entail, e.g., keeping the fluidic materials
together in the same plug, separating mixed fluidic materials into
separate plugs, etc. The fluidic materials in channel 304 are
optionally flowed via any of the fluid transport mechanisms as
described herein (e.g., electrokinetic flow, pressure driven flow,
etc.). Although not displayed in FIG. 3, it will be appreciated
that once mixed fluidic materials are separated into distinct
plugs, such plugs can be analyzed, moved, considered, etc.
separately of one another. Furthermore, it will also be appreciated
that the cross-sectional geometry of channel 304 can change over
the length of the channel, thereby producing different dispersion
rates and/or average velocities at different locations along its
length for the fluidic materials that are flowed through it.
[0066] Alternatively, or in addition, to the above, the fluidic
materials in channel 324 can be flowed into channel 306 (i.e., the
microchannel of regular cross-sectional geometry). As detailed
above, channels of the invention such as 306, have a "regular"
profile that is used to separate fluidic materials based upon their
differing dispersion rates and/or average velocities within the
channel without the use of other means of separation such as
electrophoresis, separation matrices, etc.
[0067] A nice demonstration of separation of fluidic materials as
occurs in channels such as 306 was demonstrated by the following
experiment. Mixtures of various fluidic materials (as detailed
below) were flowed through a microfluidic device comprising
microchannels of regular cross-sectional geometry (as described
herein) and the resulting separation of the various fluidic
materials due to the differences in their dispersion rates and/or
average velocity was detected via fluorescence.
[0068] In one experiment, mixtures of fluorescein (at 0.5
micromolar) and 6 micrometer diameter latex beads in a cell buffer
containing 0.1 % BSA were aspirated into a 60-90 micrometer wide by
20 micrometer deep microchannel of regular cross-sectional geometry
and flowed under negative 0.25 psi through the microfluidic device.
The resulting separation is displayed in FIG. 5. The mixture was
flowed in 10-second pulses into the channel. The transit time of
the fluorescein was 55 seconds, while the transit time of the latex
beads was 33 seconds. This conforms to the above described
properties of flow of separation of molecules by flow through
channels of regular cross-sectional geometry (see, above). In other
words, the larger beads, since their flow was governed solely by
convection, flowed through the channel faster than the small
molecule fluorescein, which was subject to Taylor-Aris
dispersion.
[0069] In another experiment demonstrating the methods and devices
of the current invention, Jurkat cells were separated from
fluorescein labeled monoclonal antibodies. See FIGS. 6A through 6E.
In FIG. 6A, fluorescein labeled monoclonal antibodies at a 1/50
dilution of a 0.5 mg/ml solution, specific for MHC-II antigens
(Ancell Corp., Bayport, Minn.), were flowed through a similar
microchannel and in a similar fashion as for the above latex bead
flow experiment.
[0070] FIG. 6B shows separation of Jurkat cells associated with
fluorescein labeled anti-MHC-II antibodies (Ancell Corp, Bayport,
Minn.) at a 1/25 dilution of a 0.5 mg/ml solution, from
unassociated fluorescein labeled anti-MHC-II antibodies, again,
based upon their differing dispersion rates and/or average
velocity. The Jurkat cells, which have approximately 200,000 MHC-II
antigens on their cell surface, were present at a concentration of
5.times.10.sup.6 per milliliter and the mixture was pulsed through
the regular cross-sectional geometry microchannel in 10 second
pulses. The graph of FIG. 6B shows the peaks of the Jurkat+labeled
antibody flowing ahead of unbound labeled antibodies. A similar run
is shown in FIG. 6c only utilizing a labeled antibody against CD3
antigen (Ancell, Corp., Bayport, Minn.) at a 1/25 dilution of a 0.5
mg/ml solution, on the Jurkat cells (Jurkat cells having
approximately 30,000 CD3 antigens present on their cell surface).
Again, the Jurkat+labeled antibodies flow ahead of the unbound
labeled antibodies.
[0071] FIG. 6D illustrates a control experiment utilizing the same
general parameters as those for the experiments displayed in FIGS.
6B and 6C, however, using anti-CD8 labeled antibodies (Ancell
Corp.) at a 1/25 dilution of a 0.2 mg/ml solution. Because Jurkat
cells do not display CD8 antigen, no cell+antibody complexes were
formed which would have separated due to their different
dispersion/average velocity rate.
[0072] FIG. 6E illustrates another control experiment wherein,
under similar conditions as for the experiment in FIG. 6C, Jurkat
cells were flowed with labeled anti-CD3 antibodies. However, in
this case, the pulses of mixed cells and antibodies (i.e., the
amount of time such mixture was flowed into the microchannel) was
100 seconds instead of 10 seconds. Because there was no "space"
(e.g., a buffer plug between the pulses), the separation of
cells+antibodies from unlabeled antibodies due to their different
dispersion/average velocity cannot be discerned.
[0073] The above examples, illustrate that the methods and devices
of the current invention are easily adaptable to many different
experimental situations and can be adapted to many different uses
(e.g., separation of many different components of mixed samples,
washing of compounds, minimization of sample plug dispersion,
keeping components of different types (e.g., cells and labeled
antibodies) together in the same fluidic plug, etc.).
INTEGRATED SYSTEMS, METHODS AND MICROFLUIDIC DEVICES OF THE
INVENTION
[0074] The microfluidic devices of the invention can include
numerous optional variant embodiments including methods and devices
for, e.g., fluid transport, temperature control, detection and the
like.
[0075] As used herein, the term "microfluidic device" refers to a
system or device having fluidic conduits or chambers that are
generally fabricated at the micron to sub-micron scale, e.g.,
typically having at least one cross-sectional dimension in the
range of from about 0. I micrometer to about 500 micrometer. The
microfluidic system of the current invention is fabricated from
materials that are compatible with the conditions present in the
specific experiments and/or separations to be performed on the
specific samples, reagents, etc. under examination, etc. Such
conditions include, but are not limited to, pH, temperature, ionic
concentration, pressure, and application of electrical fields. The
materials of the device are also chosen for their inertness to
components of the experiments to be carried out in the device. Such
materials include, but are not limited to, glass, quartz, silicon,
and polymeric substrates, e.g., plastics, depending on the intended
application.
[0076] Although the devices and systems specifically illustrated
herein are generally described in terms of the performance of a few
operations, or of one particular operation, it will be readily
appreciated from this disclosure that the flexibility of these
systems permits easy integration of additional operations into
these devices. For example, the devices and systems described will
optionally include structures, reagents and systems for performing
virtually any number of operations both upstream and downstream
from the operations specifically described herein (e.g., upstream
and/or downstream of separation of fluidic materials as described
herein, etc.). Such upstream operations include such operations as
sample handling and preparation, e.g., extraction, purification,
amplification, cellular activation, labeling reactions, dilution,
aliquotting, and the like. Similarly, downstream operations
optionally include similar operations, including, e.g., further
separation of sample components, labeling of components, assays and
detection operations, electrokinetic or pressure-based injection of
components or the like.
[0077] The microfluidic devices of the present invention can
include other features of microscale systems, such as fluid
transport systems that direct particle/fluid movement within and to
the microfluidic devices as well as the flow of fluids to and
through various channels or regions, etc. Various combinations of
fluid flow mechanisms can be utilized in embodiments of the present
invention. Additionally, various types of fluid flow mechanisms can
be utilized in separate areas of microfluidic devices of the
invention. For example, separation of fluidic materials can be
carried out in non-manipulated microchannels (i.e., regular
microchannels) using the methods of the invention and utilizing
non-electrokinetic fluid flow. While in areas of the same
microfluidic device which are not used for separation (or for other
types of separation, e.g., in manipulated, or specifically
configured, microchannels) of fluidic material using the methods of
the invention can utilize electrokinetic fluid flow. Flow of
fluidic components such as reagents, etc., can incorporate any
movement mechanism set forth herein (e.g., fluid pressure sources
for modulating fluid pressure in
microchannels/micro-reservoirs/etc.; electrokinetic controllers for
modulating voltage or current in
microchannels/micro-reservoirs/etc.; gravity flow modulators;
magnetic control elements for modulating a magnetic field within
the microfluidic device; use of hydrostatic, capillary, or wicking
forces; or combinations thereof).
[0078] The microfluidic devices of the invention can also include
fluid manipulation elements such as parallel stream fluidic
converters, i.e., converters that facilitate conversion of at least
one serial stream of reagents into parallel streams of reagents for
parallel delivery to a reaction site or reaction sites within the
device. The systems herein optionally include mechanisms such as
valve manifolds and a plurality of solenoid valves to control flow
switching, e.g., between channels and/or to control pressure/vacuum
levels in the, e.g., microchannels. Additionally, molecules, etc.
are optionally loaded into one or more channels of a microfluidic
device through one sipper capillary fluidly coupled to each of one
or more channels and to a sample or particle source, such as a
microwell plate.
[0079] In the present invention, materials such as cells, proteins,
antibodies, enzymes, substrates, buffers, or the like are
optionally monitored and/or detected, e.g., so that the presence of
a component of interest can be detected, an activity of a compound
can be determined, separation of fluidic materials can be
monitored, or an effect of a modulator, e.g., on an enzyme's
activity, can be measured. Depending upon the detected signal
measurements, decisions are optionally made regarding subsequent
fluidic operations, e.g., whether to assay a particular component
in detail to determine, e.g., kinetic information or, e.g.,
whether, when, or to what extent to shunt a portion of a fluidic
material from a main channel into a second channel (e.g., flowing a
fluidic material into a second channel once it has been separated
from a mixture of fluidic materials.
[0080] In brief, the systems described herein optionally include
microfluidic devices, as described above, in conjunction with
additional instrumentation for controlling fluid transport, flow
rate and direction within the devices, detection instrumentation
for detecting or sensing results of the operations performed by the
system, processors, e.g., computers, for instructing the
controlling instrumentation in accordance with preprogrammed
instructions, receiving data from the detection instrumentation,
and for analyzing, storing and interpreting the data, and providing
the data and interpretations in a readily accessible reporting
format. For example, the systems herein optionally include a valve
manifold and a plurality of solenoid valves to control flow
switching between channels and/or to control pressure/vacuum levels
in the channels.
[0081] Temperature Control
[0082] Various embodiments of the present invention can control
temperatures to influence numerous parameters or reaction
conditions, e.g., those in thermocycling reactions (e.g., PCR,
LCR). Additionally, the present invention can control temperatures
in order to manipulate reagent properties, etc. In general, and in
optional embodiments of the invention, various heating methods can
be used to provide a controlled temperature in the involved
miniaturized fluidic systems. Such heating methods include both
joule and non-joule heating.
[0083] Non-joule heating methods can be internal, i.e., integrated
into the structure of the microfluidic device, or external, i.e.,
separate from the microfluidic device. Non-joule heat sources can
include, e.g., photon beams, fluid jets, liquid jets, lasers,
electromagnetic fields, gas jets, electron beams, thermoelectric
heaters, water baths, furnaces, resistive thin films, resistive
heating coils, peltier heaters, or other materials, which provide
heat to the fluidic system in a conductive manner. Such conductive
heating elements transfer thermal energy from, e.g., a resistive
element in the heating element to the microfluidic system by way of
conduction. Thermal energy provided to the microfluidic system
overall, increases the temperature of the microfluidic system to a
desired temperature. Accordingly, the fluid temperature and the
temperature of the molecules within, e.g., the microchannels of the
system, are also increased in temperature. An internal controller
in the heating element or within the microfluidic device optionally
can be used to regulate the temperature involved. These examples
are not limiting and numerous other energy sources can be utilized
to raise the fluid temperature in the microfluidic device.
[0084] Non-joule heating units can attach directly to an external
portion of a chip of the microfluidic device. Alternatively,
non-joule heating units can be integrated into the structure of the
microfluidic device. In either case, the non-joule heating is
optionally applied to only selected portions of chips in
microfluidic devices (e.g., such as reaction areas, detection
areas, etc.) or optionally heats the entire chip of the
microfluidic device and provides a uniform temperature distribution
throughout the chip
[0085] A variety of methods can be used to lower fluid temperature
in the microfluidic system, through use of energy sinks. Such an
energy sink can be a thermal sink or a chemical sink and can be
flood, time-varying, spatially varying, or continuous. A thermal
sink can include, among others, a fluid jet, a liquid jet, a gas
jet, a cryogenic fluid, a super-cooled liquid, a thermoelectric
cooling means, e.g., peltier device or an electromagnetic
field.
[0086] In general, electric current passing through the fluid in a
channel produces heat by dissipating energy through the electrical
resistance of the fluid. Power dissipates as the current passes
through the fluid and goes into the fluid as energy as a function
of time to heat the fluid. The following mathematical expression
generally describes a relationship between power, electrical
current, and fluid resistance: where POWER=power dissipated in
fluid: I=electric current passing through fluid; and R=electric
resistance of fluid. POWER=I.sup.2R
[0087] The above equation provides a relationship between power
dissipated ("POWER") to current ("I") and resistance ("R"). In some
of the embodiments of the invention, wherein electric current is
directed toward moving a fluid (where such is utilized, e.g., in
areas of specially configured microchannel cross-sectional geometry
where the dispersion rate and/or average velocity of fluidic
materials are manipulated), a portion of the power goes into
kinetic energy of moving the fluid through the channel. Joule
heating uses a selected portion of the power to heat the fluid in
the channel or a selected channel region(s) of the microfluidic
device and can utilize in-channel electrodes. See, e.g., U.S. Pat.
No. 5,965,410, which is incorporated herein by reference in its
entirety for all purposes. Such a channel region is often narrower
or smaller in cross section than other channel regions in the
channel structure. The small cross section provides higher
resistance in the fluid, which increases the temperature of the
fluid as electric current passes therethrough. Alternatively, the
electric current can be increased along the length of the channel
by increased voltage, which also increases the amount of power
dissipated into the fluid to correspondingly increase fluid
temperature.
[0088] Joule heating permits the precise regional control of
temperature and/or heating within separate microfluidic elements of
the device of the invention, e.g., within one or several separate
channels, without heating other regions where such heating is,
e.g., unnecessary or undesirable. Because the microfluidic elements
involved are extremely small in comparison to the mass of the
entire microfluidic device in which they are fabricated, such heat
remains substantially localized, e.g., it dissipates into and from
the device before it affects other fluidic elements. In other
words, the relatively massive device functions as a heat sink for
the separate fluidic elements contained therein.
[0089] To selectively control the temperature of fluid or material
of a region of, e.g., a microchannel, the joule heating power
supply of the invention can apply voltage and/or current in several
optional ways. For instance, the power supply optionally applies
direct current (i.e., DC), which passes through one region of a
microchannel and into another region of the same microchannel which
is smaller in cross section in order to heat fluid and material in
the second region. This direct current can be selectively adjusted
in magnitude to complement any voltage or electric field applied
between the regions to move materials in and out of the respective
regions. In order to heat the material within a region, without
adversely affecting the movement of a material, alternating current
(i.e., AC) can be selectively applied by a power supply. The AC
used to heat the fluid can be selectively adjusted to complement
any voltage or electric field applied between regions in order to
move fluid into and out of various regions of the device.
Alternating current, voltage, and/or frequency can be adjusted, for
example, to heat a fluid without substantially moving the fluid.
Alternatively, the power supply can apply a pulse or impulse of
current and/or voltage, which will pass through one microchannel
region and into another microchannel region to heat the fluid in
the region at a given instance in time. This pulse can be
selectively adjusted to complement any voltage or electric field
applied between the regions in order to move materials, e.g.,
fluids or other materials, into and out of the various regions.
Pulse width, shape, and/or intensity can be adjusted, for example,
to heat a fluid substantially without moving the fluid or any
materials within the fluid, or to heat the material(s) while moving
the fluid or materials. Still further, the power supply optionally
applies any combination of DC, AC, and pulse, depending upon the
application. The microchannel(s) itself optionally has a desired
cross section (e.g., diameter, width or depth) that enhances the
heating effects of the current passed through it and the thermal
transfer of energy from the current to the fluid (e.g., in addition
to, or alternative to, any cross-sectional geometry used to
manipulate dispersion rate and/or average velocity of fluidic
materials).
[0090] Because electrical energy is optionally used to control
temperature directly within the fluids contained in the
microfluidic devices, the methods and devices of the invention are
optionally utilized in microfluidic systems which employ
electrokinetic material transport systems, as noted herein.
Specifically, the same electrical controllers, power supplies and
electrodes can be readily used to control temperature
contemporaneously with their control of material transport. See,
infra. In some embodiments of the invention, the device provides
multiple temperature zones by use of zone heating. On such example
apparatus is described in Kopp, M. et al. (1998) "Chemical
amplification: continuous-flow PCR on a chip" Science 280(5366):
1046-1048.
[0091] As can be seen from the above, the elements of the current
invention can be configured in many different arrangements
depending upon the specific needs of the molecules, etc. under
consideration and the parameters of the specific assays/reactions
involved. Again, the above non-limiting illustrations are only
examples of the many different configurations/embodiments of the
invention.
[0092] Fluid Flow
[0093] A variety of controlling instrumentation and methodologies
are optionally utilized in conjunction with the microfluidic
devices described herein, for controlling the transport and
direction of fluidic materials and/or materials within the devices
of the present invention by, e.g., pressure-based or electrokinetic
control, etc.
[0094] In the present system, the fluid direction system controls
the transport, flow and/or movement of samples, other reagents,
etc. into and through the microfluidic device. For example, the
fluid direction system optionally directs the movement of one or
more fluid (e.g., samples, buffers) etc. into, e.g., a microchannel
where such fluidic materials are to be separated or a microchannel
where diverse fluidic materials are to be kept together in a
"plug." The fluid direction system also optionally directs the
simultaneous or sequential movement of fluidic materials into one
or more channels, etc. Additionally, the fluid direction system can
optionally direct the shunting of portions of fluidic materials
into shunt microchannels and the like.
[0095] The fluid direction system also optionally iteratively
repeats the fluid direction movements to create high throughput
screening, e.g., of thousands of samples. Alternatively, the fluid
direction system optionally repeats the fluid direction movements
to a lesser degree of iterations to create a lower throughput
screening (applied, e.g., when the specific analysis under
observation requires, e.g., a long incubation time when a higher
throughput format would be counter productive) or the fluid
direction system utilizes a format of high throughput and low
throughput screening depending on the specific requirements of the
assay. Additionally, the devices of the invention optionally use a
multiplex format to help achieve high throughput screening, e.g.,
through use of a series of multiplexed sipper devices or
multiplexed system of channels coupled to a single controller for
screening in order to increase the amount of samples analyzed in a
given period of time. Again, the fluid direction system of the
invention optionally controls the flow (timing, rate, etc.) of
samples, reagents, buffers, etc. involved in the various optional
multiplex embodiments of the invention.
[0096] One method of achieving transport or movement of particles
through microfluidic devices is by electrokinetic material
transport. In general, electrokinetic material transport and
direction systems include those systems that rely upon the
electrophoretic mobility of charged species within an electric
field applied to the structure. Such systems are more particularly
referred to as electrophoretic material transport systems. In the
current invention, electrokinetic transport is optionally used as
the method of fluid transport when fluidic materials are
manipulated (e.g., various fluidic materials kept together in a
plug, or various materials separated from a single mixture into
distinct plugs) in a microchannel whose cross-sectional geometry is
specifically designed/configured to achieve the desired
manipulation (e.g., a channel's shape is specifically configured to
keep various fluidic materials together in a plug). However,
electrokinetic transport is not a preferred fluid transport method
when separation of fluidic materials is to be done in a
non-configured microchannel (i.e., a "regular" cross-sectional
shaped microchannel). In such cases, the preferred fluid transport
method comprises, e.g., pressure based flow, wicking based flow,
hydrostatic based flow, etc. See, below.
[0097] Electrokinetic material transport systems, as used herein,
and as optional aspects of the present invention, include systems
that transport and direct materials within a structure containing,
e.g., microchannels, microreservoirs, etc., through the application
of electrical fields to the materials, thereby causing material
movement through and among the areas of the microfluidic devices,
e.g., cations will move toward a negative electrode, while anions
will move toward a positive electrode. Movement of fluids toward or
away from a cathode or anode can cause movement of particles
suspended within the fluid (or even particles over which the fluid
flows). Similarly, the particles can be charged, in which case they
will move toward an oppositely charged electrode (indeed, it is
possible to achieve fluid flow in one direction while achieving
particle flow in the opposite direction). In some embodiments of
the present invention, the fluid and/or particles, etc. within the
fluid, can be immobile or flowing.
[0098] For optional electrophoretic applications of the present
invention, the walls of interior channels of the electrokinetic
transport system are optionally charged or uncharged. Typical
electrokinetic transport systems are made of glass, charged
polymers, and uncharged polymers. The interior channels are
optionally coated with a material which alters the surface charge
of the channel. A variety of electrokinetic controllers are
described, e.g., in Ramsey WO 96/04547, Parce et al. WO 98/46438
and Dubrow et al., WO 98/49548 (all of which are incorporated
herein by reference in their entirety for all purposes), as well as
in a variety of other references noted herein.
[0099] To provide appropriate electric fields, the system of the
current microfluidic device optionally includes a voltage
controller that is capable of applying selectable voltage levels,
simultaneously, to, e.g., each of the various microchannels and
micro-reservoirs. Such a voltage controller is optionally
implemented using multiple voltage dividers and multiple relays to
obtain the selectable voltage levels. Alternatively, multiple
independent voltage sources are used. The voltage controller is
electrically connected to each of the device's fluid conduits via
an electrode positioned or fabricated within each of the plurality
of fluid conduits (e.g., microchannels, microreservoirs, etc.). In
one embodiment, multiple electrodes are positioned to provide for
switching of the electric field direction in the, e.g.,
microchannel(s), thereby causing the analytes to travel a longer
distance than the physical length of the microchannel. Use of
electrokinetic transport to control material movement in
interconnected channel structures was described in, e.g., WO
96/94547 to Ramsey. An exemplary controller is described in U.S.
Pat. No. 5,800,690. Modulating voltages are concomitantly applied
to the various fluid areas of the device to affect a desired fluid
flow characteristic, e.g., continuous or discontinuous (e.g., a
regularly pulsed field causing the sample to oscillate its
direction of travel) flow of labeled components toward a waste
reservoir. Particularly, modulation of the voltages applied at the
various areas can move and direct fluid flow through the
interconnected channel structure of the device.
[0100] The controlling instrumentation discussed above is also
optionally used to provide for electrokinetic injection or
withdrawal of fluidic material downstream of a region of interest
to control an upstream flow rate. The same instrumentation and
techniques described above are also utilized to inject a fluid into
a downstream port to function as a flow control element.
[0101] The current invention also optionally includes other methods
of fluid transport, e.g., available for situations in which
electrokinetic methods are not desirable. See, above. For example,
fluid transport and direction, etc. are optionally carried out in
whole, or in part, in a pressure-based system to, e.g., avoid
electrokinetic biasing during sample mixing. Additionally, as
described above, pressure based fluid transport, or the like, is
used in "regular" cross-sectional shaped microchannels (i.e., ones
where the specific channel shape has not been configured to achieve
a desired result such as keeping various fluidic materials together
in a plug, or separation of fluidic materials, or the like) where,
e.g., fluidic materials are to be separated without the use of,
e.g., electrophoresis, separation matrices, etc. High throughput
systems typically use pressure induced sample introduction.
Pressure based flow is also desirable in systems in which
electrokinetic transport is also used. For example, pressure based
flow is optionally used for introducing and reacting reagents in a
system in which the products are electrophoretically separated. In
the present invention molecules are optionally loaded and other
reagents are flowed through the microchannels or microreservoirs,
etc. using, e.g., electrokinetic fluid control and/or under
pressure.
[0102] Pressure is optionally applied to the microscale elements of
the invention, e.g., to a microchannel, microreservoir, region,
etc. to achieve fluid movement using any of a variety of
techniques. Fluid flow and flow of materials suspended or
solubilized within the fluid, including cells or molecules, is
optionally regulated by pressure based mechanisms such as those
based upon fluid displacement, e.g., using a piston, pressure
diaphragm, vacuum pump, probe or the like to displace liquid and
raise or lower the pressure at a site in the microfluidic system.
The pressure is optionally pneumatic, e.g., a pressurized gas, or
uses hydraulic forces, e.g., pressurized liquid, or alternatively,
uses a positive displacement mechanism, e.g., a plunger fitted into
a material reservoir, for forcing material through a channel or
other conduit, or is a combination of such forces. Internal sources
include microfabricated pumps, e.g., diaphragm pumps, thermal
pumps, lamb wave pumps and the like that have been described in the
art. See, e.g., U.S. Pat. Nos. 5,271,724; 5,277,566; and 5,375,979
and Published PCT Application Nos. WO 94/05414 and WO 97/02347.
[0103] In some embodiments, a pressure source is applied to a
reservoir or well at one end of a microchannel to force a fluidic
material through the channel. Optionally, the pressure can be
applied to multiple ports at channel termini, or, a single pressure
source can be used at a main channel terminus. Optionally, the
pressure source is a vacuum source applied at the downstream
terminus of the main channel or at the termini of multiple
channels. Pressure or vacuum sources are optionally supplied
externally to the device or system, e.g., external vacuum or
pressure pumps sealably fitted to the inlet or outlet of channels
or to the surface openings of micro-reservoirs, or they are
internal to the device, e.g., microfabricated pumps integrated into
the device and operably linked to channels or they are both
external and internal to the device. Examples of microfabricated
pumps have been widely described in the art. See, e.g., published
International Application No. WO 97/02357.
[0104] These applied pressures, or vacuums, generate pressure
differentials across the lengths of channels to drive fluid flow
through them. In the interconnected channel networks described
herein, differential flow rates or volumes are optionally
accomplished by applying different pressures or vacuums at multiple
ports, or, by applying a single vacuum at a common waste port and
configuring the various channels with appropriate resistance to
yield desired flow rates. As discussed above, this is optionally
done with multiple sources or by connecting a single source to a
valve manifold comprising multiple electronically controlled
valves, e.g., solenoid valves.
[0105] Hydrostatic, wicking and capillary forces are also
optionally used to provide fluid flow of materials such as
reagents, buffers, etc. in the invention. See, e.g., "METHOD AND
APPARTUS FOR CONTINUOUS LIQUID FLOW IN MICROSCALE CHANNELS USING
PRESSURE INJECTION, WICKING AND ELECTROKINETIC INJECTION," by
Alajoki et al., U.S. Ser. No. 09/245,627, filed Feb. 5, 1999. In
using wicking/capillary methods, an adsorbent material or branched
capillary structure is placed in fluidic contact with a region
where pressure is applied, thereby causing fluid to move towards
the adsorbent material or branched capillary structure.
Furthermore, the capillary forces are optionally used in
conjunction with, e.g., electrokinetic or pressure-based flow in
the channels, etc. of the present invention in order to pull
fluidic material, etc. through the channels. Additionally, a wick
is optionally added to draw fluid through a porous matrix fixed in
a microscale channel or capillary. Use of a hydrostatic pressure
differential is another optional way to control flow rates through
the channels, etc. of the present invention. For example, in a
simple passive aspect, a cell suspension is deposited in a
reservoir or well at one end of a channel at sufficient volume or
height so that the cell suspension creates a hydrostatic pressure
differential along the length of the channel by virtue of, e.g.,
the cell suspension reservoir having greater height than a well at
an opposite terminus of the channel. Typically, the reservoir
volume is quite large in comparison to the volume or flow-through
rate of the channel, e.g., 10 microliter reservoirs or larger as
compared to a 100 micrometer channel cross section.
[0106] The present invention optionally includes mechanisms for
reducing adsorption of materials during fluid-based flow, e.g., as
are described in "PREVENTION OF SURFACE ADSORPTION IN MICROCHANNELS
BY APPLICATION OF ELECTRIC CURRENT DURING PRESSUE-INDUCED FLOW"
filed May 11, 1999 by Parce et al., Attorney Docket Number 01-78-0.
In brief, adsorption of components, proteins, enzymes, markers and
other materials to channel walls or other microscale components
during pressure-based flow can be reduced by applying an electric
field such as an alternating current to the material during flow.
Alternatively, flow rate changes due to adsorption are detected and
the flow rate is adjusted by a change in pressure or voltage.
[0107] The invention also optionally includes mechanisms for
focusing labeling reagents, enzymes, modulators, and other
components into the center of microscale flow paths, which is
useful in increasing assay throughput by regularizing flow
velocity, e.g., in pressure based flow, e.g., as are described in
"FOCUSING OF MICROPARTICLES IN MICROFLUIDIC SYSTEMS" by H. Garrett
Wada et al. Attorney Docket number 01-505-0, filed May 17, 1999. In
brief, sample materials are focused into the center of a channel by
forcing fluid flow from opposing side channels into the main
channel, or by other fluid manipulation.
[0108] In an alternate embodiment, microfluidic systems of the
invention can be incorporated into centrifuge rotor devices, which
are spun in a centrifuge. Fluids and particles travel through the
device due to gravitational and centripetal/centrifugal pressure
forces.
[0109] Fluid flow or particle flow in the present devices and
methods is optionally achieved using any one or more of the above
techniques, alone or in combination. For example, electrokinetic
transport can be used in one area or region of a microfluidic
device in order to, e.g., move material through a microchannel
whose cross-sectional geometry has been specifically configured to,
e.g., keep various fluidic materials (e.g., cells and enzymes or
the like) together in a plug. Additionally, pressure based flow
could be used in a different region/area of the same microfluidic
device where various fluidic materials (again, e.g., cells and
enzymes or the like) are to be separated in a microchannel that has
not been specifically configured to separate such materials (i.e.,
a "regularly" shaped microchannel). Myriad combinations of fluid
transport methods can be combined in various embodiments of the
present invention depending upon the specific needs of the
system/assay being used. Typically, the controller systems involved
are appropriately configured to receive or interface with a
microfluidic device or system element as described herein. For
example, the controller, optionally includes a stage upon which the
device of the invention is mounted to facilitate appropriate
interfacing between the controller and the device. Typically, the
stage includes an appropriate mounting/alignment structural
element, such as a nesting well, alignment pins and/or holes,
asymmetric edge structures (to facilitate proper device alignment),
and the like. Many such configurations are described in the
references cited herein.
[0110] Detection
[0111] In general, detection systems in microfluidic devices
include, e.g., optical sensors, temperature sensors, pressure
sensors, pH sensors, conductivity sensors, and the like. Each of
these types of sensors is readily incorporated into the
microfluidic systems described herein. In these systems, such
detectors are placed either within or adjacent to the microfluidic
device or one or more microchannels, microchambers, microreservoirs
or conduits of the device, such that the detector is within sensory
communication with the device, channel, reservoir, or chamber, etc.
Detection systems can be used to, e.g., discern and/or monitor
specific reactions, assays, etc. occurring within the microfluidic
device, or alternatively, or additionally, to track, e.g.,
separation of fluidic materials and/or integrity of
sample/component plugs (as occurring in the devices and methods of
the current invention). The phrase "proximal," to a particular
element or region, as used herein, generally refers to the
placement of the detector in a position such that the detector is
capable of detecting the property of the microfluidic device, a
portion of the microfluidic device, or the contents of a portion of
the microfluidic device, for which that detector was intended. For
example, a pH sensor placed in sensory communication with a
microscale channel is capable of determining the pH of a fluid
disposed in that channel. Similarly, a temperature sensor placed in
sensory communication with the body of a microfluidic device is
capable of determining the temperature of the device itself.
[0112] Many different molecular/reaction characteristics can be
detected in microfluidic devices of the current invention. For
example, various embodiments can detect such things as fluorescence
or emitted light, changes in the thermal parameters (e.g., heat
capacity, etc.) involved in the assays, etc.
[0113] Examples of detection systems in the current invention can
include, e.g., optical detection systems for detecting an optical
property of a material within, e.g., the microchannels of the
microfluidic devices that are incorporated into the microfluidic
systems described herein. Such optical detection systems are
typically placed adjacent to a microscale channel of a microfluidic
device, and optionally are in sensory communication with the
channel via an optical detection window or zone that is disposed
across the channel or chamber of the device.
[0114] Optical detection systems of the invention include, e.g.,
systems that are capable of measuring the light emitted from
material within the channel, the transmissivity or absorbance of
the material, as well as the material's spectral characteristics,
e.g., fluorescence, chemiluminescence, etc. Detectors optionally
detect a labeled compound, such as fluorographic, calorimetric and
radioactive components. Types of detectors optionally include
spectrophotometers, photodiodes, avalanche photodiodes,
microscopes, scintillation counters, cameras, diode arrays, imaging
systems, photomultiplier tubes, CCD arrays, scanning detectors,
galvo-scanners, film and the like, as well as combinations thereof.
Proteins, antibodies, or other components which emit a detectable
signal can be flowed past the detector, or alternatively, the
detector can move relative to an array to determine molecule
position (or, the detector can simultaneously monitor a number of
spatial positions corresponding to channel regions, e.g., as in a
CCD array). Examples of suitable detectors are widely available
from a variety of commercial sources known to persons of skill.
See, also, The Photonics Design and Application Handbook, books 1,
2, 3 and 4, published annually by Laurin Publishing Co., Berkshire
Common, P.O. Box 1146, Pittsfield, Mass. for common sources for
optical components.
[0115] As noted above, the present devices optionally include, as
microfluidic devices typically do, a detection window or zone at
which a signal, e.g., fluorescence, is monitored. This detection
window or zone optionally includes a transparent cover allowing
visual or optical observation and detection of the assay results,
e.g., observation of a colorimetric, fluorometric or radioactive
response, or a change in the velocity of colorimetric, fluorometric
or radioactive component.
[0116] Another optional embodiment of the present invention
involves use of fluorescence correlation spectroscopy and/or
confocal nanofluorimetric techniques to detect fluorescence from
the molecules in the microfluidic device. Such techniques are
easily available (e.g., from Evotec, Hamburg, Germany) and involve
detection of fluorescence from molecules that diffuse through the
illuminated focus area of a confocal lens. The length of any photon
burst observed will correspond to the time spent in the confocal
focus by the molecule. Various algorithms used for analysis can be
used to evaluate fluorescence signals from individual molecules
based on changes in, e.g., brightness, fluorescence lifetime,
spectral shift, FRET, quenching characteristics, etc.
[0117] The sensor or detection portion of the devices and methods
of the present invention can optionally comprise a number of
different apparatuses. For example, fluorescence can be detected
by, e.g., a photomultiplier tube, a charge coupled device (CCD) (or
a CCD camera), a photodiode, or the like.
[0118] A photomultiplier tube is an optional aspect of the current
invention. Photomultiplier tubes (PMTs) are devices which convert
light (photons) into electronic signals. The detection of each
photon by the PMT is amplified into a larger and more easily
measurable pulse of electrons. PMTs are commonly used in many
laboratory applications and settings and are well known to those in
the art.
[0119] Another optional embodiment of the present invention
comprises a charge coupled device. CCD cameras can detect even very
small amounts of electromagnetic energy (e.g., such that emitted by
fluorophores in the present invention). CCD cameras are made from
semi-conducting silicon wafers that release free electrons when
light photons strike the wafers. The output of electrons is
linearly directly proportional to the amount of photons that strike
the wafer. This allows the correlation between the image brightness
and the actual brightness of the event observed. CCD cameras are
very well suited for imaging of fluorescence emissions since they
can detect even extremely faint events, can work over a broad range
of spectrum, and can detect both very bright and very weak events.
CCD cameras are well know to those in the art and several suitable
examples include those made by: Stratagene (La Jolla, Calif.),
Alpha-Innotech (San Leandro, Calif.), and Apogee Instruments
(Tucson, Ariz.) among others.
[0120] Yet another optional embodiment of the present invention
comprises use of a photodiode to detect fluorescence from molecules
in the microfluidic device. Photodiodes absorb incident photons
which cause electrons in the photodiode to diffuse across a region
in the diode thus causing a measurable potential difference across
the device. This potential can be measured and is directly related
to the intensity of the incident light.
[0121] In some aspects, the detector measures an amount of light
emitted from the material, such as a fluorescent or
chemiluminescent material. As such, the detection system will
typically include collection optics for gathering a light based
signal transmitted through the detection window or zone, and
transmitting that signal to an appropriate light detector.
Microscope objectives of varying power, field diameter, and focal
length are readily utilized as at least a portion of this optical
train. The detection system is typically coupled to a computer
(described in greater detail below), via an analog to digital or
digital to analog converter, for transmitting detected light data
to the computer for analysis, storage and data manipulation.
[0122] In the case of fluorescent materials such as labeled cells
or fluorescence indicator dyes or molecules, the detector
optionally includes a light source which produces light at an
appropriate wavelength for activating the fluorescent material, as
well as optics for directing the light source to the material
contained in the channel. The light source can be any number of
light sources that provides an appropriate wavelength, including
lasers, laser diodes and LEDs. Other light sources are optionally
utilized for other detection systems. For example, broad band light
sources for light scattering/transmissivity detection schemes, and
the like. Typically, light selection parameters are well known to
those of skill in the art.
[0123] The detector can exist as a separate unit, but is preferably
integrated with the controller system, into a single instrument.
Integration of these functions into a single unit facilitates
connection of these instruments with a computer (described below),
by permitting the use of few or a single communication port(s) for
transmitting information between the controller, the detector and
the computer. Integration of the detection system with a computer
system typically includes software for converting detector signal
information into assay result information, e.g., integrity of
sample/component plugs comprising multiple fluidic materials;
separation of fluidic materials, concentration of a substrate,
concentration of a product, presence of a compound of interest,
interaction between various samples, or the like.
[0124] Computer
[0125] As noted above, either, or both, the fluid direction system
or the detection system, as well as other aspects of the current
invention described herein (e.g., temperature control, etc.), are
optionally coupled to an appropriately programmed processor or
computer that functions to instruct the operation of these
instruments in accordance with preprogrammed or user input
instructions, receive data and information from these instruments,
and interpret, manipulate and report this information to a user. As
such, the computer is typically appropriately coupled to one or
more of the appropriate instruments (e.g., including an analog to
digital or digital to analog converter as needed).
[0126] The computer optionally includes appropriate software for
receiving user instructions, either in the form of user input into
set parameter fields, e.g., in a GUI, or in the form of
preprogrammed instructions, e.g., preprogrammed for a variety of
different specific operations. The software then converts these
instructions to appropriate language for instructing the operation
of, e.g., the fluid direction and transport controller to carry out
the desired operation.
[0127] For example, the computer is optionally used to direct a
fluid direction system to control fluid flow, e.g., into and
through a variety of interconnected microchannels (e.g., into and
through the various microchannels of the invention, such as
specially configured cross-sectional geometry areas and/or
"regular" microchannel areas used for material separation, etc.).
Additionally, the fluid direction system optionally directs fluid
flow controlling which samples are contacted with each other and/or
with various reagents, buffers, etc. in, e.g., a detection region
or other region(s) in the microfluidic device. Furthermore, the
fluid direction system optionally controls the coordination of
movements of multiple fluids/molecules/etc. concurrently as well as
sequentially. For example, the computer optionally directs the
fluid direction system to direct the movement of at least a first
member of a plurality of molecules into a first member of a
plurality of microchannels concurrent with directing the movement
of at least a second member of the plurality of molecules into one
or more detection channel regions. Additionally or alternatively,
the fluid direction system directs the movement of at least a first
member of the plurality of molecules into the plurality of
microchannels concurrent with incubating at least a second member
of the plurality of molecules or directs movement of at least a
first member of the plurality of molecules into the one or more
detection channel regions concurrent with incubating at least a
second member of the plurality of molecules.
[0128] By coordinating channel switching, the computer controlled
fluid direction system directs the movement of at least one member
of the plurality of molecules into the plurality of microchannels
and/or one member into a detection region at a desired time
interval, e.g., greater than 1 minute, about every 60 seconds or
less, about every 30 seconds or less, about every 10 seconds or
less, about every 1.0 seconds or less, or about every 0. I seconds
or less. Each sample, with appropriate channel switching as
described above, remains in the plurality of channels for a desired
period of time, e.g., between about 0. I minutes or less and about
60 minutes or more. For example, the samples optionally remain in
the channels for a selected incubation time of, e.g., 20
minutes.
[0129] The computer then optionally receives the data from the one
or more sensors/detectors included within the system, interprets
the data, and either provides it in a user understood format, or
uses that data to initiate further controller instructions, in
accordance with the programming, e.g., such as in monitoring and
control of flow rates (e.g., as involved in separation of materials
in "regular" microchannel areas or manipulation of dispersion rates
and/or average velocity in specially configured microchannel areas,
etc.), temperatures, applied voltages, pressures, and the like.
[0130] In the present invention, the computer typically includes
software for the monitoring and control of materials in the various
microchannels, etc. For example, the software directs channel
switching to control and direct flow as described above.
Additionally the software is optionally used to control
electrokinetic, pressure-modulated, or the like, injection or
withdrawal of material. The computer also typically provides
instructions, e.g., to the controller or fluid direction system for
switching flow between channels to help achieve a high throughput
format.
[0131] In addition, the computer optionally includes software for
deconvolution of the signal or signals from the detection system.
For example, the deconvolution distinguishes between two detectably
different spectral characteristics that were both detected, e.g.,
when a substrate and product comprise detectably different
labels.
[0132] Any controller or computer optionally includes a monitor
which is often a cathode ray tube ("CRT") display, a flat panel
display (e.g., active matrix liquid crystal display, liquid crystal
display), or the like. Data produced from the microfluidic device,
e.g., fluorographic indication of separation of selected molecules,
is optionally displayed in electronic form on the monitor.
Additionally, the data gathered from the microfluidic device can be
outputted in printed form. The data, whether in printed form or
electronic form (e.g., as displayed on a monitor), can be in
various or multiple formats, e.g., curves, histograms, numeric
series, tables, graphs and the like.
[0133] Computer circuitry is often placed in a box which includes,
e.g., numerous integrated circuit chips, such as a microprocessor,
memory, interface circuits, etc. The box also optionally includes
such things as a hard disk drive, a floppy disk drive, a high
capacity removable drive such as a writeable CD-ROM, and other
common peripheral elements. Inputting devices such as a keyboard or
mouse optionally provide for input from a user and for user
selection of sequences to be compared or otherwise manipulated in
the relevant computer system.
[0134] Exemplary Use of the Devices of the Present Invention
[0135] In a conventional bead-based immunoassay, the antibody
molecules are covalently attached to beads. These bead bound
antibody molecules are then exposed to target molecules. The
mixture of the antibody/target complex and free target molecules
are separated by washing following which a fluorescently labeled
antibody conjugate is introduced and selectively bound to the
complex. The excess label molecules are removed by a secondary
washing step. Finally, the fluorescence signal from the labeled
complex is measured for the detection of the amount of bound
target.
[0136] The devices of the present invention simplify the above
process by avoiding the need to have the two washing steps and
helps to speed up the process significantly. For example, based on
the configuration of the channel structure, the bound target and
antibody complex will have a differential velocity from the free
target molecules or in the case of the second wash step, the
labeled antibody conjugate will have a differential velocity than
the free flowing label resulting in a separation of the various
species without having to introduce washing to actually cause the
separation of the species. For example, as a plug of conjugate
molecules is introduced and transported along a microfluidic
channel to flow along a plug of beads, the two species will flow
continuously down the microfluidic channel. The cross-sectional
geometry of the channel can be configured in different regions of
the channel such that the beads are initially flowing at the same
speed as the free conjugate during incubation but flowing faster
than the free conjugate after the reaction is completed. The
differential velocity of the two species will than cause the two
species to separate based on their mobility in the channel leaving
one of the species behind and essentially separating the species
without requiring an additional washing step.
[0137] Example Integrated System
[0138] FIG. 3, Panels A, B, and C and FIG. 4 provide additional
details regarding example integrated systems that optionally use
the devices of the invention and optionally are used to practice
the methods herein. As shown, body structure 302 has main channels
304 and 306 disposed therein. As stated previously, microchannels
can comprise a number of areas comprising specifically configured
cross-sectional geometry used to manipulate dispersion rates and/or
average velocity (e.g., 304) additionally, numerous microchannels
can comprise channels of "regular" cross-sectional geometry wherein
fluidic materials are separated based on their differing dispersion
rates and/or average velocity using non-electrokinetic flow (e.g.,
306).
[0139] A sample or mixture of components, e.g., typically a buffer,
sample, reagent, etc., is optionally flowed from pipettor channel
320 towards, e.g., reservoir 316, e.g., by applying a vacuum at
reservoir 316 (or another point in the system) or by applying
appropriate voltage gradients or wicking arrangements (or a
combination of such forces). Alternatively, a vacuum, or
appropriate pressure force, is applied at, e.g., reservoirs 308,
310 or through pipettor channel 320. Additional materials, such as
buffer solutions, substrate solutions, enzyme solutions, test
molecules, fluorescence indicator dyes or molecules and the like,
are optionally flowed from wells, e.g., 308 or 310 and into channel
324 and thence into channel 304.
[0140] Alternatively, a sample or mixture of components, e.g.,
typically a buffer, sample, reagent, etc., is optionally flowed
from pipettor channel 320 towards, e.g., reservoir 318 by any
non-electrokinetic flow methods as described herein. Additional
materials, such as buffer solutions, substrate solutions, enzyme
solutions, test molecules, fluorescence indicator dyes or molecules
and the like, are optionally flowed from wells, e.g., 308 or 310
and into channel 324 and thence into channel 306.
[0141] The arrangement of channels depicted in FIG. 3 is only one
possible arrangement out of many which are appropriate and
available for use in the present invention. For example, the number
and arrangement of, e.g., microchannels comprising specifically
configured cross-sectional geometry and/or "regular" microchannel
regions used for material separation can all be altered depending
upon the specific parameters of the assays to be performed, the
need for high throughput analysis, etc. Additional alternatives can
be readily devised, e.g., by combining the microfluidic elements
described herein with other microfluidic devices described in the
patents and applications referenced herein.
[0142] Samples and materials are optionally flowed from the
enumerated wells or from a source external to the body structure.
As depicted, the integrated system optionally includes pipettor
channel 320, e.g., protruding from body 302, for accessing a source
of materials external to the microfluidic system. Typically, the
external source is a microtiter dish or other convenient storage
medium. For example, as depicted in FIG. 4, pipettor channel 320
can access microwell plate 408, which, in the wells of the plate,
optionally includes, e.g., samples, buffers, fluorescence dyes,
various fluidic reagents to be interacted with the samples,
etc.
[0143] Detector 406 is in sensory communication with, e.g.,
specifically configured cross-sectional geometry microchannel 304
and/or "regular" cross-sectional geometry channel 306, detecting
signals resulting, e.g., from labeled materials flowing through the
detection region, changes in thermal parameters, fluorescence, etc.
Detector 406 is optionally coupled to any of the channels or
regions of the device where detection is desired. Detector 406 is
operably linked to computer 404, which digitizes, stores, and
manipulates signal information detected by detector 406, e.g.,
using any of the instructions described above or any other
instruction set, e.g., for determining concentration, molecular
weight or identity, interaction between samples and test molecules,
separation of fluidic materials, integrity of sample/component
plugs, or the like.
[0144] Fluid direction system 402 controls voltage, pressure, etc.
(or a combination of such), e.g., at the wells of the systems or
through the channels of the system, or at vacuum couplings fluidly
coupled to main channel 304, 306, or any other channel described
above. Optionally, as depicted, computer 404 controls fluid
direction system 402. In one set of embodiments, computer 404 uses
signal information to select further parameters for the
microfluidic system. For example, upon detecting the interaction
between a particular sample and a first reagent, the computer
optionally directs addition of a second reagent of interest into
the system to be tested against that particular sample.
[0145] Temperature control system 410 controls joule and/or
non-joule heating at, e.g., the wells of the systems or through the
channels of the system as described herein. Optionally, as
depicted, computer 404 controls temperature control system 410. In
one set of embodiments, computer 404 uses signal information to
select further parameters for the microfluidic system. For example,
upon detecting the desired temperature in a sample in, e.g.,
channel 304, the computer optionally directs addition of, e.g., a
potential binding molecule, fluorescence indicator dye, etc. into
the system to be tested against one or more samples.
[0146] Monitor 416 displays the data produced by the microfluidic
device, e.g., graphical representation of, e.g., separation or
non-separation of fluidic materials, interaction (if any) between
samples, reagents, test molecules, etc. Optionally, as depicted,
computer 404 controls monitor 416. Additionally, computer 404 is
connected to and directs additional components such as printers,
electronic data storage devices and the like.
[0147] Assay Kits
[0148] The present invention also provides kits for utilizing the
microfluidic devices of the invention. In particular, these kits
typically include microfluidic devices, systems, modules and
workstations, etc. A kit optionally contains additional components
for the assembly and/or operation of a multimodule workstation of
the invention including, but not restricted to robotic elements
(e.g., a track robot, a robotic armature, or the like), plate
handling devices, fluid handling devices, and computers (including
e.g., input devices, monitors, c.p.u., and the like).
[0149] Generally, the microfluidic devices described herein are
optionally packaged to include some or all reagents for performing
the device's functions. For example, the kits can optionally
include any of the microfluidic devices described along with assay
components, buffers, reagents, enzymes, serum proteins, receptors,
sample materials, antibodies, substrates, control material,
spacers, buffers, immiscible fluids, etc., for performing the
assays, separations, dispersion rate and/or average velocity
manipulations, etc. using the methods/devices of the invention. In
the case of prepackaged reagents, the kits optionally include
pre-measured or pre-dosed reagents that are ready to incorporate
into the assays without measurement, e.g., pre-measured fluid
aliquots, or pre-weighed or pre-measured solid reagents that can be
easily reconstituted by the end-user of the kit.
[0150] Such kits also typically include appropriate instructions
for using the devices and reagents, and in cases where reagents (or
all necessary reagents) are not predisposed in the devices
themselves, with appropriate instructions for introducing the
reagents into the channels/chambers/reservoirs/etc. of the device.
In this latter case, these kits optionally include special
ancillary devices for introducing materials into the microfluidic
systems, e.g., appropriately configured syringes/pumps, or the like
(in one embodiment, the device itself comprises a pipettor element,
such as an electropipettor for introducing material into
channels/chambers/reservoirs/etc. within the device). In the former
case, such kits typically include a microfluidic device with
necessary reagents predisposed in the
channels/chambers/reservoirs/etc. of the device. Generally, such
reagents are provided in a stabilized form, so as to prevent
degradation or other loss during prolonged storage, e.g., from
leakage. A number of stabilizing processes are widely used for
reagents that are to be stored, such as the inclusion of chemical
stabilizers (e.g., enzymatic inhibitors,
microbicides/bacteriostats, anticoagulants, etc.), the physical
stabilization of the material, e.g., through immobilization on a
solid support, entrapment in a matrix (e.g., a bead, a gel, etc.),
lyophilization, or the like.
[0151] The elements of the kits of the present invention are
typically packaged together in a single package or set of related
packages. The package optionally includes written instructions for
utilizing one or more device of the invention in accordance with
the methods described herein. Kits also optionally include
packaging materials or containers for holding the microfluidic
device, system or reagent elements.
[0152] The discussion above is generally applicable to the aspects
and embodiments of the invention described herein. Moreover,
modifications are optionally made to the methods and devices
described herein without departing from the spirit and scope of the
invention as claimed, and the invention is optionally put to a
number of different uses including the following:
[0153] The use of a microfluidic system containing at least a first
substrate and having a first channel and a second channel
intersecting the first channel, at least one of the channels having
at least one cross-sectional dimension in a range from 0.1 to 500
micrometer, in order to test the effect of each of a plurality of
test compounds on a biochemical system comprising one or more
focused cells or particles.
[0154] The use of a microfluidic system as described herein,
wherein a biochemical system flows through one of said channels
substantially continuously, providing for, e.g., sequential testing
of a plurality of test compounds.
[0155] The use of a microfluidic device as described herein to
modulate reactions within
microchannels/microchambers/reservoirs/etc.
[0156] The use of electrokinetic injection in a microfluidic device
as described herein to modulate or achieve flow in the
channels.
[0157] The use of a combination of wicks, electrokinetic injection
and pressure based flow elements in a microfluidic device as
described herein to modulate, focus, or achieve flow of materials,
e.g., in the channels of the device.
[0158] An assay utilizing a use of any one of the microfluidic
systems or substrates described herein.
[0159] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. For example, all the
techniques and apparatus described above can be used in various
combinations. All publications, patents, patent applications, or
other documents cited in this application are incorporated by
reference in their entirety for all purposes to the same extent as
if each individual publication, patent, patent application, or
other document were individually indicated to be incorporated by
reference for all purposes.
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