U.S. patent application number 10/517942 was filed with the patent office on 2006-04-27 for recirculating fluidic network and methods for using the same.
This patent application is currently assigned to FLUIDING CORPORATION. Invention is credited to JosephW Barco, IanD Manger, HanyR Nassef.
Application Number | 20060086309 10/517942 |
Document ID | / |
Family ID | 30000688 |
Filed Date | 2006-04-27 |
United States Patent
Application |
20060086309 |
Kind Code |
A1 |
Manger; IanD ; et
al. |
April 27, 2006 |
Recirculating fluidic network and methods for using the same
Abstract
The present invention provides microfluidic devices and methods
for using the same. In particular, microfluidic devices of the
present invention are useful in conducting a variety of assays and
high throughput screening. Microfluidic devices of the present
invention include elastomeric components and solid substrate
component for attaching ligand(s) on its surface. The elastomeric
layer comprises (a) a plurality of first flow channels; (b) a
plurality of second flow channels each intersecting and crossing
each of said first flow channels thereby providing a plurality of
intersecting areas formed at intersections between said first flow
channels and said second flow channels, wherein said plurality of
first flow channels and said plurality of second flow channels are
adapted to allow the flow of a solution therethrough, and wherein
said solid substrate surface is in fluid communication with at
least said intersecting areas of said plurality of first flow
channels and said plurality of second flow channels, and wherein
said plurality of first flow channels and/or said plurality of
second flow channels are capable of forming a plurality of looped
flow channels; (c) a plurality of control channels; (d) a plurality
of first control valves each operatively disposed with respect to
each of said first flow channel to regulate flow of the solution
through said first flow channels, wherein each of said first
control valves comprises a first control channel and an elastomeric
segment that is deflectable into or retractable from said first
flow channel upon which said first control valve operates in
response to an actuation force applied to said first control
channel, the elastomeric segment when positioned in said first flow
channel restricting solution flow therethrough; (e) a plurality of
second control valves each operatively disposed with respect to
each of said second flow channel to regulate flow of the solution
through said second flow channels, wherein each of said second
control valves comprises a second control channel and an
elastomeric segment that is deflectable into or retractable from
said second flow channel upon which said second control valve
operates in response to an actuation force applied to said second
control channel, the elastomeric segment when positioned in said
second flow channel restricting solution flow therethrough; (f) a
plurality of loop forming control valves each operatively disposed
with respect to each of said first and/or said second flow channels
to form said plurality of looped flow channels, wherein each of
said loop forming control valves comprises a loop forming control
channel and an elastomeric segment that is deflectable into or
retractable from said first and/or said second flow channels upon
which said loop forming control valve operates in response to an
actuation force applied to said loop forming control channel, the
elastomeric segment when positioned in said first and/or said
second flow channels restricting solution flow therethrough thereby
forming said looped flow channel; and (g) a plurality of
recirculating pumps, and wherein each recirculating pump is
operatively disposed with respect to one of said looped flow
channels such that circulation of solution through each of said
looped flow channels can be regulated by one of said recirculating
pumps.
Inventors: |
Manger; IanD; (Bronxville,
NY) ; Barco; JosephW; (San Jose, CA) ; Nassef;
HanyR; (San Mateo, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
FLUIDING CORPORATION
South San Francisco
CA
|
Family ID: |
30000688 |
Appl. No.: |
10/517942 |
Filed: |
June 23, 2003 |
PCT Filed: |
June 23, 2003 |
PCT NO: |
PCT/US03/19775 |
371 Date: |
August 4, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60391292 |
Jun 24, 2002 |
|
|
|
Current U.S.
Class: |
117/2 |
Current CPC
Class: |
F16K 99/0046 20130101;
B01L 2400/0481 20130101; B01L 2300/0636 20130101; B01L 2300/0816
20130101; B01L 2400/0415 20130101; F16K 99/0036 20130101; Y10T
436/2575 20150115; F04B 43/02 20130101; F16K 99/0026 20130101; B01L
2400/0655 20130101; B01L 2300/0861 20130101; B01L 2300/088
20130101; B81B 3/00 20130101; F16K 2099/0084 20130101; F16K 99/0059
20130101; B01L 3/502738 20130101; F16K 99/0001 20130101; F16K
99/0051 20130101; B01L 3/5025 20130101; B01L 2300/0887 20130101;
B01L 3/502707 20130101; F16K 2099/008 20130101; G01N 33/54366
20130101; G01N 33/5304 20130101; B01L 3/50273 20130101; F16K
99/0003 20130101; F16K 2099/0074 20130101; F04B 43/14 20130101;
B01L 2300/0645 20130101; F16K 2099/0078 20130101; F04B 19/006
20130101 |
Class at
Publication: |
117/002 |
International
Class: |
H01L 21/322 20060101
H01L021/322 |
Claims
1. A microfluidic device comprising: a plurality of first flow
channels and a plurality of second flow channels, each such second
flow channel intersecting multiple of the first flow channels to
define intersecting volumes and a plurality of looped flow channels
that each include segments of the flow channels between the
intersecting volumes to define a closed loop; a plurality of
control valves each such control valve having a control channel and
a deformable segment disposed to restrict flow through a respective
one of the first and second flow channels in response to an
actuation force applied to the control channel to deflect the
deformable segment a pump operatively disposed to regulate flow
through one of said looped flow channels to regulate flow by the
recirculating pump.
2. The microfluidic device of claim 1, wherein the recirculating
pumps comprises multiple control channels formed within an
elastomeric layer and separated from the looped flow channel by an
elastomeric segment deflectable into the looped flow channel in
response to an actuation force.
3. The microfluidic device of claim 1, wherein actuation of the
control valves forms a plurality of holding valves, each such
holding valve being operatively disposed to form a holding space
encapsulating one of the intersecting volumes.
4. The microfluidic device of claim 1 further comprising a solution
inlet for each of said first flow channels in fluid communication
therewith for introduction of a first solution.
5. The microfluidic device of claim 4 further comprising a second
solution inlet for each of said second flow channels in fluid
communication therewith for introduction of a second solution.
6. The microfluidic device of claim 1, wherein the flow channels
are located on an interface between a solid substrate layer and an
elastomeric layer such that one side of each flow channels is
formed by said solid substrate surface.
7. The microfluidic device of claim 1, wherein: the flow channels
are located within an elastomeric layer; and each of the
intersecting volumes comprises a via in fluid communication with a
solid substrate surface to form a well for collecting fluid.
8. The microfluidic device of claim 1 further comprising a
plurality of first flow channel pumps, each such first flow channel
pump being operatively disposed to regulate solution flow through a
respective one of the first flow channels.
9. The microfluidic device of claim 8 further comprising a
plurality of second flow channel pumps, each such second flow
channel pump being operatively disposed to regulate solution flow
through a respective one of the second flow channels.
10. The microfluidic device of claim 9, wherein each such flow
channel pumps comprises multiple control channels formed within an
elastomeric layer and separated from the respective flow channel by
an elastomeric segment deflectable into the respective flow channel
in response to an actuation force.
11. The microfluidic device of claim 1 further comprising a first
solution outlet channel in fluid communication with each of said
first flow channels to receive solution flowing from each of said
first flow channels.
12. The microfluidic device of claim 1 further comprising a second
solution outlet channel in fluid communication with each of said
second flow channels to receive solution from each of said second
flow channels.
13. The microfluidic device of claim 1, further comprising a solid
support surface having a ligand that is capable of binding to a
specific antiligand at each of the intersecting volumes.
14. A method of conducting a binding assay with a microfluidic
device having a plurality of first flow channels and a plurality of
second flow channels, each such second flow channel intersecting
multiple of the first flow channels to define intersecting volumes
and a plurality of looped flow channels that each include segments
of the flow channels between the intersecting volumes to define a
closed loop, a plurality of control valves, each such control valve
having a control channel and a deformable segment disposed to
restrict flow through a respective one of the first and second flow
channels in response to an actuation force applied to the control
channel to deflect the deformable segment, and a recirculating pump
operatively disposed to regulate flow through one of the looped
flow channels to regulate flow by the recirculating pump, the
method comprising: applying an actuating force to each control
channel of a first plurality of the control valves to restrict
solution flow through each of the second flow channels; introducing
a reagent comprising a ligand into at least one of the first flow
channels under conditions sufficient to attach the ligand
covalently to a solid substrate surface; removing the actuation
force to the each control channel of the plurality of control
valves and applying an actuation force to each control channel of a
second plurality of the control valves such that solution flow
through the each control channel of the second plurality of control
valves is restricted; and performing a binding assay by introducing
a sample solution into the second flow channels under conditions
sufficient to specifically bind an antiligand that may be present
in the sample solution to the ligand that is covalently attached to
the solid substrate surface.
15. The method of claim 14 further comprising removing any ligand
that is not attached to the solid substrate surface from the each
control channel of the second plurality of control valves prior to
introducing the sample solution into the second flow channels.
16. The method of claim 14, wherein: performing the binding assay
comprises applying an actuating force to the control valves to form
a plurality of looped flow channels; and circulating the sample
solution within each of the looped flow channels the recirculating
pumps.
17. The method of claim 14, wherein performing the binding assay
comprises applying an actuating force to the control valves after
introducing the sample solution into the second flow channels such
that a plurality of holding spaces are formed to encapsulate each
of the intersecting volumes, thereby allowing a prolonged contact
between the sample solution and the ligand that is attached to the
solid substrate surface the intersecting volumes.
18. The method of claim 14, wherein: the flow channels are located
within an elastomeric layer; and each of the intersecting volumes
comprises a via in fluid communication with the solid substrate
surface to form a well for collecting fluid.
19. The method of claim 14, wherein: the first flow channels are in
communication with a pump; and the reagent is transported through
the first flow channels under action of the pump.
20. The method of claim 19, wherein the pump comprises multiple
control channels formed within an elastomeric layer and separated
from the first flow channels by elastomeric segments deflectable
into the first flow channels in response to an actuation force,
whereby the reagent is transported along the first flow
channels.
21. The method of claim 14, wherein the second flow channels are in
communication with a pump; and the sample solution is transported
through the second flow channels under action of the pump.
22. The method of claim 21, wherein the pump comprises multiple
control channels formed within an elastomeric layer and separated
from the second flow channels by elastomeric segments deflectable
into the second flow channels in response to an actuation force,
whereby the sample solution is transported along the second flow
channels.
23. The method of claim 14, wherein performing the binding assay
comprises removing an elastomeric layer from the solid substrate
surface and determining ligand/antiligand binding at each of the
intersecting volumes with a detector.
24. The method of claim 23, wherein the detector detects an optical
signal within the intersecting volumes.
25. The method of claim 24, wherein the detector detects a
fluorescence emission, fluorescence polarization or fluorescence
resonance energy transfer.
26. The method of claim 24, wherein the detector is an optical
microscope, a confocal microscope or a laser scanning confocal
microscope.
27. The method of claim 23, wherein the detector is a non-optical
sensor selected from the group consisting of a radioactivity sensor
and an electrical potential difference sensor.
28. The method of claim 14, wherein performing the binding assay
comprises detecting binding between a substrate and a cell
receptor; a substrate and an enzyme; an antibody and an antigen; a
nucleic acid and a nucleic acid binding protein; a protein and a
protein; a small molecule and a protein; a small molecule and an
oligonucleotide; and a protein affinity tag and a metal ion.
29. The method of claim 14, wherein the assay is an assay for
detecting a toxic effect on cells or a cell death assay, or a cell
proliferation assay.
30. The method of claim 14, wherein the assay is an oligonucleotide
binding assay or a peptide binding assay.
31. The method of claim 14, wherein the assay is an antimicrobial
assay.
32. A method for producing a microfluidic device comprising:
producing a control layer, a flow layer, and a via layer from an
elastomeric polymer, wherein each of the control layer and the flow
layer comprises grooves on respective surfaces for forming control
channels and flow channels; and attaching the control layer to the
flow layer such that the grooves in the control layer are attached
to a top surface of the flow layer to form a plurality of control
channels and attaching a bottom surface of the flow layer to the
via layer to form a plurality of first flow channels and a
plurality of second flow channels, wherein each first flow channels
intersects and crosses each multiple of the second flow channels
thereby to forming a plurality of channel intersections, and
wherein a vias in the via layer is positioned at each channel
intersections.
33. The method of claim 32, wherein producing the via layer
comprises etching the via layer to produce a plurality of vias.
34. The method of claim 32, further comprising attaching the
elastomeric polymer to a solid substrate that comprises a ligand
bound to its surface or comprises a functional group capable of
attaching a ligand.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional No.
60/391,292, filed Jun. 24, 2002, which is incorporated herein by
reference in its entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to microfluidic apparatus and
methods for using the same, such as conducting a variety of
assays.
BACKGROUND OF THE INVENTION
[0003] There are several goals in the development of biological
assays, including utilization of a minimal amount of assay
components and sample, simplicity in operation and high throughput
capability. Assays preferably require a minimal amount of assay
components in order to minimize costs; this becomes a particular
issue if certain assay components are expensive and/or a large
number of assays are to be conducted. Ideally, assays require only
a minimal amount of sample because often only a very limited amount
of sample is available. The goal of simplicity of operation often
means that the assay is preferably conducted in an integrated
format in which all or most aspects of the assay can be conducted
with a single device and minimal instrumentation. The goal of high
throughput has become increasingly important in view of the trend
in current research and drug discovery efforts to screen huge
libraries of compounds to identify those that have a desired
activity.
[0004] Another area where high throughput is particularly important
is in proteomics. Proteomics is the study of the complex biological
interactions that occur between proteins within a cell. Cells
express thousands of proteins at different concentrations. The
behavior and interaction of these proteins is dependent upon the
cell type, the stage of the cell cycle, and extracellular events,
to name a few. Proteins are also chemically modified by cellular
machinery, and this modification further differentiates their
behavior and interaction with other proteins.
[0005] To address some of these problems, particularly the issue of
minimizing the amount of sample and assay agents required to
conduct an analysis, considerable effort has been invested in the
development of microfluidic devices to conduct assays. These
devices are characterized by using minute channels for the
introduction and transport of the samples and agents necessary to
conduct an assay. Unfortunately, current microfluidic devices
suffer from a number of shortcomings that limit their usefulness.
For example, current microfluidic devices often are manufactured
from silicon chips with channels being etched into different
silicon layers using established semi-conductor technologies. Such
chips, however, are brittle and the stiffness of the material often
necessitates high actuation forces. These forces and stresses can
cause layers in a multilayer chip to separate from one another. The
stiffness of the devices also imposes significant constraints on
options for controlling solution flow through the
microchannels.
[0006] Furthermore, solution flow is controlled at least in part
through the use of electrodes to generate electric fields to move
molecules and solution via electrophoresis and/or electroosmosis.
Reliance on electrodes, however, creates several problems. One
problem is that gas is often generated at the electrodes. This can
increase pressure within the device potentially causing separation
of microfabricated layers. The increased pressure and gas bubbles
can also interfere with solution flow through the channels.
Additionally, often an elaborate network of electrodes is required
in order to achieve the desired level of control over solution
transport. Fabrication of such a network can be complicated and
increases the expense of the devices. The need for such networks
also becomes particularly problematic if a device is to be prepared
that includes a large number of channels to facilitate multiplexed
and high throughput assay capabilities. Moreover, the use of
electrical fields to control solution flow necessarily requires
solutions comprising electrolytes (i.e., ionizable compounds). In
addition, the use of electric fields can be problematic for
applications involving cells as application of the electric fields
can negatively affect the cells, often killing them. Consequently,
there remains a significant need for improved microfluidic devices,
particularly those that are amendable to a wide range of high
throughput assay capabilities.
SUMMARY OF THE INVENTION
[0007] The present invention provides a variety of microfluidic
devices and methods for conducting assays and syntheses. The
devices include a solid substrate layer having a surface that is
capable of attaching ligand and/or anti-ligand, and an elastomeric
layer attached to said solid substrate surface. The elastomeric
layer comprises:
[0008] (a) a plurality of first flow channels;
[0009] (b) a plurality of second flow channels each intersecting
and crossing each of said first flow channels thereby providing a
plurality of intersecting areas formed at intersections between
said first flow channels and said second flow channels, wherein
said plurality of first flow channels and said plurality of second
flow channels are adapted to allow the flow of a solution
therethrough, and wherein said solid substrate surface is in fluid
communication with at least said intersecting areas of said
plurality of first flow channels and said plurality of second flow
channels, and wherein said plurality of first flow channels and/or
said plurality of second flow channels are capable of forming a
plurality of looped flow channels;
[0010] (c) a plurality of control channels;
[0011] (d) a plurality of first control valves each operatively
disposed with respect to each of said first flow channel to
regulate flow of the solution through said first flow channels,
wherein each of said first control valves comprises a first control
channel and an elastomeric segment that is deflectable into or
retractable from said first flow channel upon which said first
control valve operates in response to an actuation force applied to
said first control channel, the elastomeric segment when positioned
in said first flow channel restricting solution flow
therethrough;
[0012] (e) a plurality of second control valves each operatively
disposed with respect to each of said second flow channel to
regulate flow of the solution through said second flow channels,
wherein each of said second control valves comprises a second
control channel and an elastomeric segment that is deflectable into
or retractable from said second flow channel upon which said second
control valve operates in response to an actuation force applied to
said second control channel, the elastomeric segment when
positioned in said second flow channel restricting solution flow
therethrough;
[0013] (f) a plurality of loop forming control valves each
operatively disposed with respect to each of said first and/or said
second flow channels to form said plurality of looped flow
channels, wherein each of said loop forming control valves
comprises a loop forming control channel and an elastomeric segment
that is deflectable into or retractable from said first and/or said
second flow channels upon which said loop forming control valve
operates in response to an actuation force applied to said loop
forming control channel, the elastomeric segment when positioned in
said first and/or said second flow channels restricting solution
flow therethrough thereby forming said looped flow channel; and
[0014] (g) a plurality of recirculating pumps, and wherein each
recirculating pump is operatively disposed with respect to one of
said looped flow channels such that circulation of solution through
each of said looped flow channels can be regulated by one of said
recirculating pumps.
[0015] Another aspect of the present invention provides a method
for conducting a binding assay using the microfluidic devices
disclosed herein. The binding assay method generally involves:
[0016] applying an actuating force to the second control valves to
restrict solution flow through each of the second flow
channels;
[0017] introducing a reagent comprising a ligand into at least one
of the first flow channels under conditions sufficient to attach
the ligand to the solid substrate surface;
[0018] removing the actuation force to the second flow channel
control channel and applying an actuation force to the first
control channel such that solution flow through the first flow
channel is restricted; and
[0019] performing a binding assay by introducing a sample solution
into the second flow channel under conditions sufficient to
specifically bind an antiligand that may be present in the sample
solution to the ligand that is covalently attached to the solid
substrate surface.
[0020] Yet another aspect of the present invention provides a
method for producing a microfluidic device comprising a via layer.
Such a method generally involves:
[0021] producing a control layer, a flow layer, and a via layer
from an elastomeric polymer, wherein each of the control layer and
the flow layer comprises grooves on its surface for forming control
channels and flow channels, respectively;
[0022] attaching the control layer to the flow layer such that the
grooves in the control layer is attached to a top surface of the
flow layer thereby forming a plurality of control channels and
attaching the bottom surface of the flow layer to the via layer
thereby forming a plurality of first flow channels and a plurality
of second flow channels, wherein each first flow channels
intersects and crosses each of the second flow channels thereby
forming a plurality of channel intersections, and wherein each vias
in the via layer is positioned at each channel intersections;
and
[0023] optionally attaching the elastomeric polymer produced in
said step (b) to a solid substrate which is comprises a ligand
bound to its surface or comprises a functional group which is
capable of attaching a ligand.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In certain drawings, pumps are denoted with a group of three
dashed lines and valves denoted by a single dashed line.
[0025] FIGS. 1A and 1B are illustrations of an elastomeric block
and the arrangement of a control and flow channel therein.
[0026] FIG. 2A is a sectional view of an elastomeric block showing
the disposition of a flow and control channels with respect to one
another in a valve and optional electrodes for actuating the
valve.
[0027] FIG. 2B is a sectional view of an elastomeric block showing
blockage of a flow channel when a normally open valve is
actuated.
[0028] FIGS. 3A and 3B show one example of a normally-closed valve
structure.
[0029] FIG. 4 illustrates one arrangement of control and flow
channels that allow for selective blockage of certain flow
channels.
[0030] FIGS. 5A and 5B illustrate one example of a peristaltic
pump. FIG. 5A is a top schematic of the peristaltic pump. FIG. 5B
is a sectional elevation view along line 24B-24B in FIG. 5A.
[0031] FIG. 6 is a cross-sectional view that illustrates the
formation of a holding space within a flow channel upon actuation
of valves in the flow channel.
[0032] FIGS. 7A-E depicts an exemplary microfluidic device
incorporating various components. FIGS. 7A and 7C show arrangement
of each components. FIGS. 7B, 7D and 7E show an isolated close-up
view of flow channels and control channels near the intersection of
the first and the second flow channels. FIGS. 7D and 7E show
different configuration of control channels.
[0033] FIGS. 8A-8E show exemplary mold designs for each of the
corresponding control layer (808), the fluid (i.e., flow channel)
layer (804), and the via layer (812). In FIGS. 8B-D, an alignment
mask 816 is also shown.
[0034] FIG. 9 illustrates formation of a via layer and etching step
to open the vias where residual PDMS exist on the mold during the
fabrication step.
[0035] FIGS. 10A and 10B also shows exemplary designs for fluid
layer (804) and the control layer (808).
[0036] FIG. 10B shows one particular microfluidic device design
having an integrated fluid layer and control layer.
[0037] FIG. 11 shows a microfluidic device configuration used in
Example 2.
[0038] FIG. 12 is a schematic illustration of a binding assay
described in Example 2.
DETAILED DESCRIPTION
I. Definitions
[0039] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this invention belongs. The following
references provide one of skill with a general definition of many
of the terms used in this invention: Singleton et al., DICTIONARY
OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE
DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); THE
GLOSSARY OF GENETICS, 5TH ED., R. Rieger et al. (eds.), Springer
Verlag (1991); and Hale & Marham, THE HARPER COLLINS DICTIONARY
OF BIOLOGY (1991). As used herein, the following terms have the
meanings ascribed to them unless specified otherwise.
[0040] The term "elastomer" and "elastomeric" has its general
meaning as used in the art. Thus, for example, Allcock et al.
(Contemporary Polymer Chemistry, 2nd Ed.) describes elastomers in
general as polymers existing at a temperature between their glass
transition temperature and liquefaction temperature. Elastomeric
materials exhibit elastic properties because the polymer chains
readily undergo torsional motion to permit uncoiling of the
backbone chains in response to a force, with the backbone chains
recoiling to assume the prior shape in the absence of the force. In
general, elastomers deform when force is applied, but then return
to their original shape when the force is removed. The elasticity
exhibited by elastomeric materials can be characterized by a
Young's modulus. The elastomeric materials utilized in the
microfluidic devices disclosed herein typically have a Young's
modulus of between about 1 Pa-1 TPa, in other instances between
about 10 Pa-100 GPa, in still other instances between about 20 Pa-1
GPa, in yet other instances between about 50 Pa-10 MPa, and in
certain instances between about 100 Pa-1 MPa. Elastomeric materials
having a Young's modulus outside of these ranges can also be
utilized depending upon the needs of a particular application.
[0041] Some of the microfluidic devices described herein are
fabricated from an elastomeric polymer such as GE RTV 615
(formulation), a vinyl-silane crosslinked (type) silicone elastomer
(family). However, the present microfluidic systems are not limited
to this one formulation, type or even this family of polymer;
rather, nearly any elastomeric polymer is suitable. Given the
tremendous diversity of polymer chemistries, precursors, synthetic
methods, reaction conditions, and potential additives, there are a
large number of possible elastomer systems that can be used to make
monolithic elastomeric microvalves and pumps. The choice of
materials typically depends upon the particular material properties
(e.g., solvent resistance, stiffness, gas permeability, and/or
temperature stability) required for the application being
conducted. Additional details regarding the type of elastomeric
materials that can be used in the manufacture of the components of
the microfluidic devices disclosed herein are set forth in U.S.
application Ser. No. 09/605,520, filed Jun. 27, 2000, U.S.
application Ser. No. 09/724,784, filed Nov. 28, 2000, and PCT
publication WO 01/01025, all of which are incorporated herein by
reference in their entirety.
[0042] "Ligand" generally refers to any molecule that binds to an
antiligand to form a ligand/antiligand pair. Thus, a ligand is any
molecule for which there exists another molecule (i.e., the
antiligand) that specifically or non-specifically binds to the
ligand, owing to recognition of some portion or feature of the
ligand.
[0043] "Antiligand" is a molecule that specifically or
nonspecifically interacts with another molecule (i.e., the
ligand).
[0044] Exemplary ligand/antiligand pairs include antibody/antigen,
enzyme/substrate, oligonucleotide/complementary oligonucleotide,
nucleic acid/probe, drug molecule/cellular protein, drug
molecule/cell membrane, cellular protein/cellular protein, protein
affinity tag/protein, protein affinity tag/metal ion, Protein A or
G/antibody, as well as other ligand/antiligand pairs that form a
corresponding complex. It should be appreciated that the terms
ligand and antiligand for a given ligand/antiligand pair is
interchangeable. Thus, for example, either of the antibody or
antigen can be considered to be a ligand as long as the other pair
is considered to be the antiligand.
[0045] "Polypeptide," "peptides" and "protein" are used
interchangeably herein and include a molecular chain of amino acids
linked through peptide bonds. The terms do not refer to a specific
length of the product. The terms include post-translational
modifications of the polypeptide, for example, glycosylations,
acetylations, phosphorylations and the like, and also can include
polypeptides that include amino acid analogs and modified peptide
backbones.
[0046] The term "antibody" as used herein includes antibodies
obtained from both polyclonal and monoclonal preparations, as well
as the following: (i) hybrid (chimeric) antibody molecules (see,
for example, Winter et al. (1991) Nature 349:293-299; and U.S. Pat.
No. 4,816,567); (ii) F(ab')2 and F(ab) fragments; (iii) Fv
molecules (noncovalent heterodimers, see, for example, Inbar et al.
(1972) Proc. Natl. Acad. Sci. USA 69:2659-2662; and Ehrlich et al.
(1980) Biochem 19:4091-4096); (iv) single-chain Fv molecules (sFv)
(see, for example, Huston et al. (1988) Proc. Natl. Acad. Sci. USA
85:5879-5883); (v) dimeric and trimeric antibody fragment
constructs; (vi) humanized antibody molecules (see, for example,
Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988)
Science 239:1534-1536; and U.K. Patent Publication No. GB
2,276,169, published 21 Sep. 1994); (vii) Mini-antibodies or
minibodies (i.e., sFv polypeptide chains that include
oligomerization domains at their C-termini, separated from the sFv
by a hinge region; see, e.g., Pack et al. (1992) Biochem
31:1579-1584; Cumber et al. (1992) J. Immunology 149B:120-126);
and, (vii) any functional fragments obtained from such molecules,
wherein such fragments retain specific-binding properties of the
parent antibody molecule.
[0047] The terms "nucleic acid," "polynucleotide," and
"oligonucleotide" are used herein to include a polymeric form of
nucleotides of any length, including, but not limited to,
ribonucleotides or deoxyribonucleotides. There is no intended
distinction in length between these terms. Further, these terms
refer only to the primary structure of the molecule. Thus, in
certain embodiments these terms can include triple-, double- and
single-stranded DNA, as well as triple-, double- and
single-stranded RNA. They also include modifications, such as by
methylation and/or by capping, and unmodified forms of the
polynucleotide. More particularly, the terms "nucleic acid,"
"polynucleotide," and "oligonucleotide," include
polydeoxyribonucleotides (containing 2-deoxy-D-ribose),
polyribonucleotides (containing D-ribose), any other type of
polynucleotide which is an N- or C-glycoside of a purine or
pyrimidine base, and other polymers containing nonnucleotidic
backbones, for example, polyamide (e.g., peptide nucleic acids
(PNAs)) and polymorpholino (commercially available from the
Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and
other synthetic sequence-specific nucleic acid polymers providing
that the polymers contain nucleobases in a configuration which
allows for base pairing and base stacking, such as is found in DNA
and RNA.
[0048] A "probe" is an nucleic acid capable of binding to a target
nucleic acid of complementary sequence through one or more types of
chemical bonds, usually through complementary base pairing, usually
through hydrogen bond formation, thus forming a duplex structure.
The probe binds or hybridizes to a "probe binding site." The probe
can be labeled with a detectable label to permit facile detection
of the probe, particularly once the probe has hybridized to its
complementary target. The label attached to the probe can include
any of a variety of different labels known in the art that can be
detected by chemical or physical means, for example. Suitable
labels that can be attached to probes include, but are not limited
to, radioisotopes, fluorophores, chromophores, mass labels,
electron dense particles, magnetic particles, spin labels,
molecules that emit chemiluminescence, electrochemically active
molecules, enzymes, cofactors, and enzyme substrates. Probes can
vary significantly in size. Some probes are relatively short.
Generally, probes are at least 7 to 15 nucleotides in length. Other
probes are at least 20, 30 or 40 nucleotides long. Still other
probes are somewhat longer, being at least 50, 60, 70, 80, 90
nucleotides long. Yet other probes are longer still, and are at
least 100, 150, 200 or more nucleotides long. Probes can be of any
specific length that falls within the foregoing ranges as well.
[0049] The term "complementary" means that one nucleic acid is
identical to, or hybridizes selectively to, another nucleic acid
molecule. Selectivity of hybridization exists when hybridization
occurs that is more selective than total lack of specificity.
Typically, selective hybridization will occur when there is at
least about 55% identity over a stretch of at least 14-25
nucleotides, preferably at least 65%, more preferably at least 75%,
and most preferably at least 90%. Preferably, one nucleic acid
hybridizes specifically to the other nucleic acid. See M. Kanehisa,
Nucleic Acids Res. 12:203 (1984).
[0050] The term "stringent conditions" refers to conditions under
which a probe or primer will hybridize to its target subsequence,
but to no other sequences. Stringent conditions are
sequence-dependent and will be different in different
circumstances. Longer sequences hybridize specifically at higher
temperatures. Generally, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point (Tm) for
the specific sequence at a defined ionic strength and pH. In other
instances, stringent conditions are chosen to be about 20.degree.
C. or 25.degree. C. below the melting temperature of the sequence
and a probe with exact or nearly exact complementarity to the
target. As used herein, the melting temperature is the temperature
at which a population of double-stranded nucleic acid molecules
becomes half-dissociated into single strands. Methods for
calculating the T.sub.m of nucleic acids are well known in the art
(see, e.g., Berger and Kimmel (1987) Methods in Enzymology, vol.
152: Guide to Molecular Cloning Techniques, San Diego: Academic
Press, Inc. and Sambrook et al. (1989) Molecular Cloning: A
Laboratory Manual, 2nd ed., vols. 1-3, Cold Spring Harbor
Laboratory), both incorporated herein by reference. As indicated by
standard references, a simple estimate of the T.sub.m value can be
calculated by the equation: T.sub.m=81.5+0.41 (% G+C), when a
nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson
and Young, "Quantitative Filter Hybridization," in Nucleic Acid
Hybridization (1985)). Other references include more sophisticated
computations which take structural as well as sequence
characteristics into account for the calculation of T.sub.m. The
melting temperature of a hybrid (and thus the conditions for
stringent hybridization) is affected by various factors such as the
length and nature (DNA, RNA, base composition) of the probe and
nature of the target (DNA, RNA, base composition, present in
solution or immobilized, and the like), and the concentration of
salts and other components (e.g., the presence or absence of
formamide, dextran sulfate, polyethylene glycol). The effects of
these factors are well known and are discussed in standard
references in the art, see e.g., Sambrook, supra, and Ausubel,
supra. Typically, stringent conditions will be those in which the
salt concentration is less than about 1.0 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes or primers (e.g., 10 to 50 nucleotides) and at least about
60.degree. C. for long probes or primers (e.g., greater than 50
nucleotides). Stringent conditions can also be achieved with the
addition of destabilizing agents such as formamide.
[0051] A "small molecule" means a synthetic molecule having a
molecular weight of less than 1000 daltons, more typically 500
daltons or less. Such molecules include, for example,
monosaccharides, polysaccharides, polypeptides, sterols, amino
acids, lipids and nucleic acids.
[0052] The phrase "specifically binds" generally refers to binding
of a ligand and an antiligand, or vice versa, with greater affinity
and specificity than to other components in the sample. Thus, the
term refers to a binding reaction which is determinative of the
presence of the ligand in the presence of a heterogeneous
population of other biological compounds. Thus, under designated
conditions, a specified ligand binds preferentially to a particular
antiligand and does not bind in a significant amount to other
molecules present in the sample. Typically, a molecule or ligand
(e.g., an antibody) that specifically binds to an antiligand has an
association constant of at least 10.sup.3 M.sup.-1 or 10.sup.4
M.sup.-1, sometimes 10.sup.5 M.sup.-1 or 10.sup.6 M.sup.-1, in
other instances 10.sup.6 M.sup.-1 or 10.sup.7 M.sup.-1, preferably
10.sup.8 M.sup.-1 to 10.sup.9 M.sup.-1, and more preferably, about
10.sup.10 M.sup.-1 to 10.sup.11 M.sup.-1 or higher.
[0053] A difference is typically considered to be "statistically
significant" if the difference is greater than the level of
experimental error. More specifically, a difference is
statistically significant if the probability of the observed
difference occurring by chance (the p-value) is less than some
predetermined level. As used herein a "statistically significant
difference" refers to a p-value that is <0.05, preferably
<0.01 and most preferably <0.001.
[0054] The term "label" refers to a molecule or an aspect of a
molecule that can be detected by physical, chemical,
electromagnetic and other related analytical techniques. Examples
of detectable labels that can be utilized include, but are not
limited to, radioisotopes, fluorophores, chromophores, mass labels,
electron dense particles, magnetic particles, spin labels,
molecules that emit chemiluminescence, electrochemically active
molecules, enzymes, cofactors, and enzyme substrates. The term
"detectably labeled" means that an agent has been conjugated with a
label or that an agent has some inherent characteristic (e.g.,
size, shape or color) that allows it to be detected without having
to be conjugated to a separate label.
II. Introduction
[0055] Described herein are microfluidic devices and methods for
conducting a variety of different assays, such as high throughput
screening assays, cellular assays or assays involving cellular
components, and syntheses, such as combinatorial syntheses. The
microfluidic devices are characterized in part by including various
components such as flow channels, control channels, valves and/or
pumps, at least some of which are manufactured from elastomeric
materials.
[0056] In addition, microfluidic devices of the present invention
comprise a solid substrate layer. The solid substrate layer
comprises a surface to which one or more ligands are attached.
Alternatively, the solid substrate surface comprises a functional
group or is capable of being derivatized such that a ligand can be
attached to its surface. Preferably, the ligand is covalently
attached to the solid substrate surface.
[0057] The presence of a ligand on the solid substrate surface
allows a variety of assays as described in detail below.
[0058] The microfluidic devices of the present invention include a
plurality of first flow channels and a plurality of a second flow
channels. The presence of more than one flow channels allows
parallel assay on a single microfluidic device thereby increasing
the throughput. Each of the first flow channels intersects and
crosses each of the second flow channels resulting in a plurality
of intersecting areas. By introducing a fluid comprising a ligand
in the first flow channel and introducing a fluid which may
comprise an antiligand in the second flow channel, the binding
assay are achieved on the intersecting areas of the first and the
second flow channels.
[0059] By selectively activating appropriate valves, at least one
of the plurality of first flow channels or the plurality of second
flow channels are capable of forming a plurality of looped flow
channel. The valves comprise a control channel separated from a
flow channel by an elastomeric segment or membrane that can be
deflected into or withdrawn from the flow channel upon actuation of
the control channel (e.g., by applying pressure or a vacuum to the
control channel). When the elastomeric segment extends into the
flow channel, it blocks solution flow through the channel. Each of
the looped flow channels can also comprise a pump thereby providing
a means for circulating or recirculating the solution within the
looped flow channel. The pump can comprise one or more control
valves, preferably more than one control valves, and more
preferably three control valves that can be actuated in a desired
sequence.
[0060] In addition, microfluidic devices of the present invention
comprise a plurality of first control valves and a plurality of
second control valves. The each of the first control valves
regulates the flow of a solution through each of the first flow
channels and each of the second control valves regulates the flow
of a solution through each of the second flow channels. In this
manner, selective activation of control valves prevents the flow of
solution to undesired flow channels.
[0061] Some microfluidic devices of the present invention also
comprise a plurality of holding valves each forming a holding space
that encapsulates one intersecting area of the first and the second
flow channels. In this manner, the fluid flow within the
intersecting areas can be restricted thereby providing a means for
contacting the solution to the intersecting areas for a prolonged
period. The holding valve comprises two pairs of valves, one pair
in the first flow channel and another pair in the second flow
channel. In each pair of valves, the valves are positioned relative
to one another such that they are positioned in the different side
of the intersecting area. Thus, when the elastomeric segment of the
holding valves extend into the flow channel, the elastomeric
segment form a holding space within the flow channel that is
bounded by the extended elastomeric segment and encapsulating and
isolating each individual intersecting areas. In one embodiment,
the holding valves are formed from a combination of both the
plurality of first control valves and the plurality of second
control valves forms a plurality of holding valves.
[0062] Microfluidic devices of the present invention can also
include a variety of pumps, such as a pump for transporting a fluid
along the first flow channels and a pump for transporting a fluid
along the second flow channels. Certain pumps are characterized by
including a plurality, preferably at least three, control channels
that are separated from the flow channel by an elastomeric segment
that can be deflected into the flow channel when actuated. By
actuating the control channels in a staggered fashion, a
peristaltic effect can be induced.
[0063] In addition, microfluidic devices of the present invention
can also include optional reservoirs or storage areas (i.e., fluid
chambers). Such reservoirs or chambers are typically positioned at
or near the inlet portion of the flow channels to provide a storage
site for fluids prior to introducing the fluid into the first or
the second flow channels. Each flow channel can have its own
reservoir, or two or more flow channels can have one common
reservoir. Similarly, microfluidic devices of the present invention
can also include optional waste reservoirs for collecting fluids
from the flow channel outlets or the waste flow channel.
[0064] Furthermore, microfluidic devices of the present invention
can also include a waste flow channel and/or waste collection
chamber. Typically, all the outlets of the first flow channels are
in fluid communication with the first waste flow channel thereby
eliminating a need for a multiple waste flow channels for the first
flow channels. Similarly, all the outlets of the second flow
channels are in fluid communication with the second waste flow
channel thereby eliminating a need for a multiple waste flow
channels for the second flow channels. However, it should be
appreciated that the present invention is not limited to having a
single waste channel for each of the first and the second flow
channels.
[0065] The flow channels are formed within the elastomeric layer
with at least a portion of the flow channel being enclosed by the
solid substrate. In one embodiment, the solid substrate forms one
wall of the flow channel, i.e., the flow channels is located within
the interface of the solid substrate and the elastomeric layer. In
another embodiment, the entire flow channel is formed within the
elastomeric layer except for the intersecting areas of the first
and the second flow channels where the solid substrate forms one
side of the wall of the flow channel.
[0066] The microfluidic devices provided herein can be utilized in
a number of different assay applications, for example, high
throughput ligand/antiligand binding assays. By controllably
introducing different solutions into the different flow channels, a
number of different analyses or syntheses can be performed at the
same time. Thus, the microfluidic devices can be used to conduct a
number of different types of assays.
[0067] The devices disclosed herein can be utilized to screen
individual compounds and libraries of compounds to identify those
having a desired effect in various in vitro model systems. For
example, assays utilizing the microfluidic devices provided herein
can be utilized to screen libraries of compounds for those capable
of fully or partially inhibiting reactions or processes that have
undesirable consequences. For instance, libraries can be screened
to identify compounds that inhibit reactions or processes involved
in the onset of disease or particular symptoms associated with the
disease (e.g., bacterial and viral infections, hereditary diseases
and cancer). Alternatively, individual compounds and libraries of
compounds can be screened to identify particular compounds that
activate or promote reactions or processes of interest. Compounds
showing activity in initial screening can then be subjected to
other screens or modified and rescreened to identify compounds
suitable for formulation as pharmaceutical agents in treating the
disease or symptoms associated with the disease under
investigation.
[0068] The devices disclosed herein can also be utilized to
identify the concentrations of various proteins or proteins with
post-translational modifications, such as glycosylation or
phosphorylation. For example, assays utilizing the microfluidic
devices provided herein can be utilized to screen cell populations
or cell types for variations in protein concentrations. Such
information can be useful in determining phenotypes associated with
bacterial and viral infections, hereditary diseases and cancer, for
example. Such information can be useful in identifying new
pharmaceutical drug targets or for elucidating the significance of
various cellular proteins.
[0069] In general such screening methods involve attaching a ligand
(e.g., cell, enzyme, oligonucleotide, peptide, or antibody, etc.)
to the solid substrate surface within the flow channel, introducing
an antiligand (e.g., small molecule, substrate, complimentary
oligonucleotide, binding peptide, or antigen, etc., respectively)
and measuring the binding activity. The antiligand can be labeled
to further assist in detecting ligand/antiligand binding.
Alternatively, a labeled ligand can be added to form
ligand/antiligand/labeled ligand triple complex. In this manner,
ELISA (enzyme-linked immuno) and FLISA (fluorescence-linked) assays
and other conventionally known ligand/antiligand/labeled ligand
triple complex assays can be achieved.
[0070] Typically, the ligand is attached to the solid substrate
surface by introducing appropriate reagents into the microfluidic
device through the first flow channels. After the ligand is
attached, the unbound ligands and reagents are removed, e.g., by
washing the first flow channels with a washing solution.
Alternatively, when only the channel intersecting areas are of
interest, the unbound ligands can be removed by closing the first
control valve and introducing the washing solution through the
second flow channels. A sample solution which may comprising an
antiligand is then introduced through the second flow channels. The
sample solution can be circulated through the second flow channels
to afford prolonged exposure to the bound ligand or the sample
solution can be continuously flowed through the second flow
channel. Still alternatively, the sample solution can be held
within the holding space to allow prolonged contact with the bound
ligand. When a triple complex assay is performed, the unbound
ligand is removed from the second flow channels, e.g. by flushing
the second flow channels with a washing solvent. Again, when only
the channel intersecting areas are of interest, the unbound ligands
can be removed by closing the second control valve and introducing
the washing solution through the first flow channels. A ligand
solution, in which the ligand is optionally labeled, is then
introduced into the first or the second flow channels. In this
manner, the binding assay occurs at only the intersecting
areas.
[0071] One can determine the binding assay by removing the
elastomeric layer from the solid substrate layer and measuring the
appropriate binding parameter(s) at the intersecting areas.
Alternately, the binding assay can be determined with the
elastomeric layer still in contact with the solid substrate.
[0072] Similarly, the valves and pumps of the microfluidic devices
can be utilized to controllably react different reactants in the
different intersecting areas to perform combinatorial
syntheses.
III. Microfluidic Elements
[0073] A number of elements that are commonly utilized in the
microfluidic devices disclosed herein are described below. It
should be recognized that these elements can be considered modules
that can be combined in different ways to yield an essentially
unlimited number of configurations. Further, using the following
elements or modules one can tailor the microfluidic device to
include those elements useful for the particular application(s) to
be conducted with the device.
[0074] A. General
[0075] It is to be understood that the present invention is not
limited to fabrication of microfluidic devices in the manner
discussed below. Rather, other suitable methods of fabricating the
present microfluidic devices, including modifying the present
methods, are also within the scope of the present invention.
[0076] The microfluidic devices disclosed herein are typically
constructed by single and multilayer soft lithography (MLSL)
techniques and/or sacrificial-layer encapsulation methods. Both of
these methods are described in detail by Unger et al. (2000)
Science 288:113-116, in U.S. patent application Ser. No.
09/605,520, filed Jun. 27, 2000, in U.S. patent application Ser.
No. 09/724,784, filed Nov. 28, 2000, and in PCT publication WO
01/01025, all of which are incorporated herein in their entirety.
The microfluidic devices provided herein can include a variety of
different components that are described in detail infra. These
components can be arranged in a large number of different
configurations depending upon the particular application. The
following sections describe the general components that are
utilized in the devices; these sections are followed with exemplary
configurations that can be utilized in various types of assays and
high throughput screening.
[0077] As described in detail below, the elastomeric layer portion
of the flow channels can be tailored to the particular application
by modifying the internal surfaces of the elastomeric layer flow
channels.
[0078] B. Solid Substrate
[0079] The microfluidic devices of the present invention allow
conducting assay on the solid substrate. Therefore, any material
which can be derivatized to allow attachment of a ligand, or a
linker molecule, can be used as the solid substrate. Exemplary
materials suitable for the solid substrate of the present invention
include, but are not limited to, glass (including controlled-pore
glass), polystyrene, polystyrene-divinylbenzene copolymer (e.g.,
for synthesis of peptides), silicone rubber, quartz, latex,
polyurethane, gold and other derivatizable transition metals,
silicon dioxide, silicon nitride, gallium arsenide, and the like.
Solid substrate materials are preferably resistant to the variety
of undesired chemical reaction conditions to which they may be
subjected.
[0080] Individual planar solid substrate can have varied dimensions
from which a plurality of individual arrays or chips may be
fabricated. The term "array" or "chip" is used to refer to the
final product of the individual array of ligand/antiligand complex,
having a plurality of different positionally distinct
ligand/antiligand complex coupled to the surface of the solid
substrate. The size of a solid substrate is generally defined by
the number and nature of arrays that will be produced from the
solid substrate. For example, more complex arrays will generally
utilize larger areas and thus employ larger solid substrate,
whereas simpler arrays may employ smaller surface areas, and thus,
smaller solid substrate.
[0081] The size of solid substrate generally depends on the number
of ligand/antiligand complex arrays desired. Typically, however,
the solid substrate dimensions can be anywhere from about 1
cm.times.1 cm to about 30 cm.times.30 cm. In one particular
embodiment of the present invention, the solid substrate is a
standard 1''.times.3'' or 2''.times.3'' glass microscope slides, or
1''.times.1'', 1.5''.times.1.5'', or 2''.times.2'' quartz glass
windows.
[0082] Stripping and Rinsing
[0083] In order to ensure maximum efficiency in attaching a ligand
to its surface, it is generally desirable to provide a clean solid
substrate surface upon which the ligand attaching reactions are to
take place. Accordingly, in some embodiments of the present
invention, the solid substrate is stripped to remove any residual
dirt, oils or other materials which may interfere with attachment
of ligands.
[0084] The process of stripping the solid substrate typically
involves applying, immersing or otherwise contacting the solid
substrate with a stripping solution. Stripping solutions may be
selected from a number of commercially available, or readily
prepared chemical solutions used for the removal of dirt and oils,
which solutions are well known in the art. Particularly preferred
stripping solutions are composed of a mixture of concentrated
H.sub.2O.sub.2 and NH.sub.4OH. Gas phase cleaning and preparation
methods may also be applied to the solid substrate using techniques
that are well known in the art.
[0085] Derivatization
[0086] While not necessary, after the solid substrate surface has
been cleaned and stripped, the surface may be derivatized to
provide other sites or functional groups on the solid substrate
surface for attaching ligands. In particular, derivatization
provides reactive functional groups, e.g., hydroxyl, carboxyl,
amino groups or the like, to which the ligand or a linker can be
attached. For example, the solid substrate surface can be
derivatized using silane in either water or ethanol. Preferred
silanes include mono- and dihydroxyalkylsilanes, which provide a
hydroxyl functional group on the surface of the substrate.
Alternatively, aminoalkyltrialkoxysilanes can be used to provide
the initial surface modification with a reactive amine functional
group. Particularly preferred are 3-aminopropyltriethoxysilane and
3-aminopropyltrimethoxysilane ("APS"). Derivatization of the
substrate using these latter amino silanes provides a linkage that
is stable under various assaying conditions and other chemical
reaction conditions (for oligonucleotide synthesis, this linkage is
typically a phosphoramidite linkage, as compared to the
phosphodiester linkage where hydroxyalkylsilanes are used).
Additionally, this amino silane derivatization provides several
advantages over derivatization with hydroxyalkylsilanes. For
example, the aminoalkyltrialkoxysilanes are inexpensive and can be
obtained commercially in high purity from a variety of sources, the
resulting primary and secondary amine functional groups are more
reactive nucleophiles than hydroxyl groups; thus, providing a
reactive site for attaching ligands. In addition, the
aminoalkyltrialkoxysilanes are less prone to polymerization during
storage, and they are sufficiently volatile to allow application in
a gas phase in a controlled vapor deposition process. Other
suitable linkers are well known to one of ordinary skill in the
art.
[0087] Additionally, silanes can be prepared having protected or
"masked" hydroxyl groups and which possess significant volatility.
As such, these silanes can be readily purified, e.g., by
distillation, and can be readily employed in gas-phase deposition
methods of silanating solid support surfaces. After coating these
silanes onto the surface of the solid substrate, the hydroxyl
groups may be deprotected with a brief chemical treatment, e.g.,
dilute acid or base, which will not attack the solid
substrate-silane bond, so that the solid substrate can then be used
for attaching ligands or polymer synthesis. Examples of such
silanes include acetoxyalkylsilanes, such as
acetoxyethyltrichlorosilane, acetoxypropyltrimethoxysilane, which
may be deprotected after application, e.g., using vapor phase
ammonia and methylamine or liquid phase aqueous or ethanolic
ammonia and alkylamines.
[0088] The physical operation of silanation of the solid substrate
generally involves dipping or otherwise immersing the solid
substrate in the silane solution. Following immersion, the solid
substrate is generally spun laterally to provide a uniform
distribution of the silane solution across the surface of the solid
substrate. This ensures a more even distribution of reactive
functional groups on the surface of the solid substrate. Following
application of the silane layer, the silanated solid substrate may
be baked to polymerize the silanes on the surface of the solid
substrate and improve the reaction between the silane reagent and
the solid substrate surface.
[0089] Alternatively, the silane solution may be contacted with the
surface of the solid substrate using controlled vapor deposition
methods or spray methods. These methods involve the volatilization
or atomization of the silane solution into a gas phase or spray,
followed by deposition of the gas phase or spray upon the surface
of the solid substrate, usually by ambient exposure of the surface
of the solid substrate to the gas phase or spray. Vapor deposition
typically results in a more even application of the derivatization
solution than simply immersing the solid substrate into the
solution.
[0090] The efficacy of the derivatization process, e.g., the
density and uniformity of functional groups on the solid substrate
surface, may generally be assessed by adding a fluorophore which
binds the reactive groups, e.g., a fluorescent phosphoramidite such
as Fluoreprime.RTM. from Pharmacia, Corp., Fluoredite.RTM. from
Millipore, Corp. or FAM.RTM. from ABI, and looking at the relative
fluorescence across the surface of the solid support.
[0091] As described above, ligands can be attached to the solid
substrate surface prior to attaching the elastomeric layer to the
solid substrate surface. Alternatively, ligands can be attached to
the solid substrate surface after attaching the elastomeric layer
to the solid substrate. Such ligand attachment can be achieved by
introducing an appropriate reagent solution into the flow channel
under conditions sufficient to allow bond formation between the
solid substrate surface and the ligand.
[0092] C. Channels
[0093] The channels through which fluid is transported in the
microfluidic devices are typically formed at least in part from
elastomeric layer. Separated from the flow channels by an
elastomeric membrane are control channels which can be actuated to
control or regulate fluid (e.g., Solution) flow through the flow
channels. As described in greater detail below in the section on
valves, actuation of the control channel (e.g., pressurization or
pressure reduction within the flow channel) causes the elastomeric
segment separating the flow and control channel to be extended into
the flow channel, thus forming a valve that blocks solution flow in
the flow channel. Typically, the flow and control channels cross
one another at an angle.
[0094] The flow and control channels can be manufactured from two
primary techniques. One approach is to cast a series of elastomeric
layers on a micro-machined mold and then fuse the layers together.
The second primary method is to form patterns of photoresist on an
elastomeric layer in a desired configuration; in particular,
photoresist is deposited wherever a channel is desired. These two
different methods of forming the desired configuration of flow and
control channels, as well as other details regarding channel
dimensions and shape, are described in considerable detail in PCT
publication WO 01/01025, U.S. application Ser. No. 09/605,520,
filed Jun. 27, 2000, U.S. application Ser. No. 09/724,784, filed
Nov. 28, 2000, and by Unger et al. (2000) Science 288:113-116, each
of which is incorporated herein by reference in its entirety.
[0095] C. Sample Inputs
[0096] There are a number of different options for introducing a
solution into a flow channel. One option is to simply inject
solution into a flow channel using a needle, for example. One can
also pressurize a container of solution to force solution from the
container into a flow channel. A related approach involves reducing
pressure at one end of a flow channel to pull solution into a
distal opening in the flow channel.
[0097] Individual input/inlet lines can be formed that can be
loaded manually using single channel micropipettors. The
microfluidic devices can be sized according to industry
size-specifications (e.g., footprint is 127.76 0.12.times.85.47
0.12 mm) for plate readers and robotics and are designed to
interface with generic multichannel robotic pipettors/samplers with
standardized interwell spacings (pitch). Dimensional standards for
these types of plate/devices are described at
http://www.tomtec.com/Pages/platstan.hmtl and
http://www.sbsonline.com. Custom micropipettors that do not conform
to this standard can also be utilized. In some systems, an
electropipettor that is in fluid communication with a sample input
channel is utilized. Micropipettors of this type are described, for
example, in U.S. Pat. No. 6,150,180.
[0098] Inlets to the microfluidic devices disclosed herein can be
holes or apertures that are punched, drilled or molded into the
elastomeric matrix. Such apertures are sometimes referred to as
"vias." The vias can also be formed using photoresist techniques.
For example, metal etch blocking layers used in combination with
patterning of photoresist masks and the use of solvents to remove
etch blocking layers can be utilized to create vias. Vertical vias
between channels in successive elastomer layers can be formed
utilizing negative mask techniques. Vias can also be formed by
ablation of elastomer material through application of an applied
laser beam. All of these techniques are described in greater detail
in U.S. application Ser. No. 09/605,520.
[0099] Inlets can optionally be lined with couplings (e.g., made of
Teflon) to provide a seal with the pipette tips or syringe tip used
to inject a solution.
[0100] As described further below, pumps formed from elastomeric
materials can be used to transport solution through the flow
channels. For channels of known dimensions, one can precisely
regulate the volume introduced through an inlet from based upon the
number of strokes of the pump.
[0101] Any sample or solution that is chemically compatible with
the elastomeric material from which the microfluidic device is
fabricated can be introduced into the device. Once the elastomeric
material has been molded or etched into the appropriate shape, it
may be necessary to pre-treat the flow channel portion of the
material in order to facilitate operation in connection with a
particular application. For example, in order to reduce or prevent
elastomer from dissolving in the solvent or reacting with a reagent
or assaying solution, one can coat the inner walls of the flow
channels with polypropylene, polyvinylidene fluoride, Viton.RTM. or
other suitable inert materials.
[0102] D. Valves
[0103] 1. Structure
[0104] The valves of the microfluidic devices provided herein are
formed of elastomeric material and include an elastomeric segment
(or membrane or separating portion) that separates a control
channel and a flow channel. The valves have two general designs:
those that are typically open and those that are normally closed.
Valves that are typically open are actuated to block flow through a
flow channel by applying pressure to the control channel, thereby
deflecting the membrane into the flow channel to restrict flow. In
the case of valves that are normally closed, the membrane or
separating portion normally extends into the flow channel. However,
upon reduction of pressure in the control channel relative to the
flow channel, the membrane/separating portion is pulled into the
control channel, thus removing the blockage in the flow
channel.
[0105] FIGS. 1A and 1B illustrate the general elements of a valve
that is typically open. As can be seen, elastomeric structure 24
contains a control channel 32 overlying recess 21 formed from a
raised portion of a mold. When the recess in this elastomeric
structure is sealed at its bottom surface to solid substrate 14,
recess 21 forms a flow channel 30. As can be seen in FIG. 1B and
FIG. 2A, flow channel 30 and control channel 32 are preferably
disposed at an angle to one another with a small membrane 25 of
elastomeric block 24 separating the top of flow channel 30 from the
bottom of control channel 32. While these figures show control
channels that extend across the device, it should be understood
that this need not be the case. The control channel can be a recess
sufficiently large such that the membrane is able to provide the
desired level of blockage in the flow channel. FIG. 2B illustrates
the situation for a normally open elastomeric valve structure 200
in which the valve has been actuated and the flow channel is
blocked. In particular, the structure includes a control channel
120 formed within one elastomeric layer 110 that overlays another
elastomeric layer 128 which includes a flow channel 126.
Elastomeric layer 110 is attached to substrate 130. Because the
control channel has been pressurized, the membrane 122 separating
the control channel 120 and the flow channel 126 is deflected down
into the flow channel 126, thereby effectively blocking solution
flow therethrough. Once pressure is released, membrane 122 deflects
back up from the flow channel 126 to allow solution flow.
[0106] As noted above, the valves can also have a normally closed
configuration. FIG. 3A illustrates one example of a normally-closed
valve 4200 in an unactuated state. Flow channel 4202 and control
channel 4204 are formed in elastomeric block 4206. Flow channel
4202 includes a first portion 4202a and a second portion 4202b
separated by separating portion 4208. Control channel 4204 overlies
separating portion 4208. As shown in FIG. 3A, in its relaxed,
unactuated position, separating portion 4208 remains positioned
between flow channel portions 4202a and 4202b, interrupting flow
channel 4202. FIG. 3B shows a cross-sectional view of valve 4200
wherein separating portion 4208 is in an actuated position. When
the pressure within control channel 4204 is reduced to below the
pressure in the flow channel (for example by vacuum pump),
separating portion 4208 experiences an actuating force drawing it
into control channel 4204. As a result of this actuation force,
membrane 4208 projects into control channel 4204, thereby removing
the obstacle to solution flow through flow channel 4202 and
creating a passageway 4203. Upon elevation of pressure within
control channel 4204, separating portion 4208 assumes its natural
position, relaxing back into and obstructing flow channel 4202.
[0107] It is not necessary that the elastomeric layers that contain
the flow and control channels be made of the same type of
elastomeric material. For example, the membrane that separates the
control and flow channels can be manufactured from an elastomeric
material that differs from that in the remainder of the structure.
A design of this type can be useful because the thickness and
elastic properties of the membrane play a key role in operation of
the valve.
[0108] 2. Options for Actuating Valves
[0109] A variety of approaches can be utilized to open or close a
valve. If a valve is actuated by increasing pressure in a control
channel, in general this can be accomplished by pressurizing the
control channel with either a gas (e.g., air) or a fluid (e.g.,
water or hydraulic oils). However, optional electrostatic and
magnetic actuation systems can also be utilized. Electrostatic
actuation can be accomplished by forming oppositely charged
electrodes (which tend to attract one another when a voltage
differential is applied to them) directly into the monolithic
elastomeric structure. For example, referring once again to FIG. 2,
an optional first electrode 70 (shown in phantom) can be positioned
on (or in) membrane 25 and an optional second electrode 72 (also
shown in phantom) can be positioned on (or in) planar substrate 14.
When electrodes 70 and 72 are charged with opposite polarities, an
attractive force between the two electrodes will cause membrane 25
to deflect downwardly, thereby closing the "valve" (i.e., closing
flow channel 30).
[0110] Alternatively, magnetic actuation of the flow channels can
be achieved by fabricating the membrane separating the flow
channels with a magnetically polarizable material such as iron, or
a permanently magnetized material such as polarized NdFeB. Where
the membrane is fabricated with a magnetically polarizable
material, the membrane can be actuated by attraction in response to
an applied magnetic field.
[0111] Optional electrolytic and electrokinetic actuation systems
can also be utilized. For example, actuation pressure on the
membrane can be generated from an electrolytic reaction in a recess
overlying the membrane. In such an embodiment, electrodes present
in the recess are used to apply a voltage across an electrolyte in
the recess. This potential difference causes an electrochemical
reaction at the electrodes and results in the generation of gas
species, thereby giving rise to a pressure differential in the
recess. Alternatively, actuation pressure on the membrane can arise
from an electrokinetic fluid flow in the control channel. In such
an embodiment, electrodes present at opposite ends of the control
channel are used to apply a potential difference across an
electrolyte present in the control channel. Migration of charged
species in the electrolyte to the respective electrodes can give
rise to a pressure differential.
[0112] Finally, valves can be actuated the device by causing a
fluid flow in the control channel based upon the application of
thermal energy, either by thermal expansion or by production of gas
from liquid. Similarly, chemical reactions generating gaseous
products may produce an increase in pressure sufficient for
membrane actuation.
[0113] 3. Options for Selectively Actuating Valves
[0114] In order to facilitate fabrication and to reduce the number
of control channels in a microfluidic device, often a control
channel overlays a number of flow channels. In such instances,
pressurization of such a control channel could cause blockage of
all the flow channels. Often it is desired to block only selected
flow channels, rather than all the flow channels which a control
channel abuts. Selective actuation can be achieved in a number of
different ways.
[0115] One option illustrated in FIG. 4 is to control the width of
the control channels 5004, 5006 at the point at which they extend
across the flow channels 5002A and 5002B. In locations where the
control channels are wide 5004A, 5006A, pressurization of the
control channel 5004, 5006 causes the membrane separating the flow
channel and the control channel to depress significantly into the
flow channel 5002A, 5002B, thereby blocking the flow passage
therethrough. Conversely, in the locations where the control line
is narrow 5004B, 5006B, the membrane separating the channels is
also narrow. Accordingly, the same degree of pressurization will
not result in membrane becoming depressed into the flow channel
5002A, 5002B. Therefore, fluid passage thereunder will not be
blocked.
[0116] The same general effect can be obtained by varying the width
of the flow channel relative to the control channel. Incorporation
of an elastomeric support in the section of the flow channel
opposite the membrane that is deflected into the flow channel can
also prevent complete stoppage of solution flow.
[0117] Valves in certain of the figures are represented by single
dashed lines if the valve can be utilized to block solution flow
through the flow channel. A control channel that crosses a flow
channel but which does not act to block the flow channel (for the
reasons just described) is represented by a solid arch that arches
over a flow channel.
[0118] Various other methods of actuating valves are described in
the above incorporated U.S. and PCT applications.
[0119] E. Pumps
[0120] The pumps integrated within the microfluidic devices
described herein can be formed from a plurality of control channels
that overlay a flow channel. A specific example of a system for
peristaltic pumping is shown in FIGS. 5A and 5B. As can be seen, a
flow channel 30 has a plurality of generally parallel control
channels 32A, 32B and 32C passing thereover. By pressurizing
control line 32A, flow F through flow channel 30 is shut off under
membrane 25A at the intersection of control line 32A and flow
channel 30. Similarly, (but not shown), by pressurizing control
line 32B, flow F through flow channel 30 is shut off under membrane
25B at the intersection of control line 32B and flow channel 30,
etc. Each of control lines 32A, 32B, and 32C is separately
addressable. Therefore, peristalsis can be actuated by the pattern
of actuating 32A and 32C together, followed by 32A, followed by 32A
and 32B together, followed by 32B, followed by 32B and C together,
etc. Pumps of this type are denoted in shorthand form in certain of
the figures with a series of three parallel dashed lines.
[0121] External pumps can also be connected to a flow channel to
transport solutions through a channel. Alternatively, a vacuum can
be applied to a flow channel to direct fluid flow toward the region
of reduced pressure.
IV. High Throughput Screening Systems
[0122] A. General
[0123] In their simplest forms, the biochemical system models
employed in the methods and microfluidic devices of the present
invention will screen for an effect of a test compound on an
interaction between two components of a biochemical system, i.e.,
ligand-antiligand interaction, for example, antibody-antigen
interaction, enzyme-substrate interaction, and the like. In this
form, the biochemical system model will typically include the two
normally interacting components of the system for which an effector
is sought, e.g., the antibody and its protein substrate or the
enzyme and its substrate.
[0124] Determining whether a sample has an effect on this
interaction then involves contacting the ligand with the sample and
assaying for the ligand-antiligand interaction. The assayed
function can be then compared to a control, e.g., the same reaction
in the absence of the antiligand or in the presence of a known
antiligand.
[0125] Although described in terms of two-component biochemical
systems, the methods and microfluidic devices of the present
invention can also be used for more complex systems where the
result of the system is known and assayable at some level, e.g.,
enzymatic pathways, cell signaling pathways and the like.
Alternatively, the methods and microfluidic devices described
herein can be used to screen for compounds that interact with a
single component of a biochemical system, e.g., compounds that
specifically bind to a particular biochemical compound, e.g., a
receptor, enzyme, nucleic acid, etc.
[0126] Ligand can also be embodied in whole cell systems. For
example, where one is seeking to screen test compounds for an
effect on a cellular response, whole cell may be utilized by
immobilizing the cell on the solid substrate surface. Modified cell
systems can also be employed in the microfluidic devices
encompassed herein. For example, chimeric reporter systems can be
employed as indicators of an effect of a sample on a particular
biochemical system. Chimeric reporter systems typically incorporate
a heterogenous reporter system integrated into a signaling pathway
which signals the binding of a receptor to its substrate. For
example, a receptor can be fused to a heterologous protein, e.g.,
an enzyme whose activity is readily assayable. Activation of the
receptor by substrate binding then activates the heterologous
protein which then allows for detection. Thus, the surrogate
reporter system produces an event or signal which is readily
detectable, thereby providing an assay for receptor/substrate
binding. Examples of such chimeric reporter systems have been
previously described in the art.
[0127] Additionally, where one is screening for bioavailability,
e.g., transport, biological barriers can be included. The term
"biological barriers" generally refers to cellular or membranous
layers within biological systems, or synthetic models thereof.
Examples of such biological barriers include the epithelial and
endothelial layers, e.g. vascular endothelia and the like.
[0128] Biological responses are often triggered and/or controlled
by the binding of a receptor to its substrate. For example,
interaction of growth factors, i.e., EGF, FGF, PDGF, etc., with
their receptors stimulates a wide variety of biological responses
including, e.g., cell proliferation and differentiation, activation
of mediating enzymes, stimulation of messenger turnover,
alterations in ion fluxes, activation of enzymes, changes in cell
shape and the alteration in genetic expression levels. Accordingly,
control of the interaction of the receptor and its substrate can
offer control of the biological responses caused by that
interaction.
[0129] Accordingly, in one aspect, the present invention is useful
in screening for compounds that affect an interaction between a
receptor and its substrates. Thus, a receptor/substrate pair can
include a typical protein receptor, usually membrane associated,
and its natural substrate, e.g., another protein or small molecule.
Receptor/substrate pairs can also include antibody/antigen binding
pairs, complementary nucleic acids, nucleic acid associating
proteins and their nucleic acid ligands. A large number of
specifically associating biochemical compounds are well known in
the art and can be utilized in practicing the present invention. In
addition, ternary or higher binding complexes can also be assayed
using microfluidic devices of the present invention as discussed in
detail below.
[0130] Traditionally, methods for screening for effectors of a
receptor/substrate interaction have involved incubating a
receptor/substrate binding pair in the presence of a sample. The
level of binding of the receptor/substrate pair is then compared to
negative and/or positive controls. Where a decrease in normal
binding is seen, the sample is determined to be an antagonist or
inhibitor of the receptor/ligand binding. Where an increase in that
binding is seen, the substrate is determined to be an agonist of
the interaction.
[0131] A similar, and perhaps overlapping, set of biochemical
systems includes the interactions between enzymes and their
substrates. Typically, effectors of an enzyme's activity toward its
substrate are screened by contacting the enzyme with a substrate in
the presence and absence of the compound to be screened and under
conditions optimal for detecting changes in the enzyme's activity.
After a set time for reaction, the mixture is assayed for the
presence of reaction products or a decrease in the amount of
substrate. The amount of substrate that has been catalyzed is then
compared to a control, i.e., enzyme contacted with substrate in the
absence of sample or presence of a known effector. As above, a
compound that reduces the enzymes activity toward its substrate is
termed an "inhibitor" or "antagonist" whereas a compound that
accentuates that activity is termed an "agonist."
[0132] Generally, the various screening methods encompassed by the
present invention involve a simultaneous introduction of a
plurality of samples into a microfluidic device. Typically, each
sample is introduced to each of one of the plurality of first or
the second flow channels. Once injected into the device, each
sample is assayed simultaneously to provide a high throughput
screening.
[0133] As used herein, the term "sample" refers to the collection
of compound(s) that are to be screened for their ability to bind to
immobilized ligand or affect a particular immobilized biochemical
system. Samples can include a wide variety of different compounds,
including chemical compounds, mixtures of chemical compounds, e.g.,
polysaccharides, small organic or inorganic molecules, biological
macromolecules, e.g., peptides, proteins, nucleic acids, or an
extract made from biological materials such as bacteria, plants,
fungi, or animal cells or tissues, naturally occurring or synthetic
compositions. Depending upon the particular embodiment being
practiced, the samples can be provided, e.g., injected, free in
solution, or can be attached to a carrier, or a particle, e.g.,
beads. A number of suitable particles can be employed to attach the
samples. Examples of suitable particles include agarose, cellulose,
dextran (commercially available as, i.e., Sephadex, Sepharose)
carboxymethyl cellulose, polystyrene, polyethylene glycol (PEG),
filter paper, nitrocellulose, ion exchange resins, plastic films,
glass beads, polyaminemethylvinylether maleic acid copolymer, amino
acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.
Additionally, for the methods and microfluidic devices described
herein, samples can be screened individually, or in groups. Group
screening is particularly useful where hit rates for effective
samples are expected to be low such that one would not expect a
large positive result for a given group.
[0134] One exemplary area where high throughput is important is in
proteomics. Cells express thousands of proteins at different
concentrations. The behavior and interaction of these proteins is
dependent upon the cell type, the stage of the cell cycle, and
extracellular events, to name a few. Proteins are also chemically
modified by cellular machinery, and this modification further
differentiates their behavior and interaction with other proteins.
Important factors in protein studies include concentration and
post-translational modification. The differences in these factors
between cell populations can be detected by antibody detection. In
one embodiment of the present invention, an antibody (i.e., ligand)
that recognizes a particular protein of interest or the antigen
(i.e, antiligand) is immobilized on a surface. A cellular protein
sample is then washed over the immobilized antibody, which
recognizes and binds the antigen at high affinity. A second
antibody or detection antibody (i.e., a labeled ligand) that also
recognizes the antigen is added to the solution and also binds the
selected antigen. In this manner, a sandwich assay can be performed
where a chemiluminescent or fluorescent detector recognizes the
detection antibody. The bound detectors are separated from the
unbound detectors by washing. The concentration of the detectors is
proportional to the concentration of bound antigen.
[0135] B. Use of Microfluidic Devices in Highthroughput Assay
[0136] As indicated supra, the foregoing elements or modules of
microfluidic devices can be combined in a large number of
configurations and utilized in a wide variety of applications.
Exemplary designs useful for conducting certain types of assays are
described in this section. It should be understood, however, that
the microfluidic devices of the present invention are not limited
to these particular configurations.
[0137] Microfluidic devices of the present invention include a
plurality of first flow channels and a plurality of second flow
channels. Each of the first flow channels intersect with, and are
in fluid communication with, each of the second flow channels. The
devices are typically used such that ligand(s) are attached to the
solid substrate surface by introducing into each of the first flow
channels while blocking flow through the second flow channels.
Alternatively, the ligand(s) can be pre-attached to the solid
substrate surface prior to attaching the solid substrate to the
elsatomeric layer.
[0138] Once the ligand(s) are attached to the solid substrate
surface, flow through the first flow channels is blocked. Sample(s)
are subsequently introduced into each of the second flow channels,
typically such that different samples are introduced into different
second flow channels. Optionally, loop forming valves are actuated
such that each pair of the second flow channels forms a looped flow
channel. The sample(s) are then circulated through each of the
looped flow channels using a recirculating pump. Alternatively,
first control valves and the second control valves are
simultaneously actuated to form a plurality of holding spaces each
encapsulating one intersecting area. FIG. 6 illustrates a holding
space that is formed along one of the flow channels (flow channel
intersecting area is not shown). These two methods allow a
prolonged contact between the sample(s) and the solid
substrate-bound ligand. The sample(s) in the intersecting areas are
subsequently reacted with the solid substrate-bound ligand in the
intersecting areas of the first and the second flow channels.
[0139] Regardless of the particular design, a large number of
samples can be rapidly screened within a short time period because
separate reactions can simultaneously be conducted in each of the
flow channel intersecting areas.
[0140] Typically, the pumps are utilized for transporting fluids
within the flow channels.
[0141] C. Exemplary Devices
[0142] One specific example of a microfluidic device that can be
used in high throughput assaying applications is illustrated in
FIG. 7A. The device includes a plurality of first flow channels
704A-H (yellow) and a plurality of second flow channels 708A-C
(pink). These flow channels intersect one another resulting in a
plurality of channel intersections 712A-BD. It should be noted that
the second flow channels 708A-C branches off and rejoins near the
outlet, thus forming a pair of branched flow channels 708A.sub.1-2,
708B.sub.1-2, and 708C.sub.1-2. These pair of branched flow
channels form looped flow channels when control valves 724A and
724B are closed. The channel intersections 712A-BD can optionally
comprise a chamber (also called reservoir or well) which is present
when a via layer is used. See Example 1 below. The device includes
a plurality of first control valves 716A-G and a plurality of
second control valves 720A.sub.11-G.sub.19 to regulate flow of
solution through the first flow channels and the second flow
channels, respectively. In this way, the flow of solution can be
controlled to prevent the solution from one flow channel
contaminating or mixing with the solution in another flow channel.
Also shown is are loop forming control valves 724A-B and a
recirculating pump 728. In FIG. 7A, one of the loop forming control
valve 724B also functions as a pump 732, which is discussed in
detail below. The loop forming control valves 724A and 724B allow
circulation of a solution through the looped flow channel by the
action of recirculating pump 728. The device also includes optional
pumps 732 and 736, which are used to transport solutions through
the second and the first flow channels, respectively. Shown in FIG.
7C are optional chambers 740A-D for solutions that are introduced
into the second flow channels.
[0143] Referring again to FIG. 7A, in operation, to introduce
ligand(s), valves 716A-G are opened and valves
720A.sub.11-720G.sub.19 are closed, and appropriate reagent
solution(s) are introduced into the first flow channels 704A-H via
corresponding inlets to attach ligand(s) onto the solid substrate
surface. As indicated above, alternatively, ligand(s) can be
attached to the solid substrate surface prior to attaching the
elastomeric layer to the solid substrate, in which case samples are
introduced into the first flow channels. Pump 736 can be utilized
to control the rate of solution flow through the first flow
channels 704A-H. After attaching the ligands onto the solid
substrate surface (or after forming ligand/antiligand complex(s)),
the first flow channels are optionally washed to remove unattached
ligand(s) (or unbound samples).
[0144] Sample(s) to be screened (or labeled ligand(s)) are
introduced by closing first control valves 716A-G to restrict
solution flow through the first flow channels 704A-H while opening
the second control valves 720A.sub.11-G.sub.19. Sample(s) (or
labeled ligand(s)) are introduced into the second flow channels
708A-C. The sample(s) (or labeled ligand(s)) can be continuously
flowed through the second flow channels or the loop forming control
channels 724A and 724B can be closed to form a plurality of looped
flow channels from each pair of second flow channels 708. The
sample can then be circulated through the looped flow channels
using the recirculating pumps 728.
[0145] Alternatively, the first control valves 716A-G can also be
closed thereby forming a plurality of channel intersections
712A-BD. In this manner, samples (or labeled ligand(s)) are held
within the channel intersections 712A-BD to provide a prolonged
contact with the solid substrate-bound ligand (or the
ligand/antiligand complex). FIG. 7B shows position of the first and
the second control valves relative to the flow channel intersecting
areas. By actuating both of the first and the second control
valves, a holding space is formed within each flow channel
intersecting areas.
[0146] Thereafter, the second flow channels 708A-C are optionally
rinsed to remove the unbound sample(s) (or labeled ligand(s)). If
the first flow channels are used to attach ligand(s) to the solid
substrate surface, a labeled ligand can optionally be introduced to
either the first or the second flow channels to perform a sandwich
assay, such as ELISA, FLISA, etc.
[0147] After assaying is completed, the elastomeric layer is
removed from the solid substrate surface. And the solid substrate
is then analyzed with an appropriate analyzer to determine the
result.
[0148] D. Samples (i.e., Test Compounds)
[0149] The assay and screening methods described herein can be
conducted with essentially any compound. In general terms, the test
agent or test compound is potentially capable of interacting with
the component being assayed (e.g., cell, enzyme, receptor,
antibody, cellular organelle). In cellular assays, for example, the
component of the cell with which the test compound potentially
interacts can be any molecule, macromolecule, organelle or
combination of the foregoing that is located on the surface of the
cell or located within the cell. For example, if one is screening
for compounds capable of interacting with certain cellular
receptors, the test agents are selected as potentially able to
interact with the receptors of interest (e.g., binding at the
binding site of the receptor or affecting binding at the binding
site of the receptor, such as an agonist or antagonist). In certain
two-hybrid assays (see infra), the test agent is one that is
potentially able to influence the binding interaction between the
binding proteins of the two fusions (see below).
[0150] Consequently, test agents can be of a variety of general
types including, but not limited to, polypeptides; carbohydrates
such as oligosaccharides and polysaccharides; polynucleotides;
lipids or phospholipids; fatty acids; steroids; or amino acid
analogs. Further, the compounds can be growth factors, hormones,
neurotransmitters and vasodilators, for example. Likewise, the
compounds can be of a variety of chemical types including, but not
limited to, heterocyclic compounds, carbocyclic compounds,
.beta.-lactams, polycarbamates, oligomeric-N-substituted glycines,
benzodiazepines, thiazolidinones and imidizolidinones. Certain test
agents are small molecules, including synthesized organic
compounds.
[0151] Test agents can be obtained from libraries, such as natural
product libraries or combinatorial libraries, for example. A number
of different types of combinatorial libraries and methods for
preparing such libraries have been described, including for
example, PCT publications WO 93/06121, WO 95/12608, WO 95/35503, WO
94/08051 and WO 95/30642, each of which is incorporated herein by
reference.
V. Combinatorial Synthesis
[0152] A. Methods
[0153] Microfluidic devices having the general arrangement of
components as described for high throughput assays, particularly as
depicted in FIGS. 6A and 6B, can also be utilized to conduct
combinatorial chemical synthesis. The methods generally parallel
those described supra for the high throughput screening, except
that instead of ligands and samples being introduced into the flow
channels different reactants are introduced instead.
[0154] Thus, instead of attaching ligand(s), monomer(s) or
appropriate linker(s) are attached to the solid substrate and
suitably protected monomer(s) are introduced. By selective
performing deprotection, coupling reactions sequentially, one can
achieve a wide variety of combinatorial chemical synthesis.
[0155] Further guidance regarding combinatorial methods is provide
in PCT publications WO 93/06121, WO 95/12608, WO 95/35503, WO
94/08051 and WO 95/30642, each of which is incorporated herein by
reference.
[0156] B. Compounds
[0157] The compounds generated by such methods can be composed of
any components that can be joined to one another through chemical
bonds in a series of steps. Thus, the components can be any class
of monomer useful in combinatorial synthesis. Hence, the
components, monomers, or building blocks (the foregoing terms being
used interchangeably herein) can include, but are not limited to,
enzymes or enzyme modules, amino acids, carbohydrates, lipids,
phospholipids, carbamates, sulfones, sulfoxides, esters,
nucleosides, heterocyclic molecules, amines, carboxylic acids,
aldehydes, ketones, isocyanates, isothiocyanates, thiols, alkyl
halides, phenolic molecules, boronic acids, stannanes, alkyl or
aryl lithium molecules, Grignard reagents, alkenes, alkynes, dienes
and urea derivatives. The type of components added in the various
steps need not be the same at each step, although in some instances
the type of components are the same in two or more of the steps.
For example, a synthesis can involve the addition of different
amino acids at each cycle; whereas, other reactions can include the
addition of amino acids during only one cycle and the addition of
different types of components in other cycles (e.g., aldehydes or
isocyanates).
[0158] Given the diversity of components that can be utilized in
the methods of the invention, the compounds capable of being formed
are equally diverse. Essentially molecules of any type that can be
formed in multiple cycles in which the ultimate compound or product
is formed in a component-by-component fashion can be synthesized
according to the methods of the invention. Examples of compounds
that can be synthesized include polypeptides, polyketides,
oligosaccharides, polynucleotide, phospholipids, lipids,
benzodiazepines, thiazolidinones and imidizolidinones. As noted
above, the final compounds can be linear, branched, cyclic or
assume other conformations. The compounds can be designed to have
potential biological activity or non-biological activity.
VI. Variations
[0159] A. Channel Coatings
[0160] In certain methods, the flow channels are coated or treated
with various agents to enhance certain aspects of the assay. For
example, depending upon the nature of the material from which the
flow channels are formed, it can be useful to coat the flow
channels with an agent that protects against or prevents components
of the assay (for example cells, proteins, peptides, substrates,
small molecules) from adhering to the walls of the flow channels or
to the sides of the wells through which these agents are introduced
into the device. One function of these coatings is to help ensure
the biological integrity of the introduced sample. Another function
is to prevent physical interactions between cells and the walls of
the channel that might affect cellular responses or functions in
undesired ways. Examples of suitable coating agents include, but
are not limited to, TEFLON, parylene, acrylamides, polyethylene
glycol, silanes, and other agents to form self-assembled
monolayers.
[0161] Similarly, channels can be modified with a variety of agents
to achieve other purposes such as separation and sorting functions,
with the goal being to prepare the flow channels in accordance with
the particular application being conducted. More specifically, by
properly selecting the bulk matrix of the flow channel (i.e., the
particular choice of elastomers to utilize in constructing the flow
channels), surface chemistry (i.e., modification of the properties
of microchannels created within the elastomer) and the specific
modification of regions of the elastomer surface (e.g., by covalent
and/or non-covalent attachment of proteins, peptides, nucleic acids
(or their analogs), lipids, carbohydrates) can facilitate the
"tuning" of the device to a given application or combination of
applications. Methods for modification of elastomer surfaces
include, but are not limited to: (1) copolymerization with
functional groups during elastomer curing (an example of bulk
modification), (2) oxygen plasma treatment (3) modification of
plasma-treated surfaces with silanizing reagents (e.g.,
3-aminopropyltriethoxysilane,
N-(2-aminoethyl)-3-aminopropyltrimethoxysilane,
dimethylchlorosilane or hexamethyldisilazane) which form
self-assembled monolayers on the elastomer surface (which can be
used to treat individual flow channels), (4) use of photochemical
crosslinking reagents to create patterns of reactive groups on the
elastomer surface (e.g., aryl azide derivatives or quinone-based
derivatives), (5) passive modification of the elastomer surface by
adsorption.
[0162] Adsorption also enables one to create secondary or tertiary
layers of modification that offer improved properties over primary
adsorption. As a specific example, one can use antibodies against
an antigen to create a primary coating of flow channel walls. If
antigen is then bound to the bound antibody, one can then create a
secondary layer of specifically bound antigen. Antigen bound in
this way can be "presented" to the interior of the flow channel in
a more appropriate way than as a passively adsorbed primary layer.
Schemes for creating a plurality of layers composed of proteins,
nucleic acids, lipids or carbohydrates or combinations thereof will
be apparent to the skilled practitioner.
[0163] Channels can also be coated with materials that specifically
bind to assay components and/or reaction products such as products
produced by a cell or during an enzymatic assay, for instance. One
example of such a coating is one in which the channel is coated
with a metal or a metal-derivatized material. Reaction products
bearing a metal chelate tag thus become bound to the metal-coated
wall or material. Of course, a wide variety of other binding pairs
could also be utilized as substitutes for the metal chelating agent
and metal. Assays utilizing such metal-derivatized materials is
discussed in greater detail infra on the section on enzymatic
assays (see also U.S. Pat. No. 6,146,842).
[0164] B. Doping Channels with Magnetic Materials
[0165] The flow channel elastomeric walls can optionally be doped
with magnetic materials or by integration of a preformed magnet or
electromagnet into the microfluidic device. Examples of magnetic
materials that can be incorporated include magnetically polarizable
materials such as iron and permanently magnetized materials.
Inclusion of such materials within the flow channel enables
magnetic based separations to be performed. External magnets that
rotate can in some instances be used to facilitate mixing.
[0166] C. Electrodes
[0167] The flow channels can also optionally include electrodes to
provide an additional type of control over agent and solution
transport. Integration of electrodes into the devices permits
electrophoretic separations or electroosmotic flow to be integrated
with pump-driven transport. Suitable electrodes can be formed by
sputtering a thin layer of metal (e.g., gold) onto a surface in a
flow channel. Other metallization approaches such as chemical
epitaxy, evaporation, electroplating, and electroless plating can
also be utilized to form the necessary conductive material.
Physical transfer of a metal layer to the surface of the elastomer
is also available, for example by evaporating a metal onto a flat
substrate to which it adheres poorly, and then placing the
elastomer onto the metal and peeling the metal off of the
substrate. A conductive electrode can also be prepared by
depositing carbon black (e.g., Cabot Vulcan XC72R) on the elastomer
surface, either by wiping on the dry powder or by exposing the
elastomer to a suspension of carbon black in a solvent which causes
swelling of the elastomer, (such as a chlorinated solvent in the
case of PDMS).
VII. Exemplary Applications
[0168] The microfluidic devices disclosed herein can be utilized to
conduct a variety of different assays. Essentially any biological
assay or library screening application can be performed with the
microfluidic devices that are described herein, provided none of
the components of the assay or screen are incompatible with the
size of the microfluidic channels. For example, microfluidic
devices of the present invention can be used in proteomics to
identify the presence of a particular protein or to screen for any
agents that affect the activity of any class of "druggable" targets
(i.e., a target that is able to be modulated by a small molecule to
produce a desired phenotypic change in cell targets). Potential
druggable targets include, but are not limited to, G-protein
coupled receptors (GPCRs), cytokines and cytokine receptors,
nuclear receptors (ligand-dependent transcription factors),
signaling processes (e.g., receptor-ligand interactions, calcium
mobilization, kinases and phosphatases, second messengers and
transcription factors), proteases, ion channels, and determinants
of cytotoxicity (e.g., pro- and anti-apoptotic processes and cell
death). These targets can be addressed by the various types of
assays, including, for example, fluorescent detection technologies
such as fluorescence intensity determinations, fluorescence
polarization, fluorescence resonance energy transfer, time-resolved
techniques and fluorescence correlation spectroscopy.
[0169] The following include a non-exhaustive list of illustrative
assays that can be conducted with the microfluidic devices provided
herein, and illustrate the nature of the targets that can be
investigated and the types of detection schemes that can be
utilized.
[0170] A. Binding Assays
[0171] 1. General
[0172] A wide variety of binding assays can be conducted utilizing
the microfluidic devices disclosed herein. Interactions between
essentially any ligand and antiligand can be detected. Examples of
ligand/antiligand binding interactions that can be investigated
include, but are not limited to, enzyme/ligand interactions (e.g.,
substrates, cofactors, inhibitors); receptor/ligand;
antigen/antibody; protein/protein (homophilic/heterophilic
interactions); protein/nucleic; DNA/DNA; and DNA/RNA. Thus, the
assays can be used to identify agonists and antagonists to
receptors of interest, to identify ligands able to bind receptors
and trigger an intracellular signal cascade, and to identify
complementary nucleic acids, for example. Assays can be conducted
in direct binding formats in which a ligand and putative antiligand
are contacted with one another or in competitive binding formats
well known to those of ordinary skill in the art.
[0173] Because the microfluidic devices typically include a
plurality of flow channels and intersecting areas that allow
multiple analyses to be conducted at the same time, a large number
of assays can be conducted in a short period. Active ligands or
antiligands can be identified on the basis of the distinguishable
labels. For example, assays can be conducted using labels that can
be distinguished by physical, chemical, visual, radioactive, or
other means. More specifically, labels can be distinguished from
one another on the basis of different composition, size, color,
shape, magnetic properties, chemical properties, electronic
properties, fluorescent emission, for example. Specific examples of
labels that can be distinguished on the basis of different
fluorescent emissions are Luminex beads (Luminex Corporation) and
Quantum dots (Quantum Dot Corporation). Sorting and/or quantitation
is based upon label size, wavelength and/or amount of signal
generated (e.g., fluorescence).
[0174] Binding assays generally involve contacting a solution
containing ligands with a solution containing antiligands and
allowing the solutions to remain in contact for a sufficient period
such that binding partners form complexes. The ligand and/or
antiligand is usually labeled. Alternatively, and in some preferred
embodiments, a sandwich assay in which a labeled ligand is
introduced to form ligand/antiligand/labeled ligand complex. Any of
a variety of different labels can be utilized as described above.
Ligands and antiligands are typically contacted within the
intersecting areas of the flow channel. Solutions containing the
ligands and antiligands can be incubated by circulating the
solutions within the looped flow channels or holding the solution
within the holding spaces as described supra. Complexes typically
are detected by removing the solution and peeling the elastomeric
layer away from the solid substrate and analyzing the intersecting
areas of the solid substrate with an appropriate detector.
Alternatively, complexes can be detected with the elastomeric layer
still in contact with the solid substrate and analzying the
intersecting areas of the solid substrate with an appropriate
detector. The type of detector and detection method utilized
depends upon the type of label used to label the ligand or
antiligand.
[0175] 2. Exemplary Binding Assay Process
[0176] Binding assays of the present invention typically involve a
step in which complexes are separated from unreacted agents so that
labeled complexes can be distinguished from uncomplexed labeled
reactants. Often it is achieved by attaching either the ligand or
antiligand to a support. After ligands and antiligands have been
brought into contact, uncomplexed reactants are washed away and the
remaining complexes subsequently detected.
[0177] The assays performed with the microfluidic devices disclosed
herein generally involve contacting a solid substrate-bound ligand
with a solution containing an antiligand under conditions and for a
sufficient period of time to allow a ligand/antiligand complex to
form. As stated above, if neither the ligand nor the antiligand is
labeled, a labeled ligand can be also introduced under conditions
and for a sufficient period of time to allow formation of a
sandwiched complex (i.e., ligand/antiligand/labeled ligand
complex). Since the ligand or antiligand is labeled, any complexes
formed can be detected on the basis of the label in the
complex.
[0178] The assays can be conducted in a variety of ways. One
approach involves attaching the ligand of interest to the solid
substrate surface and contacting the solid substrate-bound ligand
with a solution containing antiligands. Antiligands that do not
form complexes are washed away under conditions such that complexes
that are formed remain immobilized to the solid substrate. The
detection of complexes immobilized to the solid substrate can be
accomplished in a number of ways. If the non-immobilized antiligand
is labeled, the detection of label antiligand immobilized on the
solid substrate indicates that a ligand/antiligand complex has been
formed. If, however, the non-immobilized antiligand is not labeled,
complexes can nonetheless be detected by indirect means. For
instance, a labeled ligand that specifically binds to the
antiligand can be utilized to detect complexes anchored to the
solid substrate, e.g., ELISA, FLISA, etc.
[0179] Alternatively, ligands and antiligands can be contacted in
solution. Complexes can then be separated from uncomplexed ligands
and antiligands and complexes detected. One approach for conducting
such an assay is to contact an antiligand of interest with a test
solution potentially containing a ligand that binds to the
antiligand. The resulting mixture can then be contacted with a
solid substrate-bound antibody that specifically binds to the
antiligand to immobilize any complexes that have been formed.
Labeled antibodies specific for the antiligand can then be
contacted with any immobilized complexes to detect the presence of
such complexes.
[0180] The ligand (antiligand) attached to the flow channel can be
attached directly to the solid substrate surface or via a linker.
In general, the solid substrate surface and the ligand (antiligand)
being attached to the surface need appropriate chemical
functionality such that the functional groups borne by these two
entities can react with one another and become attached. Often the
attachment is achieved by formation of a covalent bond between the
binding pair member and surface, although electrostatic, hydrogen
bond interactions and hydrophobic interactions can also act to
attach the binding pair member and surface.
[0181] A variety of linkers can be utilized to attach the ligand
(antiligand) to the solid substrate surface. The linkers typically
are polyfunctional, preferably bifunctional, with a functional
group at one end able to react with a functional group on the solid
substrate surface and a functional group on the other end able to
react with a functional group borne by the ligand (antiligand) to
be attached to the solid substrate surface. The functional groups
at each end of such linkers can be the same or different. Examples
of suitable linkers include straight or branched-chain carbon
linkers, heterocyclic linkers and peptide linkers. Exemplary
linkers that can be employed are available from Pierce Chemical
Company in Rockford, Ill. and are described in EPA 188,256; U.S.
Pat. Nos. 4,671,958; 4,659,839; 4,414,148; 4,669,784; 4,680,338,
4,569,789 and 4,589,071, Eggenweiler, H. M, Drug Discovery Today
1998, 3, 552, all of which are incorporated herein by reference in
their entirety.
[0182] Other linkers include members of a binding pair. In this
arrangement, one binding pair member is attached to the solid
substrate surface. The other member of the binding pair is attached
to the ligand (antiligand) one seeks to attach to the solid
substrate surface. Exemplary binding pair members include
biotin/avidin (or streptavidin) and antigen/antibody.
[0183] Depending upon the composition of the solid substrate, it
sometimes is necessary to derivatize the solid substrate surface so
that the binding pair member can be attached. As described supra, a
variety of agents can be used to derivatize the solid substrate
surface. Examples include, but are not limited to, silanizing
reagents (e.g., 3-aminopropyltriethoxysilane,
N-(2-aminoethyl)-3-aminopropyltrimethoxysilane,
dimethylchlorosilane or hexamethyl disilazane).
[0184] 3. Assays for Compounds that Inhibit Binding
Interactions
[0185] The microfluidic devices can also be utilized in a
competitive formats to identify agents that inhibit the interaction
between known binding partners. Such methods generally involve
preparing a reaction mixture containing the binding partners under
conditions and for a time sufficient to allow the binding partners
to interact and form a complex. In order to test a compound for
inhibitory activity, the reaction mixture is prepared in the
presence (test reaction mixture) and absence (control reaction
mixture) of the test compound. Formation of complexes between
binding partners is then detected, typically by detecting a label
borne by one or both of the binding partners. The formation of more
complexes in the control reaction then in the test reaction mixture
at a level that constitutes a statistically significant difference
indicates that the test compound interferes with the interaction
between the binding partners.
[0186] The order of addition of reactants can be varied to obtain
different binding information concerning the compounds being
tested. For example, test compounds that interfere with the
interaction between binding pair members can be identified by
conducting the reaction in the presence of the test compound, i.e.,
by introducing the test compound into the reaction mixture prior to
or simultaneously with the binding pair members. Alternatively,
test compounds capable of disrupting preformed complexes can be
identified by adding the test compound to the reaction mixture
after the complexes have been formed. This latter type analysis
enables one to identify compounds that have a higher binding
constant then one of the members of the binding pair and thus is
able to displace that binding pair member from the complex.
[0187] 4. Immunological Assays
[0188] Immunological assays are one general category of assays that
can be performed with the microfluidic devices provided herein.
Certain assays are conducted to screen a population of antibodies
for those that can specifically bind to a particular antigen of
interest. In such assays, a test antibody or population of
antibodies is contacted with the antigen which is attached to the
solid substrate surface. Other assays are conducted to examine a
sample to determine if an analyte of interest is present by
detecting binding between an antibody that specifically recognizes
the analyte and the analyte. In assays such as this, often it is
the antibody that is attached to the solid substrate surface and a
solution containing potential antigens contacted with the
immobilized antibody. In both types of assays, however, either the
antigen or antibody can be immobilized.
[0189] Sandwich Assay
[0190] Immunological assays can be conducted in a variety of
different formats. For example, the assays can involve direct
binding between antigen and antibody, the so-called sandwich assay,
enzyme linked immunosorbent assays (ELISA), fluorescent linked
immunosorbent assays (FLISA), and competitive assays. In an ELISA
or FLISA assay, for example, a capture antibody that specifically
binds to the analyte of interest is attached to the solid substrate
surface. A solution potentially containing the analyte of interest
is then introduced into the first or the second flow channels and
contacted with the immobilized capture antibody to form a binary
complex. A second antibody (a detection antibody) that recognizes
another portion of the analyte than the capture antibody is then
contacted with the binary complex through the other flow channels
to form a ternary complex at the channel intersection areas. The
detection antibody includes an assayable enzyme or a fluorescent
label.
[0191] In ELISA, formation of the ternary complex can be detected
by introducing the appropriate enzyme substrate into the flow
channel and allowed to contact any ternary complex. Signal produced
in association with the enzyme catalyzed formation of product is
detected by the detector.
[0192] In FLISA, formation of the ternary complex can be detected
by observing fluorescence at the channel intersecting areas. The
fluorescent label can be covalently bonded to one of the ligands,
thus forming a labeled ligand moiety, or a fluorescent tag can be
added to the ternary complex to form a tagged complex. Fluorescence
can be detected after removing the elastomeric layer from the solid
substrate or with the elastomeric layer in tact.
[0193] As discussed supra, capture antibodies can be attached to
the solid substrate surface via functional groups borne by the
antibody (e.g., amino, carboxyl, sulfhydryl, hydroxyl) and
complementary groups on the solid substrate surface or introduced
by derivatization.
[0194] B. Antimicrobial Assays
[0195] By contacting various microbial cells with different test
compounds, one can also utilize the devices provided herein to
conduct antimicrobial assays, thereby identifying potential
antibacterial compounds. The term "microbe" as used herein refers
to any microscopic and/or unicellular fungus, any bacteria or any
protozoan. Some antimicrobial assays involve immobilizing a cell on
the solid substrate surface and contacting it with at least one
potential antimicrobial compound. The effect of the compound can be
detected as any detectable change in the health and/or metabolism
of the cell. Examples of such changes, include but are not limited
to, alteration in growth, cell proliferation, cell differentiation,
gene expression, cell division and the like.
[0196] The following examples are provided to further illustrate
certain aspects of the invention, but are not to be construed so as
to limit the scope of the invention.
EXAMPLES
Example 1
[0197] This example illustrates a method for producing a 4-layer
microfluidic device having protein A (Staphylococcus aureus F.sub.c
binding protein) on its surface. The 4-layer device enables an
assay to be performed at a precise location on a given solid
substrate.
[0198] A fluid flow channel mold was prepared by spinning Shipley
5740 at 3600 rpm for 30 sec, soft baking for 60 s at 105.degree. C.
(measured thickness=7.8 .mu.m), and hardbaking at 140.degree. C.
for 150 sec. (final mold thickness=10 .mu.m). A control channel
mold was prepared by spinning shipley 5740 at 850 rpm for 30 sec,
followed by 5 min rest period prior to soft baking for 180 sec at
105.degree. C. (final mold thickness=20 .mu.m). A via mold was
prepared by spinning AZ PLP 100-XT at 1240 rpm for 30 sec. followed
by soft baking for 8 min at 95.degree. C. (final mold thickness=35
.mu.m).
[0199] Exemplary mold designs for each of the corresponding control
layer (808), the fluid (i.e., flow channel) layer (804), and the
via layer (812) are shown in FIGS. 8A-E. In FIGS. 8B-D, an
alignment mask 816 is also shown. Alignment mask 816 and each of
the layers also includes alignment guides 820a-e, which aids in
aligning each layers properly.
PDMS Process
[0200] Elastomeric layer was prepared as follows: [0201] 1) Mix
30:1 PDMS GE 615: 30 g part A, 1 g part B [0202] mix components at
1000 rpm under vacuum. [0203] 2) TMCS (trimethylchlorosilane) vapor
treat all three molds for 3 min. [0204] 3) Spin mixed 30:1 PDMS on
the fluid channel mold at 1090 rpm for 200 sec. [0205] PDMS
thickness is 35 .mu.m [0206] Oven bake wafer at 80.degree. C. for
30 min [0207] 4) Mix 4:1 PDMS GE 615: 80 g part A, 20 g part B
[0208] mix components at 1000 rpm under vacuum [0209] 5) Pour 4:1
PDMS on control channel mold [0210] Oven bake wafer at 80.degree.
C. for 30 min [0211] 6) Mix 30:1 PDMS GE 615: 30 g part A, 1 g part
B [0212] mix components at 1000 rpm under vacuum. [0213] 7) Spin
30:1 PDMS on the via mold at 1260 rpm for 200 sec [0214] PDMS
thickness is 30 .mu.m (5 .mu.m thinner than mold) [0215] Oven bake
wafer at 80.degree. C. for 1.5 hrs [0216] 8) Peel off PDMS from the
control channel mold [0217] Dice PDMS slab along chip borders
[0218] Punch PDMS chip at pneumatic fill marks on chip [0219] 9)
Align diced PDMS chips on to the fluid control mold [0220] Oven
bake at 80.degree. C. for 1 hr. [0221] 10) Score PDMS along chip
borders on the control channel mold [0222] 11) Peel 2 layer chips
off of the control channel mold [0223] 12) Align 2 layer chips on
the via mold [0224] Oven bake at 80.degree. C. for 3 hrs. [0225]
13) Score PDMS along chip borders on the via mold [0226] 14) Peel 3
layer chips off of the via mold Substrate Bonding
[0227] A solid substrate was prepared as follows: [0228] 1) prepare
protein A slide [0229] nitrogen dry protein A slides after removal
from 4.degree. C. refrigerator. [0230] 2) Place PDMS in a central
position on the protein A slide. [0231] 3) Oven bake at 80.degree.
C. for 30-45 min [0232] 4) Allow chip to cool to room temp. before
adding PBST. [0233] 5) Pipette in any well 0.5% PBST solution.
[0234] This device shields the samples and reagents from the
substrate except where the chemistry is to be performed on the
substrate. This shielding helps preserve the sample/reagent
concentration by preventing immobilization upstream of the intended
assay site. In addition, this device facilitates the loading of the
device and increases the bonding area between the device and the
substrate. This 3.sup.rd (i.e., via) layer effectively seals the
chip and renders the substrate bonding independent of the fluidic
network feature density. Molds are fabricated for each of the three
layers of the device. The first two molds are that of the control
and fluid layer and comprise the standard 2-layer fluidic device.
The additional 3.sup.rd mold is that of the via layer where posts
of various shapes are patterned. The height of this mold is about 5
.mu.m higher than the PDMS spun on the mold. The 2 layer chip is
assembled and is aligned to the 3.sup.rd layer. Once the 3-layer
chips is cured, the chip is dry etched from the via layer side to
open the vias as residual PDMS may exist on the mold during the
spinning step. See FIG. 9. Etching of the PDMS is done with a 2:1
mixture of C.sub.2F.sub.6 and O.sub.2 at 400 watts for 10-20
min.
[0235] While the above example is illustrated for producing a
three-layer elastomeric material comprising a via layer, by
eliminating the via layer a two-layer elastomeric material can also
be produced using the process described above. Thus, by using only
the fluid layer mold 804 and the control layer mold 808,
microfluidic devices having two-layer elastomeric material (which
forms a monolithic structure when cured) can be prepared. This
monolithic elastomeric layer is then attached directly to the solid
substrate surface for assaying. Another two-layer monolithic
elastomeric layer design and the corresponding micrfluidic device
that is fabricated is shown in FIGS. 10A and 10B.
[0236] It should be noted that while the via layer is not required,
it provides a distinct areas on the solid substrate surface that
are exposed to the assaying conditions. This is particularly useful
when the amount of sample available is extremely minute.
Example 2
[0237] This example illustrates a method for using a microfluidic
device of the present invention for conducting a FLISA assay.
[0238] Using a microfluidic device of configuration shown in FIG.
11, various assays were conducted as follows:
Fluid Inputs
[0239] Fluid inputs A, B, C, and D are the reagent inlets. Through
these inlets solutions of reagents, such as an antibody, can be
added. When the proper valves are actuated, fluid flow from these
inputs are directed into discrete areas of the chip. Fluid inputs
E, F, G, and H are the sample inlets. Through these inlets
solutions of reagents, such as analytes or antigens, can be added.
When the proper valves are actuated, fluid flow from these inputs
are directed into discrete areas of the chip. Fluid input I is a
common wash inlet. This input connects to fluid lines adjacent to
fluid inputs E-H. It may contain buffers or solutions common to all
samples. It can also contain a buffers or solution to block the
channel walls or block the substrate surface. Fluid input J is a
common wash inlet. This input connects to fluid lines adjacent to
fluid inputs A-D. It may contain buffers or solutions common to all
samples. It can also contain a buffers or solution to block the
channel walls or block the substrate surface. Fluid input K is a
common wash inlet. This input connects to fluid lines adjacent to
fluid inputs A-D. It may contain buffers or solutions common to all
samples. It can also contain a buffers or solution to block the
channel walls or block the substrate surface. Fluid input L is a
common waste inlet. This connects to common lines through which
fluid flow is directed.
[0240] Any and all of the inlets can be used to prime the chip, by
wetting the channel and the substrate.
Control Valves
[0241] Control valves are actuated at 12-16 psi. Positive
displacement fluidic pumps are activated at 60 Hz (10 Hz per step
of the cycle). Valves 1, 2, and 3 control a recirculating pump.
When activated, the valves pump fluid clockwise around the reaction
area. Valves 4, 5, and 6 control a sample through pump. When
activated, the valves pump fluid from fluid inputs E, F, G, and H,
or from fluid input I, into the reaction area. Valve 7 controls the
isolation of solutions in fluid input I. Actuating the valve
prevents a solution in fluid input I from entering the reaction
area. Opening the valve allows a solution from fluid input I into
the reaction area. Valve 8 controls the isolation of solutions in
fluid inputs A, B, C, D, E, F, G, and H. Actuating the valve
prevents samples and reagents from entering the reaction area from
fluid inputs A, B, C, D, E, F, G, and H. Opening the valve allows
samples and reagents into the reaction area from fluid inputs A, B,
C, D, E, F, G, and H. Valve 9 controls the isolation of sample in
the reaction area. Actuating the valve prevents fluid flow in the
reaction area in a vertical direction. When used in conjunction
with the recirculating pump (valves 1,2, and 3) or the sample
through pump (valves 4, 5, and 6), flow through the reaction area
occurs only horizontally. Valve 10 controls the isolation of
reagents in the sample area. Actuating the valve prevents fluid
flow in the reaction area in a horizontal direction. When used in
conjunction with the reagent through pump (valves 12, 13, and 14),
flow through the reaction area occurs only vertically. Valve 11
controls the isolation of the entire reaction area. Actuating the
valve prevents flow out of the reaction area. Actuating the valve
also prevents flow into the reaction area from fluid input L.
Valves 12,13, and 14 control a reagent through pump. When
activated, the valves pump fluid from fluid inputs A, B, C, and D,
or fluid input J, or fluid input K, into the reaction area. Valve
15 controls the isolation of fluid input J. Actuating the valve
prevents a solution from fluid input J from entering the reaction
area. Valve 16 controls the isolation of fluid input K. Actuating
the valve prevents a solution from fluid input K from entering the
reaction area.
Protocol for the Protein Microprocessor Chip
[0242] The assay can detect antigens, such as the detection of
human interleukin-6 (IL-6). As schematically illustrated in FIG.
12, the assay is a sandwich assay, which uses an immobilized mouse
anti-human IL-6 monoclonal capture antibody (mAb) and a
biotinylated polyclonal probe antibody. Immobilization of the probe
antibody is dependent upon binding of IL-6 in the sample to the
capture antibody. Dye-conjugated streptavidin is bound to the
biotinylated antibody and bound fluorescence quantified.
FLISA Scheme
[0243] In order to promote binding of the monoclonal antibody to
the glass surface in an appropriate orientation to permit
presentation of the antigen binding site to the solvent,
Staphylococcus aureus Protein A derivatized slides are used.
Protein A is a 42 KD antibody-binding protein that binds the Fc
(constant region) portion of the molecule. One protein A molecule
can bind up to 4 molecules of capture antibody. The chemistry of
Protein A binding to antibody is well known to one skilled in the
art.
[0244] Using the Chip
[0245] 1) The chip is baked onto a Protein A glass substrate for
30-45 minutes at 80.degree. C. The chip on the glass substrate is
cooled to room temperature. It is then primed by the addition of an
appropriate aqueous solution. The fluidic (i.e., flow) layer is
filled with phosphate buffered saline (PBS) containing 0.5% (v/v)
Tween-20. In a typical use, the following solutions are prepared:
TABLE-US-00001 Reagent S1: 5 S2: 2.5 S3: 1 S4: (producing Stock
ng/mL ng/mL ng/mL blank (0 organism) Concentration Stoichiometry
IL6 IL6 IL6 ng/mL) biotinylated 50 .mu.g/mL at least 3 eq 2.2 .mu.L
2.2 .mu.L 2.2 .mu.L 0 .mu.L polyclonal anti-IL6 (goat) recombinant
IL6 10 .mu.g/mL 0.5 .mu.L 0.25 .mu.L 0.1 .mu.L 0 .mu.L Cy5 labeled
1 mg/mL at least 45 eq 1 .mu.L 1 .mu.L 1 .mu.L 0 .mu.L streptavidin
dinitrophenyl- 10 .mu.g/mL 5 ng/mL 0.5 .mu.L 0.5 .mu.L 0.5 .mu.L
0.5 .mu.L Keyhole Limpet hemocyanin Alexa-Fluor 488 2 mg/mL 2
.mu.g/mL 1 .mu.L 1 .mu.L 1 .mu.L 1 .mu.L anti-dinitrophenyl Keyhole
Limpet Hemocyanin (rabbit) Rabbit whole serum 60 mg/mL 1 mg/mL
16.67 .mu.L 16.67 .mu.L 16.67 .mu.L 16.67 .mu.L PBS w/0.1% to 1 mL
978 .mu.L 978 .mu.L 979 .mu.L 982 .mu.L Tween-20 (PBST)
[0246] In addition: the following reagents are prepared. [0247] R1.
1 mg/mL Rabbit serum in PBST [0248] R2. 250 .mu.g/mL monoclonal
anti-IL6 (mouse), 2.5 .mu.g/mL anti-dinitrophenyl keyhole limpet
hemocyanin (rabbit) in PBST [0249] R3. 250 .mu.g/mL monoclonal
anti-IL6 (mouse), 2.5 .mu.g/mL anti-dinitrophenyl keyhole limpet
hemocyanin (rabbit) in PBST [0250] R4. 250 .mu.g/mL monoclonal
anti-IL6 (mouse), 2.5 .mu.g/mL anti-dinitrophenyl keyhole limpet
hemocyanin (rabbit) in PBST
[0251] Wash Solutions: [0252] W1. 1 mg/mL rabbit serum in PBST
[0253] W2. 1 mg/mL rabbit serum in PBST [0254] W3. 10 .mu.g/mL Cy3
conjugated anti-mouse IgG (goat) in PBST.
[0255] 2) Excess buffer is pipetted out of fluid inlets A, B, C, D,
E, F, G, H, I, and J. Valves 7, 8, 15, and 16 are actuated to close
off fluid inlets A, B, C, D, E, F, G, H, I, J, and K. Reagent
solutions R1, R2, R3, and R4 are added to fluid inlets A, B, C, and
D, respectively. Sample solutions S1, S2, S3, and S4 are added to
fluid inlets E, F, G, and H, respectively. Wash solution W1 is
added to fluid inlet I. Wash solution W2 is added to fluid inlet J.
Wash solution W3 is added to fluid inlet K.
[0256] 3) Reagents are then pumped into the chip. Valves 10 and 11
are actuated to allow for only vertical flow through the reaction
area. Valve 8 is opened to allow for solutions in fluid inlets A,
B, C, and D to flow into the reaction area. The reagent through
pump (valves 12, 13, and 14) is activated to direct flow to the
reaction area in a vertical direction. The pump is run for 10-20
minutes.
[0257] 4) The reaction area is washed with vertical flow. Valve 8
is actuated to prevent further flow of solutions from fluid inlets
A, B, C, and D into the reaction area. Valve 16 is opened to allow
the solution in fluid inlet J to flow through the reaction area in
a vertical direction. The reagent through pump (valves 12, 13, and
14) is activated to direct flow to the reaction area in a vertical
direction. The pump is run for 5-10 minutes (generally one half the
time of step 3).
[0258] The reagent through pump (valves 12, 13, and 14) is stopped
and valves 12, 13, and 14 are opened. Valve 16 is actuated to
prevent the solution in fluid inlet J from flowing into the
reaction area. Valve 8 remains actuated to prevent flow of
solutions from fluid inlets E, F, G, and H into the reaction area.
Valve 9 is actuated to cause flow to occur through the reaction
area in a horizontal direction. Valve 10 is opened to allow flow to
occur in a horizontal direction. Valve 11 is opened to allow
horizontal flow to occur through the reaction area to the fluid
inlet L. Valve 7 is opened to allow the solution in fluid inlet I
to flow through the reaction area in a horizontal direction. The
sample through pump (valves 4, 5, and 6) is activated to direct
flow to the reaction area in a horizontal direction. The pump is
run for 5-10 minutes.
[0259] 6) Valve 7 is actuated to prevent the solution in fluid
inlet I from flowing into the reaction area. Valve 8 is opened to
allow the solutions in fluid inlets E, F, G, and H to flow into the
reaction area in a horizontal direction. Valve 11 is opened to
allow flow to occur through the reaction area to the fluid inlet L.
The sample through pump (valves 4, 5, and 6) is activated to direct
flow to the reaction area in a horizontal direction. The pump is
run for 5 minutes.
[0260] 7) The sample through pump (valves 4, 5, and 6) is stopped
and valves 4, 5, and 6 are opened. Valve 11 is actuated to prevent
flow through the reaction area to the fluid inlet L. The
recirculating pump (valves 1, 2, and 3) is activated to direct flow
around the reaction area in a clockwise direction. The pump is run
for 30 minutes.
[0261] 8) Steps 6) and 7) can be repeated to allow for repeated
samplings of solutions from fluid inlets E, F, G, and H into the
reaction area.
[0262] The recirculating pump (valves 1, 2, and 3) is stopped and
valves 1, 2, and 3 are opened. Valve 8 is actuated to prevent the
solutions in fluid inlets E, F, G, and H from flowing into the
reaction area in a horizontal direction. Valve 11 is opened to
allow flow through the reaction area to the fluid inlet L. The
sample through pump (valves 4, 5, and 6) is activated to direct
flow to the reaction area in a horizontal direction. The pump is
run for 5-10 minutes.
[0263] 10) The sample through pump (valves 4, 5, and 6) is stopped
and valves 4, 5, and 6 are opened. Valves 10 and 11 are actuated to
allow for only vertical flow through the reaction area. Valve 9 is
opened to allow for flow into the reaction area in a vertical
direction. Valve 15 is opened to allow the solution in fluid inlet
K to flow into the reaction area. The reagent through pump (valves
12, 13, and 14) is activated to direct flow to the reaction area in
a vertical direction. The pump is run for 5-10 minutes.
[0264] At the end of the procedure, valves 10, 11, 12, 13, and 14
are opened. The chip can be removed from the substrate at this
point.
[0265] The pneumatic connections to the control layer of the chip
are removed. The chip and substrate are submerged in a solution of
PBS. The chip is removed and the substrate is washed with three
five-minute washes with fresh PBS. The slide is dried and imaged in
a fluorescence microarray scanner with at least three filter sets
to determine the signal from AlexaFluor 488, Cy3, and Cy5.
[0266] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all purposes
to the same extent as if each individual publication, patent or
patent application were specifically and individually indicated to
be so incorporated by reference.
* * * * *
References