U.S. patent application number 10/640510 was filed with the patent office on 2004-11-18 for microfluidic particle-analysis systems.
This patent application is currently assigned to Fluidigm Corporation. Invention is credited to Daridon, Antoine.
Application Number | 20040229349 10/640510 |
Document ID | / |
Family ID | 29740786 |
Filed Date | 2004-11-18 |
United States Patent
Application |
20040229349 |
Kind Code |
A1 |
Daridon, Antoine |
November 18, 2004 |
Microfluidic particle-analysis systems
Abstract
The invention provides systems, including apparatus, methods,
and kits, for the microfluidic manipulation and/or detection of
particles, such as cells and/or beads. The invention provides
systems, including apparatus, methods, and kits, for the
microfluidic manipulation and/or analysis of particles, such as
cells, viruses, organelles, beads, and/or vesicles. The invention
also provides microfluidic mechanisms for carrying out these
manipulations and analyses. These mechanisms may enable controlled
input, movement/positioning, retention/localization, treatment,
measurement, release, and/or output of particles. Furthermore,
these mechanisms may be combined in any suitable order and/or
employed for any suitable number of times within a system.
Accordingly, these combinations may allow particles to be sorted,
cultured, mixed, treated, and/or assayed, among others, as single
particles, mixed groups of particles, arrays of particles,
heterogeneous particle sets, and/or homogeneous particle sets,
among others, in series and/or in parallel. In addition, these
combinations may enable microfluidic systems to be reused.
Furthermore, these combinations may allow the response of particles
to treatment to be measured on a shorter time scale than was
previously possible. Therefore, systems of the invention may allow
a broad range of cell and particle assays, such as drug screens,
cell characterizations, research studies, and/or clinical analyses,
among others, to be scaled down to microfluidic size. Such
scaled-down assays may use less sample and reagent, may be less
labor intensive, and/or may be more informative than comparable
macrofluidic assays.
Inventors: |
Daridon, Antoine; (Belmont,
CA) |
Correspondence
Address: |
FLUIDIGM CORPORATION
7100 SHORELINE COURT
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Fluidigm Corporation
South San Francisco
CA
94080
|
Family ID: |
29740786 |
Appl. No.: |
10/640510 |
Filed: |
August 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10640510 |
Aug 12, 2003 |
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10405092 |
Mar 31, 2003 |
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60369538 |
Apr 1, 2002 |
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60378464 |
May 6, 2002 |
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Current U.S.
Class: |
435/305.2 |
Current CPC
Class: |
B01L 3/502753 20130101;
G01N 2015/0288 20130101; B01L 3/502746 20130101; B01L 2300/088
20130101; B01L 2400/0415 20130101; B01L 2200/12 20130101; B01L
2200/0652 20130101; B01L 2300/087 20130101; B01L 2400/0481
20130101; C12M 21/06 20130101; B01L 2200/10 20130101; G01N 15/1484
20130101; B01L 2300/0867 20130101; G01N 33/4833 20130101; B01L
2300/0861 20130101; B01L 2300/123 20130101; C12M 23/16 20130101;
B01L 2300/0681 20130101; B01L 2400/0655 20130101; C12M 1/34
20130101; G01N 2015/1493 20130101; B01L 2200/16 20130101; G01N
2015/008 20130101; B01L 2200/0647 20130101; B01L 2200/0668
20130101; B01L 2300/0645 20130101; G01N 2015/149 20130101; B01L
3/502738 20130101; B01L 2300/06 20130101; G02B 21/32 20130101; B01L
2300/0893 20130101; B01L 3/502761 20130101; B01L 2400/0409
20130101; B01L 2200/0636 20130101; B01L 2300/0636 20130101; B01L
2300/0887 20130101; B01L 2400/0622 20130101 |
Class at
Publication: |
435/305.2 |
International
Class: |
C12M 001/22 |
Claims
What is claimed is:
1. A microfluidic device for exposing cells to a plurality of
compounds comprising: (a) a cell inlet; (b) a plurality of cell
chambers, each in fluidic communication with each other and said
cell inlet; (c) a plurality of chamber isolation valves, said
isolation valves, when actuated, fluidicly isolating said chambers
from each other; (d) a plurality of compound inlets and outlets,
each compound inlet and outlet being in fluid communication with
one of said chambers, said compound inlets and outlets further
comprising a cell retention sieve for retaining cells within said
chambers while permitting fluid flow through said chambers so that
said cells are perfused with said fluid when said fluid is flowed
through said chambers from said compound inlets towards said
compound outlets, said compound inlets being selectively in fluid
communication with a plurality of compound sources, and said
compound outlet being in fluid communication selectively either
with a common waste receptacle or a common reagent reservoir; (e) a
plurality of compound valves in communication with said compound
inlets and outlets, said valves being capable fluidicly isolating
said chamber from said compound sources and said common receptacle
and common reagent reservoir; (f) a common waste receptacle valve,
said common waste receptacle valve being capable of fluidicly
isolating said common waste receptacle from said chamber when
actuated; and, (g) a common reagent reservoir valve, said common
reagent reservoir valve being capable of fluidicly isolating said
common waste receptable from said chamber when actuated.
2. The device of claim 1 wherein at least one of said plurality of
compounds is a cell toxic compound.
3. The device of claim 1 wherein said common reagent reservoir
contains a detection agent responsive to a toxic response of said
cells to one of said compounds.
4. A method for determining whether one of a plurality of compounds
causes a toxic response in one of a plurality of cells comprising
the steps of: providing the device of claim 1; introducing said
cells into said device of claim 1, isolating said cells within said
chambers of said device of claim 1, exposing said cells to said
plurality of said compounds while said cells are retained in said
chambers, determining whether any of said cells had a toxic
response resulting from exposure to one of said plurality of
compounds.
Description
CLAIM OF PRIORITY
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn.119(e) to provisional applications Serial Nos.
60/369,538, filed Apr. 1, 2002 and 60/378,464, filed May 6, 2002,
both of which are hereby incorporated by reference in their
entirety for all purposes and those purposes stated herein and
therein. This application further claims priority under 35 U.S.C.
.sctn.120 as a continuation-in-part of the non-provisional patent
application titled "Microfluidic Particle-Analysis Systems", by
Chou et al., filed on Mar. 31, 2003 (Atty. Docket No.: 139F.310US),
which is hereby incorporated by reference for all purposes.
CROSS-REFERENCES TO PATENT APPLICATIONS
[0002] This application incorporates by reference in their entirety
for all purposes the following U.S. patent applications: Ser. No.
09/605,520, filed Jun. 27, 2000; Ser. No. 09/724,784, filed Nov.
28, 2000; Ser. No. 09/724,967, filed Nov. 28, 2000; Ser. No.
09/796,378, filed Feb. 28, 2001; Ser. No. 09/796,666, filed Feb.
28, 2001; Ser. No. 09/796,871, filed Feb. 28, 2001; Ser. No.
09/826,583, filed Apr. 6, 2001; and Ser. No. 09/724,784, filed Nov.
28, 2001, titled MICROFABRICATED ELASTOMERIC VALVE AND PUMP
SYSTEMS, and naming Marc A. Unger, Hou-Pu Chou, Todd A. Thorsen,
Axel Scherer, Stephen R. Quake, Jian Liu, Mark L. Adams, and Carl
L. Hansen as inventors.
CROSS-REFERENCES TO OTHER MATERIALS
[0003] This application incorporates by reference in their entirety
for all purposes the following publications: Joe Sambrook and David
Russell, Molecular Cloning: A Laboratory Manual (3.sup.rd ed.
2000); and R. Ian Freshney, Culture of Animal Cells: A Manual of
Basic Technique (4.sup.th ed. 2000).
FIELD OF THE INVENTION
[0004] The invention relates to systems for the manipulation and/or
detection of particles. More particularly, the invention relates to
microfluidic systems for the manipulation and/or detection of
particles, such as cells and/or beads.
BACKGROUND OF THE INVENTION
[0005] The ability to perform molecular and cellular analyses of
biological systems has grown explosively over the past three
decades. In particular, the advent and refinement of molecular and
cellular techniques, such as DNA sequencing, gene cloning,
monoclonal antibody production, cell transfection, amplification
techniques (such as PCR), and transgenic animal formation, have
fueled this explosive growth. These techniques have spawned an
overwhelming number of identified genes, encoded proteins,
engineered cell types, and assays for studying these genes,
proteins, and cell types. As the number of possible combinations of
samples, reagents, and assays becomes nearly incalculable, it has
become increasingly apparent that novel approaches are necessary
even to begin to make sense of this complexity, especially within
reasonable temporal and monetary limitations.
[0006] One approach to these difficulties has been to reduce the
scale of assays. Accordingly, substantial effort has been directed
to developing assay methods and instrumentation for high-density
microtiter plates. However, very small assay volumes in
high-density microtiter plates, particularly assays with cells, may
suffer from a number of shortcomings. For example, cells may be
lost easily from wells, may be harmed by rapid fluid evaporation,
may contaminate nearby wells, and may be difficult to remove
efficiently from wells for additional analysis or culture. Thus,
there is a need for systems that can effectively manipulate and
analyze cells and other small particles, such as beads, in small
volumes.
SUMMARY OF THE INVENTION
[0007] The invention provides systems, including apparatus,
methods, and kits, for the microfluidic manipulation and/or
detection of particles, such as cells and/or beads.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 is a flow chart showing potential temporal
relationships between method steps for manipulation and/or
detection of particles in a microfluidic system, in accordance with
aspects of the invention.
[0009] FIG. 2A is a top plan view of a microfluidic system for
retaining and analyzing a subset of input particles, in accordance
with aspects of the invention.
[0010] FIG. 2B is a top plan view of another microfluidic system
for retaining and analyzing a subset of input particles, in
accordance with aspects of the invention.
[0011] FIG. 3 is a fragmentary, top plan view of yet another
microfluidic system for retaining and analyzing a subset of input
particles, in accordance with aspects of the invention.
[0012] FIG. 4 is a view of the system of FIG. 3 during particle
positioning and retention, illustrating the various flow paths
followed by particles, in accordance with aspects of the
invention.
[0013] FIG. 5 is a fragmentary, top plan view of a microfluidic
system for positioning and retaining a group of particles, and for
perfusing the retained group with selected reagents, in accordance
with aspects of the invention.
[0014] FIG. 6 is a photographic image of a portion of a chip
fabricated according to the system of FIG. 5, in accordance with
aspects of the invention.
[0015] FIG. 7 is a schematic rendition of the image of FIG. 6,
illustrating paths of fluid flow and particle movement relative to
a particle-retention or -capture chamber, in accordance with
aspects of the invention.
[0016] FIG. 8 is a full top plan view of the system of FIG. 5.
[0017] FIG. 9 is a photographic image of cells in a retention
chamber, after exposure to Trypan blue to stain lysed cells, but
before cell fixation, in accordance with aspects of the
invention.
[0018] FIG. 10 is another photographic image of the cells and
chamber of FIG. 9, after exposure to methanol to lyse and fix the
cells, in accordance with aspects of the invention.
[0019] FIG. 11 is yet another photographic image of the cells and
chamber of FIG. 9, after exposure to 1) methanol to lyse and fix
the cells, 2) Trypan blue to stain lysed cells, and 3) a wash
buffer to remove excess Trypan blue, in accordance with aspects of
the invention.
[0020] FIG. 11A is a fragmentary, top plan view of a microfluidic
system for measuring cell-cell communication, based on a duplicated
version of the system of FIG. 8, in accordance with aspects of the
invention.
[0021] FIG. 11B is a top plan view of selected portions of an
alternative embodiment of the system of FIG. 11A, in accordance
with aspects of the invention.
[0022] FIG. 11C is a top plan view of a two-dimensional array of
particle capture chambers that may be used in a microfluidic
system, in accordance with aspects of the invention.
[0023] FIG. 12 is a fragmentary, top plan view of a microfluidic
system for retaining and perfusing two sets of particles in
parallel, in accordance with aspects of the invention.
[0024] FIG. 13 is a view of selected portions of the system of FIG.
12, illustrating paths for fluid flow and particle movement
relative to two adjacent retention chambers, in accordance with
aspects of the invention.
[0025] FIG. 13A is a top plan view of a microfluidic system for
retaining two particles at spaced sites in a channel and perfusing
the retained particles with distinct reagents, in accordance with
aspects of the invention.
[0026] FIG. 13B is a top plan view of selected portions of the
system of FIG. 13A, in accordance with aspects of the
invention.
[0027] FIG. 13C is a top plan view of selected portions of an
alternative embodiment of the system of FIG. 13A, in accordance
with aspects of the invention.
[0028] FIG. 13D is a photograph of two beads being exposed to green
dye delivered by spaced treatment mechanisms, using a chip
constructed according to the system of FIG. 13A, in accordance with
aspects of the invention.
[0029] FIG. 13E is another photograph of the two beads of FIG. 13D
during exposure to a red dye and a green dye delivered by spaced
treatment mechanisms, in accordance with aspects of the
invention.
[0030] FIG. 13F is yet another photograph of the two beads of FIG.
13D during exposure to a red dye and a yellow dye delivered by
spaced treatment mechanisms, in accordance with aspects of the
invention.
[0031] FIG. 13G is a photograph of two cells held at separate
retention sites in a chip constructed according to the system of
FIG. 13A, in accordance with aspects of the invention.
[0032] FIG. 13H is a photograph of the two cells of FIG. 13G during
exposure to a blue dye delivered by spaced treatment mechanisms, in
accordance with aspects of the invention.
[0033] FIG. 131 is a photograph of the two cells of FIG. 13G during
treatment of only one of the cells with an organic fixative, in
accordance with aspects of the invention.
[0034] FIG. 13J is a photograph of the two cells of FIG. 13I, after
fixation of the one cell and during exposure to a blue dye,
delivered by spaced treatment mechanisms, in accordance with
aspects of the invention.
[0035] FIG. 13K is a photograph of two fluorescent beads held at
two retention sites and individually exposed to a fluorescent and a
chromophoric dye delivered by spaced treatment mechanisms, but
without the use of a spacer buffer, using a chip constructed
according to the system of FIG. 13A, in accordance with aspects of
the invention.
[0036] FIG. 13L is a fragmentary, top plan view of a microfluidic
system having separately addressable sets of linear trap arrays, in
accordance with aspects of the invention.
[0037] FIG. 14 is a top plan view of a microfluidic system for
retaining an array of particles in series and for perfusing members
of this array separately and in parallel, in accordance with
aspects of the invention.
[0038] FIG. 15 is a top plan view of selected portions of the
system of FIG. 14, illustrating fluid-layer and control-layer
networks for treating retained particles separately and in
parallel, in accordance with aspects of the invention.
[0039] FIG. 16 is a top plan view of portions of a single retention
network from the system of FIG. 14, illustrating selected paths of
fluid flow, in accordance with aspects of the invention.
[0040] FIG. 17 is a fragmentary, top plan view of a microfluidic
device for forming an array of single particles or groups of
particles, in accordance with aspects of the invention.
[0041] FIG. 18 is a pair of fragmentary, top plan schematic views
of a microfluidic device for forming an array of retained particles
that may be transferred to an array of separate sites, illustrating
particle retention and transfer configurations, on the left and
right respectively, in accordance with aspects of the
invention.
[0042] FIG. 19 is a pair of fragmentary, top plan schematic views
of another microfluidic device for forming an array of retained
particles that may be transferred to an array of separate sites,
illustrating particle retention and transfer configurations, on the
left and right respectively, in accordance with aspects of the
invention.
[0043] FIG. 20 is fragmentary, top plan schematic view of yet
another microfluidic device for forming an array of retained
particles that may be transferred to an array of separate sites, in
accordance with aspects of the invention.
[0044] FIG. 21 is a composite of top plan and sectional views
showing selected portions of a microfluidic system for retaining
particles using a particle-retention chamber that is fully spaced
from the floor of the system, in accordance with aspects of the
invention.
[0045] FIG. 22 is a composite of top plan and sectional views, and
a photographic image, showing selected portions of a microfluidic
system for retaining particles using a particle-retention chamber
that is partially spaced from the floor of the system, in
accordance with aspects of the invention.
[0046] FIG. 23 is a composite of top plan and sectional views, and
two photographic images, showing selected portions of another
microfluidic system for retaining particles using a
particle-retention chamber that is fully spaced from the floor of
the system, in accordance with aspects of the invention.
[0047] FIG. 24 is a fragmentary, top plan view of a reusable
microfluidic system for repeated retention, treatment, and release
of single particles, in accordance with aspects of the
invention.
[0048] FIG. 25 is a view of selected portions of the system of FIG.
24, particularly a particle release mechanism, in accordance with
aspects of the invention.
[0049] FIG. 26 is a fragmentary, top plan view of a reusable
microfluidic system for repeated retention, treatment, and release
of groups of particles, in accordance with aspects of the
invention.
[0050] FIG. 27 is a view of selected portions of the systems of
FIGS. 24 and 26, particularly a particle collection mechanism, in
accordance with aspects of the invention.
[0051] FIG. 28 is a fragmentary, top plan view of an input
mechanism that includes a particle suspension mechanism, in
accordance with aspects of the invention.
[0052] FIG. 29 is a fragmentary, top plan view of an adjustable
dilution mechanism, in accordance with aspects of the
invention.
[0053] FIG. 30 is a fragmentary, top plan view of another
adjustable dilution mechanism, in accordance with aspects of the
invention.
[0054] FIG. 31 is a top plan view of a microfluidic system having a
sorting mechanism based on centrifugal force, in accordance with
aspects of the invention.
[0055] FIG. 32 is a fragmentary view of the system of FIG. 31,
showing the sorting mechanism in greater detail, in accordance with
aspects of the invention.
[0056] FIG. 33 is a fragmentary, top plan view of another
microfluidic system having a sorting mechanism based on centrifugal
force, in accordance with aspects of the invention.
[0057] FIG. 34 is a top plan view of a yet another microfluidic
system having a sorting mechanism based on centrifugal force, in
accordance with aspects of the invention.
[0058] FIG. 35 is a fragmentary view of the system of FIG. 34,
showing the sorting mechanism in greater detail.
[0059] FIG. 36 is a photographic image of fluorescent beads and
particles being separated by the sorting mechanism of FIGS. 34 and
35.
[0060] FIG. 37 is a graph plotting the ratio of cells to beads over
time during sorting with the system of FIGS. 34 and 35.
[0061] FIG. 38 is a graph plotting the ratio of cells to beads over
time during sorting with the system of FIGS. 31 and 32.
[0062] FIGS. 39-43 are top plan composite views of various
cell-chamber networks for rnicrofluidic manipulation of cells, in
accordance with aspects of the invention.
[0063] FIG. 44 is a top plan view of a microfluidic system with a
parallel array of separate, isolatable cell-chamber networks, in
accordance with aspects of the invention.
[0064] FIG. 45 is a top plan view of a microfluidic system with an
isolatable cell chamber that may be fed or bypassed by a parallel
fluidic circuit, in accordance with aspects of the invention.
[0065] FIG. 46 is a top plan view of a microfluidic system having a
cell chamber that forms a loop, in accordance with aspects of the
invention
[0066] FIG. 47 is a top plan view of a microfluidic system in which
particle and reagent networks intersect at a common cell chamber,
in accordance with aspects of the invention.
[0067] FIGS. 48 and 49 are photographic images of filtering
mechanisms with size-selective channels that are included in the
reagent networks of chips fabricated according to the system of
FIG. 47.
[0068] FIG. 50 is a composite of two photographic images showing
cells cultured in a cell chamber of a chip fabricated according to
the system of FIG. 47.
[0069] FIG. 50A is a fragmentary, top plan view of a system for
depositing cells in a cell chamber, based on a nonlinear,
asymmetrical flow path, in accordance with aspects of the
invention.
[0070] FIG. 50B is a fragmentary, top plan view of a modified
version of the system of FIG. 50A, in which reagent(s) may be
recirculated through the cell chamber, in accordance with aspects
of the invention.
[0071] FIG. 50C is a top plan view of a cell chamber having two
distinct compartments connected by a set of radially arrayed,
size-selective channels, in accordance with aspects of the
invention.
[0072] FIG. 50D is a top plan view of a version of the cell chamber
of FIG. 50C, modified to interconnect the two compartments more
fully, in accordance with aspects of the invention.
[0073] FIG. 51 is an isometric schematic view of a microfluidic
system for performing electrophysiological analysis on an array of
cells, in accordance with aspects of the invention.
[0074] FIG. 52 is a top plan view of a microfluidic system for
performing electrophysiological analysis on a single cell, in
accordance with aspects of the invention.
[0075] FIG. 53 is a fragmentary top plan view of a microfluidic
system related to the system of FIG. 52, showing a modified
focusing mechanism, in accordance with aspects of the
invention.
[0076] FIG. 54 is a top plan view of selected portions of the
system of FIG. 52 with a retained cell, in accordance with aspects
of the invention.
[0077] FIG. 55 is a top plan view of selected portions of the
system of FIG. 52 during perfusion of a retained cell, in
accordance with aspects of the invention.
[0078] FIG. 56 is another top plan view of selected portions of the
system of FIG. 52, in accordance with aspects of the invention.
[0079] FIG. 57 is yet another top plan view of selected portions of
the system of FIG. 52, in accordance with aspects of the
invention.
[0080] FIG. 58 is a photographic image of a portion of a chip
fabricated according to the system of FIG. 52.
[0081] FIG. 59 is an abstracted view of a microfluidic device for
performing patch-clamp analysis of cells, in accordance with
aspects of the invention.
[0082] FIG. 60 is a fragmentary top plan view of a microfluidic
device for performing patch-clamp analysis of multiple individual
cells, in accordance with aspects of the invention.
[0083] FIG. 61 is a graph showing 95% probability of successfully
obtaining an electrophysiological reading as a function of both the
number of apertures (channels) analyzed and the fraction of
individual apertures that give a successful reading.
[0084] FIG. 62 is a fragmentary side elevation view of a
microfluidic mold spin-coated with a first layer of patternable,
selectively removable material, in accordance with aspects of the
invention.
[0085] FIG. 63 is a fragmentary side elevation view of the mold of
FIG. 62 after patterned removal of the first layer, in accordance
with aspects of the invention.
[0086] FIG. 64 is a fragmentary side elevation view of the mold of
FIG. 63 spin-coated with a second layer of patternable, selectively
removable material, in accordance with aspects of the
invention.
[0087] FIG. 65 is a fragmentary side elevation view of the mold of
FIG. 64 after patterned removal of the second layer, in accordance
with aspects of the invention.
[0088] FIG. 66 is a fragmentary side elevation view of the mold of
FIG. 65 after heating at elevated temperatures to round remaining
portions of the second layer, in accordance with aspects of the
invention.
[0089] FIG. 67 is a fragmentary side elevation view of the mold of
FIG. 66 spin-coated with a third layer of patternable, selectively
removable material, in accordance with aspects of the
invention.
[0090] FIG. 68 is a fragmentary side elevation view of the mold of
FIG. 67 following patterned removal of the third layer, in
accordance with aspects of the invention.
[0091] FIG. 69 is a fragmentary side elevation view of the mold of
FIG. 68 acting to mold complementary surface features of a
fluid-layer membrane, in accordance with aspects of the
invention.
[0092] FIG. 70 is a composite of photographic images of 1) a
fluid-layer mold formed using the method depicted in FIGS. 62-68
and 2) a corresponding molded chip formed from the fluid-layer
mold, in accordance with aspects of the invention.
[0093] FIG. 71 is a composite of photographic images of 1) a
fluid-layer mold formed using the method depicted in FIGS. 62-68
and 2) a corresponding molded chip formed partially from the
fluid-layer mold, in accordance with aspects of the invention.
[0094] FIG. 71A is a graph of fluorescence emission versus time for
a fluorophore being excited at different light intensities, in
accordance with aspects of the invention.
[0095] FIG. 71 is a schematic diagram of an embodiment of a method
for increasing the signal-to-noise ratio of a detected signal by
modulation of an exciting light source and demodulation of the
detected signal, based on the modulation, in accordance with
aspects of the invention.
[0096] FIG. 71C is a pair of graphs of time-dependent measured
noise and measured signal plus noise without (top) and with
(bottom) implementation of the modulation-demodulation method of
FIG. 71B in a microfluidic system, in accordance with aspects of
the invention.
[0097] FIG. 71D is a graph of measured fluorescence intensity
versus time prior to and during cycles of exposure of a
biotinylated bead to a streptavidin-dye conjugate in a microfluidic
system, in accordance with aspects of the invention.
[0098] FIG. 71E is a graph of measured fluorescence intensity
versus time prior to and during exposure of ionomcyin to a retained
cell that was preloaded with a calcium-sensor dye, using the method
of FIG. 71B in a microfluidic system, in accordance with aspects of
the invention.
[0099] FIG. 71F is a graph of measured fluorescence intensity
versus time at a position in a microfluidic system prior to and
during exposure to a dye, in accordance with aspects of the
invention.
[0100] FIG. 72 is a time-lapse set of photographic images recording
size-selective flow of blood cells through a microfluidic system,
in accordance with aspects of the invention.
[0101] FIG. 73 is diagram showing the structure of biotin and its
mode of binding to streptavidin.
[0102] FIG. 74 is a time-lapse set of photographic images recording
interaction of specific binding pairs on beads in a microfluidic
system, in accordance with aspects of the invention.
[0103] FIG. 75 is a time-lapse set of photographic images recording
stimulation of ion flux in a microfluidic system, in accordance
with aspects of the invention.
[0104] FIG. 76 is a time-lapse set of photographic images recording
apoptosis and necrosis in a microfluidic system, in accordance with
aspects of the invention.
[0105] FIGS. 77 and 78 are diagrams showing the structures and
excitation/emission spectra for membrane dyes used in the analysis
of Example 22.
[0106] FIG. 79 is a photographic image recording successful
staining of a cell's membrane in a non-microfluidic
environment.
[0107] FIG. 80 is a time-lapse set of photographic images recording
retention of a single cell at a preselected site in a microfluidic
system, in accordance with aspects of the invention.
[0108] FIG. 81 is a time-lapse set of photographic images recording
retention of a group of cells at a preselected site in a
microfluidic system, in accordance with aspects of the
invention.
[0109] FIG. 82 is a time-lapse set of photographic images recording
entry of a fluorescent cell into a retention chamber already
holding several cells, in accordance with aspects of the
invention.
[0110] FIG. 83 is a time-lapse set of photographic images recording
fixation and staining of a retained cell in a microfluidic system,
in accordance with aspects of the invention.
[0111] FIG. 84 is a top plan view of a microfluidic system for
analyzing a size-selected set of cells, in which the system
includes serially disposed filtration and retention mechanisms, a
perfusion mechanism, and a flow-based detection mechanism, in
accordance with aspects of the invention.
[0112] FIG. 85 is another top plan view of the microfluidic system
of FIG. 84, showing identifying labels for reservoirs and valves,
in accordance with aspects of the invention.
[0113] FIG. 86 is a top plan view of selected portions of the
system of FIG. 84, illustrating selected aspects including a
filtration mechanism, in accordance with aspects of the
invention.
[0114] FIG. 87 is another top plan view of selected portions of the
system of FIG. 84, in accordance with aspects of the invention.
[0115] FIG. 88 is yet another top plan view of selected portions of
the system of FIG. 84, in accordance with aspects of the
invention.
[0116] FIG. 89 is a top plan view of a perfusion device for
exposing particles to an array of different reagents or different
reagent concentrations.
[0117] FIGS. 90 through 94 depict a top plan view of a device being
used to measure chemotactic response of cells to a
chemoattractant.
[0118] FIG. 95 is a close-up top plan view of a perfusion chamber
with associated valving system.
DETAILED DESCRIPTION
[0119] The invention provides systems, including apparatus,
methods,.and kits, for the microfluidic manipulation and/or
analysis of particles, such as cells, viruses, organelles, beads,
and/or vesicles. The invention also provides microfluidic
mechanisms for carrying out these manipulations and analyses. These
mechanisms may enable controlled input, movement/positioning,
retention/localization, treatment, measurement, release, and/or
output of particles. Furthermore, these mechanisms may be combined
in any suitable order and/or employed for any suitable number of
times within a system. Accordingly, these combinations may allow
particles to be sorted, cultured, mixed, treated, and/or assayed,
among others, as single particles, mixed groups of particles,
arrays of particles, heterogeneous particle sets, and/or
homogeneous particle sets, among others, in series and/or in
parallel. In addition, these combinations may enable microfluidic
systems to be reused. Furthermore, these combinations may allow the
response of particles to treatment to be measured on a shorter time
scale than was previously possible. Therefore, systems of the
invention may allow a broad range of cell and particle assays, such
as drug screens, cell characterizations, research studies, and/or
clinical analyses, among others, to be scaled down to microfluidic
size. Such scaled-down assays may use less sample and reagent, may
be less labor intensive, and/or may be more informative than
comparable macrofluidic assays.
[0120] Further aspects of the invention are described in the
following sections: (I) microfluidic systems, (ID physical
structures of fluid networks, (HII) particles, (IV) input
mechanisms, (V) positioning mechanisms, (VI) retention mechanisms,
(VII) treatment mechanisms, (VIII) measurement mechanisms, (IX)
release mechanisms, (X) output mechanisms, (XI) cell culture
mechanisms, (XII) particle-based manipulations, and (XIII)
examples.
Microfluidic Systems
[0121] Definitions and Overview
[0122] Particle manipulations and analyses are performed in
microfluidic systems. A microfluidic system generally comprises any
system in which very small volumes of fluid are stored and
manipulated, generally less than about 500 .mu.L, typically less
than about 100 .mu.L, and more typically less than about 10 .mu.L.
Microfluidic systems carry fluid in predefined paths through one or
more microfluidic passages. A microfluidic passage may have a
minimum dimension, generally height or width, of less than about
200, 100, or 50 .mu.m. Passages are described in more detail below
in Section II.
[0123] Microfluidic systems may include one or more sets of
passages that interconnect to form a generally closed microfluidic
network. Such a microfluidic network may include one, two, or more
openings at network termini, or intermediate to the network, that
interface with the external world. Such openings may receive,
store, and/or dispense fluid. Dispensing fluid may be directly into
the microfluidic network or to sites external the microfluidic
system. Such openings generally function in input and/or output
mechanisms, described in more detail in Sections IV and X below,
and may include reservoirs, described in more detail in Section II
below.
[0124] Microfluidic systems also may include any other suitable
features or mechanisms that contribute to fluid, reagent, and/or
particle manipulation or analysis. For example, microfluidic
systems may include regulatory or control mechanisms that determine
aspects of fluid flow rate and/or path. Valves and/or pumps that
may participate in such regulatory mechanisms are described in more
detail below in Section II. Alternatively, or in addition,
microfluidic systems may include mechanisms that determine,
regulate, and/or sense fluid temperature, fluid pressure, fluid
flow rate, exposure to light, exposure to electric fields, magnetic
field strength, and/or the like. Accordingly, microfluidic systems
may include heaters, coolers, electrodes, lenses, gratings, light
sources, pressure sensors, pressure transducers, microprocessors,
microelectronics, and/or so on. Furthermore, each microfluidic
system may include one or more features that act as a code to
identify a given system. The features may include any detectable
shape or symbol, or set of shapes or symbols, such as
black-and-white or colored barcode, a word, a number, and/or the
like, that has a distinctive position, identity, and/or other
property (such as optical property).
[0125] Materials
[0126] Microfluidic systems may be formed of any suitable material
or combination of suitable materials. Suitable materials may
include elastomers, such as polydimethylsiloxane (PDMS); plastics,
such as polystyrene, polypropylene, polycarbonate, etc.; glass;
ceramics; sol-gels; silicon and/or other metalloids; metals or
metal oxides; biological polymers, mixtures, and/or particles, such
as proteins (gelatin, polylysine, serum albumin, collagen, etc.),
nucleic acids, microorganisms, etc.; and/or the like.
[0127] Exemplary materials for microfluidic systems are described
in more detail in the patent applications listed above under
Cross-References, which are incorporated herein by reference.
[0128] Methods of Fabrication
[0129] Microfluidic systems, also referred to as chips, may have
any suitable structure. Such systems may be fabricated as a unitary
structure from a single component, or as a multi-component
structure of two or more components. The two or more components may
have any suitable relative spatial relationship and may be attached
to one another by any suitable bonding mechanism.
[0130] In some embodiments, two or more of the components may be
fabricated as relatively thin layers, which may be disposed
face-to-face. The relatively thin layers may have distinct
thickness, based on function. For example, the thickness of some
layers may be about 10 to 250 .mu.m, 20 to 200 .mu.m, or about 50
to 150 .mu.m, among others. Other layers may be substantially
thicker, in some cases providing mechanical strength to the system.
The thicknesses of such other layers may be about 0.25 to 2 cm, 0.4
to 1.5 cm, or 0.5 to 1 cm, among others. One or more additional
layers may be a substantially planar layer that functions as a
substrate layer, in some cases contributing a floor portion to some
or all microfluidic passages.
[0131] Components of a microfluidic system may be fabricated by any
suitable mechanism, based on the desired application for the system
and on materials used in fabrication. For example, one or more
components may be molded, stamped, and/or embossed using a suitable
mold. Such a mold may be formed of any suitable material by
micromachining, etching, soft lithography, material deposition,
cutting, and/or punching, among others. Alternatively, or in
addition, components of a microfluidic system may be fabricated
without a mold by etching, micromachining, cutting, punching,
and/or material deposition.
[0132] Microfluidic components may be fabricated separately,
joined, and further modified as appropriate. For example, when
fabricated as distinct layers, microfluidic components may be
bonded, generally face-to-face. These separate components may be
surface-treated, for example, with reactive chemicals to modify
surface chemistry, with particle binding agents, with reagents to
facilitate analysis, and/or so on. Such surface-treatment may be
localized to discrete portions of the surface or may be relatively
nonlocalized. In some embodiments, separate layers may be
fabricated and then punched and/or cut to produce additional
structure. Such punching and/or cutting may be performed before
and/or after distinct components have been joined.
[0133] Exemplary methods for fabricating microfluidic systems are
described in more detail in the patent applications identified
above under Cross-References, which are incorporated herein by
reference.
Physical Structures of Fluid Networks
[0134] Overview
[0135] Microfluidic systems may include any suitable structure(s)
for the integrated manipulation of small volumes of fluid,
including moving and/or storing fluid, and particles associated
therewith, for use in particle assays. The structures may include
passages, reservoirs, and/or regulators, among others.
[0136] Passages
[0137] Passages generally comprise any suitable path, channel, or
duct through, over, or along which materials (e.g., fluid,
particles, and/or reagents) may pass in a microfluidic system.
Collectively, a set of fluidically communicating passages,
generally in the form of channels, may be referred to as a
microfluidic network. In some cases, passages may be described as
having surfaces that form a floor, a roof, and walls. Passages may
have any suitable dimensions and geometry, including width, height,
length, and/or cross-sectional profile, among others, and may
follow any suitable path, including linear, circular, and/or
curvilinear, among others. Passages also may have any suitable
surface contours, including recesses, protrusions, and/or
apertures, and may have any suitable surface chemistry or
permeability at any appropriate position within a channel. Suitable
surface chemistry may include surface modification, by addition
and/or treatment with a chemical and/or reagent, before, during,
and/or after passage formation.
[0138] In some cases, passages, and particularly channels, may be
described according to function. For example, passages may be
described according to direction of material flow in a particular
application, relationship to a particular reference structure,
and/or type of material carried. Accordingly, passages may be inlet
passages (or channels), which generally carry materials to a site,
and outlet passages (or channels), which generally carry materials
from a site. In addition, passages may be referred to as particle
passages (or channels), reagent passages (or channels), focusing
passages (or channels), perfusion passages (or channels), waste
passages (or channels), and/or the like.
[0139] Passages may branch, join, and/or dead-end to form any
suitable microfluidic network. Accordingly, passages may function
in particle positioning, sorting, retention, treatment, detection,
propagation, storage, mixing, and/or release, among others.
[0140] Further aspects of passages are included throughout this
Detailed Description, and in the patent applications identified
above under Cross-References, which are incorporated herein by
reference.
[0141] Reservoirs
[0142] Reservoirs generally comprise any suitable receptacle or
chamber for storing materials (e.g., fluid, particles and/or
reagents), before, during, between, and/or after processing
operations (e.g., measurement and/or treatment). Reservoirs, also
referred to as wells, may include input, intermediate, and/or
output reservoirs. Input reservoirs may store materials (e.g.,
fluid, particles, and/or reagents) prior to inputting the materials
to a microfluidic network(s) portion of a chip. By contrast,
intermediate reservoirs may store materials during and/or between
processing operations. Finally, output reservoirs may store
materials prior to outputting from the chip, for example, to an
external processor or waste, or prior to disposal of the chip.
[0143] Further aspects of reservoirs are included in the patent
applications identified above under Cross-References, which are
incorporated herein by reference.
[0144] Regulators
[0145] Regulators generally comprise any suitable mechanism for
generating and/or regulating movement of materials (e.g., fluid,
particles, and/or reagents). Suitable regulators may include
valves, pumps, and/or electrodes, among others. Regulators may
operate by actively promoting flow and/or by restricting active or
passive flow. Suitable functions mediated by regulators may include
mixing, sorting, connection (or isolation) of fluidic networks,
and/or the like.
[0146] Further aspects of regulators, particularly the structure,
fabrication, and operation of valves and pumps, are included in the
patent applications identified above under Cross-References, which
are incorporated herein by reference, and in Section XIII,
particularly Example 8.
Particles
[0147] Overview
[0148] Microfluidic systems may be used to manipulate and/or
analyze particles. A particle generally comprises any object that
is small enough to be inputted and manipulated within a
microfluidic network in association with fluid, but that is large
enough to be distinguishable from the fluid. Particles, as used
here, typically are microscopic or near-microscopic, and may have
diameters of about 0.005 to 100 .mu.m, 0.1 to 50 .mu.m, or about
0.5 to 30 .mu.m. Alternatively, or in addition, particles may have
masses of about 10.sup.-20 to 10.sup.-5 grams, 10.sup.-16 to
10.sup.-7 grams, or 10.sup.-14 to 10.sup.-8 grams. Exemplary
particles may include cells, viruses, organelles, beads, and/or
vesicles, and aggregates thereof, such as dimers, trimers, etc.
Cells
[0149] Overview
[0150] Cells, as used here, generally comprise any
self-replicating, membrane-bounded biological entity, or any
nonreplicating, membrane-bounded descendant thereof. Nonreplicating
descendants may be senescent cells, terminally differentiated
cells, cell chimeras, serum-starved cells, infected cells,
nonreplicating mutants, anucleate cells, etc.
[0151] Cells used as particles in microfluidic systems may have any
suitable origin, genetic background, state of health, state of
fixation, membrane permeability, pretreatment, and/or population
purity, among others. Origin of cells may be eukaryotic,
prokaryotic, archae, etc., and may be from animals, plants, fungi,
protists, bacteria, and/or the like. Cells may be wild-type;
natural, chemical, or viral mutants; engineered mutants (such as
transgenics); and/or the like. In addition, cells may be growing,
quiescent, senescent, transformed, and/or immortalized, among
others, and cells may be fixed and/or unfixed. Living or dead,
fixed or unfixed cells may have intact membranes, and/or
permeabilized/disrupted membranes to allow uptake of ions, labels,
dyes, ligands, etc., or to allow release of cell contents. Cells
may have been pretreated before introduction into a microfluidic
system by any suitable processing steps. Such processing steps may
include modulator treatment, transfection (including infection,
injection, particle bombardment, lipofection, coprecipitate
transfection, etc.), processing with assay reagents, such as dyes
or labels, and/or so on. Furthermore, cells may be a monoculture,
generally derived as a clonal population from a single cell or a
small set of very similar cells; may be presorted by any suitable
mechanism such as affinity binding, FACS, drug selection, etc.;
and/or may be a mixed or heterogeneous population of distinct cell
types.
[0152] Eukaryotic Cells
[0153] Eukaryotic cells, that is, cells having one or more nuclei,
or anucleate derivatives thereof, may be obtained from any suitable
source, including primary cells, established cells, and/or patient
samples. Such cells may be from any cell type or mixture of cell
types, from any developmental stage, and/or from any genetic
background. Furthermore, eukaryotic cells may be adherent and/or
nonadherent. Such cells may be from any suitable eukaryotic
organism including animals, plants, fungi, and/or protists.
[0154] Eukaryotics cells may be from animals, that is, vertebrates
or invertebrates. Vertebrates may include mammals, that is,
primates (such as humans, apes, monkeys, etc.) or nonprimates (such
as cows, horses, sheep, pigs, dogs, cats, marsupials, rodents,
and/or the like). Nonmammalian vertebrates may include birds,
reptiles, fish, (such as trout, salmon, goldfish, zebrafish, etc.),
and/or amphibians (such as frogs of the species Xenopus, Rana,
etc.). Invertebrates may include arthropods (such as arachnids,
insects (e.g., Drosophila), etc.), mollusks (such as clams, snails,
etc.), annelids (such as earthworms, etc.), echinoderms (such as
various starfish, among others), coelenterates (such as jellyfish,
coral, etc.), porifera (sponges), platyhehninths (tapeworms),
nemathelminths (flatworms), etc.
[0155] Eukaryotic cells may be from any suitable plant, such as
monocotyledons, dicotyledons, gymnosperms, angiosperms, ferns,
mosses, lichens, and/or algae, among others. Exemplary plants may
include plant crops (such as rice, corn, wheat, rye, barley,
potatoes, etc.), plants used in research (e.g., Arabadopsis,
loblolly pine, etc.), plants of horticultural values (ornamental
palms, roses, etc.), and/or the like.
[0156] Eukaryotic cells maybe from any suitable fungi, including
members of the phyla Chytridiomycota, Zygomycota, Ascomycota,
Basidiomycota, Deuteromycetes, and/or yeasts. Exemplary fungi may
include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia
pastoralis, Neurospora crassa, mushrooms, puffballs, imperfect
fungi, molds, and/or the like.
[0157] Eukaryotic cells may be from any suitable protists
(protozoans), including amoebae, ciliates, flagellates, coccidia,
microsporidia, and/or the like. Exemplary protists may include
Giardia lamblia, Entamoeba. histolytica, Cryptosporidium, and/or N.
fowleri, among others.
[0158] Particles may include eukaryotic cells that are primary,
that is, taken directly from an organism or nature, without
subsequent extended culture in vitro. For example, the cells may be
obtained from a patient sample, such as whole blood, packed cells,
white blood cells, urine, sputum, feces, mucus, spinal fluid,
tumors, diseased tissue, bone marrow, lymph, semen, pleural fluid,
a prenatal sample, an aspirate, a biopsy, disaggregated tissue,
epidermal cells, keratinocytes, endothelial cells, smooth muscle
cells, skeletal muscle cells, neural cells, renal cells, prostate
cells, liver cells, stem cells, osteoblasts, and/or the like.
Similar samples may be manipulated and analyzed from human
volunteers, selected members of the human population, forensic
samples, animals, plants, and/or natural sources (water, soil, air,
etc.), among others.
[0159] Alternatively, or in addition, particles may include
established eukaryotic cells. Such cells may be immortalized and/or
transformed by any suitable treatment, including viral infection,
nucleic acid transfection, chemical treatment, extended passage and
selection, radiation exposure, and/or the like. Such established
cells may include various lineages such as neuroblasts, neurons,
fibroblasts, myoblasts, myotubes, chondroblasts, chondrocytes,
osteoblasts, osteocytes, cardiocytes, smooth muscle cells,
epithelial cells, keratinocytes, kidney cells, liver cells,
lymphocytes, granulocytes, and/or macrophages, among others.
Exemplary established cell lines may include Rat-1, NIH 3T3, HEK
293, COS1, COS7, CV-1, C2C12, MDCK, PC12, SAOS, HeLa, Schneider
cells, Junkat cells, SL2, and/or the like.
[0160] Prokaryotic Cells
[0161] Particles maybe prokaryotic cells, that is,
self-replicating, membrane-bounded microorganisms that lack
membrane-bound organelles, or nonreplicating descendants thereof.
Prokaryotic cells may be from any phyla, including Aquificae,
Bacteroids, Chlorobia, Chrysogenetes, Cyanobacteria, Fibrobacter,
Firmicutes, Flavobacteria, Fusobacteria, Proteobacteria,
Sphingobacteria, Spirochaetes, Thermomicrobia, and/or Xenobacteria,
among others. Such bacteria may be gram-negative, gram-positive,
harmful, beneficial, and/or pathogenic. Exemplary prokaryotic cells
may include E. coli, S. typhimurium, B subtilis, S. aureus, C.
perfringens, V. parahaemolyticus, and/or B. anthracis, among
others.
[0162] Viruses
[0163] Viruses may be manipulated and/or analyzed as particles in
microfluidic systems. Viruses generally comprise any
microscopic/submicroscopic parasites of cells (animals, plants,
fingi, protists, and/or bacteria) that include a protein and/or
membrane coat and that are unable to replicate without a host cell.
Viruses may include DNA viruses, RNA viruses, retroviruses,
virions, viroids, prions, etc. Exemplary viruses may include HIV,
RSV, rabies, hepatitis virus, Epstein-Barr virus, rhinoviruses,
bacteriophages, prions that cause various diseases (CJD
(Creutzfeld-Jacob disease, kuru, GSS
(Gerstmann-Straussler-Scheinker syndrome), FFI (Fatal Familial
Insomnia), Alpers syndrome, etc.), and/or the like.
[0164] Organelles
[0165] Organelles may be manipulated and/or analyzed in
microfluidic systems. Organelles generally comprise any particulate
component of a cell. For example, organelles may include nuclei,
Golgi apparatus, lysosomes, endosomes, mitochondria, peroxisomes,
endoplasmic reticulum, phagosomes, vacuoles, chloroplasts, etc.
[0166] Beads
[0167] Particle assays may be performed with beads. Beads generally
comprise any suitable manufactured particles. Beads may be
manufactured from inorganic materials, or materials that are
synthesized chemically, enzymatically and/or biologically.
Furthermore, beads may have any suitable porosity and may be formed
as a solid or as a gel. Suitable bead compositions may include
plastics (e.g., polystyrene), dextrans, glass, ceramics, sol-gels,
elastomers, silicon, metals, and/or biopolymers (proteins, nucleic
acids, etc.). Beads may have any suitable particle diameter or
range of diameters. Accordingly, beads may be a substantially
uniform population with a narrow range of diameters, or beads may
be a heterogeneous population with a broad range of diameters, or
two or more distinct diameters.
[0168] Beads may be associated with any suitable materials. The
materials may include compounds, polymers, complexes, mixtures,
phages, viruses, and/or cells, among others. For example, the beads
may be associated with a member of a specific binding pair (see
Section VI), such as a receptor, a ligand, a nucleic acid, a member
of a compound library, and/or so on. Beads may be a mixture of
distinct beads, in some cases carrying distinct materials. The
distinct beads may differ in any suitable aspect(s), such as size,
shape, an associated code, and/or material carried by the beads. In
some embodiments, the aspect may identify the associated material.
Codes are described further in Section XII below.
[0169] Vesicles
[0170] Particles may be vesicles. Vesicles generally comprise any
noncellularly derived particle that is defined by a lipid envelope.
Vesicles may include any suitable components in their envelope or
interior portions. Suitable components may include compounds,
polymers, complexes, mixtures, aggregates, and/or particles, among
others. Exemplary components may include proteins, peptides, small
compounds, drug candidates, receptors, nucleic acids, ligands,
and/or the like.
Input Mechanisms
[0171] Overview
[0172] Microfluidic systems may include one or more input
mechanisms that interface with the microfluidic network(s). An
input mechanism generally comprises any suitable mechanism for
inputting material(s) (e.g., particles, fluid, and/or reagents) to
a microfluidic network of a microfluidic chip, including selective
(that is, component-by-component) and/or bulk mechanisms.
[0173] Internal/External Sources
[0174] The input mechanism may receive material from internal
sources, that is, reservoirs that are included in a microfluidic
chip, and/or external sources, that is, reservoirs that are
separate from, or external to, the chip.
[0175] Input mechanisms that input materials from internal sources
may use any suitable receptacle to store and dispense the
materials. Suitable receptacles may include a void formed in the
chip. Such voids may be directly accessible from outside the chip,
for example, through a hole extending from fluidic communication
with a fluid network to an external surface of the chip, such as
the top surface. The receptacles may have a fluid capacity that is
relatively large compared to the fluid capacity of the fluid
network, so that they are not quickly exhausted. For example, the
fluid capacity may be at least about 1, 5, 10, 25, 50, or 100
.mu.L. Accordingly, materials may be dispensed into the receptacles
using standard laboratory equipment, if desired, such as
micropipettes, syringes, and the like.
[0176] Input mechanisms that input materials from external sources
also may use any suitable receptacle and mechanism to store and
dispense the materials. However, if the external sources input
materials directly into the fluid network, the external sources may
need to interface effectively with the fluid network, for example,
using contact and/or noncontact dispensing mechanisms. Accordingly,
input mechanisms from external sources may use capillaries or
needles to direct fluid precisely into the fluid network.
Altematively, or in addition, input mechanisms from external
sources may use a noncontact dispensing mechanism, such as
"spitting," which may be comparable to the action of an inkjet
printer. Furthermore, input mechanisms from external sources may
use ballistic propulsion of particles, for example, as mediated by
a gene gun.
[0177] Facilitating Mechanisms
[0178] The inputting of materials into the microfluidics system may
be facilitated and/or regulated using any suitable facilitating
mechanism. Such facilitating mechanisms may include gravity flow,
for example, when an input reservoir has greater height of fluid
than an output reservoir. Facilitating mechanisms also may include
positive pressure to push materials into the fluidic network, such
as mechanical or gas pressure, or centrifugal force; negative
pressure at an output mechanism to draw fluid toward the output
mechanism; and/or a positioning mechanism acting within the fluid
network. The positioning mechanism may include a pump and/or an
electrokinetic mechanism. Positioning mechanisms are further
described below, in Section V. In some embodiments, the
facilitating mechanism may include a suspension mechanism to
maintain particles such as cells in suspension prior to inputting,
for example, as described in Example 7.
Positioning Mechanisms
[0179] Overview
[0180] Microfluidic systems may include one or more positioning
mechanisms. A positioning mechanism generally comprises any
mechanism for placing particles at preselected positions on the
chip after inputting, for example, for retention, growth,
treatment, and/or measurement, among others. Positioning mechanisms
may be categorized without limitation in various ways, for example,
to reflect their origins and/or operational principles, including
direct and/or indirect, fluid-mediated and/or non-fluid-mediated,
external and/or internal, and so on. These categories are not
mutually exclusive. Thus, a given positioning mechanism may
position a particle in two or more ways; for example, electric
fields may position a particle directly (e.g., via electrophoresis)
and indirectly (e.g., via electroosmosis).
[0181] The positioning mechanisms may act to define particle
position longitudinally and/or transversely. The term "longitudinal
position" denotes position parallel to or along the long axis of a
microfluidic channel and/or a fluid flow stream within the channel.
In contrast, the term "transverse position" denotes position
orthogonal to the long axis of a channel and/or an associated main
fluid flow stream. Both longitudinal and transverse positions may
be defined locally, by equating "long axis" with "tangent" in
curved channels.
[0182] The positioning mechanisms may be used alone and/or in
combination. If used in combination, the mechanisms may be used
serially (i.e., sequentially) and/or in parallel (i.e.,
simultaneously). For example, an indirect mechanism such as fluid
flow may be used for rough positioning, and a direct mechanism such
as optical tweezers may be used for final positioning (and/or
subsequent retention, as described elsewhere). The remainder of
this section describes without limitation a variety of exemplary
positioning mechanisms, sorted roughly as direct and indirect
mechanisms.
[0183] Direct Positioning Mechanisms
[0184] Direct positioning mechanisms generally comprise any
mechanisms in which a force acts directly on a particle(s) to
position the particle(s) within a microfluidic network. Direct
positioning mechanisms may be based on any suitable mechanism,
including optical, electrical, magnetic, and/or gravity-based
forces, among others. Optical positioning mechanisms use light to
mediate or at least facilitate positioning of particles. Suitable
optical positioning mechanisms include "optical tweezers," which
use an appropriately focused and movable light source to impart a
positioning force on particles. Electrical positioning mechanisms
use electricity to position particles. Suitable electrical
mechanisms include "electrokinesis," that is, the application of
voltage and/or current across some or all of a microfluidic
network, which may, as mentioned above, move charged particles
directly (e.g., via electrophoresis) and/or indirectly, through
movement of ions in fluid (e.g., via electroosmosis). Magnetic
positioning mechanisms use magnetism to position particles based on
magnetic interactions. Suitable magnetic mechanisms involve
applying a magnetic field in or around a fluid network, to position
particles via their association with ferromagnetic and/or
paramagnetic materials in, on, or about the particles.
Gravity-based positioning mechanisms use the force of gravity to
position particles, for example, to contact adherent cells with a
substrate at positions of cell culture.
[0185] Indirect Positioning Mechanisms
[0186] Indirect positioning mechanisms generally comprise any
mechanisms in which a force acts indirectly on a particle(s), for
example, via fluid, to move the particle(s) within a microfluidic
network, longitudinally and/or transversely.
[0187] Longitudinal Indirect Positioning Mechanisms
[0188] Longitudinal indirect positioning mechanisms generally may
be created and/or regulated by fluid flow along channels and/or
other passages. Accordingly, longitudinal positioning mechanisms
may be facilitated and/or regulated by valves and/or pumps that
regulate flow rate and/or path. In some cases, longitudinal
positioning mechanisms may be facilitated and/or regulated by
electroosmotic positioning mechanisms. Alternatively, or in
addition, longitudinal positioning mechanisms may be input-based,
that is, facilitated and/or regulated by input mechanisms, such as
pressure or gravity-based mechanisms, including a pressure head
created by unequal heights of fluid columns.
[0189] Transverse Indirect Positioning Mechanisms
[0190] Transverse indirect positioning mechanisms generally may be
created and/or regulated by fluid flow streams at channel
junctions, laterally disposed regions of reduced fluid flow, and/or
channel bends. Channel junctions may be unifying sites or dividing
sites, based on the number of channels that carry fluid to the
sites relative to the number that carry fluid away from the sites.
Transverse indirect positioning mechanisms may be based on laminar
flow, stochastic partitioning, and/or centrifugal force, among
others.
[0191] Laminar Flow-Based Transverse Positioning Mechanisms
[0192] Transverse positioning of particles and/or reagents in a
microfluidic system may be mediated at least in part by a laminar
flow-based mechanism. Laminar flow-based mechanisms generally
comprise any positioning mechanism in which the position of an
input flow stream within a channel is determined by the presence,
absence, and/or relative position(s) of additional flow streams
within the channel. Such laminar flow-based mechanisms may be
defined by a channel junction(s) that is a unifying site, at which
inlet flow streams from two, three, or more channels, flowing
toward the junction, unify to form a smaller number of outlet flow
streams, preferably one, flowing away from the junction. Due to the
laminar flow properties of flow streams on a microfluidic scale,
the unifying site may maintain the relative distribution of inlet
flow streams after they unify as laminar outlet flow streams.
Accordingly, particles and/or reagents may remain localized to any
selected one or more of the laminar flow streams, based on which
inlet channels carry particles and/or reagents, thus positioning
the particles and/or reagents transversely.
[0193] The relative size (or flow rate) and position of each inlet
flow stream may determine both transverse position and relative
width of flow streams that carry particles and/or reagents. For
example, an inlet flow stream for particles/reagents that is
relatively small (narrow), flanked by two larger (wider) flow
streams, may occupy a narrow central position in a single outlet
channel. By contrast, an inlet flow stream for particles/reagents
that is relatively large (wide), flanked by a comparably sized flow
stream and a smaller (narrower) flow stream, may occupy a wider
position that is biased transversely toward the smaller flow
stream. In either case, the laminar flow-based mechanism may be
called a focusing mechanism, because the particles/reagents are
"focused" to a subset of the cross-sectional area of outlet
channels. Laminar flow-based mechanisms may be used to individually
address particles and/or reagents to plural distinct retention
sites. Exemplary laminar flow-based positioning mechanisms are
further described below, in Examples 24, 7, 9, 11, and 26, among
others.
[0194] A laminar flow-based mechanism may be a variable mechanism
to vary the transverse position of particles/reagents. As described
above, the relative contribution of each inlet flow stream may
determine the transverse position of particles/reagents flow
streams. Altered flow of any inlet flow stream may vary its
contribution to the outlet flow stream(s), shifting
particles/reagents flow streams accordingly. In an extreme case,
referred to as a perfusion mechanism, a reagent (or particle) flow
stream may be moved transversely, either in contact with, or spaced
from, retained particles (reagents), based on presence or absence
of flow from an adjacent inlet flow stream. Such a mechanism also
may be used to effect variable or regulated transverse positioning
of particles, for example, to direct particles to retention sites
having different transverse positions. Exemplary variable or
regulated transverse positioning mechanisms, referred to as
perfusion mechanisms, are further described below, in Examples 2-4,
6, 7, 11, and 26, among others.
[0195] Stochastic Transverse Positioning Mechanisms
[0196] Transverse positioning of particles and/or reagents in a
microfluidic system may be mediated at least in part by a
stochastic (or portioned flow) positioning mechanism. Stochastic
transverse positioning mechanisms generally comprise any
positioning mechanism in which an at least partially randomly
selected subset of inputted particles or reagent is distributed
laterally away from a main flow stream to a region of reduced fluid
flow within a channel (or, potentially, to a distinct channel). The
region of reduced flow may promote particle retention, treatment,
detection, minimize particle damage, and/or promote particle
contact with a substrate. Stochastic positioning mechanisms may be
determined by dividing flow sites and/or locally widened channels,
among others.
[0197] Dividing flow sites may effect stochastic positioning by
forming regions of reduced fluid flow rate. Dividing flow sites
generally include any channel junction at which inlet flow streams
from one (preferably) or more inlet channels are divided into a
greater number of outlet channels, including two, three, or more,
channels. Such dividing sites may deliver a subset of particles,
which may be selected stochastically and/or based on a property of
the particles (such as mass), to a region of reduced flow rate or
quasi-stagnant flow formed at or near the junction. The fraction of
particles represented by the subset may be dependent upon the
relative flow directions of the outlet channels relative to the
inlet channels. These flow directions may be generally orthogonal
to an inlet flow stream, being directed in opposite directions, to
form a "T-junction." Alternatively, outlet flow directions may form
angles of less than and/or greater than 90.degree.. Exemplary
reduced-velocity, dividing-flow positioning mechanisms are further
described below, in Examples 1, 2, 3, 4, 6, 7, and 26, among
others.
[0198] The dividing-flow positioning mechanism, with two or more
outlet channels, may be used as a portioned-flow mechanism.
Specifically, fluid, particles, and/or reagents carried to the
channel junction may be portioned according to fluid flow through
the two or more outlet channels. Accordingly, the fractional number
or volume of particles or reagent that enters the two or more
channels may be regulated by the relative sizes of the channels
and/or the flow rate of fluid through the channels, which in turn
may be regulated by valves, or other suitable flow
regulatory-mechanisms. In a first set of embodiments, outlet
channels may be of very unequal sizes, so that only a small
fraction of particle and/or reagents are directed to the smaller
channel. In a second set of embodiments, valves may be used to
forms desired dilutions of reagents. In a third set of embodiments,
valves may be used to selectively direct particles to one of two or
more fluid paths. Examples of these three sets of embodiments are
further described below, in Examples 11, 8, and 7,
respectively.
[0199] Locally widened channels may promote stochastic positioning
by producing regions of decreased flow rate lateral to a main flow
stream. The decreased flow rate may deposit a subset of inputted
particles at a region of decreased flow rate. Such widened channels
may include nonlinear channels that curve or bend at an angle.
Alternatively, or in addition, widened regions may be formed by
recesses formed in a channel wall(s), chambers that intersect
channels, and/or the like, particularly at the outer edge of a
curved or bent channel. Exemplary locally widened channels that
promote stochastic transverse positioning are described further in
Example 10.
[0200] Centrifugal-force-based Transverse Positioning
Mechanisms
[0201] Transverse positioning of particles and/or reagents also may
be mediated at least in part by a centrifugal positioning
mechanism. In centrifugal positioning mechanisms, particles may
experience a centrifugal force determined by a change in velocity,
for example, by moving through a bend in a fluid path. Size and/or
density of particles may determine the rate of velocity change,
distributing distinct sizes and/or densities of particle to
distinct transverse positions. Exemplary centrifugal positioning
mechanisms are further described below, in Example 9.
Retention Mechanisms
[0202] Overview
[0203] Microfluidic systems may include one or more retention
mechanisms. A retention mechanism generally comprises any suitable
mechanism for retaining (or holding, capturing, or trapping)
particles at preselected positions or regions of microfluidic
networks, including single or plural mechanisms, operating in
series and/or in parallel. Retention mechanisms may act to overcome
the positioning force exerted by fluid flow. Furthermore, retention
mechanisms, also referred to as capture or trapping mechanisms, may
retain any suitable number of particles, including single particles
or groups/populations of particles. Suitable retention mechanisms
may be based on physical barriers coupled with flow, chemical
interactions, vacuum forces, fluid flow in a loop, gravity,
centrifugal forces, magnetic forces, electrical forces, and/or
optically generated forces, among others.
[0204] Retention mechanisms may be selective or nonselective.
Selective mechanisms may be fractionally selective, that is,
retaining less than all (a subset of) inputted particles.
Fractionally selective mechanisms may rely at least in part on
stochastic positioning mechanisms, such as that exemplified in
Example 2. Alternatively, or in addition, selective mechanisms may
be particle-dependent, that is, retaining particles based on one or
more properties of the inputted particle, such as size, surface
chemistry, density, magnetic character, electrical charge, optical
property (such as refractive index), and/or the like.
[0205] Physical Barrier-Based Retention Mechanisms
[0206] Retention mechanisms may be based at least partially on
particle contact with any suitable physical barrier(s) disposed in
a microfluidic network. Such particle-barrier contact generally
restricts longitudinal particle movement along the direction of
fluid flow, producing flow-assisted retention. Flow-assisted
particle-barrier contact also may restrict side-to-side/orthogonal
(transverse) movement. Suitable physical barriers may be formed by
protrusions that extend inward from any portion of a channel or
other passage (that is, walls, roof, and/or floor). For example,
the protrusions may be fixed and/or movable, including columns,
posts, blocks, bumps, walls, and/or partially/completely closed
valves, among others. Some physical barriers, such as valves, may
be movable or regulatable. Alternatively, or in addition, a
physical barrier may be defined by a recess(es), formed in a
channel or other passage, or by a fluid-permeable membrane. Other
physical barriers may be formed based on the cross-sectional
dimensions of passages. For example, size-selective channels may
retain particles that are too large to enter the channels.
(Size-selective channels also may be referred to as filter
channels, microchannels, or particle-restrictive or
particle-selective channels.)
[0207] Further aspects of physical barriers and size-selective
channels are described below in Section XIII, and in the patent
applications listed in the Cross-References, which are incorporated
herein by reference.
[0208] Chemical Retention Mechanisms
[0209] Chemical retention mechanisms may retain particles based on
chemical interactions. The chemical interactions may be covalent
and/or noncovalent interactions, including ionic, electrostatic,
hydrophobic, van der Waals, and/or metal coordination interactions,
among others. Chemical interactions may retain particles
selectively and/or nonselectively. Selective and nonselective
retention may be based on specific and/or nonspecific chemical
interactions between particles and passage surfaces.
[0210] Chemical interactions may be specific. Specific mechanisms
may use specific binding pairs (SBPs), for example, with first and
second SBP members disposed on particles and passage surfaces,
respectively. Exemplary SBPs may include biotin/avidin,
antibody/antigen, lectin/carbohydrate, etc. These and additional
exemplary SBPs are listed below in Table 1, with the designations
of first and second being arbitrary. SBP members may be disposed
locally within microfluidic networks before, during and/or after
formation of the networks. .For example, surfaces of a substrate
and/or a fluid layer component may be locally modified by
adhesion/attachment of a SBP member before the substrate and fluid
layer component are joined. Alternatively, or in addition, an SBP
member may be locally associated with a portion of a microfluidic
network after the network has been formed, for example, by local
chemical reaction of the SBP member with the network (such as
catalyzed by local illumination with light).
1TABLE 1 Representative Specific Binding Pairs First SBP Member
Second SBP Member Antigen antibody Biotin avidin or streptavidin
Carbohydrate lectin or carbohydrate receptor DNA antisense DNA or
DNA-binding protein enzyme substrate or enzyme inhibitor Histidine
NTA (nitrilotriacetic acid) IgG protein A or protein G RNA
antisense RNA
[0211] Chemical interactions also may be relatively nonspecific.
Nonspecific interaction mechanisms may rely on local differences in
the surface chemistry of microfluidic networks. Such local
differences may be created before, during and/or after
passage/microfluidic network formation, as described above. The
local differences may result from localized chemical reactions, for
example, to create hydrophobic or hydrophilic regions, and/or
localized binding of materials. The bound materials may include
poly-L-lysine, poly-D-lysine, polyethylenimine, albumin, gelatin,
collagen, laminin, fibronectin, entactin, vitronectin, fibrillin,
elastin, heparin, keratan sulfate, heparan sulfate, chondroitin
sulfate, hyaluronic acid, and/or extracellular matrix
extracts/mixtures, among others.
[0212] Other Retention Mechanisms
[0213] Other retention mechanisms may be used alternatively, or in
addition to, physical barrier-based and/or chemical
interaction-based retention. Some or all of these mechanisms,
and/or the mechanisms described above, may rely at least partially
on friction between particles and passages to assist retention.
[0214] Retention mechanisms may be based on vacuum forces, fluid
flow, and/or gravity. Vacuum-based retention mechanisms may exert
forces that pull particles into tighter contact with passage
surfaces, for example, using a force directed outwardly from a
channel. Application of a vacuum, and/or particle retention, may be
assisted by an aperture/orifice in the wall of a channel or other
passage. By contrast, fluid flow-based retention mechanisms may
produce fluid flow paths, such as loops, that retain particles.
These fluid flow paths may be formed by a closed channel-circuit
having no outlet (e.g., by valve closure and active pumping),
and/or by an eddy, such as that produced by generally circular
fluid-flow within a recess. Gravity-based retention mechanisms may
hold particles against the bottom surfaces of passages, thus
combining with friction to restrict particle movement.
Gravity-based retention may be facilitated by recesses and/or
reduced fluid flow rates. Further aspects of vacuum-based and fluid
flow-based retention mechanisms are described below in Examples 11
and 12, and Example 10, respectively.
[0215] Retention mechanisms may be based on centrifugal forces,
magnetic forces, and/or optically generated forces. Retention
mechanisms based on centrifugal force may retain particles by
pushing the particle against passage surfaces, typically by
exerting a force on the particles that is generally orthogonal to
fluid flow. Such forces may be exerted by centrifugation of a
microfluidic chip and/or by particle movement within a fluid flow
path (see Example 9). Magnetic force-based retention mechanisms may
retain particles using magnetic fields, generated external and/or
internal to a microfluidic system. The magnetic field may interact
with ferromagnetic and/or paramagnetic portions of particles. For
example, beads may be formed at least partially of ferromagnetic
materials, or cells may include surface-bound or internalized
ferromagnetic particles. Electrical force-based retention
mechanisms may retain charged particles and/or populations using
electrical fields. By contrast, retention mechanisms that operate
based on optically generated forces may use light to retain
particles. Such mechanisms may operate based on the principal of
optical tweezers, among others.
[0216] Another form of retention mechanism is a blind-fill channel,
where a channel has a inlet, but no outlet, either fixedly or
transiently. For example, when the microfluidic device is made from
a gas permeable material, such as PDMS, gas present in a dead-end
channel can escape, or be forced out of the channel through the gas
permeable material when urged out by the inflow of liquid through
the inlet. This is a preferred example of blind-filling.
Blind-filling can be used with a channel or chamber that has an
inlet, and an outlet that is gated or valved by a valve. In this
example, blind filling of a gas filled channel or chamber occurs
when the outlet valve is closed while filling the channel or
chamber through the inlet. If the inlet also has a valve, that
valve can then be closed after the blind fill is complete, and the
outlet can then be opened to expose the channel or chamber contents
to another channel or chamber. If a third inlet is in communication
with the channel or chamber, that third inlet can introduce another
fluid, gas or liquid, into the channel or chamber to expel the
blind-filled liquid to be expelled from the channel or chamber in a
measured amount. The result is similar to a sample loop system of
an HPLC.
Further Aspects of Retention Mechanisms are Described in Sections V
and XIII
Treatment Mechanisms
[0217] Overview
[0218] Treatment mechanisms generally comprise any suitable
mechanisms for exposing a particle(s) to a reagent(s) and/or a
physical condition(s), including fluid-mediated and
non-fluid-mediated mechanisms.
[0219] Reagents
[0220] Particles may be exposed to reagents. A reagent generally
comprises any chemical substance(s), compound(s), ion(s),
polymer(s), material(s), complex(es), mixture(s), aggregate(s),
and/or biological particle(s), among others, that contacts a
particle or particle population in a microfluidic system. Reagents
may play a role in particle analysis, including operating as
chemical/biological modulators (interaction reagents),
detection/assay reagents, solvents, buffers, media, washing
solutions, and/or so on.
[0221] Chemical modulators or biological modulators may include any
reagent that is being tested for interaction with particles.
Interaction generally includes specific binding to particles and/or
any detectable genotypic and/or phenotypic effect on particles (or
modulators). Further aspects of interactions and
genotypic/phenotypic effects that may be suitable are described
below in Section XII.
[0222] Chemical modulators may include ligands that interact with
receptors (e.g., antagonists, agonists, hormones, etc.). Ligands
may be small compounds, peptides, proteins, carbohydrates, lipids,
etc. Further aspects of ligands and receptors, and their use in
measuring interaction, or effects on signal transduction pathways,
are described below in Section XII.
[0223] Alternatively, or in addition, chemical modulators may be
nucleic acids. The nucleic acids may be DNA, RNA, peptide nucleic
acids, modified nucleic acids, and/or mixtures thereof, and may be
single, double, and/or triple-stranded. The nucleic acids may be
produced by chemical synthesis, enzymatic synthesis, and/or
biosynthesis, and may be plasmids, fragments, sense/antisense
expression vectors, reporter genes, vectors for genomic
integration/modification (such as targeting nucleic acids/vectors
(for knockout/-down/-in)), viral vectors, antisense
oligonucleotides, dsRNA, siRNA, nucleozymes, and/or the like.
Nucleic acid reagents may also include transfection reagents to
promote uptake of the nucleic acids by cells, such as lipid
reagents (e.g., lipofectamine), precipitate-forming agents (such as
calcium phosphate), DMSO, polyethylene glycol, viral coats that
package the nucleic acids, and/or so on.
[0224] Modulators may be miscellaneous chemical materials and/or
biological entities. Miscellaneous chemical modulators may be ions
(such as calcium, sodium, potassium, lithium, hydrogen (pH),
chloride, fluoride, iodide, etc.), dissolved gases (NO, CO.sub.2,
O.sub.2, etc.), carbohydrates, lipids, organics, polymers, etc. In
some embodiments, biological modulators may be exposed to cells,
for example, to infect cells, to measure cell-cell interactions,
etc. Biological modulators may include any cells, viruses, or
organelles, as described above in Section III.
[0225] Reagents may be detection/assay reagents. Detection/assay
reagents generally comprise any reagents that are contacted with
particles to facilitate processing particles (or particle
components) for detection of a preexisting or newly created aspect
of the particles (or components). Detection/assay reagents may
include dyes, enzymes, substrates, cofactors, and/or SBP members
(see Table 1 of Section VI above), among others. Dyes, also
referred to as labels, generally include any optically detectable
reagent. Suitable dyes may be luminophores, fluorophores,
chromogens, chromophores, and/or the like. Such dyes may be
conjugated to, or may be, SBP members; may act as enzyme
substrates; may inherently label cells or cell structures (e.g.,
DNA dyes, membrane dyes, trafficking dyes, etc.); may act as
indicator dyes (such as calcium indicators, pH indicators, etc.);
and/or the like. Enzymes may operate in particle assays by
incorporating dyes into products and/or by producing a product that
may be detected subsequently with dyes, among others. Suitable
enzymes may include polymerases (RNA and/or DNA), heat-stable
polymerases (such as Taq, VENT, etc.), peroxidases (such as HRP),
phosphatases (such as alkaline phosphatase), kinases, methylases,
ligases, proteases, galactosidases (such as beta-galactosidase,
glucuronidase., etc.), transferases (such as chloramphenicol
acetyltransferase), oxidoreductases (such as luciferase), and/or
nucleases (such as DNAses, RNAses, etc.), among others. SBP
members, such as antibodies, digoxigenin, nucleic acids, etc., may
be directly conjugated to dyes, enzymes, and/or other SBP members;
may be noncovalently bound to dyes and/or enzymes (either pre-bound
or bound in an additional exposure step); and/or so on. Further
aspects of detection/assay reagents, including the types of assays
in which these reagents may be used, are described below in Section
XII.
[0226] Fluid-mediated Mechanisms
[0227] Treatment mechanisms may use fluid-mediated mechanisms to
expose particles to reagents. The reagents may be brought to the
particles, for example, when the particles are retained, or the
particles may be brought to the reagents, for example, when the
reagents are present (and optionally retained) in specific portions
of fluid networks.
[0228] Fluid-mediated mechanisms may be flow-based, field-based,
and/or passive, among others. Flow-based treatment mechanisms may
operate by fluid flow, mediated, for example, by gravity flow or
active flow (pumping), to carry reagents to particles, or vice
versa. In some embodiments, the flow-based treatment mechanisms may
operate by regulated transverse (side-to-side) positioning, as
described above/below in Sections V and XIII, to precisely regulate
exposure of reagents (or particles) to particles (or reagents). By
contrast, field-based mechanisms may combine particles and reagents
by moving reagents (or particles) with electric fields. The
electric fields may produce any suitable electrokinetic effects,
such as electrophoresis, dielectrophoresis, electroosmosis, etc.
Alternatively, or in addition, reagents may be combined with
particles by diffusion of the reagents.
[0229] Non-flow-mediated Mechanisms
[0230] Particles in microfluidic systems may be exposed to physical
modulators/conditions using non-fluid-mediated mechanisms. However,
these "non-fluid-mediated" mechanisms may use properties of fluid
to assist in their operation, such as transfer of thermal energy or
pressure to particles via fluid. The physical modulators/conditions
may be applied to particles from sources that are external and/or
internal to the microfluidic systems. Exemplary physical
modulators/conditions may include thermal energy (heat), radiation
(light), radiation (particle), an electric field, a magnetic field,
pressure (including sound), a gravitational field, etc.
[0231] Treatment Targets
[0232] Treatment mechanisms may act on any suitable particles,
including any of the particles described above in Section III. The
particles may be intact, permeabilized, and/or lysed. Accordingly,
treatment mechanisms may act on released cell components. Particles
may be treated in arrays, either serially, for example, using a
shared treatment mechanism, and/or in parallel, for example, using
distinct and/or shared treatment mechanisms.
[0233] Further aspects of treatment mechanisms are described above
in Section V (positioning reagents/fluid/particles) and below in
Section XIII.
Measurement Mechanisms
[0234] Overview
[0235] Particles manipulated by a microfluidic system may be
analyzed by one or more measurement mechanisms at one or more
measurement sites. The measurement mechanisms generally comprise
any suitable apparatus or method for detecting a preselected
particle or particle characteristic (provided, for example, by the
particle, a particle component, and/or an assay product, among
others). The measurement sites generally comprise any suitable
particle position or positions at which a measurement is performed,
internal and/or external to the system.
[0236] Detection Methods
[0237] The measurement mechanism may employ any suitable detection
method to analyze a sample, qualitatively and/or quantitatively.
Suitable detection methods may include spectroscopic methods,
electrical methods, hydrodynamic methods, imaging methods, and/or
biological methods, among others, especially those adapted or
adaptable to the analysis of particles. These methods may involve
detection of single or multiple values, time-dependent or
time-independent (e.g., steady-state or endpoint) values, and/or
averaged or (temporally and/or spatially) distributed values, among
others. These methods may measure and/or output analog and/or
digital values.
[0238] Spectroscopic methods generally may include detection of any
property of light (or a wavelike particle), particularly properties
that are changed via interaction with a sample. Suitable
spectroscopic methods may include absorption, luminescence
(including photoluminescence, chemiluminescence, and
electrochemiluminescence), magnetic resonance (including nuclear
and electron spin resonance), scattering (including light
scattering, electron scattering, and neutron scattering),
diffraction, circular dichroism, and optical rotation, among
others. Suitable photoluminescence methods may include fluorescence
intensity (FLINT), fluorescence polarization (FP), fluorescence
resonance energy transfer (FRET), fluorescence lifetime (FLT),
total internal reflection fluorescence (TIRF), fluorescence
correlation spectroscopy (FCS), fluorescence recovery after
photobleaching (FRAP), fluorescence activated cell sorting (FACS),
and their phosphorescence and other analogs, among others.
[0239] Electrical methods generally may include detection of any
electrical parameter. Suitable electrical parameters may include
current, voltage, resistance, capacitance, and/or power, among
others.
[0240] Hydrodynamic methods generally may include detection of
interactions between a particle (or a component or derivative
thereof) and its neighbors (e.g., other particles), the solvent
(including any matrix), and/or the microfluidic system, among
others, and may be used to characterize molecular size and/or
shape, or to separate a sample into its components. Suitable
hydrodynamic methods may include chromatography, sedimentation,
viscometry, and electrophoresis, among others.
[0241] Imaging methods generally may include detection of spatially
distributed signals, typically for visualizing a sample or its
components, including optical microscopy and electron microscopy,
among others.
[0242] Biological methods generally may include detection of some
biological activity that is conducted, mediated, and/or influenced
by the particle, typically using another method, as described
above. Suitable biological methods are described below in detail in
Section XII.
[0243] Detection Sites
[0244] The measurement mechanism may be used to detect particles
and/or particle characteristics at any suitable detection site,
internal and/or external to the microfluidic system.
[0245] Suitable internal detection sites may include any site(s) in
or on a microfluidic system (a chip). These sites may include
channels, chambers, and/or traps, and portions thereof. Particles
or particle characteristics may be detected while the particles (or
released components/assay products) are stationary or moving.
Stationary particles may be encountered following particle
retention, for example, cells growing in a cell chamber. Moving
particles may be encountered before and/or after particle
retention, or upon confinement to a region. In particular,
particles may be moved past a detection site by any suitable
positioning mechanism, for example, by fluid flow (flow-based
detection).
[0246] Suitable external detection sites may include any site(s)
away from or independent of a microfluidic system. External
detection sites may be used to detect a particle or particle
characteristic after removal of particles (or particle components)
from a microfluidic system. These external sites may be used
instead of and/or in addition to internal sites, allowing particles
(or particle components) to be further manipulated and/or detected.
These further manipulations and/or detection methods may overlap
with, but preferably complement, the manipulations and/or methods
performed in the microfluidic system, including mass spectrometry,
electrophoresis, centrifugation, PCR, introduction into an
organism, use in clinical treatment, and/or cell culture, among
others.
[0247] Detected Characteristics
[0248] The measurement method may detect and/or monitor any
suitable characteristic of a particle, directly and/or indirectly
(e.g., via a reporter molecule). Suitable characteristics may
include particle identity, number, concentration, position
(absolute or relative), composition, structure, sequence, and/or
activity among others. The detected characteristics may include
molecular or supramolecular characteristics, such as the
presence/absence, concentration, localization,
structure/modification, conformation, morphology, activity, number,
and/or movement of DNA, RNA, protein, enzyme, lipid, carbohydrate,
ions, metabolites, organelles, added reagent (binding), and/or
complexes thereof, among others. The detected characteristics also
may include cellular characteristics, such as any suitable cellular
genotype or phenotype, including morphology, growth, apoptosis,
necrosis, lysis, alive/dead, position in the cell cycle, activity
of a signaling pathway, differentiation, transcriptional activity,
substrate attachment, cell-cell interaction, translational
activity, replication activity, transformation, heat shock
response, motility, spreading, membrane integrity, and/or neurite
outgrowth, among others.
[0249] Further aspects of detected characteristics and their use in
particle assays are described below in Sections XII and XIII.
Release Mechanisms
[0250] Overview
[0251] A microfluidic system may include any suitable number of
particle release mechanisms. A release mechanism generally
comprises any mechanism(s) for allowing a retained particle to move
away from a preselected site/area at which it is retained,
including removing, overcoming, and/or rendering ineffective the
retention mechanism(s) that retains the particle. Release
mechanisms that are suitable may be selected based, at least
partially, on the retaining force. After release, particles (or
particle components) may have any suitable destination.
[0252] Removing the Retaining Force
[0253] A release mechanism may operate by removing the retaining
force. Accordingly, particles that are retained by a specific
mechanism may be released by terminating that mechanism. For
example, particles retained by a chemical interaction/bond may be
released by cleaving the bond, such as with a protease(s) (e.g.,
trypsin), or otherwise disrupting the interaction, such as with
altered ionic conditions (e.g., with EDTA) or pH, or with an excess
of a SBP member. Similarly, particles retained by a physical
barrier, such as a closed valve, may be released by moving/removing
the barrier. Furthermore, particles retained by fluid flow, a
vacuum, light, an electrical field, a magnetic field, and/or a
centrifugal force may be released by removing/redirecting the
corresponding flow, force, field, etc.
[0254] Overcoming the Retaining Force
[0255] A release mechanism may operate by overcoming a retaining
force with a greater force. Accordingly, particles may be released
by any positioning mechanism(s) that applies a force greater than
the retaining force. For example, retained particles may be
released by a releasing flow. The releasing flow may be an
increased flow rate in the direction of bulk fluid flow, for
example, when a particle is weakly retained (such as by
gravity/friction, or weak chemical interactions). Alternatively,
the releasing flow may act counter to a retaining flow, for example
orthogonal or opposite to the retaining flow. For example, the
releasing flow may reposition particles to be out of contact with a
retaining physical barrier (see Example 7). Alternatively, or in
addition, retained particles may be released by any other suitable
positioning mechanism(s), as described above in Section V, that is
capable of generating sufficient force.
[0256] Rendering Ineffective the Retaining Force
[0257] A release mechanism may operate by rendering ineffective the
retaining force on a particle. Such a release mechanism may operate
by releasing components of the particle. For example, retained
cells may be lysed to release intracellular components, producing a
lysate, or beads may be treated to release associated materials
and/or to fragment/disintegrate the beads. Lysis generally includes
any partial or complete disruption of the integrity of a
cell-surface membrane, and may be produced via temperature, a
detergent, a ligand, chemical treatment, a change in ionic
strength, an electric field, etc.
[0258] Destination of Released Particles/Components
[0259] Released particles and/or particle components may have any
suitable destination(s). Suitable immediate destinations may
include a positioning mechanism and/or fluid surrounding the
particles. After release, particles may be repositioned with a
positioning mechanism, either nonselectively or selectively.
Selective positioning may position the particle based on a measured
characteristic. Positioning may be to a second retention mechanism
(and/or a culture chamber), to a detection mechanism (such as a
flow-based mechanism), and/or to an output mechanism. Fluid
surrounding the particles may be a suitable destination for
particle components (such as cells lysates and/or bead components)
to be contacted with detection/assay reagents. Alternatively, cell
lysates and/or bead components may be repositioned as with intact
particles.
[0260] Further aspects of release mechanisms and destinations of
released particles/components are described below in Section
XIII.
[0261] Output Mechanisms
[0262] Microfluidic systems may include one or more output
mechanisms that interface with the microfluidic network(s). An
output mechanism generally comprises any suitable mechanism for
outputting material(s) (e.g., fluid, particles, and/or reagents)
from a microfluidic system, or portions thereof, including
selective and/or bulk mechanisms. The output mechanism may direct
outputted material to any suitable location, such as an internal
and/or external sink. A sink generally comprises any receptacle or
other site for receiving outputted materials, for disposal (e.g., a
waste site) or for further study or manipulation (e.g., a
collection site). The outputting of materials from the
microfluidics system may be facilitated and/or regulated using any
suitable facilitating mechanism, such as sources of internal
pressure and/or external vacuum. The output mechanism may include a
selection mechanism, such as a filter, that selects outputted
materials based on some criterion, such as whether the material is
a particle or a fluid.
Cell Culture Mechanisms
[0263] Overview
[0264] Cells may be cultured using a cell culture mechanism in
microfluidic systems. The cell culture mechanism generally
comprises any suitable mechanism for growing cells, including
maintenance and/or propagation. Suitable cells are described above
in Section III.
[0265] Structural Matters
[0266] A cell culture mechanism of a microfluidic system may
include one or more culture chambers in which to culture cells.
Culture chambers may have any suitable size, shape, composition,
and/or relationship to other aspects of microfluidic systems, based
on the number of cells to be cultured, size of cells, assays to
performed on the cells, and/or growth characteristics of the cells,
among others. The size of a culture chamber may be only large
enough to hold one cell, several cells or more (2 to 50), or many
cells (50 to 1000 or more) of a given cell size. Accordingly,
culture chambers may be defined by a selected portion of a passage,
an entire passage, or a set of passages. In some embodiments,
culture chambers may be formed by substantially enlarged channels.
Culture chambers may have any suitable height that allows cells of
interest to enter the chamber. This height may be greater than,
less than, and/or equal to other portions of the microfluidic
network. Some or all of the surfaces of a culture chamber, such as
the walls, roof, and/or substrate, may be treated or modified to
facilitate aspects of cell culture, particularly specific or
nonspecific cell attachment, cell survival, cell growth, and/or
cell differentiation (or lack thereof), among others. Suitable
methods of passage treatment and treatment agents are described
above in Section VI, relative to chemical retention mechanisms.
[0267] Culture Conditions
[0268] The cell culture mechanism may culture cells under any
suitable environmental conditions using any appropriate
environmental control mechanisms. Suitable environmental conditions
may include a desired gas composition, temperature, rate and
frequency of media exchange, and/or the like. Environmental control
mechanisms may operate internal and/or external to a microfluidic
system. Internal mechanisms may include on-board heaters, gas
conduits, and/or media reservoirs. External mechanisms may include
an atmosphere- and/or temperature-controlled incubator/heat source,
and/or a media source external to the system. An
atmosphere-controlled incubator may be more suitable when the
system is at least partially formed of a gas-permeable material,
such as PDMS. Media, including gas-conditioned media, may be
introduced from an external source by any suitable input mechanism,
including manual pipetting, automated pipetting, noncontact
spitting, etc. In some embodiments, the chip may be preincubated
with media, which may then be discarded, prior to the introduction
of cells and/or other biological materials.
[0269] Further aspects of cell culture mechanisms, culture
chambers, and culture conditions are described below in Example 10,
and the materials listed in Cross-References, particularly R. Ian
Freshney, Culture of Animal Cells: A Manual of Basic Technique
(4.sup.th ed. 2000), which is incorporated herein by reference.
Particle-Based Manipulations
[0270] Overview
[0271] Microfluidic systems are used for particle manipulations.
Particle manipulations generally comprise any suitable sequence of
unitary operations, for performing a desired function or assay.
Unitary operations may be performed by each of the mechanisms
described above in Sections IV to X, among others.
[0272] Exemplary Sequences of Operations
[0273] FIG. 1 shows an exemplary method 100 for microfluidic
manipulation and analysis of particles with systems of the
invention. Each step of method 100 may be repeated any suitable
number of times and in any appropriate order, as described below,
based on the application. Exemplary sequences of steps are
indicated by arrows.
[0274] Particles typically are initially inputted in an input step,
shown at 101. Particle input introduces particles to a microfluidic
system and may be mediated by any of the input mechanisms described
above in Section IV.
[0275] Particles next are typically positioned, shown at 102.
Positioning moves particles to selected positions along passages
(longitudinal positioning), and/or to selected positions along one
or more axes generally orthogonal to the long axis (transverse
positioning). Suitable positioning mechanisms that mediate one or
both of these particle movements are described above in Section
V.
[0276] Particle positioning may lead to one of two paths, shown at
103 and 104. Path 103 leads to particle output, shown at 105.
Particle output may be mediated by one of the output mechanisms
described above in Section X, and may be used to discard, collect,
and/or transfer particles for further analysis, among others. Path
104 leads to one or more of three operations, particle retention
106, particle treatment 107, and/or particle measurement/detection
108. These operations may be conducted in any suitable order, for
any desired number of times. Particle retention mechanisms,
treatment mechanisms, and measurement mechanisms are described
above in Sections VI, VII, and VIII, respectively.
[0277] The steps of treating and/or measuring particles may be
carried out with or without particle retention. Accordingly, the
steps of treating and/or measuring particles may be followed
directly by additional positioning 102, or first may use a release
step, shown at 109, if particles have been retained. Suitable
release mechanisms are described above in Section IX.
Alternatively, microfluidic systems may be discarded before
particle release, additional positioning, and/or output.
[0278] Particles that have returned to the positioning step after
entering path 104 may be manipulated further. Some or all of these
particles may be repositioned to path 103 to be outputted 105.
Alternatively, or in addition, some or all of these particles may
be directed back to path 104 to be further treated, retained,
and/or measured. Therefore, method 100 enables any suitable
sequence of particle manipulations and analyses at one or plural
positions within a microfluidic system.
[0279] Exemplary sequences of operations may be illustrated further
as follows. For the following discussion, the operations performed
by the steps of method 100 are abbreviated with the following
single underlined letters: Input, Position, Retain, Treat, Measure,
rElease, and Output.
[0280] A basic manipulation of microfluidic analyses is IP. This
sequence of steps may lead to output (IPO) or to (path 104),
resulting in the basic retention sequence IPR, flow-based
measurement, IPM, or flow-based treatment, IPT.
[0281] Retained particles may be subjected to any suitable
additional steps. The particles may be treated (IPRT), measured
(IPRM), repeatedly measured over time (IPRMMM . . . ), treated and
then measured (IPRTM), or repeatedly treated and measured
(IPRTMTMTM . . . ). Retained particles may be released (IPR . . .
E) after optional treatment and/or measurement. Released particles
may be repositioned and then outputted (IPR . . . EPO); measured
during flow (IPR . . . EPM); treated (IPR . . . EPT); treated and
measured (IPR . . . EPTM); retained and treated (IPR . . . EPRT);
retained, treated, and measured, (IPR . . . EOPRTM); and/or so
on.
[0282] Cell-based Assays/Methods
[0283] The microfluidic systems of the invention may be used for
any suitable cell assays or methods, including any combinations of
cells, cell selection(s) (by selective retention), treatment(s),
and/or measurement(s), as described above in Sections III, VI, VII,
and VIII, respectively.
[0284] The cell assays may characterize cells, either with or
without addition of a modulator. Cell assays may measure cell
genotypes, phenotypes, and/or interactions with modulators. These
assays may characterize individual cells and/or cell
populations/groups of any suitable size. Cells may be characterized
in the absence of an added modulator to define one or more
characteristics of the cells themselves. Alternatively, or in
addition, cell may be characterized in the presence of an added
modulator to measure interaction(s) between the cells and the
modulator. Moreover, cells may be exposed to a selected
concentration of a reagent, or a plurality of concentrations of a
reagent. In other embodiments, cells are exposed to a gradient of
concentrations of reagent to determine whether such cells will be
attracted or repelled by increasing amounts of such reagent.
[0285] In other embodiments, a quantity of cells may be measured
out by first filling a measuring chamber having at least one inlet,
the inlet having at least one valve, where the valve is opened,
cells are introduced into the chamber, preferably by blind filling
a dead-end chamber, or by opening up an outlet valve to an outlet
in communication with the chamber, the outlet having a retention
mechanism for preventing the cells from exiting the chamber. The
measure amount of cells is then displaced to a culturing region for
culturing.
[0286] In other embodiments, a first type of cell is grown in fluid
communication with a second type of cell, wherein the first type of
cell is affected by the presence of the second type of cell,
preferably as a co-culture or feeder type relationship. The cells
of the first type and the cells of the second type are kept
separate from each other by a retention mechanism, although fluid,
preferably liquid, is permitted to be in joint contact with each
type of cell so that sub-cellular or biochemical materials may be
exchanged between cell types.
[0287] Genotypic Assays
[0288] Genotypic assays may be conducted on cells in microfluidic
systems to measure the genetic constitution of cells. The genotypic
assays may be conducted on any suitable cell or cell populations,
for example, patient samples, prenatal samples (such as embryonic,
fetal, chorionic villi, etc.), experimentally manipulated cells
(such as transgenic cells), and/or so on. Such genotypic aspects
may include copy number (such as duplication, deletion,
amplification, and/or the like) and/or structure (such as
rearrangement, fusion, number of repeats (such as dinucleotide,
triplet repeats, telomeric repeats, etc.), mutation,
gene/pseudogene, specific allele,
presence/absence/identity/frequency of single nucleotide
polymorphisms, integration site, chromosomal/episomal, and/or the
like) of a nuclear and/or mitochondrial gene(s), genomic region(s),
and/or chromosomal region (s) (such as telomeres, centromeres,
repetitive sequences, etc.). Methods for genotypic assays may
include nucleic acid hybridization in situ (on intact cells/nuclei)
or with DNA released from cells, for example, by lysing the cells.
Nucleic acid hybridization with nucleic acids may be carried out
with a dye-labeled probe, a probe labeled with a specific binding
pair (see Section VI), a stem-loop probe carrying an energy
transfer pair (such as a "molecular beacon"), and/or with a probe
that is labeled enzymatically after hybridization (such as by
primer extension with a polymerase, modification with terminal
transferase, etc). Alternatively, or in addition, methods for
genotypic assays may include polymerase-mediated amplification of
nucleic acids, for example, by thermal cycling (PCR) or by
isothermal strand-displacement methods. In some embodiments,
genotypic assays may use electrophoresis to assist in analysis of
nucleic acids. Related gene-based assays may measure other aspects
of gene regions, genes, chromosomal regions, whole chromosomes, or
genomes, using similar assay methods, and suitable probes or DNA
dyes (such as propidium iodide, Hoechst, etc.). These other aspects
may include total DNA content (for example 2N, 4N, 8N, etc., to
measure diploid, tetraploid, or polyploid genotypes and/or cell
cycle distribution), number or position of specific chromosomes,
and/or position of specific genes (such as adjacent the nuclear
membrane, another nuclear structure, and so on).
[0289] Phenotypic Assays
[0290] Phenotypic assays may be conducted to characterize cells in
microfluidic systems, based on genetic makeup and/or environmental
influences, such as presence of modulators. These assays may
measure any molecular or cellular aspect of whole cells, cellular
organelles, and/or endogenous (native) or exogenous (foreign) cell
constituents/components.
[0291] Aspects of a whole cell or whole cell population may include
number, size, density, shape, differentiation state, spreading,
motility, translational activity, transcriptional activity, mitotic
activity, replicational activity, transformation, status of one or
more signaling pathways, presence/absence of processes,
intact/lysed, live/dead, frequency/extent of apoptosis or necrosis,
presence/absence/efficiency of attachment to a substrate (or to a
passage), growth rate, cell cycle distribution, ability to repair
DNA, response to heat shock, nature and/or frequency of cell-cell
contacts, etc.
[0292] Aspects of cell organelles may include number, size, shape,
distribution, activity, etc. of a cell's (or cell population's)
nuclei, cell-surface membrane, lysosomes, mitochondria, Golgi
apparatus, endoplasmic reticulum, peroxisomes, nuclear membrane,
endosomes, secretory granules, cytoskeleton, axons, and/or
neurites, among others.
[0293] Aspects of cell constituents/components may include
presence/absence or level, localization, movement, activity,
modification, structure, etc. of any nucleic acid(s),
polypeptide(s), carbohydrate(s), lipid(s), ion(s), small molecule,
hormone, metabolite, and/or a complex(es) thereof, among others.
Presence/absence or level may be measured relative to other cells
or cell populations, for example, with and without modulator.
Localization may be relative to the whole cell or individual cell
organelles or components. For example, localization may be
cytoplasmic, nuclear, membrane-associated, cell-surface-associated,
extracellular, mitochondrial, endosomal, lysosomal, peroxisomal,
and/or so on. Exemplary cytoplasmic/nuclear localization may
include transcription factors that translocate between these two
locations, such as NF-.kappa.B, NFAT, steroid receptors, nuclear
hormone receptors, and/or STATs, among others. Movement may include
intracellular trafficking, such as protein targeting to specific
organelles, endocytosis, exocytosis, recycling, etc. Exemplary
movements may include endocytosis of cell-surface receptors or
associated proteins (such as GPCRs, receptor tyrosine kinases,
arrestin, and/or the like), either constitutively or in response to
ligand binding. Activity may include functional or optical
activity, such as enzyme activity, fluorescence, and/or the like,
for example, mediated by kinases, phosphatases, methylases,
demethylases, proteases, nucleases, lipases, reporter proteins (for
example beta-galactosidase, chloramphenicol acetyltransferase,
luciferase, glucuronidase, green fluorescent protein (and related
derivatives), etc.), and/or so on. Modification may include the
presence/absence, position, and/or level of any suitable covalently
attached moiety. Such modifications may include phosphorylation,
methylation, ubiquitination, carboxylation, and/or farnesylation,
among others. Structure may include primary structure, for example
after processing (such as cleavage or ligation), secondary
structure or tertiary structure (e.g., conformation), and/or
quaternary structure (such as association with partners in, on, or
about cells). Methods for measuring modifications and/or structure
may include specific binding agents (such as antibodies, etc.), in
vivo or in vitro incorporation of labeled reagents, energy transfer
measurements (such as FRET), surface plasmon resonance, and/or
enzyme fragment complementation or two-hydrid assays, among
others.
[0294] Nucleic acids may include genomic DNA, mitochondrial DNA,
viral DNA, bacterial DNA, phage DNA, synthetic DNA, transfected
DNA, reporter gene DNA, etc. Alternatively, or in addition, nucleic
acids may include total RNAs, hnRNAs, mRNAs, tRNAs, siRNAs, dsRNAs,
snRNAs, ribozymes, structural RNAs, viral RNAs, bacterial RNAs,
gene-specific RNAs, reporter RNAs (expressed from reporter genes),
and/or the like. Methods for assaying nucleic acids may include any
of the techniques listed above under genotypic assays. In addition,
methods for assaying nucleic acids may include ribonuclease
protection assays.
[0295] Polypeptides may include any proteins, peptides,
glycoproteins, proteolipids, etc. Exemplary polypeptides include
receptors, ligands, enzymes, transcription factors, transcription
cofactors, ribosomal components, regulatory proteins, cytoskeletal
proteins, structural proteins, channels, transporters, reporter
proteins (such as those listed above which are expressed from
reporter genes), and/or the like. Methods for measuring
polypeptides may include enzymatic assays and/or use of specific
binding members (such as antibodies, lectins, etc.), among others.
Specific binding members are described in Section VI.
[0296] Carbohydrates, lipids, ions, small molecules, and/or
hormones may include any compounds, polymers, or complexes. For
example, carbohydrates may include simple sugars, di- and
polysaccharides, glycolipids, glycoproteins, proteoglycans, etc.
Lipids may include cholesterol and/or inositol lipids (e.g.,
phosphoinositides), among others; ions may include calcium, sodium,
chloride, potassium, iron, zinc, hydrogen, magnesium, heavy metals,
and/or manganese, among other; small molecules and/or hormones may
include metabolites, and/or second messengers (such as cAMP or
cGMP, among others), and/or the like. Concentration gradients
and/or movement of ions may provide electrical measurements, for
example, by patch-clamp analysis, as described in Examples 11 and
12.
[0297] Interaction Assays
[0298] Interaction generally comprises any specific binding of a
modulator to a cell or population of cells, or any detectable
change in a cell characteristic in response to the modulator.
Specific binding is any binding that is predominantly to a given
partner(s) that is in, on, or about the cell(s). Specific binding
may have a binding coefficient with the given partner of about
10.sup.-3 M and lower, with preferred specific binding coefficients
of about 10.sup.-4 M, 10.sup.-6 M, or 10.sup.-8 M and lower.
Alternatively, interaction may be any change in a phenotypic or
genotypic characteristic, as described above, in response to the
modulator.
[0299] Interaction assays may be performed using any suitable
measurement method. For example, the modulator may be labeled, such
as with an optically detectable dye, and may be labeled secondarily
after interaction with cells. Binding of the dye to the cell or
cells thus may be quantified. Alternatively, or in addition, the
cell may be treated or otherwise processed to enable measurement of
a phenotypic characteristic produced by modulator contact, as
detailed above and in Section VIII.
[0300] Cells and/or cell populations may be screened with libraries
of modulators to identify interacting modulators and/or modulators
with desired interaction capabilities, such as a desired phenotypic
effect (such as reporter gene response, change in expression level
of a native genelprotein, electrophysiological effect, etc.) and/or
coefficient of binding. A library generally comprises a set of two
or more members (modulators) that share a common characteristic,
such as structure or function. Accordingly, a library may include
two or more small molecules, two or more nucleic acids, two or more
viruses, two or more phages, two or more different types of cells,
two or more peptides, and/or two or more proteins, among
others.
[0301] Signal Transduction Assays
[0302] Microfluidic assays of cells and/or populations may measure
activity of signal transduction pathways. The activity may be
measured relative to an arbitrary level of activity, relative to
other cells and/or populations (see below), and/or as a measure of
modulator interaction with cells (see above).
[0303] Signal transduction pathways generally comprise any flow of
information in a cell. In many cases, signal transduction pathways
transfer extracellular information, in the form of a ligand(s) or
other modulator(s), through the membrane, to produce an
intracellular signal. The extracellular information may act, at
least partially, by triggering events at or near the membrane by
binding to a cell-surface receptor, such as a G Protein-Coupled
Receptor (GPCR), a channel-coupled receptor, a receptor tyrosine
kinase, a receptor serine/threonine kinase, and/or a receptor
phosphatase, among others. These events may include changes in
channel activity, receptor clustering, receptor endocytosis,
receptor enzyme activity (e.g., kinase activity), and/or second
messenger production (e.g., cAMP, cGMP, diacylglcyerol,
phosphatidylinositol, etc.). Such events may lead to a cascade of
regulatory events, such as phosphorylation/dephosphorylation,
complex formation, degradation, and/or so on, which may result,
ultimately, in altered gene expression. In other cases, modulators
pass through the membrane and directly bind to intracellular
receptors, for example with nuclear receptors (such as steroid
receptors (GR, ER, PR, MR, etc.), retinoid receptors, retinoid X
receptor (RXRs), thyroid hormone receptors, peroxisome
proliferation-activating receptors (PPARs), and/or xenobiotic
receptors, among others). Therefore, any suitable aspect of this
flow of information may be measured to monitor a particular signal
transduction pathway.
[0304] The activity measured may be based at least partially, on
the type of signal transduction pathway being assayed. Accordingly,
signal transduction assays may measure ligand binding; receptor
internalization; changes in membrane currents; association of
receptor with another factor, such as arrestin, a small G-like
protein such as rac, or rho, and/or the like; calcium levels;
activity of a kinase, such as protein kinase A, protein kinase C,
CaM kinase, myosin light chain kinase, cyclin dependent kinases,
PI3-kinase, etc.; cAMP levels; phosholipase C activity; subcellular
distribution of proteins, for example, NF-.kappa.B, nuclear
receptors, and/or STATs, among others. Alternatively, or in
addition, signal transduction assays may measure expression of
native target genes and/or foreign reporter genes that report
activity of a signal transduction pathway(s). Expression may be
measured as absence/presence or level of RNA, protein, metabolite,
or enzyme activity, among others, as described above.
[0305] Comparison of Cells and/or Cell Populations
[0306] Cell-based assays may be used to compare genotypic,
phenotypic, and/or modulator interaction of cells and/or
populations of cells. The cells and/or populations may be compared
in distinct microfluidic systems or within the same microfluidic
system. Comparison in the same microfluidic system may be conducted
in parallel using a side-by-side configuration, as exemplified by
Example 3, in parallel at isolated sites, as exemplified by Example
4, and/or in series, as exemplified by Example 5.
[0307] Single-Cell Assays
[0308] Microfluidic systems may be used to perform single-cell
assays, which generally comprise any assays that are preferably or
necessarily performed on one cell at a time. Examples of single
cell assays include patch-clamp analysis, single-cell PCR,
single-cell fluorescence in situ hybridization (FISH), subcellular
distribution of a protein, and/or differentiation assays
(conversion to distinct cell types). In some cases, single-cell
assays may be performed on a retained group of two or more cells,
by measuring an individual characteristic of one member of the
group. In other cases, single-cell assays may require retention of
a single cell, for example, when the cell is lysed before the
assay.
[0309] Sorting/Selection
[0310] Microfluidic systems may be used to sort or select single
cells and/or cell populations. The sorted/selected cells or
populations may be selected by stochastic mechanisms (see Example
2), size, density, magnetic properties, cell-surface properties
(that is, ability to adhere to a substrate), growth and/or survival
capabilities, and/or based on a measured characteristic of the
cells or populations (such as response to a ligand, specific
phenotype, and/or the like). Cells and/or populations may be sorted
more than once during manipulation and/or analysis in a
microfluidic system. In particular, heterogeneous populations of
cells, such as blood samples or clinical biopsies, partially
transfected or differentiated cell populations, disaggregated
tissues, natural samples, forensic samples, etc. may be
sorted/selected. Additional aspects of cell sorting and suitable
cells and cell populations are described above in Section III and
below in Examples 9, 15, 23, and 26.
[0311] Storage/Maintenance
[0312] Microfluidic systems may perform storage and/or maintenance
functions for cells. Accordingly, cells may be introduced into
microfluidic systems and cultured for prolonged periods of time,
such as longer than one week, one month, three months, and/or one
year. Using microfluidic systems for storage and/or maintenance of
cells may consume smaller amounts of media and space, and may
maintain cells in a more viable state than other
storage/maintenance methods. Additional aspects of storing and
maintaining cells in microfluidic systems are included in Section
XI above and Example 10 below.
[0313] Assays/Methods with Other Particles
[0314] Microfluidic systems may be used for any suitable virally
based, organelle-based, bead-based, and/or vesicle-based assays
and/or methods. These assays may measure binding (or effects) of
modulators (compounds, mixtures, polymers, biomolecules, cells,
etc.) to one or more materials (compounds, polymers, mixtures,
cells, etc.) present in/on, or associated with, any of these other
particles. Alternatively, or in addition, these assays may measure
changes in activity (e.g., enzyme activity), an optical property
(e.g., chemiluminescence, fluorescence, or absorbance, among
others), and/or a conformational change induced by interaction.
[0315] In some embodiments, beads may include detectable codes.
Such codes may be imparted by one or more materials having
detectable properties, such as optical properties (e.g., spectrum,
intensity, and or degree of fluorescence excitation/emission,
absorbance, reflectance, refractive index, etc.). The one or more
materials may provide nonspatial information or may have discrete
spatial positions that contribute to coding aspects of each code.
The codes may allow distinct samples, such as cells, compounds,
proteins, and/or the like, to be associated with beads having
distinct codes. The distinct samples may then be combined, assayed
together, and identified by reading the code on each bead. Suitable
assays for cell-associated beads may include any of the cell assays
described above.
[0316] Suitable protocols for performing some of the assays
described in this section are included in Joe Sambrook and David
Russell, Molecular Cloning: A Laboratory Manual (3.sup.rd ed.
2000), which is incorporated herein by reference.
EXAMPLES
[0317] The following examples describe selected aspects and
embodiments of the invention, including methods of fabricating,
integrating, and using microfluidic systems, and devices, and
mechanisms for manipulation and analysis of particles. These
examples are included for illustration and are not intended to
limit or define the entire scope of the invention.
[0318] Many of the examples presented below include figures showing
molds, fluid layers, and/or control layers that are color-coded.
Since molds and fluid or control layers have complementary
patterns, the color-coded schemes generally represent both molds
and fluid or control layers, although one or the other is often
designated in the corresponding description. Throughout these
examples, the colors of molds and/or fluidic layers have the
following meanings: regions in red have a height of approximately
20 .mu.m, and a rectangular cross-sectional geometry; regions in
blue have a height of about 20 .mu.m, and a semi-circular/arcuate
cross-sectional geometry; regions in turquoise have a height of
about 5 .mu.m and a rectangular cross-sectional geometry; and
regions in white are not raised from the general surface of the
mold and/or form a portion of the substrate-contacting surface of a
fluid layer. The widths of these regions are generally cited in the
text.
[0319] Dimensions and cross-sectional geometries presented in these
examples are exemplary only, being designed for particles of about
8 to 12 82 m in diameter. Accordingly, any absolute or relative
dimensions or cross-sectional geometries may be selected based the
application and the size of input particles being analyzed. Thus,
the regions in red and blue may have a height of about 0.5 to 100,
1 to 75, or 2 to 50 .mu.m. Regions in turquoise may have a height
of about 0.1 to 50, 0.2 to 25, or about 0.5 to 20 .mu.m. In
addition, these regions may have any suitable cross-sectional
geometries based on the application. Furthermore, regions in red
and blue may have any suitable width based on their function. For
example, regions in red used for particle positioning may have
widths of at least about 2, 10, 20, or 50 .mu.m. By contrast,
regions in red used for reagent dispensing may have smaller widths
of at least about 0.2, 1, 2, or 5 .mu.m. Regions in blue may have
widths of at least about 5, 10, 20, or 50 .mu.m.
Example 1
Cell Positioning and Retention Mechanisms
[0320] This example describes microfluidic systems for positioning
and/or retaining single particles or groups of particles, based, at
least in part, on divergent flow paths; see FIGS. 2-4.
[0321] Background
[0322] There are many cell analyses that benefit from or require
the precise positioning and retention of a single cell or a small
group of cells. In particular, positioned and retained cells may be
treated and observed in real time. However, currently available
mechanisms for positioning and retaining cells are either expensive
and labor intensive, or imprecise and deleterious to cells. For
example, micromanipulators enable a user to select and precisely
position a single cell. However, micromanipulators are expensive,
and require that users observe the cell throughout the
micromanipulation. Hence, the user can only position one cell at a
time. At the other extreme, filters offer a crude, but much cheaper
and faster mechanism for positioning and retaining cells. However,
filters have a number of disadvantages. For example, they are easy
to clog, difficult to control (particularly with regard to the
number of retained cells), and potentially harmful to particles
such as cells due to the pressure drop across the filter.
Therefore, there is a need for cell positioning and retention
systems that are economical, guided automatically without optical
monitoring, and/or able to gently manipulate cells without
substantially damaging them.
[0323] Description
[0324] This example describes mechanisms for positioning and/or
retaining particles such as cells and/or beads without requiring
optical monitoring. Once retained, the particles may be analyzed by
any suitable method, including optical and electrical methods,
among others. The described mechanisms use a microfluidic flow path
that diverges to form a quasi-stagnant fluidic region at the
position of divergence. Particles entering this quasi-stagnant
fluidic region from a microfluidic stream experience a reduction in
velocity, which may be exploited to effect their "soft landing" in
a suitable retention structure or trap. Accordingly, the retained
particles are more likely to be undamaged and suitable for
subsequent analyses.
[0325] Embodiment 1
[0326] FIG. 2A shows a system 10 for microfluidic manipulation
and/or analysis of particles, in accordance with aspects of the
invention. System 110 includes (1) an input reservoir 112, (2) a
microfluidic network 114 having three fluidic channels 116, 118,
120, and (3) two output or waste reservoirs 122, 124. Particles are
loaded, generally in suspension, into input reservoir 112. The
loaded particles may enter network 114 in response to net fluid
flow, shown as flow streams 126, 128, 130, between the input and
waste reservoirs. The net fluid flow may be determined by active
and/or passive flow, mediated, for example, by pumping and/or
gravity, respectively.
[0327] The bifurcation of fluid flow stream 126 into flow streams
128, 130 creates a positioning mechanism 132. This positioning
mechanism uses a reduced-velocity flow stream 134, shown as a
dotted arrow, to gently position a fraction of particles through an
extension of flow stream 126.
[0328] Particles may be carried by flow stream 134 into a suitable
retention mechanism 136. In system 110, this retention mechanism
includes a recess 138 formed in opposing wall 140, near a terminal
end of reduced-velocity flow stream 134. Recess 138 may have a
width and depth that accommodates one particle or a group of two or
more particles. Recess 138 includes retention structures 142 that
block movement of retained particles, generally in the direction of
flow streams 128, 130. The depth of recess 138, coupled with any
extension of retention structures 142, generally away from wall
140, may determine the number of particles retained and their
associated retention efficiency. Thus, retention mechanism 136 may
effect stable or transient retention of particles. Transient
retention may provide an average time of occupancy that is suitable
for treatment and/or analysis, followed by stochastic loss and
replacement of a particle or particles by other particles entering
along reduced-velocity flow stream 134.
[0329] Particles retained by retention mechanism 136 may be treated
and/or analyzed. In some embodiments, retained particles are
analyzed electrically, for example, using an electrode 143.
Alternatively, or in addition, retained particles may be treated
and/or analyzed and then removed by a suitable release mechanism
144. For example, in system 110, the release mechanism applies a
dislodging pressure on retained cells that opposes flow stream 134.
Release mechanisms are described further in Section IX above and in
Examples 7 and 26 below.
[0330] Embodiment 2
[0331] FIG. 2B shows another system 110' for microfluidic
manipulation and/or analysis of particles, in accordance with
aspects of the invention. The operational principles for system
110' of FIG. 2B are similar to those for system 110 of FIG. 2A.
However, channels 118' and 120' diverge less than 90.degree. from
channel 116' in system 110', in contrast to their orthogonally
directed counterparts in system 110. Consequently, a greater
fraction of particles may be positioned in flow stream 134' in
system 110' than in flow stream 134 in system 110, but a greater
dislodging force also may be present. In other embodiments, the
output channels may have any suitable angles of divergence,
including greater than 90.degree., and/or they may have unequal
angles of divergence. The angles of divergence and any asymmetry in
the two fluid paths may be alterable to select the number of
particles trapped and/or retained and their positions within the
trap.
[0332] Embodiment 3
[0333] FIG. 3 shows yet another system 170 for microfluidic
manipulation and/or analysis of particles, in accordance with
aspects of the invention. System 170 includes (1) a fluidic network
172 of channels 174, 176, 178 and (2) a retention mechanism or trap
180. A flow stream 181 brings input sample and fluid to a
T-junction 182, at which stream 181 is divided into orthogonally
directed, primary flow-streams 184, 186. As in systems 110, 110' of
FIGS. 2A and 2B, a reduced velocity, positioning flow-stream 188
extends from stream 181, between primary streams 184, 186, toward
opposing wall 188. However, unlike systems 110 and 110', system 170
also includes partitions 192, 194 ("P" and "Q", respectively) in
the form of rectangular blocks. Partitions 192, 194 divide the main
channels to create secondary channels 196, 198, which extend
generally parallel to main channels 176, 178. These secondary
channels divide positioning flow-stream 188 and direct it
orthogonally in opposite directions, as shown by secondary flow
streams 200, 202. Secondary flow streams transport fluid at a lower
velocity than primary streams 184, 186 because of their position
within network 172.
[0334] FIG. 4 shows system 170 during particle input, after
positioning and retention of a single particle 204 between
partitions 192, 194 by trap 180. Particles 206 entering network 172
may travel along flow stream 181, generally in both central and
lateral positions within channel 174. Laterally positioned cells,
such as cells 208, follow primary flow streams 184, 186 along
channels 176, 178. In contrast, centrally positioned cells, such as
cells 210, follow positioning stream 188 toward a slot or gap 212
between partitions 192, 194. In this embodiment, gap 212 is
slightly wider than the diameter of cells 206, so that it will
accept only one cell. In other embodiments, and/or for other cells,
gap 212 may be wide enough to accept two or more cells. Whatever
the width of gap 212, wall 190 and partitions 192, 194, form a
retention site 214 at which cell 204 or cells may be stably
retained. Once cell 204 is positioned at the retention site by trap
180, its presence may tend to block or diminish fluid flow along
secondary streams 200, 202, through secondary channels 196, 198
(see FIG. 3). Accordingly, secondary streams 200, 202 have
diminished capacity to draw additional cells between partitions
192, 194. As a result, in some embodiments, trap 180 may
preferentially retain only one cell automatically, without any need
for optical monitoring during positioning and/or retention. Thus,
retention site 214 may be dimensioned based on the size of cells to
be retained. For example, eukaryotic cells typically are about 2 to
10 .mu.m in diameter, so gap 212 may be slightly wider than this
diameter, whereas secondary channels 196, 198 may be slightly
narrower than this diameter, to prevent entry of cells into these
channels.
[0335] Retained cell 204 may be treated and/or analyzed using any
suitable method, such as optical and/or electrical detection of
cell characteristics, as described above in Section VIII. This
treatment and/or analysis may be facilitated by a microchannel 216
that extends outward from wall 190 into chamber 218. Microchannel
216 is smaller than the diameter of retained cell 204 and may be
used to exert positive and/or negative pressure on the retained
cell, or apply and/or measure an electrical potential and/or
current across the retained cell, among others, as described below
in Examples 11 and 12.
Example 2
Microfluidic Systems for Trapping and Perfusing Particles
[0336] This example describes microfluidic systems that position
and retain single particles or sets of particles, and allow rapid,
precise perfusion of the retained particles or sets of particles
with reagents; see FIGS. 5-11C.
[0337] Background
[0338] Many cell studies benefit from analysis of a population of
cells. The population may provide discrete information from
individual cells of the population and averaged information from
the entire population. Accordingly, a population of cells may allow
concurrent analysis of distinct types of cells when the population
is heterogeneous, or a range of cell phenotypes or responses when
the population is homogeneous or clonal. Therefore, studies of
cells in a microfluidic environment would benefit from microfluidic
systems that automatically position and/or retain a set of cells at
a preselected site on a microfluidic chip. Furthermore, these
studies would benefit from mechanisms that allow the retained set
of cells to be perfused with selected reagents, such as drugs, test
compounds, or labels, in a controllable and definable manner.
[0339] Description
[0340] This example describes microfluidic systems that enable a
user to trap multiple cells within a cell retention chamber, and
perfuse the trapped cells with reagents for controlled intervals.
These systems may be formed by any suitable method, including
multilayer soft lithography involving multiple layers of
photoresist, for example, using molds fabricated as described below
in Example 13 and elsewhere in this Detailed Description, and in
the patent applications listed above under Cross-References and
incorporated herein by reference. Accordingly, in some embodiments,
the cross-sectional geometry of fluidic channels may vary between
rectangular in flow channels and arcuate at the position of
valves.
[0341] Embodiment 1
[0342] FIGS. 5-11 show a system 250 for microfluidic analysis of
cell populations. This system is described in detail below,
including (a) system description, (b) system production, (c) system
operation, and (d) system protocols.
[0343] System Description
[0344] FIG. 5 shows a portion of a system 250 for microfluidic
analysis of cell populations. System 250 includes a microfluidic
layer 252 and a control layer 254. Microfluidic layer 252 forms a
microfluidic network 256 of interconnected channels, depicted in
blue and orange. Control layer 254 is positioned over, and
abutting, the microfluidic layer, and includes valves and pumps
(see also FIG. 8), depicted in purple. Exemplary dimensions
presented below for system 250 are based on cell diameters of about
8 to 12 .mu.m.
[0345] The microfluidic layer includes microfluidic channels with
distinct geometries and functions. Blue, flow channels 258 have a
semi-circular or arcuate cross-sectional profile and are positioned
generally upstream and downstream of mechanisms for cell
positioning, retention, and/or treatment, which are described
below. These flow channels have cross-sectional profiles that allow
the channels to be acted upon effectively by valves and pumps
present in control layer 254. In this example, flow channels are
about 200 .mu.m wide and 20 .mu.m high. In contrast, orange, cell
channels 260 have a rectangular profile. In this example, cell
channels are about 100 .mu.m wide and 20 .mu.m high. Because
channel height does not restrict lateral movement, at least to
first order, the cells or particles can travel freely within the
cell channel, following the walls or more central positions based
on the particular laminar flow stream that carries a particular
cell or particle. Thus, these cell channels are used to position
cells to preselected laminar flow streams and preselected regions
of the microfluidic network. Perfusion channels 262, described more
fully below, also are shown in orange and function to controllably
perfuse retained cells. In this particular example, perfusion
channels are about 10 .mu.m wide.
[0346] System 250 includes an input mechanism 263, a positioning
mechanism 264, a retention mechanism 266, and a perfusion mechanism
268. The positioning and retention mechanisms function together to
position and trap cells in a retention or capture chamber 270. The
perfusion mechanism functions to effect delivery of reagents to the
cells in retention chamber 270, typically after cell retention.
[0347] Input mechanism 263 introduces particles into the system,
using an input reservoir or well, as described below (see FIG.
8).
[0348] Positioning mechanism 264 operates to increase the
probability that input cells will enter the retention chamber.
Mechanism 264 operates through convergent flow streams that join
but remain segregated in a laminar distribution. Input flow streams
272, 274, 276 carry fluid along flow channels 278, 280, 282,
respectively. However, channel 280 also may carry cells, whereas
flanking channels 278, 282 generally do not. As a result, at
confluence 284, flow stream 274 occupies a central portion, flanked
by flow streams 272, 276. Accordingly, the accompanying cells are
focused to a central portion of combined stream 286. In some
embodiments, additional flow streams may be included, and/or cells
may be included in other flow streams, as exemplified below in
Example 3.
[0349] FIGS. 6 and 7 show, respectively, corresponding actual and
schematic views of the retention mechanism or trap 266 of FIG. 5.
The retention mechanism includes a partially closed retention or
capture chamber 270. Chamber 270 may have a size of about 60-100
.mu.m long, 50-100 .mu.m wide, and 20 .mu.m high. Chamber 270 is
formed by opposing channel wall 288, front wall 290, side walls
292, 294, and top and bottom walls (not shown). Front wall includes
an aperture 296 through which cells enter the chamber from a
reduced-velocity stream 298, extending from combined stream 286.
The reduced-velocity stream may be less damaging to cells that
enter the chamber, increasing viability and the probability of a
fruitful analysis. Aperture 296 is about 5-20 .mu.m wide and may
have a height corresponding to some or all of the channel height.
Fluid entering aperture 296 as part of stream 298 may pass through
side-wall channels 300. In this example, each side wall includes
three side-wall channels 300, which have a rectangular profile
about 10 .mu.m wide and 5 .mu.m high. In general, the side-wall
channels are dimensioned to selectively retain cells or particles
of interest, while allowing fluid or smaller cells or particles to
pass through. Thus, chamber 270 functions as a filter. However, in
contrast to standard filters, only a fraction of input cells enter
chamber 270. The fraction may be less than about 1 in 10, 1 in 100
or 1 in 1,000, among others, depending on the design of the
retention chamber, the speed of the input fluid stream, and the
size and density of particles, among others.
[0350] FIG. 7 shows a focused stream of cells 302 flowing toward
chamber 270. Cells 302 either enter aperture 296 or are carried
orthogonally by channels 304, 306. Within chamber 270 microstreams
308 connect chamber 270 with side-wall channels 300.
[0351] Perfusion mechanism 268 provides precisely controlled
exposure to reagents for trapped cells in chamber 270. FIG. 5 shows
the general design of the perfusion mechanism. Trapped cells are
selectively exposed to buffer or reagent streams carried by one of
two or more perfusion channels 310, 312. Fluid, such as media,
buffer, and/or reagent, flows through perfusion channels 310 and/or
312 and joins focusing buffer stream 314. During perfusion,
focusing buffer stream 314 is produced by input fluid from one or
more input reservoirs "B," described more fully below, flowing
past.chamber 270 in a single stream. Thus, the stream no is longer
split as occurs during cell positioning and retention, as shown in
FIG. 7. Due to laminar flow and the position of perfusion channels
310, 312, fluid from either one of these channels enters to join
main flow stream 314 on the side of the main flow stream nearest
chamber 270. Therefore, the trapped cells are exposed to fluid from
perfusion channel 310 or 312. However, if fluid is flowing from
both perfusion channels, fluid from perfusion channel 312 shields
trapped cells from fluid flowing from perfusion channel 310, such
as a reagent. Accordingly, the contents of perfusion channel 312
may be referred to as a shield liquid or shield buffer. With
concurrent flow from both perfusion channels, cells may be rapidly
exposed to a reagent from perfusion channel 310 by stopping flow
from channel 312. Stopping the flow of the perfusion buffer may
expose cells to reagent within a very short time, in some cases
about 150 msec after stopping flow. Therefore, cell analyses that
require precise control of reagent exposure to measure rapid cell
responses may be conducted reproducibly with the rapid response
times afforded by this microfluidic system.
[0352] Perfusion mechanism 268 may be modified to achieve similar
perfusion or to change the exposure response time. For example,
similar perfusion may be obtained by disposing perfusion channels
on opposing sides of transverse channel 316, or disposing both
perfusion channels on opposing wall 288. Alternatively, or in
addition, the exposure time may be increased or reduced by moving
perfusion channel 310 closer to, or farther from, main flow stream
314. Example 3 shows a perfusion channel that empties directly into
the focusing buffer stream.
[0353] FIG. 8 shows additional aspects of microfluidic system 250.
These additional aspects include macrofluidic reservoirs, and
valves and/or pumps of the control layer that control fluid flow
within the microfluidic network.
[0354] Macrofluidic reservoirs allow system 250 to interface with
the macroscopic world. Each reservoir or well functions as a
fluidic inlet or outlet connected directly to at least one
microfluidic channel. Fluidic inlet-well A, shown at 330, provides
for particle input, generally as a cell suspension. Fluidic
inlet-well B, shown at 332, holds a focusing buffer, which is split
into two focusing channels, 334, 336, that ultimately form
converging flow streams 272, 276. Fluidic outlet-well C, shown at
338, holds output liquid, generally waste liquid, that flows
through the system. Well C accepts fluid from one or both of fluid
channels 340, 342. Fluidic inlet-wells D and E, shown at 344 and
346, may hold first and second reagents for exposure to trapped
cells. Fluidic inlet-well F, shown at 348, holds the shield buffer
that blocks exposure of the reagents until desired.
[0355] Control layer interfaces are numbered one through eleven.
Each interface acts as a gas inlet to regulate opening and closing
of one or more valves. Interface seven controls cell input valve
350. Similarly, interface eight controls fluid channel 340,
determining whether main flow stream 314 bifurcates or is a single
stream. Interfaces nine, ten, and eleven control valves 352, 354,
356, which regulate inflow of reagent or shield buffer from fluidic
inlets D, E, and F, respectively. Interfaces 1 through 3 and 4
through 6 control sets of values, shown at 358 and 360,
respectively. Valves within each set are actuated in a defined
sequence to pump liquid by peristalsis from inlets B (valve set
360) or D-F combined (valve set 358).
[0356] System Production
[0357] System 250 may be formed using any suitable method. In an
exemplary approach, the system is formed by layering and fusing
microfluidic layer 252, control layer 254, and a substrate layer,
formed, for example, by a cover slip (not shown). Specifically, in
this approach, the microfluidic and control layers are molded by
soft lithography and then fused. Next, the resulting fused
multilayer structure is bonded to the cover slip substrate layer.
Finally, microfluidic channels are wetted with deionized water.
[0358] System Operation
[0359] System 250 may be used to load, position, and/or retain
particles, such as cells, using any suitable method. In an
exemplary approach, valves 7, 9, 10, 11 are closed, and the
remaining valves, including the pump valves, are opened. Wells B
and F are loaded with focusing and shield buffers, respectively,
wells D and E are loaded with reagents, and well A is loaded with a
cell suspension. Valve 7 is then opened, after ensuring that waste
well C is at least partly empty, enabling cells to flow towards
well C. At this point, no liquid flows from wells D, E, and F.
Buffer flows from well B to well C, and cells flow from well A to
well C. The cells flowing out of well A are focused in the center
of combined flow stream 286 (see FIG. 7) by focusing fluid streams
coming from well B, thereby flanking cells flowing from well A. The
focusing fluid streams 272, 276 increase the likelihood that input
cells will enter retention chamber 270, which is placed near where
focusing occurs. The focused stream of cells is split into two
streams adjacent the retention chamber. Each stream flows in a
direction orthogonal to the focused stream and opposite to each
other, as described above. The trap is placed at a point of the
flow stream below where the stream splits, so that the velocity of
flow is lower than in the rest of the channel, therefore increasing
the likelihood that retained cells are viable. Once a sufficient
number of cells are captured, valve 7 is closed to stop the flow of
cells from well A.
[0360] System Protocols
[0361] System 250 may be used for any suitable protocols or
procedures involving positioned and/or retained particles. In a
exemplary protocol, cells are exposed to reagents in wells D and/or
E, as described below. This protocol is exemplified by successive
exposure of retained cells to first and second reagents, such as a
cell stain specific for dead/fixed cells and a cell fixative,
respectively; see FIGS. 9-11.
[0362] The system is readied for perfusion as follows. First, valve
8 is closed, so that the flow of focusing buffer from well B no
longer is split adjacent retention chamber 270. As a result, the
focusing buffer moves predominantly or exclusively along main flow
stream 314, which is unbranched (see FIG. 5). Next, pumps that
control valve sets 354, 356 are activated and run through the
entire protocol. A suitable frequency for valve closure is about 60
Hz.
[0363] Shield buffer flow is initiated as follows. Initially,
valves 7-11 are in a closed position, so that only focusing buffer
from well B flows towards waste well C. Then, valve 11 is opened,
so that shield buffer flows from F to C and focusing buffer flows
from B to C.
[0364] Flow of the first reagent, in this case Trypan blue, is
initiated as follows. Valve 9 is opened, so that fluid flows
through both valves 9 and 11. Valves 7, 8, and 10 are maintained in
their closed positions. Since the shield buffer is flowing, the
first reagent is spaced from the cell retention chamber by the
shield buffer. Therefore, this configuration readies the system for
perfusion and may be used to wash the fluidic network without
exposing the cells to either of the first and second reagents.
[0365] Perfusion of the first reagent is initiated as follows. Once
the fluid lines are washed with the first reagent, the shielding
buffer is turned off, and the cells are exposed rapidly to the
already flowing first reagent. Specifically, valve 11 is closed,
joining already-closed valves 7, 8, and 10. In contrast, valve 9
remains open. In this way, the shield buffer no longer separates
the flow stream of the first reagent and the cell retention
chamber, allowing the first reagent to perfuse the cells.
[0366] After a suitable exposure time, the first reagent is washed
out of the cell retention chamber as follows. Valve 11 is opened to
restart flow of the shield buffer. In addition, valve 9 is closed
to stop flow of the first reagent, joining already-closed valves 7,
8, and 10. In some cases, valve 9 may be left open to facilitate
repeated exposure of the cells to the first reagent over a short
time interval. FIG. 9 shows about twenty Jurkat cells 380 in
retention chamber 270 after exposure to a dye, Trypan blue, that
stains fixed cells and a shield buffer to wash away the dye. Debris
382 is stained, but cells 380 are unstained.
[0367] Flow of the second reagent, in this case methanol, is
initiated as follows. Valve 10 is opened, joining already-open
valve 11. Valves 7, 8, and 9 remain closed. This configuration is
used to wash the fluidic network with the second reagent without
exposing the trapped cells to this reagent.
[0368] Perfusion of the second reagent is initiated as follows.
Valve 11 is closed to turn off flow of the shielding buffer,
joining already closed valves 7, 8, and 9. Valve 10 remains open,
to expose cells 380 to the second reagent, in this case methanol,
thus fixing the cells. FIG. 10 shows cells 380 being perfused with
methanol. There is an optically detectable boundary 384 between the
methanol 386 and the focusing buffer 388, caused by their distinct
indexes of refraction.
[0369] After a suitable exposure time, the second reagent is washed
out of the cell retention chamber as follows. Valve 11 is opened to
initiate flow of the shield buffer. In contrast, valve 10 is
closed, to join already-closed valves 7, 8, and 9.
[0370] Cells 380 are then exposed for a second time to the first
reagent, followed by washing with the shield buffer, as follows.
The sequence of valve manipulations are as described above, except
that valve 9 is left open during washing with shield buffer to show
a shielded flow path of the first reagent. Now, since the cells
have been fixed and permeabilized by methanol, they stain with the
dye carried in the first reagent. FIG. 11 shows cells 380 stained
blue after their second exposure to Trypan blue and subsequent
washing with shield buffer. The shielded flow path 390 of the first
reagent, Trypan blue, is visible focused between shield buffer 392
and focusing buffer 388.
[0371] The microfluidic system demonstrated here can be used for
any suitable assay, such as screening compounds against a small
population of cells, with the size of the small population be
selected to be statistically representative of cell behavior. The
particles may include cells and/or beads, among others. The cells
may be nonadherent and/or adherent cells, either in suspension or
attached to a substrate provided by the microfluidic system. The
beads similarly may be nonadherent or adherent, and may be used to
carry samples, reagents, and/or cells, among others.
[0372] Embodiment 2
[0373] FIGS. 11A and 11B show a system 400 for measuring
interaction between separated, but proximate particles. Such
interaction may be provided by diffusible materials released by a
first particle (or particle population) and received by a second,
separated particle (or particle population). These diffusible
materials may include cell-secreted hormones, viral particles, cell
components released by cell lysis, and/or so on. The diffusible
materials may produce changes in the second particle or particle
population that are related to any measurable particle or
population characteristic, such as cell identity, gene expression,
apoptosis, hormone secretion, growth, and/or the like.
Alternatively, or in addition, such communication may include long,
thin processes extending from cells, such as axons and/or
dendrites. Exemplary particle characteristics are described further
in Sections VIII and XII above.
[0374] System 400 may be formed by disposing two versions of system
250 in a tail-to-tail configuration. Accordingly, each individual
subsystem 250 may include a retention mechanism 266, an
individually controlled perfusion mechanism 268 for introducing
reagents to each group of captured particles, and an input flow
stream 274 for carrying particles and/or buffer to the retention
mechanism. However, system 400 also includes communication passages
402 that provide fluidic communication between each retention
mechanism 266 and retention chamber 270.
[0375] Communication passages 402 may be size-selective channels
configured to prevent movement of retained particles, generally
cells, between each subsystem 250. However, passages 402 are
configured to allow movement or passage of any smaller material
released from the retained particles (such as molecules, polymers,
molecular complexes, and/or smaller particles, such as viruses), or
of processes, such as axons and/or dendrites, extending to, from,
and/or between retained cells. Furthermore, perfusion mechanisms
268 may be used to determine the effect of reagents on cell-cell
communication mediated by passages 402.
[0376] FIG. 11B shows an alternative embodiment of paired retention
mechanisms 266, mechanism 404, that may be included in system 400.
Mechanism 404 includes paired retention mechanisms 406, dimensioned
to trap single particles 408. Retention mechanisms 406 are
fluidically coupled through communication passages 402.
Accordingly, communication between single-cells may be analyzed
using mechanism 404.
[0377] Embodiment 3
[0378] FIG. 11C shows a retention mechanism 410 that maybe used in
system 250 or any other suitable microfluidic system to form a
positioned, two-dimensional array of retained particles. Mechanism
410 includes an array of individual traps 412 oriented to receive
particles from inlet channel 414. Traps 412 form a two-dimensional
array of particle retention sites. Traps 412 may have any suitable
configuration, including staggered rows, as shown here,
orthogonally arranged rows and columns, or irregular
configurations. (In some embodiments, some of traps 412 may be
positioned in alternative planes (e.g., in front of and/or behind
the plane of the drawing) to form three-dimensional arrays of
retained particles.) Each trap 412 may be dimensioned to hold one
or plural particles and may include size-selective channels or
similar features to allow fluid to flow through portions of the
traps. Traps 412 may be disposed within a common chamber 416 having
an single or plural outlet channels 418 (such as chamber 270,
described above, or chamber 1970 of Example 10 below), within a
chamber having no outlet besides an inlet channel, or within a
channel, such as transverse channel 316, described above, among
others.
Example 3
Microfluidic Systems for Parallel Retention and/or Treatment of
Particles
[0379] This example describes microfluidic mechanisms and systems
that position a plurality of particles and/or reagents at discrete
transverse regions and flow paths within a channel or flow stream;
see FIGS. 12-13K. This positioning may allow parallel retention of
distinct particles at adjacent, but distinct, sites and/or parallel
exposure of particles at these sites to distinct reagents.
[0380] Background
[0381] Biological analyses benefit from a capability to directly
compare the phenotypes of two or more cells or groups of cells,
under similar or distinct treatment regimens. However, in the
macroscopic world, such cells or group of cells often are treated
at distinct, relatively widely spaced sites, such as different
tissue culture dishes or wells of a microtiter plate, potentially
exposing the cells to undesired differences in treatment
conditions. Accordingly, such analyses may need to be averaged over
many experiments to achieve meaningful results. Therefore, it would
be desirable to have a microfluidic system that positions, treats,
and analyzes particles or groups of particles adjacent one another
at a microscopic level, to allow more consistent and efficient
side-by-side comparisons.
[0382] Description
[0383] The microfluidic systems described in this example position
a plurality of particles or (particle populations) and/or reagents
along distinct, transversely disposed flow paths or regions within
a channel or flow stream. The transversely disposed flow paths may
be defined by introducing the particles and/or reagents into the
channel along distinct laminar flow paths, by joining separate
inlet channels (or inlet flow streams) carrying the particles
and/or reagents. These flow paths may abut one another or may be
spaced apart by one or plural spacer fluids, such as buffers. These
spacer fluids may follow one or plural interposed flow paths formed
by one or plural inlet channels interposed between the inlet
channels that carry the particles and/or reagents.
[0384] The transversely disposed flow paths may be used to carry
distinct (or similar) particles to distinct retention sites or
chambers within the channel. The distinct retention sites may
retain distinct (or similar) particles for exposure to the same
reagent. For example, the distinct particles may be exposed to
reagents, such as modulators and/or labels, to compare
characteristics of the particles, such as response to the
modulators, labeling characteristics, and/or so on. Thus, the
position of each retention site may be used to identify the
corresponding particle(s) retained at that position. For example,
one retention site may be used to hold a control particle(s), as a
reference, and another retention site may be used to hold a
particle(s) of interest, allowing the control particle(s) and the
particle(s) of interest to be compared directly. Alternatively, one
retention site may hold a bead(s) carrying a reagent, and another
site may hold a cell(s) to be analyzed. In this approach, cell
components released by cell lysis or secretion then may be analyzed
for interaction with the reagent held by the bead.
[0385] Alternatively, or in addition, transversely disposed flow
paths may be used to expose similar (or distinct) particles to
distinct reagents and to identify each reagent or exposed particle
based on position. Particles may be retained at positionally
distinct retention sites, either inputted from distinct reservoirs
or a single reservoir. Next, the retained particles may be
contacted with distinct reagents carried to the distinct sites by
transversely disposed flow paths. The transversely disposed flow
paths may be formed by a set of inlet channels distinct from,
and/or overlapping with, inlet channels that introduced the
particles. Position of the retained particles identifies each of
the distinct reagents exposed to the particles. In some
embodiments, the distinct reagents may include a compound with a
known activity that acts as a reference, and one or more test
compounds for comparison.
[0386] The microfluidic systems of this example may allow more
efficient and meaningful use of microfluidic space for comparative
analysis of particles and/or reagents.
[0387] In certain embodiments, a junction between two inlets and an
outlet may be used to transiently expose or perfuse particles,
preferably cells, with selected reagents. By alternating the inlet
flow between plus and minus reagent flows, the downstream
conditions of the outlet will change in proportion to the rate of
flow between both inlets.
[0388] Embodiment 1
[0389] FIGS. 12 and 13 show a microfluidic system 420 (Embodiment
1) for retaining separate populations of particles, and exposing
the populations to one or more selected reagents.
[0390] Description of Embodiment 1
[0391] System 420 is formed by multilayer soft lithography,
generally as described above (for system 250) in Example 2 and
below in Example 13. Here, particle positioning region 422 is shown
as red rectangles, input/focusing channels 424 as blue regions, and
perfusion channels 426 as red lines. The dimensions of each region
or channel and/or the number of channels may be selected based on
particle size, reagent delivery volume, and/or the number of
separate populations to be retained, among others.
[0392] System 420 differs from system 250 of Example 2 in several
aspects. First, system 420 includes more than one reservoir for
holding and introducing particles. Thus, inlets 1 and 2, shown at
428, 430, respectively, connect to particle input channels 432,
434. Second, system 420 includes three focusing channels 436, 438,
440, and corresponding reservoirs or inlets for holding buffer (not
shown). The focusing channels, also referred to as spacer channels,
may be used to flank and separate the particle input channels.
Third, system 420 has more than one retention chamber 442, with the
chambers generally positioned adjacent each other below confluence
444, where input flow streams 446 join. Fourth, system 420 spaces
retention chambers 442 from wall 448, thus forming proximal and
distal diverging flow streams 450 and 452, respectively.
[0393] Applications of Embodiment 1
[0394] System 420 may be used as follows. Inlets 1 and 2 are loaded
with distinct suspensions of particles, such as different cell
types, and inlets corresponding to focusing channels 436, 438, 440
are loaded with focusing buffer. A pump(s) is started that drives
flow of the focusing buffer through the focusing channels. Valves
that control the flow of particles from inlets 1 and 2 are opened.
Particles enter confluence 444, but are focused to spaced,
intermediate, laminar flow streams 454, 456, shown in FIG. 13, by
flow from the focusing or spacer channels. Apertures 458, 460 of
the retention chambers are aligned with particle flow streams 454,
456, respectively, to receive one or more particles from the
corresponding flow stream. By taking advantage of the laminar flow
properties of fluids in system 420, the five streams flow together
but remain substantially distinct. Mixing of the fluids is limited
to diffusion, which in the case of large particles, such as beads
or cells, is very slow.
[0395] FIG. 13 shows the laminar flow pattern extending from
confluence 444 through divergence junction 462. Focusing flow
streams 464, 466, 468 flank and separate particle streams 454, 456,
thus guiding particles carried by these streams toward retention
chambers 442. Flow streams in junction 462 may diverge above (464,
468), below (466), and/or within (470) retention chambers 442.
Microchannels 472 within each retention chamber pass fluid but
retain particles.
[0396] After a sufficient number of particles have entered each
retention chamber 442, analysis of the particles may begin. Flow
from inlets 1 and 2 may be terminated, and flow may be converted
from a divergent pattern to a unitary flow path, by closing valve
474, as described above for operation of system 250 in Example 2.
Next, the trapped particles may be perfused with buffer/reagents
from perfusion channels 426. In system 420, perfusion channel 476
discharges fluid directly upstream of the retention chambers. This
configuration may provide more rapid perfusion of trapped particles
with reagents than system 250 of Example 2 above, because the
outlet end of channel 476 is very close to the retention chambers,
feeding more directly into the unitary flow path produced by the
focusing buffers.
[0397] System 420 may be modified by changing various parameters.
For example, the number of particle input-streams and/or focusing
streams may be varied, along with the number of retention chambers,
to trap additional particle populations or individual particles.
Thus, three or more particle input-streams may be used to trap
three or more types of particles in three or more retention
chambers. These three or more retention chambers may be disposed in
any suitable arrangement, including linear and staggered (e.g.,
triangular configurations). In some embodiments, the size of the
retention chambers may be varied, for example, so that only one or
a very small number of particles are trapped in each chamber (see
embodiment 2 of this example, and Examples 4-7, 11, and 12 below).
Furthermore, as described below, focusing streams and spacer
channels may be eliminated in some cases without substantial
cross-contamination of particles between particle streams and
retention sites.
[0398] Embodiments 2 and 3
[0399] FIGS. 13A-C shows two alternative embodiments of system 420,
systems 480 (Embodiment 2) and 480' (Embodiment 3), for retaining
and treating particles at separate, but adjacent sites. Similar to
system 420 described above, system 480 or 480' may be used to
selectively input and retain one or plural particles at each of
plural retention sites positioned at discrete positions transverse
to a flow direction within a channel. However, system 480 or 480'
also may be used to separately contact retained particles with
distinct reagents at distinct retention sites.
[0400] Description f Embodiments 2 and 3
[0401] System 480 includes an input mechanism 482, a focusing or
transverse positioning mechanism 484, a retention mechanism 486, an
output mechanism 488, a plurality of individually controllable and
distinct treatment mechanisms 490, 492, and a release mechanism
494; see FIGS. 13A and 13B.
[0402] Input mechanism 482 includes particle input channels 496,
498 and focusing or spacer channels 1762, 1764, 1766, similar to
those described above for system 420. Particles, such as cells, may
be inputted from input reservoirs "Cell 1" and "Cell 2" along
particle inlet channels 496, 498, to positioning channel 1768.
Input mechanism 482 also may introduce focusing or spacer fluid,
preferably buffer, from buffer reservoirs 1770, 1772, 1774 ("Buffer
1," "Buffer 2," and "Buffer 3," respectively) along spacer channels
1762, 1764, 1766, respectively, to positioning channel 1768.
[0403] Transverse positioning mechanism 484 may be determined by
inlet channels. More specifically, the relative spatial
configuration in which the inlet channels 496, 498, 1762-1766 join
positioning channel 1768, along with relative sizes of, and/or flow
rates from, these inlet channels, provides transverse positioning
mechanism 482. Positioning mechanism 484 places each individual
flow stream from each inlet channel in a laminar flow path based on
this spatial configuration. Accordingly, particles from reservoirs
Cell 1 and Cell 2 are spaced from each other centrally in
positioning channel 1768 by buffer from inlet channel 1764 and
laterally from each channel wall by buffer from inlet channels
1762, 1766, as described above for system 420.
[0404] Retention mechanism 486 includes a plurality of
single-particle retention sites, here referred to as "Trap A" and
Trap B" (see FIG. 13B). Trap A and Trap B each are positioned to
retain a particle introduced by one of the two particle reservoirs,
Cell 1 and Cell 2, and carried at correspondingly distinct,
transverse positions along positioning channel 1768; see FIG. 13B.
Accordingly, Trap A is positioned to retain a particle introduced
from Cell 1, and Trap B from Cell 2. Particles not retained may be
carried past retention mechanism 486 to output mechanism 488, along
central outlet (waste) channel 1776 or flanking outlet (waste)
channels 1778.
[0405] Treatment mechanisms 490, 492 provide exposure of retained
particles to distinct reagents, indicated as Reagents 1-4; see FIG.
13B. A particle retained in Trap A may be exposed to Reagent 1
and/or 2 (controlled by valves V2 and V3), and a particle retained
in Trap B may be exposed to Reagent 3 and/or 4 (controlled by
valves V6 and V7). These reagents may be stored and delivered
(sequentially and/or simultaneously, in any desired proportion and
for any desired time) using any suitable mechanism, such as those
described above in Example 2 and below in Example 8. Reagents from
each treatment mechanism may be separately addressed to a
corresponding retention site, by transverse positioning of reagent
flow streams entering positioning channel 1768. Reagents flow
toward central outlet channel 1776, but occupy a discrete portion
of the entire flow stream within positioning channel 1768 and
transverse channel 1780 due to laminar flow. Accordingly, reagents
from treatment mechanism 490 may be restricted to the left side of
positioning channel 1768 in FIG. 13B (and thus Trap A), whereas
reagents from treatment mechanism 492 may be restricted to the
right half of the channel (and thus Trap B). Optionally, spacer
buffer from central reservoir 1772, Buffer 2, may flow between
reagents delivered by the treatment mechanisms, reducing the
probability of any reagent crossing over, and thus contaminating,
the noncorresponding retention site.
[0406] Release mechanism 494 enables release of retained particles.
After release, the released particles may be analyzed further
and/or collected, and/or the retention sites may accept a new set
of particles for another round of treatment and analysis. Release
mechanism 494, may be operated by valve V4, to produce a localized
reverse or dislodging flow that propels the retained particles out
of the retention sites. Release mechanism 494 is similar to the
release mechanism described below in Example 7. However, in
contrast to the release mechanism described below, retention sites
in the present example are spaced from reverse flow channels
1782.
[0407] FIG. 13C shows selected portions of a modified version of
system 480, system 480'. System 480' is distinct from system 480 in
at least two aspects. First, retention mechanism 1784 includes
retention chambers 1786, 1788 that are larger than the retention
sites of system 480, and thus are capable of holding plural
particles. Second, treatment mechanisms 1790, 1792 include reagent
inlet channels 1794, 1796 that introduce reagents into transverse
channel 1798, rather than positioning channel 1800. This altered
position of the reagent inlet channels moves the reagents farther
from retained particles, but may facilitate washing out reagents
toward outlet channels 1802 after exposure. However, during
treatment, reagents from inlet channels 1794, 1796 are still
positioned transversely relative to the general direction of fluid
flow toward central outlet channel 1804. Accordingly, reagent inlet
channels may deliver reagents at any suitable sites that allow
laminar flow-based localization of reagents.
[0408] Systems 480 and 480' may be modified in any suitable aspect.
For example, a single population of particles, such as from a
single input reservoir, may be retained at plural distinct
retention sites, such as Trap A and Trap B, and then the sites
separately exposed to distinct reagents introduced by distinct
treatment mechanisms. Alternatively, or in addition, inlet channels
provided by treatment mechanisms and particle input mechanisms may
overlap or converge upstream of a common positioning channel, such
as positioning channel 1768 or 1800.
[0409] Applications of Embodiment 2
[0410] Exemplary operation of system 480 is described below using
cells. System 480 may be readied for operation by loading the input
reservoirs with cells and buffers and equilibrating channels with
the buffers, as described in other examples.
[0411] Trap A and Trap B may be loaded as follows. Valves V1, V4,
and V5 are opened, and valves V2, V3, V6, and V7 are closed. Five
flow streams coming from each of the five reservoirs meet before
Trap A and Trap B in positioning channel 1768. The cells from
reservoirs Cell 1 and Cell 2 are directed to their respective Traps
A and B. Fluid and unretained cells flow past retention sites along
divergent flow paths toward a plurality of outlet channels 1776,
1778.
[0412] Once a cell (or cells) is retained in each retention site,
valve V4 is left open, and valves V1 and V5 are closed. Closing
valve V1 blocks input of additional cells, and stops flow from
lateral buffer reservoirs 1770, 1774. Closing valve V5 stops
divergent flow, so that buffer (from central buffer reservoir 1772
(Buffer 2)) flows to central outlet channel 1776 along a unitary
path.
[0413] Distinct reagents may be delivered to the retained cells as
follows. Valve V4 is left open, and all other valves remain closed.
Both pumps are running. Valve V2 and/or valve V3 may be opened to
address Reagent 1 and/or 2 to Trap A. Valve V6 and/or valve V7 may
opened to address Reagent 3 and/or 4 to Trap B. Valves may be
partially opened as described in Example 8 to provide a desired
mixture of reagents. Buffer from reservoir 1772 flows past Traps A
and B to outlet channel 1776 and may be used as a barrier between
the streams of reagents addressed to Traps A and B. At any suitable
time, valve V5 may be closed to release the retained cells.
[0414] Exemplary Results with Chips Produced According to
Embodiment 2
[0415] System 480 was tested as described below. Microfluidic chips
were fabricated according to system 480 of FIG. 13A and used for
analysis of flow patterns and particle treatment efficacy with
colored and/or fluorescent dyes.
[0416] FIGS. 13D-F show dye patterns formed by colored dyes
introduced using each treatment mechanism and a flowing spacer
buffer to separate reagents. In each figure, Trap A holds a 10
.mu.m bead, and Trap B two 6 .mu.m beads. FIG. 13D shows a dye
pattern formed by green dye delivered from each treatment mechanism
and an orange dye-labeled spacer buffer delivered by reservoir
1772. The orange spacer buffer separate the two green dyes, and
each green dye flows from its corresponding inlet channel 1806,
1808 to outlet channel 1776. Some green dye also travels slowly
along transverse channel 1780. FIG. 13E shows a dye pattern formed
by red dye delivered from treatment mechanism 490, green dye from
mechanism 492, and orange dye from buffer reservoir 1772. FIG. 13F
shows a dye pattern formed by red dye delivered from treatment
mechanism 490, yellow dye from mechanism 492, and orange dye from
buffer reservoir 1772.
[0417] FIGS. 13G-13J show an analysis of treatment efficacy of
single Jurkat cells captured in each of Traps A and B. FIG. 13G
shows the two retained cells 1810, 1812 prior to treatment. FIG.
13H shows exposure of each cell to Trypan blue dye delivered by
distinct treatment mechanisms. The spacer buffer forms an uncolored
column of fluid 1814 between the two blue regions surrounding Traps
A and B. Membranes of both cells are intact so neither stains
efficiently with the dye. FIG. 13I shows exposure of cell 1810 in
Trap A to methanol, to fix the cell, while cell 1812 in Trap B is
addressed with buffer. FIG. 13J shows the two cells being exposed
to the blue dye after fixation of cell 1810. Cell 1810 can no
longer exclude the blue dye and is stained blue. Cell 1812 has not
been in contact with methanol and is not stained.
[0418] FIG. 13K demonstrates that spacer buffers may not be
required to prevent cross contamination of particles and/or
reagents during particle loading and/or exposure to reagents. Each
trap has been loaded with a fluorescent bead 1816, 1818. Bead 1816
is addressed with a fluorescent dye, fluorescein, and bead 1818
with Trypan blue, using treatment mechanisms 490, 492,
respectively. No spacer buffer stream separates the two reagent
streams, but the reagents do not substantially cross over and
contaminate the other trap. It should be noted that the time for
diffusion of reagents (or particles) transverse to their laminar
flow streams is limited by the relatively short time that the
laminar flow streams are in contact before passing Traps A and B.
Accordingly, analyses may be conducted with or without spacer
streams, with spacer streams being used to lower the probability of
cross-contamination.
[0419] Embodiment 4
[0420] FIG. 13L shows a portion of microfluidic system 1820 that
may be used to separately address particles and/or reagents to sets
of particle traps. System 1820 includes a plurality of serially
arrayed sets 1822, 1824 of particle traps 1826. Each set 1822, 1824
is disposed to a discrete transverse position with a fluid flow
stream, in this case defined by a channel 1828. Accordingly,
laminar flow streams carrying particles (1830, 1832) or reagents
(1834, 1836) may be segregated to discrete transverse regions of
channel 1828, so that each set 1822, 1824 is individually
addressed. In alternative embodiments, traps. 1826 are disposed in
a transverse channel, such as channel 1798 or a chamber, such as a
cell chamber with size-selective channel around its perimeter.
Example 4
Microfluidic System for Multiplexed Analysis of Particles in an
Array
[0421] This example describes a microfluidic system that loads
particles in a serially distributed set of particle retention
sites, and separately addresses reagents to each of these sites in
parallel; see FIGS. 14-16.
[0422] Background
[0423] Cell analyses often involve the use of arrays of cells or
cell populations. These arrays may be formed in microtiter plates,
so that individual wells within the array can be treated
distinctly, for example, with distinct test compounds. During or
after treatment, the microplate arrays are analyzed in multiplex to
measure properties of cells within each individual well. However,
such arrays are difficult to form reproducibly with microtiter
plates when single cells or a small group of cells are placed in
each well. Even if formed in microtiter plates, rapidly treating
the cells in such microtiter plates, and measuring short-term
consequences of such treatments, poses substantial technical
hurdles. Therefore, a microfluidic system is needed that forms more
reproducible arrays of individual cells or small groups of cells at
distinct positions, and that allows separate, rapid treatment and
analysis of the cells at the distinct positions.
[0424] Description
[0425] This example describes a microfluidic system that serially
traps small sets of particles at preselected positions within the
system, allowing treatment of the trapped particles in parallel
with desired reagents. Due to serial trapping of input particles, a
single loading of particles into one inlet may be used to supply
particles to an entire array of traps. Thus, this design may be
used to integrate a large number of traps into a single system.
This microfluidic system also reduces the number of control lines
required, as single control lines regulate sets of fluidic
channels, such as perfusion channels, that individually interface
with each of the traps. Accordingly, single control lines provide
parallel control for fluidic delivery to, or output from, each of
the traps. Such parallel control allows similar particles that are
retained by each trap to be individually treated with distinct
reagents. Furthermore, such parallel control allows all traps to be
fluidically connected during particle loading, but then fluidically
isolated during particle treatment and measurement. This
arrangement of the traps enables the fabrication of larger
microfluidic systems that may be suitable for use in
high-throughput drug discovery. For example, system 510 has a
footprint of 2 by 4 cm. By increasing this density somewhat and
increasing the number of traps over twenty-fold, at least 128 traps
may be disposed on a single substrate of 8 by 12 cm, allowing each
of the 128 traps to be addressed by two distinct reagents, with a
total of 256 reagents per substrate.
[0426] FIG. 14 shows a microfluidic system 510 for forming and
analyzing an array of particles. System 510 may be formed by any
suitable technique, such as multilayer soft lithography, to include
at least two distinct layers: (1) a microfluidic network layer 512,
shown in blue and orange, and (2) a control layer 514, shown in
pink. Channels having distinct widths and/or cross-sectional shapes
may be formed within each layer using molds fabricated, for
example, as described in Example 17.
[0427] Microfluidic layer 512 includes two orthogonally directed
networks. Particle loading network 516 is used to input and
position particles, so that the particles are retained at a linear
array of particle traps 518. Particle treatment system 520 is an
array of parallel, individual perfusion networks 522 that intersect
loading network 516 at individual particle traps 518.
[0428] Particle loading network 516 includes an inlet 524, an
outlet 526, and a loading channel 528 extending there between.
Inlet well 524, labeled C, is a reservoir that receives and holds a
particle suspension to be introduced into network 516. Outlet well
526, labeled W, is a waste reservoir that receives and holds fluid
and unretained particles that have traveled through network 516.
Loading channel 528 carries particles between inlet well 524 and
outlet well 526 to each of a plurality of particle traps 518
disposed along channel 528. Fluid is actively transported along
network 516 by a three-valve pump 530, labeled "pump 1," which is
positioned near the terminus of network 516 to pull fluid through
the network. Positioning the pump after the traps delays potential
damage to fragile particles, for example, due to compression under
closing valves, until particles have passed all particle traps
518.
[0429] Each perfusion network 522 directs fluid between perfuision
inlets 532, traps 518, and treatment outlets 534. Perfusion inlets
532 are of two main types: buffer inlet-wells 536, labeled "B," and
reagent inlet-wells 538, labeled "R.sub.xy." The buffer inlet-wells
hold a buffer or other washing or maintenance liquid, such as water
or a solvent. Based on their positions within particle treatment
system 520, the buffer inlet-wells are either a terminal inlet-well
540 or an intermediate inlet-well 542. Terminal inlet-wells 540
feed fluid to only one trap, whereas intermediate inlet-wells 542
are shared between two adjacent traps. Based on whether they are
intermediate or terminal inlet-wells, buffer inlet-wells feed a
main stream and/or a shielding stream. The control and function of
these two streams are described further below. The reagent
inlet-wells hold one of two (or more) reagents (or reagent
mixtures) that may be precisely exposed to an individual trap.
Reagent inlet-wells are labeled "R.sub.xy," with "x" referring to
trap assignment relative to the array of traps 518, and "y"
referring to one of the two reagents that can be directed to a
given trap. For example, reagent inlet-well R.sub.12 feeds the
first of the plurality of traps (closest inlet C) with the second
of two reagent choices for that trap. Fluid that passes each trap
518 may be directed to a corresponding treatment outlet-well 534 or
waste well, labeled here as W1-W6. For example, reagents from
reagent inlets R.sub.41, and R.sub.42 flow past and/or through trap
number 4 and are collected in waste well W.sub.x, where x=4.
[0430] Control layer 514 regulates fluid flow from perfusion
inlet-wells 532 with a limited number of control lines that act on
many fluid channels 544 in parallel; see FIGS. 14 and 15. A
three-valve pump 546, "pump 2," acts simultaneously on all inlet
channels 544 that extend from perfusion inlet-wells 532, to
actively drive fluid from these inlet-wells to and past traps 518,
and on to waste outlet-wells 534. Opening or closing each of four
perfusion valves, V1-V4, determines whether fluid actually flows
through each of the specific types of inlet channels 544 within the
perfusion system. Valve V1 regulates control line 548, which
includes a plurality of individual valves positioned over each of a
corresponding plurality of focusing channels 550 included among
inlet channels 544. Similarly, valve V2 regulates control line 552,
which includes valves that control each of a plurality of
first-reagent channels 554, valve V3 regulates line 556, which
controls each of a corresponding plurality of second-reagent
channels 558, and valve V4 regulates line 560, which controls each
of a corresponding plurality of shield channels 562. Thus, opening
or closing each of valves V1-V4 provides unified, parallel control
over flow of individual inlets to each of the plurality of
traps.
[0431] FIG. 15 shows a portion of system 510, including traps 2, 3,
and 4, to illustrate in more detail the design and rationale for
the switching valves. Insulation valves 564 function in the control
layer to mediate switching between particle loading network 516 and
particle treatment system 520. Insulation valve V5 controls a set
of valves that block flow along loading channel 528 at a position
downstream of particle inlet 524 (inlet C) and of the traps. Thus,
activation of valve V5 fluidically isolates each trap and converts
system 510 from a particle-loading configuration to a perfusion
configuration. In contrast, insulation valve V6 controls a set of
valves blocking flow to each individual treatment outlet 534,
preventing diversion of particles to treatment outlets during
particle loading, when valve V6 is closed. Therefore, valves V5 and
V6 are primary determinants of parallel versus serial use of system
510.
[0432] FIGS. 15 and 16 show details of the loading mechanism.
Loading channel 528 forms a divided flow path 564 at each trap 518.
Thus, particle stream 566 diverges directly upstream of each trap
518, at a T-junction 568, following divided flow path 564, and then
converging to form reunited particle stream 566. At each T-junction
568, a subset of particles do not follow divided flow path 564, but
flow instead directly into trap 518. Accordingly, each trap is
loaded using a divergent-flow mechanism, as described above in
Example 2, but, in system 510, without the use of focusing-buffer
streams during particle loading to focus particle flow within
channel 528. In this example, trap 518 includes a retention chamber
similar to retention chamber 270 of FIG. 5-8 in Example 2. However,
any suitable traps may be used, such as single-particle traps
described below in Examples 4-7, 11, and 12.
[0433] The subsequent perfusion of trapped particles uses shielding
and perfusion mechanisms analogous to those of Example 2. Buffer
flow from each buffer inlet 536 flows along focusing channels 550,
into loading channel 528, and past trap 518 in a unitary flow path
572, shown in FIG. 16 as a dashed path, analogous to focusing
buffer stream 314 of FIG. 5. Unitary flow path 572 may perform a
variety of functions, such as bathing trapped particles during
treatment, providing a retaining force on trapped particles during
perfusion, and focusing inflowing reagents and shield buffer, in
their laminar flow streams, toward the trapped particles.
Similarly, combined first and second reagent channel 554/558 and
shield channel 562 determine precise exposure to first and second
reagents, as described above in Example 2.
[0434] Applications
[0435] An exemplary use of system 510 to load particles and expose
the particles to different reagents is described below. System 510
is formed and readied for use as described elsewhere in this
Detailed Description.
[0436] Loading particles into each of traps 518 may be conducted as
follows. Valves 1-4 and 6 are closed, and valve 5 is open. Pump 1
is running, and pump 2 is not. The buffer inlet-wells B, shown at
536, are loaded with buffer, each of inlet-wells R.sub.xy is loaded
with a reagent, and inlet-well C is loaded with a cell suspension.
After making sure that the waste inlet-wells 526 are empty, pump 1
is allowed to pull the particles to the traps.
[0437] Conversion from a loading to a perfusion configuration may
be carried out as follows. Once each of the traps has its desired
occupancy and/or is full, pump 1 is stopped and valve V5 is closed.
Each trap is now isolated. Next, Valve V6 is opened to allow
fluidic access to waste outlet-wells 534. Then, valve V1 is opened
to permit flow of buffer from each inlet-well 536.
[0438] Trapped particles are perfused with each of the first and
second reagents as follows. Pump 2 is started, running at a
frequency of about 60 Hz. This pump is running throughout the
following treatments. Pumping action of pump 2 drives buffer
through focusing channels 550, along unitary flow path 572 past
each trap 518, toward waste outlet-wells 534. Prior to perfusion,
valves V2, V3 and V4 are closed, so that only no fluid flows from
along shield channel 562 or reagent channels 554, 558. Flow of the
first reagent and the shield buffer is initiated by opening valves
V2 and V4, while valve V3 remains closed. This valve configuration
is used to wash the fluidic network without exposing the trapped
particles to the first reagents, because the shield buffer directs
the first reagent stream to a spaced flow path separated from the
trapped particles. Once the fluid lines are washed with each of the
first reagents, valve V4 is closed to stop from of the shield
buffer, allowing each of the first reagents to contact trapped
particles. After a desired duration of exposure to each first
reagent, valve V2 is closed, allowing the shield buffer to wash
away reagent one, and rapidly terminating exposure. Trapped
particles may be exposed to each second reagent in parallel by
following a comparable series of steps, but opening and then
closing valve V3 instead of V2. In alternative perfusion
strategies, particles may be exposed to both the first and second
reagents simultaneously, by opening both valves V2 and V3 together.
Furthermore, particles may be exposed to any desired ratio of first
and second reagents by partially closing valves V2 and/or V3, as
described below in Example 7.
Example 5
Microfluidic Device for Forming and Analyzing a Particle Array
Using a "Cell Comb"
[0439] This example describes a microfluidic device for forming and
analyzing arrays of small number of particles, such as cells; see
FIGS. 17-20.
[0440] Background
[0441] In many applications, it is necessary to form an array of
cell-analysis chambers, with each chamber containing the same
number of cells. These chambers allow multiple experiments, such as
drug screens, to be conducted in parallel, in a consistent and
comparable fashion. Currently, standard analyses use wells of
microtiter plates as cell chambers, distributing an equal volume of
a cell suspension to each of the wells. The size of these chambers
and thus the number of cells analyzed has been decreasing in
response to efforts to reduce the use of space, reagents, and cells
in these analyses. Unfortunately, results from these analyses
become increasingly variable as the average number of cells per
well decreases. For example, with 96-well microtiter plates, there
generally are about 3000 to 5000 cells at the bottom of a well;
with 384-well plates, this number drops to about 1000 cells; and,
as researchers push for smaller and smaller assay volumes, such as
with 1536-well plates, this number drops further to only about 250
cells. These small average numbers of cells may lead to variations
in the actual number of cells among wells of as high as 20%. Such
variations lead to huge errors in the detected reaction signals.
Accordingly, with even fewer cells per well, for example, with
single cell assays or when cells of interest are in limited supply,
microtiter plates do not provide an adequate cell-analysis chamber
unless cells are counted to place an equal number per well. Even
then, microtiter plates are deficient for performing rapid
experimental manipulations. For example, early responses to
treatment with a drug are difficult to measure with microtiter
plates, because adding and mixing steps cannot be performed very
rapidly. Therefore, many cell-analyses would benefit from systems
for efficiently loading, rapidly treating, and analyzing small
numbers of cells.
[0442] Description
[0443] FIG. 17 shows a microfluidic device 610 for forming an array
of single particles or small groups of particles. Device 610
includes an input channel 612, a waste channel 614, and an array of
filter channels 616 extending between the input and waste channels.
Device 610 also includes a fixed-volume particle chamber 618 formed
in each filter channel 616, and a set of valves for sample handling
(see below). Device 610 may be referred to as a "cell comb" because
the path for cell (particle) flow takes the shape of a comb, with
chambers 614 representing the teeth of the comb.
[0444] The components of a cell comb each have a distinct function.
Input channel 612 carries input particles, such as a particle 620,
to each filter channel 616. A filter 622 is disposed within, or
adjoining, each filter channel. Filter 622 allows fluid to pass
into waste channel 614, but retains particles 620 in a portion of
filter channel 616 that corresponds to chamber 618.
[0445] Filter 622 may take various forms, provided as a
component(s) separate from the walls of filter channel 616 and/or
integral to these walls. For example, filter 622 may be formed by a
porous membrane that is specific for each chamber 618 or that is
shared by two or more or all chambers 618. Alternatively, filter
622 may be formed by smaller, "leak" channels within filter channel
616, or by posts, obstacles, or protrusions that extend into a
portion of filter channel 616, or that are disposed adjoining or
adjacent an end of the filter channel. The diameter of the smaller
channels, or the spacing of the posts/obstacles, determines the
size of particle retained in chamber 618. Thus, as long as the
diameters of these smaller channels, or the maximum spacing between
these posts/obstacles, are sufficiently less than the diameter of a
particle to be retained, the particle will be confined to chamber
618 while fluid will pass readily into waste channel 614. In
addition, the passage of fluid through the filter provides a
retaining force to reduce or prevent backflow of particles into
input channel 612.
[0446] The capacity and retention ability of each chamber 618 is
defined at least in part by filter channel 616 and filter 622. The
diameter and length of filter channel 616, coupled with the
position of filter 622 relative to filter channel 616, define the
capacity of chamber 618. Accordingly, chamber 618 may be
dimensioned to receive a fixed number of input particles 620, such
as a single particle. Such input particles may have a common size,
such as cells from a homogeneous cell population, or they may have
a range of sizes, such as cells from blood. In some embodiments,
the diameter of filter channel 616 allows size-selective retention
of a single particle. For example, the diameter may be large enough
to receive certain particles in a heterogeneous-particle
population, such as red blood cells, but small enough to exclude
others, such as white blood cells. Filter 622 also acts size
selectively, as described above, so in combination with chamber
618, individual filter channels 616 may be designed to retain a
single cell within a defined size range. Alternatively, individual
filter channels may be designed to retain a group of two or more
cells, with each cell having a minimum size that is retained by
filter 622.
[0447] Pressure differences within device 610 create positioning
and retaining forces for particles 620. Flow between input channel
612 and waste channel 614 creates a positive pressure difference
between the input channel and the waste channel across filter
channel 616. As a result, particles are carried into chambers 618
by fluid and fill each of the chambers very rapidly. After the
particles have filled some or all of chamber 618, a set of valves
may be used to isolate each chamber 618 (see below). In particular,
the closure of such valves may transform each cell chamber into an
isolated reaction chamber, with a fixed number of particles for
analysis.
[0448] FIGS. 18-20 show valves, additional filters, and analysis
sites that may be used with, or added to, device 610 for
manipulating the contents of individual chambers 618.
[0449] FIG. 18 shows a device 630 that is similar to device 610,
but that includes a separate analysis site 632 opposing each
chamber 618. A site valve 634 controls access to analysis site 632,
and a pair of input valves 636 isolates each chamber 618 along
input channel 612. The left panel of FIG. 18 shows a loading
configuration for each of valves 634, 636. Here, site valve 634 is
closed (indicated by an "X") to prevent input particles 620 from
entering analysis site prematurely, and input valves 636 are open
to allow particles to access each chamber 618. The right panel of
FIG. 18 shows repositioning of retained particle 620 to analysis
site 632. Here, site valve 634 is open, but input valves 636 are
closed. Particle 620 is displaced from chamber 618, by fluid
flowing in reverse across filter channel 616 from waste channel
614, rather than input channel 612. Since input valves 636 are
closed, fluid and particle 620 flow orthogonally to input channel
612, into analysis site 632. After particle 620 is delivered to
analysis site 632, site valve 634 is closed to isolate the particle
fluidically from other particles. In other embodiments, additional
fluidic lines may be used to deliver reagents to analysis site 632,
or analysis site 632 may be a blind channel that is preloaded with
such reagents.
[0450] FIG. 19 shows a device 650 that is similar to device 630 of
FIG. 18, but that includes switchable filters 652. Switchable
filters 652 may be switched between a closed, filtering position,
shown on the left, and an open, nonfiltering position, shown on the
right. After particle loading, switchable filters 652 are opened to
direct particle 620 to an analysis site. Such a switchable-filter
design allows unidirectional flow across filter channel 616 to both
retain and release particle 620. Accordingly, fluid flow from input
channel carries out each both retention and release, using
particle-laden fluid during retention, and particle-free fluid
during release. Waste valves 654 are closed before switchable
filter 652 is opened to direct particle 620 to analysis site 656.
Switchable/regulatable filters may be formed by size-selective
channels that are formed on valve membranes. With this arrangement,
deflection of the valve membranes may move the size-selective
channels in or out of filtering position by pressure exerted
through a control layer. Alternatively, or in addition,
size-selective channels may be adjacent to, or flanking, valve
membranes, as described below in Example 26.
[0451] FIG. 20 shows another device 660 with a switchable filter
652. In device 660, waste channel 614 includes a series of waste
filters 662 that function in place of waste valves 654 in device
650. Waste filters 662 play a dual role in allowing waste to flow
down waste channel 664, while directing particle 620 toward
analysis site 666. The passages of analysis sites 666 may serve as
waste channels.
[0452] Applications
[0453] Cell combs, described in this example, may be useful in a
variety of applications. For example, cell combs may be useful in
drug discovery, serving as replacements for microtiter plates in
cell assays to provide tighter control of the cell numbers. With
current technology, the fabrication of each cell chamber in a cell
comb device can be carried out with precision. Therefore, cell
assays may be performed with an array of cells formed using this
device, with reduced signal variation from chamber to chamber, even
with single-cell assays. Cell combs may, more generally, be used
with a variety of micron-sized particles, in addition to cells,
such as fluorescently or enzymatically coated beads. This device
also can operate in gas phase, as long as the size of the particles
of interest is larger than the pore size of the filter units. Cell
combs also can be cascaded so that objects of different sizes are
filtered out at different stages.
Example 6
Particle-Retention Mechanisms
[0454] This example describes mechanisms for retaining particles,
using particle traps that are spaced from their corresponding
substrates; see FIGS. 21-23.
[0455] Background
[0456] One goal of microfluidic systems is the capability of
retaining particles at preselected positions for subsequent
treatment and analysis. Traps that perform such retention functions
may perform optimally if they have minimal effects on fluid flow;
otherwise, flow patterns around the traps may be disrupted, slowing
or reducing particle and reagent entry into the traps. Examples 1
and 2 above describe traps that may be used to retain single
particles or groups of particles. However, these traps have limited
flow through the traps themselves. For example, trap 180 of Example
1 includes blocks P and Q that reduce or prevent cross-flow on
either side of a single retained particle. Similarly, retention
chamber 270 of Example 2 includes relatively narrow microchannels
300 that may restrict fluid flow substantially. Thus, there is a
need for an alternative trap that may be positioned closer to
particle input flow streams without disrupting flow patterns, while
allowing quicker and more efficient access by reagent and washing
flow streams.
[0457] Description
[0458] This example describes retention mechanisms having improved
fluid flow properties. These mechanisms are positioned downstream
of a particle flow stream, near the point at which the particle
flow-stream diverges at a T-junction. These mechanisms have been
dimensioned to trap a single particle; however, they alternatively
may be dimensioned to trap two or more particles. The microfluidic
system with respect to which each retention mechanism is
illustrated, particularly positioning mechanism 264 and perfusion
mechanism 268, is described above in Example 2. This earlier
example describes suitable fluid flow paths, and the operation of
the positioning and perfusion mechanisms. However, the retention
mechanisms presented in this example may be combined with any other
suitable microfluidic mechanisms for particle analysis.
[0459] Embodiment 1
[0460] FIG. 21 shows a microfluidic system 710 for positioning,
retaining, and/or perfusing a single particle, in accordance with
aspects of the invention. Portions of system 710 that are molded
from distinct photoresist layers are shown as distinct colors, as
described above (see introductory section of Examples). Retention
mechanism 712 includes a trap 713, shown in turquoise, positioned
centrally in T-junction 714, in a spaced relation from distal wall
716. Here, view 718, on the top right, is a schematic
representation of trap 713, with points of sectional view
indicated; view 720, on the middle right, is a horizontal sectional
view near the top of retention mechanism 712; and view 722, on the
bottom right, is a vertical sectional view nearer the side of
retention mechanism 712. Trap 713 extends downward from roof 724 as
a U-shaped block 726. This block includes a recess 728 that acts as
a retention site for a single particle. The block extends toward
substrate 730, in this case formed of glass, but remains in a
spaced relation, in this case about 5 .mu.m apart from the
substrate, to form a flow channel 732 that extends under all of
block 726. Thus block 726 forms a stalactite-based trap with a
potential flow stream below its entire bottom surface 734.
[0461] Embodiment 2
[0462] FIG. 22 shows another microfluidic system 740 for
positioning, retaining, and/or perfusing a single particle, in
accordance with aspects of the invention. View 742 shows a
color-coded schematic of a system 740, whereas view 744 shows a
photograph of an actual microfluidic system formed according to
view 742, but flipped horizontally. System 740 includes a trap 746
positioned centrally at T-junction 714. Trap 746 is spaced from
distal wall 716, disposing any retained particle quite close to
perfusion channel 748 for very rapid exposure to reagents (see
Example 2 for a more complete description of the perfusion
mechanism). Trap 746 includes a retention site 750 for holding a
particle, flanked by trap channels 752, shown in turquoise, that
extend to the edges of trap 746. Thus, fluid can enter retention
site 750 and flow laterally out trap channels. View 754 shows the
structure of trap 746 schematically. Trap 746 includes three
rectangular columns 756 that extend down to substrate 730, bridged
by channel forming portion 758, shown in dotted outline in view
754, which extends down to 5 .mu.m from substrate 730.
Cross-sectional views 762, 764, 766 show the structure of trap 746
in more detail.
[0463] Embodiment 3
[0464] FIG. 23 shows yet another microfluidic system 790 for
positioning, retaining, and/or perfusing a single particle, in
accordance with aspects of the invention. System 790 includes a
particle retention mechanism, trap 792, that abuts distal wall 716,
in alignment with particle stream 794 focused down input channel
796. Trap 792 includes a retention site 798, which is twenty .mu.m
in height, and flanked by retention blocks 800 that are spaced from
substrate 730 by about 5 .mu.m. View 802 shows a line
representation of trap 792, but includes a portion 804 of
microfluidic system outside of distal wall 716. Sectional views
806, 808 show how retention blocks 800 extend outward and downward
from distal wall 716 and channel roof 810, but form a trap channel
812 that extends under entire bottom surface 814 of the trap. Thus,
trap 792 is structured as a stalactite.
[0465] Views 816, 818 are two photographs taken of trap 792 at
different depths of focus. In view 816, the focal plane is near the
substrate surface, showing sharp lines at comers 820, where the
microfluidic layer 822 contacts substrate 730. The bottom perimeter
824 of blocks 800 is blurry because bottom surface 814 is raised
above substrate 730 (see also views 806, 808). In view 818, the
focal plane is slightly higher, raised about 5 .mu.m, placing
bottom perimeter 824 in focus. Now, comers 818 are out of
focus.
Example 7
Mechanisms for Reusable Microfluidic Systems
[0466] This example describes mechanisms that promote reuse of
microfluidic systems, including mechanisms for release, collection,
and/or resuspension of particles; see FIGS. 24-28.
[0467] Background
[0468] Microfluidic systems often are designed for single use. Such
single-use systems may be used to retain and analyze a single cell
or multiple cells, but they then are not or cannot be used again
because the cell or cells interfere with analysis of newly
introduced cells. Thus, these single-use systems then are
discarded, and additional single-use systems must be initialized
for additional analysis. This approach is not an efficient use of
the single-use systems. Moreover, this approach wastes macroscopic
volumes of cells and reagents, and is time consuming for
initialization. Thus, there is a need for a reusable microfluidic
system that releases retained particles after their analysis,
freeing the system (or cells) for additional analysis.
[0469] Description
[0470] This example describes microfluidic mechanisms that enable
formation of reusable microfluidic systems. These microfluidic
mechanisms include (1) a particle release mechanism, (2) a particle
collection mechanism, and (3) a particle suspension mechanism. The
particle release mechanism removes a particle(s) from a trap,
generally after treatment and/or analysis in the trap. The release
mechanism may provide a force that propels particles out of the
trap at any selected time. The particle collection mechanism may be
used to collect particles discharged by the release mechanism.
Collected particles may be cultured, measured, treated, and/or
discarded. The particle suspension mechanism reduces particle
settling in an inlet well, so that a single loading of particles
into the inlet well produces a relatively constant particle flow
from the inlet well over time. These three mechanisms alone, or in
any suitable combination, may enable more efficient and economical
use of microfluidic systems for particle analysis.
[0471] Embodiment 1
[0472] FIG. 24 shows a microfluidic system 850 having a particle
release mechanism 852 and a particle collection mechanism 854, in
accordance with aspects of the invention. The general design of
system 850 is as described in Example 2, and elsewhere in this
Detailed Description, including a particle focusing mechanism 856,
a particle retention mechanism or trap 858, and a perfusion
mechanism 860. These particle focusing and perfusion mechanisms are
at least substantially equivalent to positioning and perfusion
mechanisms 264, 268, respectively, shown in FIG. 5 of Example 2.
System 850 may be formed as described elsewhere in this Detailed
Description. The meaning of each colored region of system 850 also
has been described above, and therefore will not be repeated
here.
[0473] FIG. 25 shows trap 858 in more detail. Trap 858 may be
dimensioned for capturing a single particle and is similar to trap
746 of FIG. 22, described above, except that trap 858 disposes
channel 862 against distal wall 864, in contrast to trap 746, which
spaces channel 752 away from distal wall 716.
[0474] Particle retention and treatment are essentially as
described for Example 2 above, but the operation of a slightly
different control layer 866 is described here for clarity. Control
layer 866 includes valves V1-V4. Valve V1 corresponds to valve 8 of
FIG. 8, described above, and is used to convert between divided and
unified flow paths. Valve V2 controls particle release mechanism
852; its function is described below. Valves V3 and V4 control
fluidic flow to waste reservoir 868 and particle collection
mechanism 854, respectively. During particle loading into trap 858,
valves V1, V2, and V3 are open, and valve V4 is closed. During
reagent delivery by perfusion mechanism 860, valves V1 and V4 are
closed, and valves V2 and V3 are open.
[0475] Particle release mechanism 852 may be used at any time to
release particles, particularly after use of perfusion mechanism
860 and/or measurement of trapped particles. Release mechanism 852
operates by a dislodging flow to propel retained particles out
their confinement in trap 858; see FIGS. 24 and 25. The dislodging
flow originates in a reservoir channel 870 that is fluidically
connected to trap 858 using a size-selective channel 872.
Size-selective channel 872 has a diameter that prevents entry of
particles but that does not restrict passage of fluid to, or from,
reservoir channel 870.
[0476] Fluid flow through size-selective channel 872, and thus
particle release, is controlled by valve V2 (see FIG. 24). Valve V2
is a control-layer valve disposed over reservoir channel 870. When
valve V2 is closed, reservoir channel is compressed, forcing fluid
outward through size-selective channel 872 into trap 858. This
releases trapped particles, propelling them out of trap 858 into a
flow stream, such as main flow stream 874, shown in FIG. 25, which
carries the particles away from trap 858. Typically, in use, the
focusing buffer pump is running, the reagent valves are closed, and
the shield buffer is running. Thus, the main flow stream goes from
the buffer wells to the cell culture area, described below. When
valve V2 is opened, reservoir channel 870 expands, bringing fluid
in through size-selective channel 868 and refilling the reservoir
channel.
[0477] Embodiment 2
[0478] FIG. 26 shows a system 880 for retaining and releasing
groups of particles, in accordance with aspects of the invention.
System 880 generally is similar to system 850 (compare with FIG.
25), but with several exceptions. First, trap 882 includes a much
larger retention site 884 than trap 858, capable of holding a group
of particles. Thus, walls 886 extend substantially into cross
channel 888, and each wall includes three size-selective channels
890, rather than the one present in trap 858. Moreover, trap 882 is
wider than trap 858, so multiple expulsion channels 892 are used to
release particles from confinement in trap 882, rather than one.
Second, perfusion channel 894 has been moved slightly away from
focusing channel 896 to ensure effective delivery of reagents to
all particles in trap 882.
[0479] Released particles generally may be discarded or saved for
further treatment and/or analysis, for any trap size or
configuration. Particles to be discarded may be carried toward
waste reservoir 868 by opening valve V3 and closing valves V1 and
V4 (see FIG. 24). Alternatively, particles to be saved may be
carried toward particle collection mechanism 854 by opening valve
V4 and closing valves V3 and V1 during particle release. Thus,
valves V3 and V4 provide a sorting mechanism 898 to selectively
discard or collect each individual particle or group of
particles.
[0480] Once a retained particle has been released, system 850 may
be readied to trap another particle. Toward this end, valve V4 is
closed, if it-was opened during particle release, and valves V1,
V2, and V3 are opened. System 850 then is ready to receive another
particle.
[0481] Embodiment 3
[0482] FIGS. 24 and 27 show a particle collection mechanism 854, in
accordance with aspects of the invention. Collection mechanism 854
includes an inlet channel 904, a retention area 906, filter
channels 908, and an outlet 910. Inlet channel 904 carries released
particles toward retention area 906 when valve V4 is open during
release. Fluid flows through retention area 906 to outlet 910 by
passing through filter channels 908, which act as size-selective
channels that prevent released particles from flowing to the
outlet. Thus, released particle are collected in retention area
906. When the collected particles are cells, the retention area may
be used to culture cells to promote cell growth, differentiation,
and/or response to a treatment, such as by perfusion mechanism 860.
Alternatively, the retention area may be operatively connected to a
measurement system for particle analysis, and/or may be a site of
particle lysis or further treatment. In some embodiments, inlet
channel 904 may be connected to other channels (not shown) that
allow reagents to be introduced to retention area 906 separate from
particle retention, treatment, and analysis at trap 858.
Alternatively, or in addition, reagents may be introduced by
perfusion mechanism 860 and/or focusing channel 896. Particles
collected in retention area 906 may be released by reversed flow to
send them up inlet channel 904 or by forming collection mechanism
854 so that a valve (or valves) replaces some of the filter
channels.
[0483] Embodiment 4
[0484] Standard particle input mechanisms, such as inlet-well 330
of FIG. 8, are sufficient for single-use microfluidic systems.
However, these mechanisms may be inadequate for reusable systems.
In reusable systems, it may be desirable to load a suspension of
particles into an inlet-reservoir(s) at the beginning of an
analysis, and then to use that same suspension as a source for
multiple particle loadings and analyses. Unfortunately, during such
extended analyses, particles typically settle out of the
suspension, so that the particle input concentration decreases with
time, increasing the amount of time required to load particles.
Thus, there is a need for a mechanism for maintaining particles in
suspension in an inlet reservoir during extended analyses, to allow
repeated loading and analysis of particles from this
suspension.
[0485] FIG. 28 shows a particle suspension mechanism 920 that may
be integrated into reusable microfluidic systems, such as systems
850 and 880 described above. This suspension mechanism helps to
maintain particles in suspension and/or helps to resuspend settled
particles during the course of analyses with a reusable
microfluidic system. Mechanism 920 includes an inlet reservoir 922,
recirculation channels 924, and pumping valves 926. Inlet reservoir
922 receives and stores particle suspensions during analyses. Thus,
reservoir 922 may be an interface with the macroscopic world.
Recirculation channels 924 are joined at each end 928 to the base
of reservoir, but are spaced from the reservoir at an intermediate
portion 930. Pumping valves 926 are regulated by the control layer,
and are coordinated to peristaltically pump fluid through
recirculation channels 924, as described elsewhere in this Detailed
Description. Accordingly, fluid in reservoir 922 flows away from,
and then back to, reservoir 922, continuously acting to mix the
contents of reservoir 922 and thus to maintain the particles in
suspension. Therefore, a more stable concentration of particle
flows from outlet 932 over time.
Example 8
Microfluidic Mechanisms for Adjustable Reagent Delivery
[0486] This example describes mechanisms for adjustably diluting
reagents so that reagents may be delivered to particles at a range
of reagent concentrations, for example, as a gradient; see FIGS.
29-30.
[0487] Background
[0488] Studies of cells frequently involve dose-response analyses
to determine how the cells respond to a range of concentrations of
a reagent, such as a drug. These dose-response analyses may be used
to determine a variety of qualitative and/or quantitative
information, including an effective dose, a half-maximal response
dose, a lethal dose, a dose to produce a more specific response,
and so on. In many analyses, a reagent of interest is prepared as a
high concentration stock solution, and then various volumes of the
reagent are dispensed to provide a range of doses. However, this
approach may not be suitable with microfluidic systems, because it
may not be practical to dispense metered volumes in a microfluidic
system and because it may require a mixer to mix and thus dilute
such a dispensed volume. Thus, there is a need for a microfluidic
mechanism that dispenses a premixed reagent at a range of selected
concentrations, using a small number of reagent stocks.
[0489] Description
[0490] This section describes two exemplary dilution mechanisms,
having independent (Embodiment 1) and coordinated (Embodiment 2)
control.
[0491] Embodiment 1
[0492] FIG. 29 shows an adjustable dilution mechanism 960 for
combining first and second reagents at a range of concentrations,
in accordance with aspects of the invention. Dilution mechanism 960
includes a microfluidic layer 962 having first and second reagent
reservoirs 964, 966, and first and second controllable flow
channels 968, 970 acting as outlets for the reservoirs. The
controllable flow channels narrow and meet at a junction 972 to
form a common mixing channel 974. Reagents are mixed in mixing or
diffusion channel 974, generally by diffusion of reagents into the
adjacent flow stream(s). Thus, mixing channel 974 may be
substantially narrower than flow channels 968, 970, generally about
1 to 20 .mu.m. In contrast, flow channels 968, 970 are wide enough
to be controlled by valves, with an arcuate cross-section. Here,
fluid flow from each reservoir is independently controlled by
control layer 976, via three-valve pumps 978, and shutoff valves
980; however, fluid flow in other embodiments may be controlled by
other control mechanisms.
[0493] Dilution mechanism 960 is used to combine first and second
reagents, R1 and R2, in a desired ratio based on the rate at which
each pump moves fluid through flow channels 968, 970. Thus, reagent
R1 may be introduced, for example, at 100%, 50%, 20%, 10% and 0% of
reservoir 964 concentration, by running pumps 976 and 978 at
relative pumping flow rates of 1:0, 1:1, 1:4, 1:9, and 0:1,
respectively. Valves 980 may be used to override the pump and/or to
modulate the effect of a specific pump rate, as described below. To
improve control, the adjustable dilution mechanism may use
relatively precise control of pump speed and a large number of
control lines in the control layer.
[0494] Embodiment 2
[0495] FIG. 30 shows another adjustable dilution mechanism 990 for
combining first and second reagents at a range of concentrations,
in accordance with aspects of the invention. Dilution mechanism 990
is structured similarly to dilution mechanism 960, as indicated by
components with identical numbering. However, dilution mechanism
990 uses a single pump 978, generally at a constant pumping rate,
to coordinately drive flow of both reagents. Furthermore, mechanism
990 uses adjustable valves 994, 996, rather than shutoff valves.
Closure of adjustable valves is controllable by regulating the
pressure used to deflect the adjustable valves. Thus, each
adjustable valve may be independently adjusted with a suitable
pressure to provide a desired partial obstruction to flow channels
968, 970, and thus a desired flow rate and reagent mixture in
diffusion channel 974. A simple dilution of a first reagent may be
carried out by using an appropriate solvent or buffer as the second
reagent.
[0496] Applications
[0497] The dilution mechanisms described above may be used as
part(s) of any suitable microfluidic device, for any suitable
applications. For example, dilution mechanism 990 may be used in
microfluidic system 250 in FIG. 8 of Example 2 to prepare and
deliver a desired mixture of reagents for particle perfusion, by
providing empirically determined pressures to valves 9 and 10.
Example 9
Microfluidic Sorting Mechanisms Based on Centrifugal Forces
[0498] This example describes mechanisms for sorting particles
based on their mass, density, and/or other properties; see FIGS.
31-38.
[0499] Background
[0500] Microfluidic analyses of particles may benefit from or even
require sorting crude or heterogeneous input populations of
particles into their components. For example, the input population
may be a mixture of single cells, cell clusters, and/or cell
debris. Alternatively, or in addition, the input population may be
a mixed population of distinct cell types. In these cases, sorting
may separate single cells from clusters and debris, and cells of
one type from cells of another type. Optical systems may be used to
actively sort individual particles according to their different
optical properties, such as fluorescence intensity. However, these
optical systems require that the input particles be constantly
monitored and actively directed to distinct sorting bins based on
optical properties. Thus, there is a need for a microfluidic
sorting mechanism that separates distinct particles, potentially
passively, based on different physical properties of the distinct
particles.
[0501] Description
[0502] This example describes mechanisms for passively sorting
particles based on physical differences between the particles, such
as mass, density, shape, and/or surface characteristics, among
others. These mechanisms are passive, exploiting the centrifugal
forces exerted on flowing particles during a sharp change of
direction, rather than active monitoring and switching. These
mechanisms are described and demonstrated as part of simplified
fluidic systems lacking valves and other functional mechanisms.
Instead, fluids are moved through these systems by pressure
differences produced by liquid columns having different heights in
input and output reservoirs. However, these sorting mechanisms may
be integrated into any suitable microfluidic system.
[0503] Embodiment 1
[0504] FIGS. 31 and 32 show a microfluidic system 1020 having a
sorting mechanism 1022 that separates particles according to
physical differences between the particles, in accordance with
aspects of the invention. Here, mechanism 1022 sorts particles from
inlet reservoir 1024 into one of three outlet or sorting channels
1026. These sorting channels lead to distinct outlet reservoirs
1028, labeled here as outlets 1-3. The sorting channels in this
embodiment have a minimum width of about 50 .mu.m and a height of
about 17-18 .mu.m. However, more generally, mechanism 1022 may be
formed with any suitable dimensions. Furthermore, mechanism 1022
may sort particles from any suitable source, such as a microfluidic
treatment or analysis, into any desired number of outlet channels
and/or other microfluidic mechanisms or structures, such as culture
chambers, retention mechanisms, perfusion mechanisms, and/or the
like.
[0505] Mechanism 1022 includes structures that act sequentially
along a flow stream. First, hydrodynamic focusing region 1030 acts
to focus particles from particle inlet channel 1032 into a narrow
stream. Two side reservoirs 1034, 1036, each filled with a focusing
fluid, such as a buffer, are connected to inlet channel 1032 using
focusing channels 1038, 1040. Focusing channels 1038, 1040 may have
different widths, and thus different flow rates, to asymmetrically
position the narrow stream in the inlet channel. Second,
acceleration region 1042 narrows the width of the channel to
increase the flow velocity and further focus particles into a
single stream. Third, curved region 1044 bends sharply to give the
input particles an angular velocity and a radial acceleration.
Fourth, a separation region 1046 is positioned after curved region
1044. Separation region 1046 widens into a larger chamber with a
number of receiving or sorting channels 1026 that act as sorting
bins to segregate sorted particles. In separation region 1046,
particles are distributed based on their mass (weight). The
tendency of particles to continue moving in a straight line
increases with mass, so that heavier particles move to the outside
of the flow stream, and lighter particles remain closer to the
center of the flow stream. Accordingly, in this embodiment, the
heaviest particles tend to distribute more to receiving channel
1048, the lightest particles to receiving channel 1050, and the
intermediate-mass particles to receiving channel 1052. In some
cases, other physical properties of the particles, such as density,
shape, and/or surface properties, among others, also may contribute
to the relative distributions of particles between these receiving
channels.
[0506] The sorting capabilities of sorting mechanism 1022 may be
modified by altering one or more of several potential sorting
parameters. These sorting parameters may include the extent of
narrowing of the acceleration region, the radius of curvature of
the curved region, the angle of broadening of the separation
region, and/or the number of receiving channels/bins, among others.
These parameters may impart such capabilities as improved
resolution, separation into a different number of sorting channels
(bins) and/or resolution of a different range of particle weights,
densities, etc.; among others.
[0507] Embodiment 2
[0508] FIG. 33 shows a microfluidic system 1060 having a sorting
mechanism 1062 with modified sorting parameters, in accordance with
aspects of the invention. Sorting mechanism 1062 has a narrower
acceleration region 1064 than acceleration region 1042 of sorting
mechanism 1022, potentially imparting greater velocity to the
particles, and thus better focusing. In addition, sorting mechanism
1062 has a curved region 1066 with a distinct radius of curvature
relative to curved region 1044 of sorting mechanism 1022.
Furthermore, sorting mechanism 1062 has a separation region 1068
having a greater angle of separation (subtended angle) than
separation region 1046 of sorting mechanism 1022, connected to
four, rather than three, sorting channels 1070.
[0509] Embodiment 3
[0510] FIGS. 34 and 35 show another microfluidic system 1080 having
a sorting mechanism 1082 with modified sorting parameters, in
accordance with aspects of the invention. Sorting mechanism 1082
has a narrower acceleration region 1084 than either region 1042 or
region 1064, providing even greater velocity and focusing. In
addition, sorting mechanism 1082 has a curved region 1086 with a
smaller radius of curvature than curved regions 1044 and 1066 of
FIGS. 31-33. Furthermore, sorting mechanism 1082 has a separation
region 1088 with an even greater angle of separation, compared to
regions 1046 and 1068.
[0511] Applications
[0512] FIGS. 36-38 show experimental results demonstrating the
ability of systems 1020 and 1060 to sort a mixed population of
particles. In these experiments, the mixed population of particles
was formed, prior to loading into an input reservoir, using two
sizes (and types) of particles: beads with an average diameter of
about 1 .mu.m, and Jurkat cells with an average diameter of about
10 .mu.m. These two sizes of particles are distinguishably labeled
with distinct fluorescent dyes: the beads emit green light, and the
cells emit red light.
[0513] FIG. 36 shows an image of particles being sorted using a
sorting mechanism as described in this example. The particles are
split into two streams 1100 in the separation region. The lower
stream is enriched for cells (red), and the upper stream is
enriched for beads (green). Flow of particles through the system is
powered by a 1-cm high column of fluid in the inlet reservoir.
[0514] FIGS. 37 and 38 show graphs of data obtained with systems
1080 and 1020, respectively, as each sorted the mixed population of
beads and cells, described above. These graphs were generated by
counting the relative numbers of particles that entered each of two
receiving channels. The graphs each plot the fraction of cells
(blue diamonds) and beads (pink squares) that distribute to the
lower receiving channel, either sorting channel 1102 or 1048,
respectively. The ratio of cells to beads in the lower receiving
channel is plotted in yellow. In both system 1080 and 1020, a
greater fraction of cells than beads are entering the lower
receiving channel. In system 1080, about twice as many cells as
beads entered the lower receiving channel. In system 1020, this
ratio was slightly lower and more variable.
[0515] Summary
[0516] The systems shown in this example have the ability to
passively enrich particles based on sorting mechanisms that
distinguish physical properties of particles. The approximately
two-fold enrichment obtained using these systems may be sufficient
to facilitate or improve some microfluidic analyses. Furthermore,
each of these systems may be modified and refined, and/or connected
in series to improve enrichment of desired particles.
Example 10
Microfluidic Systems for Manipulating Sets of Particles
[0517] This example describes microfluidic systems having
relatively large chambers, in which larger sets of particles, such
as adherent and/or nonadherent cells, can be retained, stored,
cultured, treated, and/or released; see FIGS. 39-50D.
[0518] Background
[0519] The introduction and/or removal of particles into and out of
microfluidic systems, at macroscopic/microscopic interfaces, may
inefficient and/or harmful. For introduction, particles must be
placed in suspension and often are introduced through an inlet
reservoir. During this loading process, a substantial fraction of
the particles may be lost, which may be problematic if the
particles are expensive and/or in limited supply, such as with
cells from a clinical or forensic sample. Furthermore, during
introduction and/or removal, particles may be contaminated, for
example, by exposure to contaminating microorganisms, and/or
damaged, for example, by evaporation of inlet- or outlet-reservoir
liquid. Accordingly, it is desirable to avoid repeatedly
introducing and removing particles from microfluidic systems during
a sequential set of assays. Therefore, there is a need for chambers
for storing, treating, maintaining, measuring, and/or in
particular, amplifying (i.e., culturing) particles, such as cells,
particularly for serial analyses of particle populations. With such
chambers, these serial analyses could be conducted without
transferring the populations to a macroscopic environment between
analyses.
[0520] However, such chambers need to address a number of problems
or issues related to their use with cells. First, these chambers
may need a ceiling height that does not interfere with cell
movement within the chambers. In particular, the ceiling of larger
chambers, particularly those formed of elastomeric materials, may
tend to sag, obstructing cell movement. Second, these chambers may
need a substrate that promotes adhesion, survival, and growth of
adherent cells, when such cells are being used. Many adherent cells
do not behave normally unless they are attached to a substrate.
Third these chambers may need to pass media and/or reagents over
cells in the chambers, without loss of, or damage to, the cells.
Pumps that circulate fluid may crush fragile eukaryotic cells, and
some filters that restrict cell movement may be clogged by cells
and/or allow cells to pass. Fourth, these chambers may require an
ability for gas to diffuse into cell chambers, to maintain a proper
pH during cell growth.
[0521] Description
[0522] This example describes various microfluidic systems that
address and solve some or all of the problems and issues cited
above. These microfluidic systems may be formed using multilayer
soft lithography, as described elsewhere in this Detailed
Description and in the Cross-References. Channels or chambers for
particle storage, treatment, analysis, and cell growth are formed
using molds fabricated as described generally in Example 13, using
plural layers of photoresist, when needed. Such molds may be used
to construct channels large enough for cell entry and growth, for
example, about 200 .mu.m wide by about 20-35 .mu.m high.
Furthermore, as described below, such molds may be used to form
particle chambers of various dimensions. These channels and/or
chambers may be integrated into microfluidic systems that include
valves, pumps, rotary mixers, filters, sorters, multiplexers,
perfusion mechanisms, and/or additional particle retention sites,
among others, to perform any suitable analysis of particles.
[0523] Embodiment 1
[0524] FIGS. 39-43 illustrate exemplary microfluidic networks 1130
that include relatively large chambers 1132 for retaining
particles, in accordance with aspects of the invention. These
networks have been fabricated using multilayer soft lithography,
with large chambers that did not collapse. These chambers have a
height of about 36 microns. The chambers were formed by a modified
process using molds in which two layers, each of about 18 microns,
were sequentially layered on top of a substrate, and selectively
retained at the positions where the cell chambers were formed. The
chambers were rounded. This process produces a generally arcuate
(arch-like) cross-sectional configuration that may enhance
stability. As a result, this process allows formation of chambers
with width-to-height ratios less than about 10:1 that do not
collapse. In contrast, microfluidic channels having width-to-height
ratios greater than 10:1 formed by a standard soft lithography
process may collapse more frequently.
[0525] The large chambers may be connected to an input reservoir
1134 and an output reservoir 1136. The input reservoir may connect
to an inlet channel 1138 that bifurcates, as shown at 1140, to
direct flow into each of two channels 1142. Outlet channels 1144
extend from each pair of chambers to join and carry fluid to output
reservoir 1136. For more efficient use of space and input
reservoirs, some systems, such as system 1146, share a common inlet
reservoir 1148 for two pairs of chambers. Thus, particles may be
loaded into inlet reservoir 1148 to distribute the particles to
each of four chambers. In other embodiments, an input reservoir may
be fluidically connected to one, two, three, four, or more chambers
using any suitable number of channels. The channels may extend
directly between a particle reservoir and a cell chamber, or they
may branch any desired number of times at any desired number of
positions. The movement of fluid through these chambers may be
controlled by any suitable mechanism, such as valves and/or pumps,
among others. For example, FIG. 44 shows a system 1150, in which an
array of networks 1130 are controlled in parallel by control lines
1152, 1154 that regulate valves 1156 flanking each chamber 1132. In
this case, each of the eight valves shown is opened or closed in
parallel through actuation at control port 1158, either providing
an open chamber for particle loading, or a closed chamber for
particle isolation, respectively.
[0526] Chambers 1132 may have any desired shape and size. Suitable
cross-sectional shapes may include diamonds 1160 (FIGS. 39 and 41),
rectangles 1162 (FIGS. 39, 42, and 43), squares 1164 (FIG. 39),
circles 1166 (FIG. 39 and 40), ellipses or elongated circles 1168
(FIGS. 39, 40, and 41), and/or the like. Suitable sizes are about
100 microns to about 1 centimeter in diameter, depending on
particle type, assay, and so on. Specific chambers shown in FIGS.
39-43 that have been constructed successfully have diameters of
from about 0.9 mm to 2.6 mm.
[0527] Chambers may be completely isolated from the substrate in
their interiors, or they may be supported by columns, posts, or
other structures. These columns or posts may project downward from
the roof of the channel to contact the substrate, generally being
integrally formed in the microfluidic layer during fabrication of
this layer. Alternatively, or in addition, these columns or posts
may project upward from the substrate, being formed as a portion of
the substrate or an addition to the substrate. To be effective, the
columns or posts should be spaced adequately to avoid obstructing
cell movement through the chambers, although more tightly spaced
structures could be used to form a cell pen or other
subchamber.
[0528] Embodiment 2
[0529] FIG. 45 shows a microfluidic system 1180 having a
microfluidic network 1130 through which fluid flow is more flexibly
controlled. Specifically, fluid flow through chamber 1132 is
controllable by two nested sets of flanking control valves 1182,
1184 that sit to both sides of chamber 1132. A parallel pumping
circuit 1186 is disposed as an parallel fluid path 1188, having
pump 1190 and extending from upstream and downstream cell chamber
1132, at an intermediate nested-position between nested valve sets
1182, 1184.
[0530] System 1180 may be operated as follows. During cell
(particle) loading, nested valve sets 1182, 1184 are opened and
fluid flows passively from input reservoir 1134 to output reservoir
1136, bringing cells to chamber 1132. When a desired number of
cells have entered chamber 1132, one or both of valve sets 1182,
1184 are closed to isolate chamber 1132. If only valve set 1182 is
closed, pump 1190 may be activated to circulate fluid through a
loop that include chamber 1132 and alternate fluid path 1188, to
prevent cell adhesion to the substrate, or to maintain a fluid flow
over cells that have adhered. Alternatively, only valve set 1184
may be closed, allowing fluid to flow between input and output
reservoirs using alternate, parallel fluid path 1188, to the
exclusion of a path through chamber 1132. Thus, fluid channels may
be flushed and re-equilibrated with any desired reagent. Once the
fluid channels have been re-equilibrated, the desired reagent,
valve set 1182 may be closed and the desired valve set 1184 may be
opened, to actively pump the desired reagent in a closed loop that
includes chamber 1132. For example, the reagent may be a mixture of
trypsin and EDTA, or another suitable detaching reagent. Pumping
the mixture of trypsin and EDTA through the closed loop detaches
adhered cells. Opening valve set 1182 then allows the detached
cells to be flushed from the system, either to output reservoir
1136 or to any additional microfluidic mechanism or set of
mechanisms, as described throughout this Detailed Description.
[0531] Embodiment 3
[0532] FIG. 46 shows a microfluidic system 1210 with a cell chamber
1212 formed as a looped channel or ring structure, in accordance
with aspects of the invention. Cells (or particles) are introduced
into chamber 1212 and retained there, either by balancing fluid
height between input and output reservoir 1214, 1216, respectively,
or by closing one or more valves 1218 that interconnect these
reservoirs. Partial closure of valves 1218, particularly valves
adjacent or within chamber 1212, may be used to permit fluid flow,
while preventing cell flow, past the valves. Once cells are loaded
into chamber 1212, four valves 1220 may be actuated in an
appropriate order to move fluid around chamber 1212
[0533] Embodiment 4
[0534] FIGS. 47-49 shows another microfluidic system 1240 with a
chamber 1242 formed as a looped channel or ring structure, in
accordance with aspects of the invention. System 1240 offers
distinct networks for particle inflow/outflow--particle network
1244--and for reagent inflow/outflow--reagent network 1246. These
distinct networks overlap at chamber 1242.
[0535] Particle network 1244 is used to load particles into chamber
1242 and to receive particles flowing from chamber 1242. Particles
are loaded initially into input reservoir 1248, which feeds the
particles into input channel 1250. Input channel 1250 flows into
chamber 1242 Chamber 1242 bifurcates and rejoins at outlet channel
1252. Outlet channel 1252 carries fluid to output reservoir 1254.
Fluid flow between reservoirs 1248 and 1254 can be terminated at
any selected time by closing one or both of valves 1256 and 1258.
Closing both valves fluidically isolates chamber 1242 from the
remainder of particle network 1244.
[0536] Reagent network 1246 is used to move fluid, particularly
fluid carrying reagents, through chamber 1242, while selectively
retaining particles. Reagent network 1246 directs fluid and
reagents from one or more reagent reservoirs 1260 through inlet
channel 1262 into chamber 1242. Flow from each reagent reservoir
1260 is independently regulated by valves 1264, which control flow
of a single reagent or a mixture of reagents. Desired ratios and/or
dilutions of reagents may be formed by precisely controlling flow
rate through each valve, for example, as described above in Example
8. Reagents entering chamber 1242 from inlet channel 1262 follow a
bifurcated path that rejoins at outlet channel 1266. Outlet channel
1266 carries fluid to waste reservoir 1268. Inflow or outflow can
be regulated with valves 1270, 1272, respectively, which may be
closed to isolate chamber 1242 from reagent network 1246,
particularly during particle loading and/or removal. Furthermore, a
reagent pump 1274 may be used to pull reagents from reagent
reservoirs 1260 to waste reservoir 1268.
[0537] Reagent network 1246 blocks exit (and entry) of particles
from (and to) chamber 1242, based on particle size. To achieve
this, reagent network 1246 interfaces with chamber 1242 using
filtering mechanisms 1276. FIGS. 48 and 49 show photographs of
size-selective channels 1278 disposed in outlet channel 1266,
adjacent chamber 1242.
[0538] Chamber 1242 includes a chamber pump 1280 (see FIG. 47).
Chamber pump 1280 is used to circulate fluid through chamber 1242,
for example, (1) to suspend cells (such as during detachment of
adhered cells with trypsin), (2) to move cells away from filtering
mechanism 1276, reducing or preventing clogging of the mechanism,
(3) to promote mixing within chamber 1242, and/or the like.
[0539] An exemplary method for feeding cells in chamber 1242 is a
follows. One of reagent reservoirs 1260 is loaded with about 20
.mu.L media, and waste reservoir 1268 is loaded with about 10 .mu.L
media (or buffer). These reservoirs have the same diameter, so this
asymmetrical loading gives reagent reservoir 1260 a fluid head of
about 10 .mu.L. Flow to equalize fluid heights subsequently
transfers about 5 .mu.L of media through chamber 1242 to waste
reservoir 1268 over the course of about 30 min. Particle network
1244 may be used instead, or in addition, if the cells in chamber
1242 are adherent.
[0540] System 1240 allows extended culture of adherent cells. FIG.
50 shows NIH 3T3 cells 1290 that are alive and adherent in chamber
1242, 3 weeks after they were seeded. The field of cells shown has
been tested for viability (top panel) and visualized for general
morphology by bright field illumination (bottom panel). A
substantial majority of cells was determined to be alive, as
evidenced by lack of ethidium homodimer staining (Molecular Probes;
Live/Dead Viability Assay Kit), and to have normal morphology.
During the 3-week incubation, cells 1290 were subjected to the
passive-flow feeding regimen described above, repeated once every 2
days.
[0541] Embodiment 5
[0542] FIG. 50A shows a system 1910 for depositing cells (or other
particles) in a microfluidic chamber 1912, based on an
asymmetrically disposed flow path. Particles and fluid flow into
chamber 1912 from inlet channel 1914. The particles and fluid may
follow plural distinct flow paths 1916, 1918 toward outlet channels
1920, 1922, respectively. One or more valves 1924 may be used to
select one or both of the flow paths.
[0543] Selection of asymmetrically disposed flow path 1916 allows a
subset of inputted cells to be deposited in chamber 1912. Main flow
path 1916 may be both asymmetrically disposed and nonlinear. Such a
flow path defines a highest velocity main stream corresponding to
main flow path 1916. However, some of the fluid also follows
lower-velocity auxiliary streams (weaker flow streams) disposed
more distally in chamber 1912, in quasi-stagnant region 1926.
Accordingly, the subset of cells that follows the auxiliary streams
within chamber 1912 tend to be deposited in chamber 1912 by
settling out and contacting a substrate defined by the chamber.
Such contact diminishes the ability of fluid flow to move the
settled cells and may promote additional interactions between the
settled cells and the substrate, such as formation of a secreted
extracellular matrix. In other embodiments, the subset of cells
that are deposited may be determined by varying any suitable
parameters including degree of nonlinearity of flow path 1916,
location of flow path 1916 relative to the chamber, chamber
dimensions, fluid flow rate, and/or the like.
[0544] Embodiment 6
[0545] FIG. 50B shows a system 1930 that is based on system 1910
but includes additional mechanisms and features. System 1930
includes an input mechanism 1932, an output mechanism 1934, and a
treatment mechanism 1936. Input mechanism 1932 includes an input
reservoir 1938 for introducing cells and/or fluid, such as buffer
or media. Output mechanism 1934 includes an output reservoir 1940
that may receive fluid from outlet channels 1942 and/or 1944,
provided by flow paths 1918 and/or 1916, respectively. Valve 1924
may be operated to block flow along path 1918, whereas valve 1948
may be operated to block flow to output reservoir 1940 from either
flow path. Treatment mechanism 1936 may include plural reagent
reservoirs 1950, valves 1952 that regulate flow from each reagent
reservoir, and a valve 1954 to regulate communication between
entire treatment mechanism 1936 and chamber 1912.
[0546] System 1930 may be used to deposit cells as follows. Cells
are inputted by input mechanism 1932, generally with valve 1948
opened, and valve 1924 closed. Cells travel along flow path 1916,
with a subset following auxiliary flow streams to be deposited in
quasi-stagnant region 1926, as described above.
[0547] Once a sufficient number of cells have been deposited within
chamber 1912, the deposited cells may be manipulated further as
follows. Valve 1956 may be closed and the contents of input
reservoir 1938 replaced with media to achieve a fluid head that is
approximately equal to that of output reservoir 1940, to produce no
net flow between reservoirs (a "balanced flow" condition), and then
valve 1956 may be reopened. The deposited cells may be incubated a
suitable time period, such as overnight, during which time they may
adhere by interaction with a substrate defined by the chamber. Such
adhered cells are retained within chamber 1926. Alternatively,
nonadherent cells may be used without attachment to chamber
1912.
[0548] Adhered (or nonadhered) cells may be treated with reagents
from reagent reservoirs 1950 by operating treatment mechanism 1936.
First, reagents may be introduced into chamber 1912 by opening one
or more valves 1952, and valve 1954, to direct selected reagents
along flow path 1958, along a reverse of flow path 1916, and/or
along outlet channel 1944. Next, chamber 1912 may be placed within
a closed loop by closing valves 1948, 1954, and 1956. Pump 1960 may
be started to circulate reagent around the closed loop, providing a
mixing action that continuously perfuses cells in chamber 1912 with
reagent.
[0549] Embodiment 7
[0550] FIG. 50C shows a cell chamber 1970 that may be used to
deposit (and retain) cells in one or two compartments 1972, 1974.
Compartments 1972, 1974 may be connected by radially arrayed,
size-selective channels 1976 to form a "spoked wheel" structure.
Cells (or other particles) may be inputted from first input channel
1978 and deposited in compartment 1972. Fluid may flow through
size-selective channels 1976 to second input channel 1980.
Alternatively, or in addition, additional cells, such as a distinct
cell type, may be inputted from second input channel 1980 to be
deposited in outer compartment 1974, with fluid flowing toward
first input channel 1978. With each of the two compartments
occupied by distinct cell populations, cell-cell communication may
be analyzed by passage of released cell components (or extended
cell structures) through the size-selective channels between the
two compartments. In alternative embodiments, the first and second
compartments may have any suitable geometry, such as interdigitated
fingers or intermeshed spirals, among others, to increase the area
of communication between the two compartments. Furthermore,
additional compartments may be added to measure interactions
between additional cell types.
[0551] Embodiment 8
[0552] Cell chamber 1990 is a modified version of chamber 1970 that
includes an overflow capability. Here, inner compartment 1972 acts
as a chamber that is connected to overflow compartment 1992 by
transverse passages 1994, in addition to size-selective channels
1976. Accordingly, input channel 1978 may be used to direct most of
inputted cells (or other particles) into inner compartment 1972
using entrance 1996. However, once inner compartment 1972 becomes
filled, additional cells may travel along transverse passages,
through overflow compartment 1992 and out outlet channel 1998.
[0553] Applications
[0554] The microfluidic systems described here may be used for the
manipulation of adherent and nonadherent cells. For example, after
introduction to a chamber, NIH 3T3 cells adhere to the substrate to
retain the cells effectively within the chamber. Once adhered,
these cells remain attached to the substrate as fluidic flows are
directed over them passively and/or actively. These cells remain
viable at a range of flow rates and valve closure pressures.
However, cell viability may be compromised when higher valve
actuation pressures are used, because higher pressures lead to
complete valve closure. A valve that closes upon a cell can crush
it. In particular, at high pumping frequencies, all cells within a
population inside a ring may be crushed, since they have a high
probability of being crushed. In this case, the ring may become
filled with cell debris, which may be a starting point for assays
on cell components. The nuclear membrane may or may not be
compromised by this treatment.
[0555] In general, manipulation of adherent cells on the chips is
achieved in the following manner. Adherent cells are prepared from
seed flasks by releasing the cells from the flasks, for example, by
trypsinization, followed by washing, centrifugation, and
resuspension in a standard tissue culture medium, such as DMEM or
RPMI. Once a desired concentration has been achieved, cells are
loaded using a manual pipettor into the input well and cells flow
into the microfluidic channel structures under the head flow
generated by the column of liquid. Once adhered, adherent cells can
be resuspended in the microfluidic channel by addition of
trypsin-EDTA or other cell-detaching agents.
[0556] The microfluidic layer and substrate may be treated (or left
untreated) to promote cell flow, cell viability, cell adhesion or
nonadhesion, cell growth, and/or the like. Fluidic channels and/or
the substrate may be treated with a nonionic detergent, such as
TWEEN; a serum protein, such as a serum albumin (e.g., BSA); whole
or fractionated serum from any suitable animal; extracellular
matrix extracts, components, or mixtures, such as collagen,
polylysine, SIGMACOTE, MATRIGEL, etc.; and/or the like.
Example 11
Systems for Electrophysiological Analysis of Cells in a
Microfluidic Environment
[0557] This example describes microfluidic systems for positioning,
retaining, treating, and/or measuring cells, particularly for
electrophysiological analyses; see FIGS. 51-58.
[0558] Background
[0559] Cell-surface membranes are an essential part of all cells,
defining their extent, and separating and maintaining the
differences between the cell interior (cytoplasm) and the
extracellular milieu. Accordingly, controlling membrane
permeability and the selectivity of ion movement across membranes,
mediated by ion channels and transporters, is fundamental to cell
survival, cell physiology, and signal transduction mechanisms,
particularly neurotransduction. Thus, many cell-surface receptors
couple to ion channels and transporters, making measurement of
membrane currents a very rapid and sensitive indicator of cell
physiology and receptor activity. Therefore, many drug assays
benefit from or, in some cases, require a measurement of the
effects of drugs on ion currents, referred to as
electrophysiology.
[0560] The preferred method for conducting electrophysiological
analyses of cells membranes is the "patch-clamp" analysis of
individual cells. Typically, in this approach, a glass electrode
with a diameter of about 0.1-1 .mu.m is electrically sealed against
the membrane of a single cell, surrounding a membrane "patch" on
the cell. The patch then may be left intact, separated from the
cell, "perforated" with channel-forming agents, or penetrated,
based on the type of information desired. With both intact patches
and patches separated from a cell, the size of the patch and the
density of channels in the membrane determine the number of
channels being analyzed. Thus, different sizes of patches may allow
"single-channel recordings" from small regions of membrane, or
recordings from many of channels in "macropatch recording."
Alternatively, membrane patches can be perforated or penetrated to
measure electrical properties of the entire cell membrane, in
"whole-cell" patch-clamp studies. Perforated patches introduce a
channel-forming agent, such as the antibiotics nystatin or
amphotericin B, into the membrane. Perforated patches enable whole
cell recording of channel activity with loss of larger cytoplasmic
components. Penetrated patches place an electrode inside a cell, so
that the electrode and the cell's cytoplasm are continuous.
Accordingly, penetrated patches also enable whole-cell patch-clamp
recording.
[0561] Despite the importance of electrophysiology as an assay tool
and the variety of patch-clamp methods available for measuring
electrical activity at membranes, these methods require substantial
time and skill for their proper execution. In particular, each of
these methods generally is carried out manually, by a
highly-skilled electrophysiologist. The electrophysiologist must
precisely position an electrode against the membrane of each cell,
and manipulate the electrode and/or cell additionally to form a
gigaseal and/or penetrate the cell. Accordingly, the
electrophysiologist must devote considerable time and energy to the
execution of patch-clamp methods, making them expensive and
ill-suited to screening applications in which many samples must be
studied. Thus, there is a need for a more automated system that
simplifies cell manipulation and at least partially automates patch
formation.
[0562] Description
[0563] This example describes microfluidic devices that allow
measurements of ion channel activity. These devices position a
single cell in abutment with an aperture, so that the cell's
membrane forms a high resistance, gigaohm seal, termed a gigaseal,
around the aperture. The gigaseal allows channel currents across
the cell membrane to be measured, by "whole cell" patch-clamp
recording. Measurement of currents in the presence and absence of
potential modulators of channel activity, such as agonists and
antagonists of receptors that couple with channels, provides a
rapid and sensitive method for testing these modulators. Since
changes in channel currents often are transient, the device also
facilitates rapid perfusion of the cell with potential modulators
and wash solutions. This allows rapid exposure and removal of the
modulators. The device may be configured as a system that
simultaneously and/or sequentially analyzes more than one single
cell (see, among others, Example 12).
[0564] Embodiment 1
[0565] FIG. 51 shows a microfluidic device 1310 for measuring ion
currents, in accordance with aspects of the invention. Device 1310
includes a planar patch clamp electrode consisting generally of
three layers: a substrate layer 1312, a fluidic layer 1314, and a
base layer 1316.
[0566] Substrate layer 1312 includes one or more patchable orifices
1318, of about 0.1-5 .mu.m, or about 1-5 .mu.m in diameter. The
perimeter of each orifice forms a gigaseal with the membrane of a
single cell being analyzed. Accordingly, substrate layer 1312 may
be fabricated from any nonconducting material capable of forming a
highly resistant seal, and may be relatively hard. Suitable
materials for the substrate layer include glass, silicon, and/or
plastic, among others.
[0567] The substrate layer separates fluidic layer 1314 and base
layer 1316. The fluidic and base layers each are filled with one or
more buffer solutions that mimic the external and internal ionic
environments, respectively, of single cells being analyzed. These
buffer solutions may be referred to as external and internal
buffers, respectively. The movement of ions through the cell
membrane, effectively between the fluidic and base layers, creates
currents that can be measured using sensitive amplification
equipment. The fluidic layer may be formed by any suitable
technique, such as multilayer soft lithography, for example, as
described elsewhere in this Detailed Description. The fluidic layer
may be controlled by any suitable control mechanism, such as an
overlying microfluidic control layer 1320. The base layer may be
formed out of any suitable material, such as glass, plastic, and/or
an elastomeric material, among others. The base layer may be cut
(punched), molded, etched, and/or embossed, among others, to (1)
form a tight seal with substrate layer 1312, and (2) form a
reservoir holding internal buffer that is in fluidic contact with
each orifice and that accepts an electrode and/or electrode plate,
typically connected to suitable stimulation and recording
equipment. In preferred embodiments, the bore of the patch clamp
channel may be large enough to permit dislocation or dislodging of
the particle from the patch clamp when fluid flow is reversed
through the bore of the patch clamp channel.
[0568] Embodiment 2
[0569] FIGS. 52-58 shows a microfluidic system 1340 for single-cell
patch-clamp recordings, in accordance with aspects of the
invention. System 1340 includes a fluid-layer network 1342 and a
fluid control layer 1344, both formed by multilayer soft
lithography, for example, as described elsewhere in this Detailed
Description. Network 1342 and control layer 1344 position a single
cell over a patchable orifice or aperture formed by a substrate
layer (see below). Positioning the single cell establishes an
appropriate buffer gradient between fluid-layer network 1342 and a
base-layer fluidic chamber, as described above for FIG. 51. Once a
high-resistance seal is formed between the positioned cell and the
substrate, around the orifice, system 1340 allows the positioned
cell to be perfused with one or more of a set of reagents, such as
drugs, ligands (for the case of ligand-gated channels), buffers
with distinct ionic compositions, and/or wash solutions. Perfusion
of these reagents permits rapid measurement of the effect of these
reagents on the electrical activity of the cell.
[0570] To carry out these functions, system 1340 includes several
mechanisms that cooperate serially and/or in parallel. A cell
manipulation mechanism 1346 inputs, positions, and retains single
cells. A cell perfusion mechanism 1348 exposes and washes the
retained single cells in a precisely controlled manner using a set
of reagent-input networks. An electrical monitoring mechanism 1350
electrically contacts both the fluid-layer network 1342 and a
base-layer fluidic chamber (not shown) to measure current, voltage,
and/or resistance of retained single cells before, during, and/or
after exposure to desired reagents and/or electrical
manipulations.
[0571] Cell manipulation mechanism 1346 itself includes a set of
mechanisms, including a cell input mechanism 1352, a cell
positioning mechanism 1354, and a cell retention mechanism 1356.
These mechanisms act in a coordinated fashion to manipulate single
cells for patch-clamp experiments.
[0572] Cell input mechanism 1352 generally comprises any mechanism
that acts through an input reservoir 1358 to introduce cells into
fluid-layer network 1342. Input mechanism 1352 is similar to input
mechanism 263 of Example 2. Other suitable input mechanisms are
described above, in Section IV.
[0573] Cell positioning mechanism 1354 generally comprises any
mechanism that acts to position single cells within microfluidic
network 1342. In addition to simple flow channels, the
cell-positioning mechanism may include a focusing mechanism 1360.
Focusing mechanism 1360 places input cells in an input stream 1362
at a central portion of inlet channel 1364, labeled "E1," flanked
by focusing flow streams from focusing reservoirs 1366, 1368,
labeled "F1" and "F2." Mechanism 1360 directs fluid from input and
focusing reservoirs 1358, 1366, 1368 to junction 1370 from three
orthogonal directions. FIG. 53 shows an alternative cell-focusing
mechanism 1372, in which cell-input and focusing streams join at
acute angles, forming an "arrowhead" configuration. Focusing
mechanisms 1360 and 1372 are similar to aspects of positioning
mechanism 263 of Example 2.
[0574] Cell positioning mechanism 1354 stochastically segregates
single cells using a divided-flow mechanism 1374, downstream from
focusing mechanism 1360 or 1372; see FIG. 54. Specifically, focused
cells are directed down inlet channel E1 and encounter a divided
flow path 1376. Divided flow path 1376 directs fluid to a waste
reservoir 1378 (see FIGS. 52 and 53) through outlet channels 1380,
1382 (labeled "W1" and "W2," respectively, in FIG. 54). These
outlet channels include a narrowed portion 1384 and a
size-restrictive channel 1386 that determine the relative flow rate
through each corresponding outlet channel. Narrowed portion 1384
has a substantially larger diameter than size-selective channel
1386, so that most of the flowing fluid (and cells) passes through
narrowed portion 1384. However, some fluid passes through
size-restrictive channel 1386, eventually bringing a single cell
1388 to the mouth of the channel.
[0575] Cell retention mechanism 1356 generally comprises any
mechanism for retaining a cell at a desired position, generally
adjacent an orifice and/or electrode(s). Here, the cell retention
mechanism functions at the channel mouth; see FIGS. 54 and 57. In
particular, cell 1388 cannot enter size-restrictive channel 1386
because the cell is too large. However, the pressure drop across
size-restrictive channel 1386 pulls cell 1388 against the channel
mouth, holding cell 1388 in position. Positioned cell 1388 may
restrict or block flow through size-restrictive channel 1386, so
that additional cells no longer are urged toward channel 1386. Cell
1388 also is positioned over an orifice 1390 (see FIG. 56) defined
by the substrate layer. In alternative embodiments, single cells
may be positioned and retained over an orifice by any suitable
positioning and/or retention mechanisms, for example, those
described elsewhere in this Detailed Description.
[0576] With cell 1388 in position over orifice 1390, flow from
input reservoir 1358 is terminated, but flow from focusing
reservoir F1 and/or F2 continues. Continued flow from F1 and/or F2
may be used to prevent additional cells from stopping near cell
1388, which might interfere with measurements. In addition,
continued flow from F1 and/or F2 ensures that buffer in the region
surrounding cell 1388 is refreshed. To perform whole-cell
recordings, reservoirs F1 and/or F2, and generally input reservoir
1358, are filled with external buffer, so that all of fluidic
network 1342 is equilibrated with external buffer. In contrast,
base-layer chamber, below orifice 1390, is filled with internal
buffer from a lower face (or side) of the base layer, generally
prior to cell input. The contents of these reservoirs could be
reversed, if the cell is positioned on the opposite side of the
aperture, or for reasons of experimental design.
[0577] Positioned cell 1388 is pulled against orifice 1390 by
applying a vacuum from the base-layer chamber. This establishes a
highly resistant seal, the formation of which can be measured as an
increase in resistance between fluid-layer network 1342 and the
base-layer chamber (below orifice 1390) using electrodes in each
chamber. Generally, fluid-layer network 1342 serves as a ground,
and a recording electrode is positioned in the base-layer chamber.
Once the seal is formed, the resulting patched cell can be measured
for its baseline electrical activity or properties.
[0578] After establishing this baseline, and/or using an average or
calculated baseline, the effect of reagents, such as drugs, may be
tested using perfuision mechanism 1348. FIG. 52 shows the general
layout of mechanism 1348, which includes a shield or wash reservoir
1394, and a series of reagent reservoirs 1396, in this case five
reservoirs, labeled D1-D5. Flow through inlet channels 1398
extending from reservoirs 1394, 1396 is actively promoted by a pump
1400 in control layer 1344. Pump 1400 acts in concert on all inlet
channels 1398 to provide a uniform force for delivering the
reagents and wash buffer. In contrast, flow through each individual
inlet channel 1398 is regulated by a corresponding control valve
1402 that determines whether fluid flows through the inlet channel
1398. Valves 1402 are shown in more detail in FIGS. 53, 54, 56-58,
where these valves are labeled V.sub.W, and V1-V5, corresponding to
control of wash reservoir ("W") and reagent reservoirs D1-D5,
respectively.
[0579] FIG. 55 show perfusion mechanism 1348 in more detail.
Perfusion mechanism 1348 controls exposure of cell 1388 to each
selected reagent using a regulatable fluid sheath or shield,
similar to that described for perfusion mechanism 268 of Example 2.
Wash reservoir W is filled with external buffer, and the buffer is
flowed past cell 1388 from wash inlet-channel 1404 by opening valve
V.sub.W. Specifically, focusing buffer from F1 and/or F2 entering
chamber E1 pushes the wash buffer in a laminar flow pattern or
sheath flow 1406 over cell 1388, against wall 1408. Because wash
inlet-channel 1404 is closer to cell 1388 than any of the reagent
inlet channels 1398, sheath flow 1406 spaces and prevents contact
of reagents flowed from any of the reagent inlet channels. Upon
closing valve V.sub.W, any flowing reagent rapidly contacts the
cell, and recordings can be made as desired. Accordingly, cell 1388
may be exposed rapidly to any reagents in reservoirs D1-D5 in a
controlled manner by selective opening and closing valves V.sub.W
and V1-V5, allowing measurement of electrical responses in a
correspondingly rapid time frame. Therefore, ligands introduced
through reservoirs D1-D5 may be used to study their antagonist or
agonist activity on ligand gated channels, among others.
[0580] Microfluidic system 1340 may be configured in many suitable
ways. For example, reagent inlet channels may unite, entering
chamber E1 through a common port, as shown in system 250 of Example
2 (see FIG. 8). In this way, each reagent is equally spaced by
sheath flow 1406 of the wash buffer and thus will reach cell 1388
at the same time when the sheath flow is terminated. Furthermore,
such a design would allow reagent mixing and dilution, as described
above in Example 8. Alternatively, or in addition, a pump may be
included to drive flow from input reservoir 1358 and focusing
reservoirs 1366, 1368. Furthermore, system 1340 may be modified to
be reusable by including a cell removal mechanism, as described in
Example 7. System 1340 may be modified additionally or
alternatively to include a parallel or serial array of
retention/analysis sites, for example, as described above in
Examples 3-5, or below in Example 12.
Example 12
Microfluidic System for Multiplexed Analysis of Cells by Patch
Clamp
[0581] This example describes microfluidic systems for performing
electrophysiological analysis on one or more cells out of a set of
single cells; see FIGS. 59-61.
[0582] Background
[0583] Patch clamping is an electrophysiological method that relies
on the formation of a seal between a biological membrane (for
instance, a cell) and an aperture. This seal may facilitate the
measurement of small currents created by the passage of ions across
the membrane. However, the seal generally should be tight, since
current leakage around the seal may interfere with, or prevent,
measurement of the small currents across the membrane.
[0584] The efficiency of seal formation is an important issue for
the development of automated, high-throughput devices for screening
drugs based on electrophysiological effects on cells. In manual
patch-clamp systems, the efficiency with which cells can be
successfully analyzed varies, but very skilled technicians
typically achieve properly sealed patches at an efficiency of only
about 50%. A similar efficiency achieved by an automated device
would require the device to "cherry-pick" wells containing properly
sealed patches for use in drug screens, limiting the utility of
such a device. Furthermore, even when properly sealed patches are
formed, more than one cell may need to be analyzed to identify a
typical or average cell response. Thus, there is a need for an
automated device that more efficiently forms sealed patches on
cells, facilitating averaged analysis of multiple cells and
reducing problems associated with cell-to-cell variation in
electrophysiological response.
[0585] Description
[0586] This example provides a multiplexed version of a
single-aperture microfluidic device, with a defined number ("n") of
individually controllable apertures. Each individually controllable
aperture may be used to analyze a single cell by patch-clamp
methods. Because only one patched cell is required to form an
effective seal for each experiment, the use of multiple apertures
increases the probability of forming this seal with the device. In
addition, the device allows each aperture, and its associated cell,
to be included in, or excluded from, an analysis. Thus, signals may
be obtained from each individual cell that is successfully sealed
by electrically isolating each corresponding aperture.
Alternatively, or in addition, an "averaged" signal may be obtained
from two or more of the individually controllable apertures, either
by averaging separate measurements or measuring from two or more
apertures concurrently. Averaged signals may improve the robustness
of any data obtained.
[0587] Single-aperture Embodiment
[0588] FIG. 59 shows a one-aperture device 1430 to illustrate how
each of the n apertures is structured. Device 1430 directs a single
cell 1432 into abutment with an aperture 1434. Aperture 1434
connects chambers 1436, 1438. These internal and external chambers,
1436 and 1438, respectively, carry buffers whose compositions
resemble that of the internal (cytoplasm) and external
(extracellular) environments, respectively, of cell 1432. A vacuum
may be applied to internal chamber 1436 to pull cell 1432 toward
aperture 1434, forming a seal between the cell and aperture.
Sealing and rupture of the cell membrane (whole cell entry) make
the inside of cell 1432 electrically continuous with internal
chamber 1436. In other embodiments, the membrane may be left
unruptured but perforated, for example, by addition of
channel-forming agents to internal chamber 1436, or the membrane
may be left unruptured and unperforated.
[0589] Electrical measurements then may be obtained. External
chamber 1438 may be connected to ground, while internal chamber
1436 may carry a recording electrode, generally connected to an
amplifier. Ions passing through the membrane of cell 1432 create a
current that may be measured following amplification with the
amplifier. Device 1430 may be used to measure changes in ion
channel-associated and/or transporter-associated currents in the
presence of potential drug candidates or other modulators.
[0590] Multi-aperture Embodiment
[0591] FIG. 60 shows a microfluidic device 1450 that is a
multiplexed version of device 1430, in accordance with aspects of
the invention. Device 1450 may include a shared internal chamber
1452 that extends around the perimeter of device 1450. Internal
chamber 1452 may connect to a shared external chamber 1454 using a
plurality of apertures 1456, in this case, four. Each aperture may
be isolatable, both electrically and fluidically, using control
valves 1458 (V.sub.N, V.sub.S, V.sub.E, and V.sub.W). In addition,
each aperture may be disposed immediately adjacent a cell retention
mechanism, such as retention site or trap 1460. Traps 1460 may be
arranged so as to facilitate parallel loading from a single
suspension of cells (one reservoir) or from plural suspensions of
cells (plural reservoirs). Internal chamber 1452 may be connected
to a vacuum supply, and a recording electrode and ground may be
connected to external and internal chambers, 1452 and 1454,
respectively.
[0592] Device 1450 may be readied and used as follows. First,
internal chamber 1452 may be loaded with internal buffer at
internal-chamber port 1462 (Port I), so that internal buffer is
loaded up to apertures 1456. Next, open valves V.sub.N, V.sub.S,
V.sub.E, and V.sub.W may be closed, and cells may be loaded as a
suspension using an input mechanism at a common input port 1464
(Port C). Then, the cell suspension may flow from Port C to output
reservoirs 1466 ("outlet"). Single cells may be positioned and
retained at each trap 1460 (N, S, E, W) using any suitable
positioning and retention mechanisms, such as those described
elsewhere in this Detailed Description, for example, Examples 1-3.
Once a desired number of cells are retained by retention
mechanisms, device 1450 may be used for cell analysis. The vacuum
supply may be turned on, and one or more valves at a time may be
opened to form an electrical connection between the internal and
external chambers, through the corresponding aperture 1456. The
resistance of the connection may be used to determine if a
sufficient seal has been produced at the aperture, with the
membrane of the retained cell. If so, recording may be
commenced.
[0593] Device 1450 may be modified in any suitable fashion,
incorporating any suitable microfluidic mechanisms, such as those
described in this Detailed Description. For example, device 1450
may be structured to load cells serially and/or in parallel, as
described above in Examples 3-5. Furthermore, device 1450 may be
included in an array of such devices to form a microfluidic array.
Alternatively, or in addition, device 1450 may include a perfusion
mechanism, such as that described in Examples 2 and 8, to allow
precise delivery of selected reagents, to individual cells or to a
plurality of cells, serially or in parallel. Similarly, device 1450
may measure electrical parameters of cells serially, that is, by
using one aperture at a time, or in parallel, by using two or more
apertures at a time, to obtain a summed reading of all connected
apertures.
[0594] FIG. 61 shows data from a simple statistical analysis
illustrating a few of the advantage of a multiplexed patch-clamp
system, such as system 1450. The fractional probability of
successfully obtaining a seal in a well containing n apertures,
P.sub.n, is related to the fractional probability of failed seal
formation, P.sub.f, at a single aperture by the equation
P.sub.n=1-P.sub.f.sup.n. The probability of successful seal
formation for a single aperture, P.sub.s, is related to P.sub.f by
the equation P.sub.f+P.sub.s=1. Therefore, if a seal is obtained
successfully in 50% of attempts, then with 4 apertures,
P.sub.4=1-(0.5).sup.4=1-0.0625- =0.9375. This corresponds to a
93.75% chance of obtaining at least one seal among the four
apertures. FIG. 61 graphs the relationship between n (x-axis) and
P.sub.s (y-axis), with curve 1474 indicating (n, P.sub.s) pairs
that give a 95% probability of at least one of the n apertures
forming a successful seal. (Apertures are called "channels" in FIG.
61.) P.sub.n approaches unity, as P.sub.s and/or n are
increased.
Example 13
Multilayer Mold-Fabrication Method of Varying Height and/or
Cross-Sectional Geometries of Molded Microfluidic Structures
[0595] This example describes a method for producing, by soft
lithography, microfluidic devices in which the cross-sectional
geometry and/or height of structures within and/or between
microfluidic networks vary; see FIGS. 62-71.
[0596] Background
[0597] A microfluidic network may include structures having a
variety of functions. For example, regulatable channels may include
deflectable valves, acting to partially or completely close the
channels and/or to propel fluid through the channels. These
channels generally are formed with a semicircular or arcuate
cross-sectional geometry to enable efficient valve closure. By
contrast, particle-positioning channels may act primarily as
conduits for particles carried by fluid. These particle-positioning
channels generally have a height sufficient to allow particle
movement. Accordingly, particle-positioning channels may benefit
from a rectangular cross section to enable particles to move
unrestrictedly from side-to-side (transversely) within the
channels. Such unrestricted movement may allow particles to occupy
a greater proportion of the width of the channels, rather than just
the central portion, as with arcuate channels. Other channels may
be size-selective or particle-restrictive, preventing entry of
particles greater than a given size. These particle-restrictive
channels may have a height that is less than the diameter of
particles of interest. Furthermore, microfluidic networks may
include cell/culture chambers with roof heights that are greater
than more narrow channels, as described in Example 10, to improve
the functionality of the chambers. Therefore, these and other
structures described elsewhere in this Detailed Description may
benefit from, or require, roof height to vary in order to function
properly.
[0598] Single-layer molds often are formed using a desired
thickness of photoresist on a substrate. The photoresist is
patterned using a corresponding template that allows selective
light exposure and photosensitization of patterned regions of the
photoresist. Depending on whether the photoresist is positive or
negative, the selectively exposed regions are either resistant or
sensitive, respectively, to subsequent removal during development
with a suitable developing agent. This development nonspecifically
removes all sensitive regions, generally down to the substrate. The
resistant regions are generally rectangular in cross-section, but
may be heated to round their edges into an rounded/arcuate
configuration. Accordingly, these remaining regions of the
resulting mold may produce microfluidic channels of complementary
structure using soft lithography. In other embodiments, multiple
layers of photoresist may be built up by sequential coating,
masking, and
[0599] Despite the importance of varying height and/or
cross-sectional shape across a microfluidic network, molds formed
from a single layer of selectively removable material, such as
photoresist, may not allow sufficient flexibility in the structure
of a microfluidic network formed from the mold. For example, the
depth to which the single layer may be removed cannot be varied
readily, producing features of a single height, generally equal to
the thickness of the single layer. Similarly, cross-sectional
geometry may be difficult to vary within a single layer of the
mold. Treatments that alter cross-sectional geometry, such as
heating, also may act nonselectively across the single layer.
Therefore, a method is needed for forming a mold using plural
selectively removable layers.
[0600] Description of Method
[0601] The method described in this example may be used to form
channels with different cross-sectional geometries and/or heights
at distinct positions within a microfluidic network. A mold is
fabricated using plural layers of photoresist that are each
individually patterned, selectively removed according to the
pattern, and optionally rounded by heating. Thus, each of the
plural layers may contribute only a subset of the resulting mold,
so that the mold's relief pattern is the sum of the remaining
portions from each of the plural layers. Using the mold to form a
microfluidic network allows various types of channels or other
passages to be formed. Channels with a rounded/arcuate
cross-sectional shape may be formed in sections of the network
where valves are needed. These sections may be connected with other
portions of the network that are formed to have a rectangular
profile, to promote particle movement and to enable precise
delivery of one or more particles to a specific area of a
microfluidic network. The specific area can be as small as the
dimension of a single particle, such as a cell. These structures
and other suitable microfluidic structures may be produced using
the method described below. This method focuses on formation of a
fluid layer, but may be suitable for any portion(s) of a
microfluidic system, including a control layer or a base layer (see
Example 11).
[0602] A fluid-layer mold is fabricated in a first series of steps
by micromachining techniques. The fluid-layer mold may be used
subsequently in a second series of steps, as described below, to
mold a complementary microfluidic layer by soft lithography. FIGS.
62-68 illustrate how fluid-layer mold 1480 may be formed by
sequentially disposing, patterning, and selectively removing three
layers of photoresist on or above a silicon wafer. Each layer is
formed at a desired thickness by applying the photoresist, and then
rotating the wafer according to a defined rotational profile to
produce the structure of FIGS. 62, 64, and 67. Next, the
photoresist is baked, patterned by exposure to UV light, and then
developed to selectively remove portions of each layer, shown in
FIGS. 63, 65, and 68. To mold closable channels, a photoresist
layer may be baked at high temperature to round remaining portions,
shown in FIG. 66. Each individual step is detailed further
below.
[0603] The first layer may be applied directly to a bare silicon
wafer (the substrate). The first layer may have any suitable
thickness, in this case 5 .mu.m, and may be formed with any
suitable material, such as a negative photoresist, SU 82005
(Microchem, Newton, Mass.). After application of the negative
photoresist, the wafer may be rotated according to a suitable
rotational protocol to achieve a desired thickness and consistency.
For example, the wafer may be rotated as follows: rotate to 500 rpm
over 5 sec, maintain at 500 rpm for 5 sec, ramp to 3000 rpm over 8
sec, and then maintain at this speed for 30 sec. Then the rotation
may be halted and the wafer heated according to a suitable heating
protocol. For example, the wafer may be heated for 1 min at
65.degree. C., 2 min at 95.degree. C., and finally 30 sec at
65.degree. C. This he process may drive off the solvent in which
the photoresist may be supplied. FIG. 62 shows mold 1480 with
substrate 1482 carrying first layer 1484. The relative sizes of
components here and in related FIGS. 63-69 are not drawn to
scale.
[0604] The first layer may be patterned and selectively removed as
follows. A desired template may be positioned in contact with the
first layer and then exposed to UV light, 160 J/cm.sup.2. Next, the
substrate/first layer may be subjected to a suitable post-exposure
heating protocol, such as: 1 min at 65.degree. C., 2 min 30 sec at
95.degree. C., and 30 sec at 65.degree. C. U (unexposed) first
layer may be washed away with any suitable developer, such as that
supplied by Microchem, followed by washing with acetone and then
isopropanol. Then, the first layer may be subjected to a suitable
post-development heating protocol, such as 1 min at 65.degree. C.,
5 min at 95.degree. C., and then 30 sec at 65.degree. C. This
heating protocol may be followed by a post-development exposure
with UV light, 400 J/cm.sup.2. FIG. 63 shows mold 1480 with first
layer 1484 contributing first-layer relief-structure 1486 (residual
first layer), which may have a height of 5 .mu.m.
[0605] The second layer may be added next and may have any suitable
thickness, in this case a thickness of 20 .mu.m formed by spin
coating. First, mold 1480 may be treated with hexamethyldisilazane
(HMDS) for 10 min. Next, a suitable patternable material, such as a
positive photoresist, PLP 100 (AZ Electronic Materials/Clariant
Corporation) may be applied. Application may be by spin coating,
using any suitable protocol, such as the following: spin the wafer
at 500 rpm, dispense the positive photoresist to the wafer/residual
first layer over 14 sec, spin 15 sec, ramp to 2000 rpm over 5 sec,
and maintain at this speed for 30 sec. Rotation then may be
stopped, and the second layer may be baked for 2 min at 100.degree.
C. FIG. 64 shows mold 1480, at this intermediate stage, carrying
second layer 1488, which covers first-layer relief-structure
1486.
[0606] The second layer may be patterned and selectively removed as
follows. Any suitable template may be positioned in contact with
the second layer and exposed to UV light, 450 J/cm.sup.2. Next, the
second layer may be developed (selectively removed) by any suitable
protocol, such as 3 min. in AZ 400K 1/3 with deionized water. FIG.
65 shows mold 1480 after patterned removal of both first and second
layers 1484, 1488. First-layer relief-structure 1486 and a
second-layer relief-structure 1490 may have distinct heights based
on the thickness of photoresist from which they are formed.
[0607] Second-layer relief-structure 1490 may be rounded by any
suitable heating protocol. For example structure 1490 may be
rounded by the following heating protocol: ramp from 70.degree. C.
to 100.degree. C. (1.degree. C./min), maintain 60 min at
100.degree. C., ramp to 200.degree. C. (1.degree. C./min), maintain
60 min at 200.degree. C., and ramp down to 40.degree. C. (1.degree.
C./min). FIG. 66 shows how this heating protocol may convert
rectangular second-layer relief-structure 1490 (FIG. 65) to rounded
second-layer relief-structure 1492.
[0608] A third layer may be added next and may have any suitable
thickness, for example, a thickness of 20 .mu.m. A suitable
selectively removable material, such as negative photoresist SU8
2050 (Microchem), may be applied to the wafer carrying the residual
first and second layers. Spin coating may be achieved by the
following protocol: the wafer is ramped to 500 rpm over 5 sec,
maintained at this speed for 5 sec, ramped to 5000 rpm over 17 sec,
and maintained at this higher speed for 30 sec. The rotation is
stopped. Next, the third layer may be heated by any suitable, such
as: 2 min. at 65.degree. C., 3 min. at 95.degree. C., and 30 sec at
65.degree. C. FIG. 67 shows third layer 1494, which covers
first-layer and second-layer relief-structures 1486, 1492 at this
stage.
[0609] The third layer may be patterned and selectively removed as
follows. A desired template may be positioned in contact with the
third layer and exposed to UV light, 310 J/cm.sup.2. The exposed
layer may be heated by any suitable protocol, such as 1 min. at
65.degree. C., 4 min. at 95.degree. C., and 30 sec at 65.degree. C.
Next, the third layer may be selectively removed with a suitable
developer, such as that of Microchem, and then may be washed with
acetone followed by isopropanol. Subsequently, the third layer may
be subjected to a suitable post-development heating protocol, such
as 1 min. at 65.degree. C., 5 min. at 95.degree. C., and 30 sec at
65.degree. C. Finally, the third layer may be exposed to UV light
in a post-development exposure of 500 J/cm.sup.2. FIG. 68 shows
mold 1480 having a third-layer relief-structure 1496.
[0610] Any suitable aspects of the method described above may be
modified, and any patternable, selectively removable material may
be used. In addition, any suitable number of layers may be used.
Furthermore, each layer may have any desired thickness, according
to the height of a desired relief structure. When optically
patternable layers are used, each layer may be negative or positive
photoresist, and may be used to form a rectangular or rounded
cross-sectional profile. Relief structures formed by distinct
layers may be nonoverlapping, partially overlapping, and/or
completely overlapping in specific regions or all regions of the
mold. Accordingly, relief structures may represent the sum of
plural selectively removed layers.
[0611] An exemplary method for forming a control-layer mold is as
follows. The mold may be fabricated from a single layer of positive
photoresist. A 20-.mu.m layer of suitable photoresist, such as
positive photoresist PLP 100, may be applied, patterned,
selectively removed, and rounded as described above for the second
layer of the fluid-layer mold.
[0612] The fluid-layer and control-layer molds fabricated above may
be used to mold a microfluidic chip using any suitable material,
particularly an elastomeric material, such as polydimethylsiloxane
(PDMS). Exemplary PDMS elastomers are General Electric Silicones
RTV 615, produced from a two-component mixture of a
prepolymer/catalyst and a crosslinker. In this two-component
mixture, the prepolymer/catalyst (component A) is a
polydimethylsiloxane bearing vinyl groups and a platinum catalyst,
and the crosslinker (component B) bears silicon hydride (Si--H)
groups. Using these specific components, components A and B may
function optimally at a ratio of about 10:1 (A:B). However,
"off-ratios" above and below this ratio may be used for the
fluid-layer membrane and the control layer to promote subsequent
bonding. For example, the control layer may be formed at a ratio of
about 4:1, to provide rigidity and thus mechanical stability, and
the fluid-layer membrane at a ratio of about 30:1. The excess of
either component A or B in these two layers remain reactive near
the membrane surface. Accordingly, these two layers may be abutted
and bonded by post-curing with baking to fuse these layers into a
monolithic structure (see below).
[0613] The fluid-layer and control-layer molds may be fabricated
and joined as follows. After treatment with trichloromethylsilane
(TCMS), a relatively thin PDMS membrane, for example, about 50-150
.mu.m, may be spun on completed fluid-layer mold 1480. FIG. 69
shows a membrane 1498 being formed on fluid-layer mold 1480. In
addition, a thicker PDMS layer, for example, approximately 5-10 mm,
may be formed on the control-layer mold. After suitable first-step
curing, such as 90 min at 80.degree. C., the control layer may be
detached from the mold, cut, and punched to interface properly with
control lines of the control layer. Then, this control layer may be
aligned with the fluid layer, while the fluid-layer membrane 1498
is still attached to the fluid-layer mold. Once assembled, the
fluid and control layers may be cured a second time to chemically
bond them, using a post-curing step of heating for about 3 hours at
80.degree. C. After post-curing, the resulting chip may be detached
from the fluid-layer mold, cut, and punched to create fluid
reservoirs that interface at desired positions with channels.
Finally, the chip may be bonded to a suitable substrate, such as a
glass cover slip, to complete the fluid channels.
[0614] The post-curing step may be modified to enhance
compatibility with cells. Lower ratios of PDMS components A and B,
such as 4:1 (A:B), tend to be toxic to cells, particularly during
cell culture. This toxicity may be due to a diffusible, toxic
material(s) in the control layer. Thus, when a much thicker control
layer, formed at a ratio of 4:1, is fused to a thin fluid-layer
membrane, formed at a ratio of 30:1, the resulting monolithic
structure may have the toxic characteristics of a 4:1 layer, even
within the fluid-layer portion. However, suitable treatment of the
control layer, either alone in contact with the fluid layer
membrane, reduces or eliminates this toxic characteristic. Suitable
treatments that remove or modify the toxic material may include
exposure to heat, a chemical (such as a gas, a liquid, a plasma,
etc.), radiation, light, and/or the like. (Such treatments also may
reduce the movement of fluids within the channel, or components
thereof, into the chip.) In some embodiments, longer post-curing at
elevated temperature may remove or modify the toxic material(s),
enhancing the effectiveness of the resulting chips for cell
experiments. Such a longer post-curing step may be conducted for
about 6 hours, 12 hours, or more preferably about 24 hours or more
at about 80.degree. C.
[0615] Images of Molds and Chips
[0616] FIGS. 70 and 71 show photographic images of fluid-layer
molds and the corresponding microfluidic chips formed with these
molds. The microfluidic networks represented here, have been shown
and described in system 1340 of Example 11 (FIG. 70) and in a
modified form in system 850 of Example 7 (FIG. 71). Distinct
regions of each mold and fluid layer are indicated by letters A, B,
and C. Area A corresponds to rounded second-layer relief-structures
1492 described above. These areas are color-coded in blue on many
of the figures presented above. Channels of area A are about 200
.mu.m wide and approximately 20 .mu.m high. Area A may be used to
form valves and pumps by overlapping control lines from a control
layer with this area, such as valve 1500 in FIG. 71. Area B
corresponds to third-layer relief-structure 1496. These areas are
color-coded in red on many of the figures presented above. Channels
of area B have a rectangular profile, approximately 100 .mu.m wide
and 20 .mu.m high. These channels enable precise particle control,
because they allow particles to distribute across the width of the
channel, following the walls and/or the center of a fluid
stream(s). Such channels may be used to drive particles to precise
areas of each chip. Area C corresponds to first-layer relief
structure 1486. These areas are color-coded in turquoise on several
of the figures presented above. These channels have a rectangular
profile, 10 .mu.m wide and 5 .mu.m high. Small channels of this
type are used in combination with channels of area A or B to trap
cells or beads. Fluid may flow in these channels entry of cells or
beads may be restricted.
Example 14
Detection System for Kinetic Analyses in Microfluidic Systems
[0617] This example describes a detection system, including a
modulation-demodulation method and the use of tracer materials, for
analysis of kinetic reactions involving particles in microfluidic
systems; see FIGS. 71A-F.
[0618] Background
[0619] Microfluidic systems may be used to measure the kinetics of
many aspects of cellular metabolism. However, metabolic processes
of physiological significance can occur at substantially different
rates, with characteristic times that may range from microseconds
(10.sup.-6 sec) or less to days (10.sup.5 sec) or more. Therefore,
detection methods are needed to measure cellular events that occur
at these vastly differing rates.
[0620] Time-resolved fluorescence spectroscopy has been one of the
most popular approaches to cellular kinetics studies. Typically,
dye molecules are introduced into cells, and emission from the
molecules is produced by excitation with an intense light source
(such as an arc lamp or laser). The intensity of this emission is
monitored over the course of the analysis to infer the kinetics of
a process under study. However, the emission intensity of the dye
molecules may be reduced or extinguished over time by
photobleaching. As a result, some cellular processes that occur
over relatively longer time periods may be more difficult to
monitor in a microfluidic system due to this photobleaching.
[0621] Because the rate of photobleaching is related to the
intensity of exciting light, a weaker light source may be used to
reduce this rate. For example, FIG. 71A shows a comparison of
photobleaching rates versus time using a relatively stronger laser
(1.6 mW) and a relatively weaker laser (1.6 .mu.W). However, the
exciting light source produces a reduced emission signal and
signal-to-noise ratio, since the emission signal is proportional to
the illumination intensity. Therefore, microfluidic analyses would
benefit from a detection system that reduces photobleaching,
increases the ratio of signal-to-noise, and/or allows kinetic
analysis of both fast and slow processes.
[0622] Description of Detection System
[0623] This example describes an exemplary detection system for use
with microfluidic assays, in accordance with aspects of the
invention. The detection system may include a
modulation-demodulation mechanism; see FIGS. 71B-71E. This
mechanism may improve signal-to-noise ratios, allowing use of
weaker light sources, and/or reduce photobleaching, allowing use of
stronger light sources. The detection system also may include a
method using tracer dyes to measure initiation of rapid kinetic
reactions with particles; see FIG. 71 F.
[0624] Light Detection Device
[0625] FIG. 71B shows an exemplary system 2010 for detecting an
optical signal from a sample. System 2010 may include a light
source 2012, optics 2014, a detector 2016, a digital storage device
2018, and a modulation-demodulation mechanism 2020.
[0626] Light source 2012 may be used to illuminate one or more
particles with light to visualize the particle and/or to perform an
assay. The light source may generally may include any mechanism for
producing light having the desired characteristics, including
time-dependent and/or continuous light sources. Suitable examples
may include a laser, a light-emitting diode (LED), or a lamp, among
others.
[0627] Optics 2014 may be used to receive light from light source
2012 and direct the light at the particles and/or to receive light
from the particles and direct it to detector 2016. Optics may
mediate any suitable alteration of light to facilitate analysis,
including refraction, reflection, diffraction, polarization,
attenuation, spectral alteration, and/or scattering, among others.
Suitable optics may include lenses, mirrors, fiber optics, filters,
gratings, etalons, and/or the like. Exemplary optics may include a
conventional microscope or other suitable optical device that is
separate from, or partially or wholly integrated with, a
microfluidic system.
[0628] Modulation-demodulation mechanism 2020 may include a
modulator 2022 and/or a demodulator 2024. Modulator 2022 generally
comprises any mechanism to provide time-dependent variation in the
intensity of exposure of sample to source 2012. This variation may
be intrinsic and/or extrinsic to the light source. Intrinsic
modulation occurs when the light source itself changes in
intensity, as with a pulsed or strobe laser (such as a diode
laser). Such a pulsed laser may be pulsed very rapidly, up to
millions of pulses per second, allowing for high-frequency
illumination of particles. Extrinsic modulation occurs when the
light source is continuous (or quasi-continuous), but a downstream
mechanism alters the intensity of light before it is incident on
the sample. Suitable extrinsic modulators include optical chopper
wheels, Pockels cells, Kerr cells, acousto-optic modulators, and/or
electro-acoustic and other modulation devices. By contrast,
demodulators generally comprise any mechanism for interpreting
signals from detector 2016 based on the activity of the modulator.
The control and interplay between the modulator and demodulator may
be performed using any suitable mechanism, such as lock-in
amplification using custom-designed and/or commercial devices.
[0629] Detector 2016 may be used to detect light, rapidly and/or
repeatedly, and convert the detected light into representative
electrical signals. Such a detector may include a photomultiplier
tube, avalanche photodiode, and/or other photodetector that
provides the ability to rapidly detect light signals produced by a
source 2012 illuminating the particles. Collecting light emitted
through optical filters into photomultiplier tubes or other
photodetectors may enable conversion of photons to electrons for
collection of quantitative information.
[0630] Digital storage device 2018 may digitize and/or store
electrical signals received from detector 2016. These stored
signals may be retrieved, corrected, and/or otherwise converted or
manipulated, and printed or displayed, as desired.
Exemplary Results using a Modulation-Demodulation Mechanism for
Microfluidic Analysis
[0631] FIG. 71C shows a comparison of signal-to-noise ratios over
time without (top) and with (bottom) source and signal
modulation-demodulation- . In this example, an embodiment of
modulation-demodulation mechanism 2020 boosts the signal-to-noise
ratio by a factor of over 2000-fold. Accordingly, weaker light
sources may be used and an emitted fluorescence signal may be
measured over a longer time course.
[0632] FIG. 71D shows use of an embodiment of mechanism 2020 to
determine the rate at which a reagent-particle interaction occurs
in a single experiment. Here, a biotinylated bead has been loaded
into a trap on a microfluidic chip, such as a chip designed
according to system 250 of Example 2. Dye-labeled streptavidin
(reagent) is exposed to the bead in a pulsatile fashion, using
cycles of staining and washing controlled by automated operation of
control valves. In this case, each ten-second cycle includes a
two-second exposure to reagent, followed by an eight-second
exposure to wash buffer. Each cycle produces a spike in
fluorescence intensity. However, the average fluorescence intensity
achieves a near-maximal level in about twenty cycles. Accordingly,
maximal staining occurred in about forty seconds (twenty cycles
times two seconds per cycle). Therefore, flow-based exposure and
washing may be optimized to avoid time-and labor-intensive labeling
and washing steps, and to minimize use of reagent. The pulsatile
exposure illustrated here may be used with any suitable particle
and dye combination to measure the rate at which interaction
occurs.
[0633] FIG. 71E shows the ability of an embodiment of the
microfluidic detection system to measure a kinetic response of
signal transduction in a cell. A calcium sensor dye, Fluo-3, was
loaded into a cell, and the cell was trapped in a microfluidic
chip, such as a chip designed according to system 250 of Example 2.
The trapped cell was stimulated with ionomycin, at about time=120
sec, to promote release of intracellular calcium. The graph shows
intensity of fluorescence, corresponding to intracellular calcium
concentrations, versus time. Such an analysis measures the response
of an individual cell, so compensatory oscillations in calcium
levels are visible.
[0634] Method Using Tracer Dyes
[0635] Most rapid reactions or events are difficult or impossible
to measure unless their starting points can be precisely defined.
Accordingly, a tracer material, such as a tracer dye, may be
included in a reagent of interest to indicate the time at which
fluid containing the tracer dye and reagent contacts a particle(s).
Thus, first detection of the tracer dye in contact with the
particle defines a zero time point at which a reaction or event was
initiated.
[0636] The tracer dye may have any optically detectable property
and may be inert or reactive. Suitable optically detectable
properties are described above in Section VIII. Inert dyes
generally do not contribute directly to a detected assay result.
Therefore, inert dyes generally do not affect cellular metabolism,
and may not interfere optically or chemically with reagent dyes
used to measure information about particles. Inert dyes may be
nonbinding or binding. Nonbinding dyes do not bind to particles and
may simply mark fluid volumes. Binding dyes may bind to particles,
but do not contribute directly to a detected result from particles.
By contrast, reactive dyes react with particles and contribute to a
detected result. Suitable reactive dyes may be detectable when
first combined with particles, but may show a change in an optical
property during an assay. Inert or reactive dyes may be excluded
from cells, may partition into particles, or may be transported
into the interior of cells. Inert and reactive dyes that may be
suitable are sold by Molecular Probes, Eugene, Oreg.
[0637] Rapid perfusion mechanisms, such as perfusion mechanism 268
of Example 2 above, coupled with a tracer dye and detection system
described in this example, may allow very rapid analyses to be
performed on particles. Such rapid analyses may measure events that
occur in less than about 2 sec, 1 sec, or 500 msec. Furthermore,
these rapid analyses may be performed on living cells to measure
cell responses that are not detectable readily by other
methods.
[0638] FIG. 71F shows use of an embodiment of
modulation-demodulation mechanism 2020 and a tracer dye in a
microfluidic system to measure the rate at which reagent is exposed
to particles. A perfusion mechanism, such as mechanism 268, was
used to expose a retention site to a fluorescent dye. The resulting
increase in fluorescence was measured over time. At time "T," an
electrical signal was sent to a valve controller. After a short
mechanical delay of about 5 msec, fluorescence measured at the
retention site begins to increase, reaching a maximum value in less
than 100 milliseconds. Accordingly, rapid kinetic analyses on a
millisecond time scale may be performed using microfluidic systems
described herein.
Example 15
Microfluidic Analysis of a Heterogeneous Particle Population--Part
I
[0639] This example describes microfluidic systems for sorting and
analyzing heterogeneous populations of particles, particularly
cells, based on differences in particle size; see FIG. 72.
[0640] Background
[0641] Heterogeneous cell populations, such as blood, present a
challenge for rapid analysis. Cells of interest in blood generally
need to be separated from other cells that are of less interest to
avoid interference from these other cells. Accordingly, blood may
need to be treated/manipulated to selectively lyse, coagulate,
pellet, bind, and/or modify, among others, specific cells within
the blood. Such manipulations add to the time and expense required
for analysis of blood, because they involve trained personnel,
expensive equipment, lengthy incubations, repeated transfer of
relatively large volumes of reagent or sample, and/or the like. In
addition, such manipulations expose personnel to increased risk of
exposure to infectious agents in the blood. As a result, many
diagnostic procedures using whole blood are expensive and slow.
Therefore, integrated systems are needed that automatically sort
and analyze heterogeneous cell populations on a microfluidic
scale.
[0642] Description
[0643] This example describes microfluidic systems that sorts blood
cells and other heterogeneous particle populations according to
diameters of individual particles. With these systems very small
volumes of blood may be sufficient for statistically significant
diagnoses or prognoses. Such systems may facilitate analysis of
patient samples with improved speed, accuracy, safety, and/or cost,
among others.
[0644] FIG. 72 shows a microfluidic system 1520 sorting cells.
System 1520 is based on system 250 of Example 2 and includes
positioning and retention mechanisms 264, 266 described in that
example. A blood sample was introduced into system 1520 and
directed toward retention chamber 270. Cells 1522 of this sample
include red blood cells and platelets, but do not include
detectable white blood cells, which would be retained by the
retention mechanism due to their larger diameters. Cells 1522 enter
chamber 270 but exit through size-selective side-wall channels 300.
FIGS. 72 A-D show time-lapse video images that include cells in
chamber 270 and in channels 300. White blood cells such as
lymphocytes, monocytes, and granulocytes (neutrophils, eosinophils,
and basophils), when present, would be retained in chamber 270.
These white blood cells are too large to pass through channels 300.
Therefore, system 1520 may be used to separate red blood cells and
platelets from white blood cells, for selective analysis of the
white blood cells (or red blood cells) in the system.
[0645] System 1520 may be modified to select plural populations of
particles of different size. For example, the system may be
modified to include a serial set of retention mechanisms. Outflow
through size-selective channels 300 for each retention mechanism
270 may be directed partially or completely toward an input site of
a successive retention mechanism. Each successive mechanism may
have a reduced diameter of channel 300, so that a reduced diameter
of particle is retained in each successive mechanism. With this
arrangement, larger particles are retained earlier in the series of
mechanisms, whereas smaller particles are retained later in the
series. Any suitable retention mechanism may be used at each
position in the series.
[0646] Particles retained in the retention mechanism of system 1520
or related systems may be treated and analyzed. Particles may be
treated by exposing them to desired reagents, for example, using
perfusion mechanism 268 of Example 2, or by introducing reagents
from any other reservoirs included in system 1520. Thus, particles
retained in distinct retention mechanisms may be isolated and
exposed to distinct reagents, as described in Example 4. Systems
such as system 1520 may enable on-chip staining and washing,
eliminating any need for multiple pipeting and/or centrifugation
steps during manipulation and detection.
[0647] Suitable characteristics of retained particles may be
detected by flow or scanning cytometry, among others. In flow
cytometry, particles are detected while flowing past a detection
mechanism, such as a light source coupled to a photodetector.
Accordingly, particles may be released from each retention
mechanism, for example, using a release mechanism, such as
described above in Example 7, to flow past a detector.
Alternatively, or in addition, characteristics of particles may be
detected or otherwise detected while the particles are relatively
stationary, such as when localized in chamber 270. Photons may be
converted to electrons using photomultiplier tubes, avalanche
photodiodes, CCDs, or similar technologies. Light emitted from dyes
may be bright enough to detect using a single CCD, and scattered
light may yield enough structural information from particles, when
combined with functional information, to identify specifically the
type and state of particles.
[0648] Additional aspects of sorting a heterogeneous particle
population are described below in Example 26.
Example 16
Microfluidic Interaction of Specific Binding Pairs on Beads
[0649] This example describes detection of interaction between a
specific binding pair, biotin and avidin, on beads in a
microfluidic system; see FIGS. 73-74.
[0650] Background
[0651] Beads are used frequently by pharmaceutical and
biotechnology companies as carriers for drug targets, drug
candidates, chemical syntheses, immunoassays, chromatography,
and/or so on. However, small numbers of beads are difficult to
manipulate, particularly to detect reactions that occur rapidly. As
a result, using currently available technology, assays with beads
generally are conducted on a relatively large scale, wasting
valuable reagents and/or may measuring a reaction endpoint that
misses valuable earlier reaction information. Therefore, systems
are needed to study interaction, including rapid interactions,
using small numbers of beads.
[0652] A specific binding pair, biotin/streptavidin, was selected
for interaction on beads; see FIG. 73. Biotin is a vitamin with a
molecular weight of 244 daltons. Its partner, avidin, binds biotin
with fierce tenacity, being the strongest non-covalent attachment
known, with an association constant of 10.sup.15 M.sup.-1. This
binding reaction has been studied intensively for many decades, and
there is a rich literature. The great strength of this binding
suggests that it might be a good model system for the study of
biological binding reactions in general. It has also formed the
basis for many detection and signal amplification strategies for
both research and clinical labs.
[0653] Avidin and streptavidin are vertebrate and bacterial biotin
partners, respectively. Avidin is a protein with a molecular weight
of about 68 kilodaltons, including four identical subunit chains,
each 128 amino acids long. Avidin is found predominantly in the egg
white of birds, amphibia, and reptiles. The protein streptavidin,
produced by the bacterium Streptomyces avidinii, has a structure
very similar to avidin, also binding biotin tightly. However,
streptavidin often exhibits lower nonspecific binding, and thus is
frequently used in place of avidin.
[0654] Method
[0655] Materials for measuring biotin/avidin interaction were as
follows. A microfluidic chip was fabricated based on system 250 of
Example 2. Beads, 6.7-micron biotinylated polystyrene microspheres,
were obtained from Spherotech Corporation. Other buffers and
reagents included phosphate-buffered saline (PBS) containing 0.5%
BSA (sterile filtered), and the streptavidin conjugated
fluorophores streptavidin-Alexa 350, streptavidin-Alexa 488, and
streptavidin-PE (phycoerythryn), each obtained from Molecular
Probes. Binding reactions were monitored with an inverted
fluorescent microscope connected to a video camera.
[0656] The analysis was conducted according to the following
numbered steps.
[0657] The fluid network of the chip was washed with water, then
with PBS/BSA/Tween-20.
[0658] Beads were captured on the chip using its retention
chamber.
[0659] Streptavidin-conjugates were loaded into reagent-wells on
the chip (2 .mu.L of each conjugate in 1 mL PBS).
[0660] The captured beads were exposed to each of the
conjugates.
[0661] A 63X oil-immersion lens on the inverted microscope was used
to maximize fluorescent signal. Blue and green/red filter sets were
used.
[0662] In some cases the rate of photobleaching by the detection
mechanism exceeded the rate at which fluorescent conjugates were
captured by the beads. In these cases, the procedure was repeated
without constant exposure to UV, opening the UV shutter only long
enough to document binding.
[0663] Results
[0664] FIG. 74 shows the results of portions of the analysis as
selected video frames during exposure of streptavidin-Alexa 488
conjugate to retained beads. In FIG. 74A, the beads have been
loaded in chamber 270, but have not bound detectable amounts of the
conjugate and are not detectable. In FIG. 74B, beads 1550 are
detectable above background. In FIG. 74C, they have become readily
detectable, after unbound conjugate is washed out of the chamber.
FIG. 74D shows beads 1550 under bright field illumination to
localize the beads and demonstrate that all beads in the chamber
are stained with conjugate.
[0665] Similar exposures to the other conjugates gave less intense
staining. Detectable staining with streptavidin-Alexa 350 was
visible, but streptavidin-PE did not yield a detectable signal.
However, more sensitive detection mechanisms, such as a laser
scanning cytometer may allow detection of streptavidin-PE
binding.
Example 17
Measuring Ion Flux in Cells using a Microfluidic System
[0666] This example describes analysis of intracellular ion
concentrations, such as calcium ion concentrations, using a
microfluidic system; see FIG. 75.
[0667] Background
[0668] Calcium is a very important intracellular ion. It plays a
vital role in the transduction of signals from the cell membrane to
the cell cytoplasm and nucleus. A change in intracellular calcium
levels is an indication that the cell is responding to a stimulus.
Many stimuli cause mobilization of calcium, either as an influx
from the extracellular medium or by release from intracellular
pools. Fluorescent calcium indicators allow this mobilization to be
observed.
[0669] Method
[0670] Materials used for measuring intracellular calcium levels
were as follows. A microfluidic chip was constructed based on a
modified version of system 850 of Example 7. Fluo 3/AM, a
fluorescent Ca.sup.+2 indicator dye was obtained from Calbiochem,
and used as a 5 mM stock. Ionomycin, free acid form, was also
obtained from Calbiochem. Cells were Jurkat T-cells and were grown
in RPMI media.
[0671] The analysis was conducted according to the following
numbered steps.
[0672] Cells were cultured in RPMI media.
[0673] Cells/media (5 mL) were pelleted at 1000 rpm for 5 min.
[0674] The cells were resuspended in RPMI containing 5 .mu.M Fluo-3
(10 mL RPMI plus 8 .mu.L FLUO-3 AM).
[0675] The cell/Fluo-3 mixture was incubated at 37.degree. C. for
30 min to load the cells with indicator dye.
[0676] The cells were pelleted and washed twice with Hanks'
balanced salt solution (HBBS) containing 20 mM HEPES (200 .mu.L 1M
Hepes in 10 mL HBBS).
[0677] The cells were placed in the input reservoir of the
chip.
[0678] The microscope and video camera were set up.
[0679] HBBS/Hepes buffer was pumped across cells, acting as a
shield buffer to regulate exposure to reagent.
[0680] HBBS/Hepes containing ionomycin was pumped past the cells,
but in a layer spaced from the cells by the shield buffer.
[0681] The flow of shield buffer flow was terminated, exposing the
cells to ionomycin.
[0682] Calcium flux was recorded with the video camera as ionomycin
contacted the cells.
[0683] Results
[0684] FIG. 75 shows the results of the analysis, as selected video
frames, before and after exposure of Jurkat cells, loaded with
indicator dye, to ionomycin. FIG. 75A shows two cells 1570 captured
in retention site 1572 and visualized under bright field
illumination. In FIG. 75B, these cells lack fluorescence before
ionomycin exposure. In contrast, FIG. 75C reveals fluorescence
(green signal) of cells 1570 very soon after ionomycin exposure. A
negative control demonstrated that ionomycin was required for this
fluorescence (not shown).
Example 18
Microfluidic Analysis of Cell-Surface Markers
[0685] This example describes a method for detection of
cell-surface markers, such as CD4 and CD8, on cultured T-cells
using labeled antibodies.
[0686] Background
[0687] The CD4 molecule recognizes an antigen that interacts with
class II molecules of the major histocompatibility complex (MHC)
and is the primary receptor for the human immunodeficiency virus
(HIV)(Dalgleish et al., 1984; Maddon et al., 1986). The cytoplasmic
portion of the antigen is associated with the protein tyrosine
kinase p56.sup.lck (Rudd et al., 1989). The CD4 antigen may
regulate the function of the CD3 antigen/T-cell antigen receptor
(TCR) complex (Kurrle et al., 1989). The CD4 antibody reacts with
monocytes/macrophages that have an antigen density lower than that
on helper/inducer T lymphocytes (Wood et al., 1983).
[0688] The CD8 antigen is present on the human suppressor/cytotoxic
T-lymphocyte subset (Evans, et al., 1981; Ledbetter et al., 1981)
as well as on a subset of natural killer (NK) lymphocytes (Lanier
et al., 1983). The CD8 antigenic determinant interacts with class I
MHC molecules, resulting in increased adhesion between the
CD8.sup.+ T lymphocytes and the target cells (Anderson et al.,
1987; Eichmann et al., 1987; Gallagher et al., 1988). Binding of
the CD8 antigen to class I MHC molecules enhances the activation of
resting T lymphocytes. CD8 recognizes an antigen expressed on the
32-kDa a-subunit of a disulfide-linked bimolecular complex
(Moebius, 1989). The cytoplasmic domain of the .alpha.-subunit of
the CD8 antigen is associated with the protein tyrosine kinase
p56.sup.lck (Rudd et al., 1989; Gallagher et al., 1989).
[0689] Determining the percentages of CD4+ and CD8+ lymphocytes may
be useful in monitoring the immune status of patients with immune
deficiency diseases, autoimmune diseases, or immune reactions. The
relative percentage of the CD4+ subset is depressed and the
relative percentage of the CD8.sup.+ subset is elevated in many
patients with congenital or acquired immune deficiencies such as
severe combined immunodeficiency (SCID) and acquired
immunodeficiency syndrome (AIDS)(Schmidt, 1989; Giorgi, 1990).
[0690] The percentage of suppressor/cytotoxic lymphocytes can be
outside the normal reference range in some autoimmune diseases
(Antel et al., 1986) and in certain immune reactions such as acute
graft-versus-host disease (GVHD) and transplant rejection (Gratama
et al., 1984; Bishop et al., 1986). The relative percentage of the
CD8.sup.+ lymphocyte population may often be decreased in active
systemic lupus erythematosus (SLE) but can also be increased in SLE
patients undergoing steroid therapy (Wolde-Mariani et al.,
1984).
[0691] The CD4.sup.+/CD8.sup.+ (helper/suppressor) lymphocyte
ratio, quantified as the ratio of CD4 fluorescein isothiocyanate
(FITC)-positive lymphocytes to CD8 phycoerythrin (PE)-positive
lymphocytes, has been used to evaluate the immune status of
patients with, or suspected of developing, autoimmune disorders or
immune deficiencies (Antel et al., 1986; Wolde-Mariam et al., 1984;
Smolen et al., 1982). In many cases, the relative percentages of
helper lymphocytes decline and suppressor lymphocytes increase in
immune deficiency states. These states may also be marked by T-cell
lymphopenia (Ohno et al., 1988). In addition, the ratio has been
used to monitor bone marrow transplant patients for onset of acute
GVHD (Gratama et al., 1984).
[0692] The Jurkat cell, a human mature leukemic cell line,
phenotypically resembles resting human T lymphocytes and has been
widely used to study T cell physiology. These cells are round,
growing singly or in clumps in suspension. They were established
from a human T cell leukemia in the peripheral blood of a
14-year-old boy with acute lymphoblastic leukemia (ALL) at first
relapse in 1976. This cell line is also called "JM" (JURKAT and JM
are derived from the same patient and are sister clones).
Occasionally JM may be a subclone with somewhat divergent features
confirmed as human with IEF of AST, LDH, and NP. Jurkat cells have
the following general restriction properties: CD2+, CD3+, CD4+,
CD5+, CD6+, CD7+, CD8-, CD13-, CD19-, CD34+, TCRalpha/beta+, and
TCRgamma/delta-.
[0693] Method
[0694] Materials used for analysis of CD4 and CD8 were as follows.
Microfluidic chips was constructed based on a modified version of
system 850 of Example 7. Jurkat T-cells were cultured in RPMI.
Fluorophore-conjugated antibodies, CD4-fluorescein isothiocyanate
(FITC) and CD8-phycoerythryn (PE), were used. Buffer for dilution,
focusing, washing, etc. was PBS containing 0.5% BSA. Data were
collected with an inverted fluorescent microscope equipped with a
video camera.
[0695] The analysis was conducted according to the following
numbered steps.
[0696] Jurkat cells were grown in RPMI and then pelleted (10 mL of
media/cells).
[0697] The cells were resuspended in 1 mL PBS containing 0.5%
BSA.
[0698] Anti-CD4 -FITC and anti-CD8-PE- antibody-conjugates were
diluted 1:100 in PBS containing 0.5% BSA.
[0699] The chip was prepared by running deionized water through the
microfluidic network and then was mounted on an inverted
fluorescent microscope. The 100.times. or 63.times. oil-immersion
lens was used to maximize fluorescent signal.
[0700] Cells were loaded onto the chip, positioned, and
retained.
[0701] The diluted antibody-conjugates were loaded into separate
reagent input-wells of the chip.
[0702] Exposure to light from the UV lamp was minimized to avoid
photobleaching.
[0703] Anti-CD4-FITC was exposed to cells for 2 min.
[0704] The valve regulating CD4 antibody-conjugate flow was
closed.
[0705] The shield-buffer flow line was opened to remove unbound
antibodies.
[0706] The UV excitation shutter was opened and cell fluorescence
was recorded.
[0707] When fluorescence was dim or invisible, the UV shutter was
closed and steps 8 through 11 were repeated.
[0708] Step 12 was repeated until fluorescence was observed and
documented.
[0709] As a negative control, steps 8 through 12 were repeated
using anti-CD8-PE.
[0710] Results
[0711] Anti-CD8 antibody-conjugate did not bind to Jurkat cells,
and therefore little or no red fluorescence was visible in the time
frame needed to visualize the green fluorescence of the anti-CD4
antibody-conjugate. The procedure may be repeated with continuous
UV exposure to observe antibody binding in real-time.
[0712] References
[0713] Maddon P, Dalgleish A, McDougal J, Clapham P, Weiss R, Axel
R. The T4 gene encodes the AIDS virus receptor and is expressed in
the immune system and the brain. Cell. 1986;47:333-348.
[0714] Dalgleish A, Beverly P, Clapham P, Crawford D, Greaves M,
Weiss R. The CD4 (T4) antigen is an essential component of the
receptor for the AIDS virus. Nature.
1984;312(December):763-767.
[0715] Rudd C, Burgess K, Barber E, Schlossman S. Monoclonal
antibodies to the CD4 and CD8 antigens precipitate variable amounts
of CD4/CD8-associated p56-lck activity. In: Knapp W, Dorken B,
Gilks W R, et al, eds. Leucocyte Typing IV: White Cell
Differentiation Antigens. Oxford: Oxford University
Press;1989:326-327.
[0716] Kurrle R. Cluster report: CD3. In: Knapp W, Dorken B, Gilks
W R, et al, eds. Leucocyte Typing IV: White Cell Differentiation
Antigens. Oxford: Oxford University Press;1989:290-293.
[0717] Wood G, Warner N, Warnke R. Anti-Leu-3/T4 antibodies react
with cells of monocyte/macrophage and Langerhans lineage. J
Immunol. 1983;131(1):212-216.
[0718] Evans R, Wall D, Platsoucas C, et al. Thymus-dependent
membrane antigens in man: Inhibition of cell-mediated lympholysis
by monoclonal antibodies to the TH.sub.2 antigen. Proc Natl Acad
Sci USA. 1981;78(1):544-548.
[0719] Ledbetter J A, Evans R L, Lipinski M, Cunningham-Rundles C,
Good R A, Herzenberg L A. Evolutionary conservation of surface
molecules that distinguish T lymphocyte helper/inducer and T
cytotoxic/suppressor subpopulations in mouse and man. J Exp Med.
1981;153(February):310-323.
[0720] Lanier L L, Le A M, Phillips J H, Warner N L, Babcock G F.
Subpopulations of human natural killer cells defined by expression
of the Leu-7 (HNK-1) and Leu-11 (NK-15) antigens. J Immunol.
1983;131(4):1789-1796.
[0721] Anderson P, Blue M-L, Morimoto C, Schlossman S.
Cross-linking of T3 (CD3) with T4 (CD4 ) enhances the proliferation
of resting T lymphocytes. J Immunol. 1987;139:678-682.
[0722] Eichmann K, Johnson J, Falk I, Emmrich F. Effective
activation of resting mouse T lymphocytes by cross-linking
submitogenic concentrations of the T-cell antigen receptor with
either Lyt-2 or L3T4. Eur J Immunol. 1987;17:643-650.
[0723] Gallagher P, Fazekas de St. Groth B, Miller J. CD4 and CD8
molecules can physically associate with the same T-cell receptor.
Proc Natl Acad Sci USA. 1989;86:10044-10048.
[0724] Moebius U. Cluster report: CD8. In: Knapp W, Dorken B, Gilks
W R, et al, eds. Leucocyte Typing IV: White Cell Differentiation
Antigens. Oxford: Oxford University Press;1989:342-343.
[0725] Bernard A, Boumsell L, Hill C. Joint report of the First
International Workshop on Human Leucocyte Differentiation Antigens
by the investigators of the participating laboratories: T2
protocol. In: Bernard A, Boumsell L, Dausett J, Milstein C,
Schlossman S, eds. Leucocyte Typing. Berlin:
Springer-Verlag;1984:25-60.
[0726] Schmidt R. Monoclonal antibodies for diagnosis of
immunodeficiencies. Blut. 1989;59:200-206.
[0727] Centers for Disease Control. Guidelines for the performance
of CD4.sup.+ T-cell determinations in persons with human
immunodeficiency virus infection. MMWR. 1992;41(No. RR-8):1-17.
[0728] Giorgi J, Hultin L. Lymphocyte subset alterations and
immunophenotyping by flow cytometry in HIV disease. Clin Immunol
Newslett. 1990;10(4):55-61.
[0729] Antel J, Bania M, Noronha A, Neely S. Defective suppressor
cell function mediated by T8.sup.+ cell lines from patients with
progressive multiple sclerosis. J Immunol. 1986;137:3436-3439.
[0730] Gratama J. Naipal A, Oljans P, et al. T lymphocyte
repopulation and differentiation after bone marrow transplantation:
Early shifts in the ratio between T4.sup.+ and T8.sup.+ T
lymphocytes correlate with the occurrence of acute
graft-versus-host disease. Blood. 1984;63(6):1416-1423.
[0731] Bishop G, Hall B, Duggin G, Horvath J, Sheil A, Tiller D.
Immunopathology of renal allograft rejection analyzed with
monoclonal antibodies to mononuclear cell markers. Kidney Internat.
1986;29:708-717.
[0732] Wolde-Mariam W, Peter J. Recent diagnostic advances in
cellular immunology. Diagnost Med. 1984;7:25-32.
[0733] Smolen J, Chused T, Leiserson W, Reeves J, Alling D,
Steinberg A. Heterogeneity of immunoregulatory T-cell subsets in
systemic lupus erythematosus: Correlation with clinical features.
Am J Med. 1982;72:783-790.
[0734] Ohno T, Kanoh T, Suzuki T, et al. Comparative analysis of
lymphocyte phenotypes between carriers of human immunodeficiency
virus (HIV) and adult patients with primary immunodeficiency using
two-color immunofluorescence flow cytometry. J Exp Med.
1988;154:157.
Example 19
Measuring Cell Lysis in a Microfluidic System
[0735] This example describes capture, lysis, and staining of
cells.
[0736] Background
[0737] Acridine orange (AO) was used for staining. AO binds to
single stranded nucleic acids as a dimer, which fluoresces red in
color, and to double stranded nucleic acids as a monomer, which
fluoresces green. This difference in fluorescent wavelength is
caused by differential accessibility of AO molecules to the nucleic
acid binding sites. AO fluorescence is also pH sensitive, staining
acidic organelles, such as lysosomes, orange.
[0738] Method
[0739] Materials used for measuring lysis were as follows.
Microfluidic chips was constructed based on system 250 of Example
2. Jurkat T-cells were cultured in RPMI. Acridine Orange was
dissolved at 5 .mu.g/ml in PBS. Solutions or liquids to lyse cells
included PBS containing 0.05% hydrogen peroxide, deionized water,
PBS containing 2% TWEEN 20 (0.2 .mu.m filtered), and WINDEX. Data
were collected on an inverted fluorescent microscope equipped with
a video camera.
[0740] The analysis was conducted according to the following
numbered steps.
[0741] Jurkat cells were grown in RPMI and pelleted (10 mL of
culture media/cells).
[0742] The cells were resuspended in 5 mL PBS containing 5 .mu.g/ml
Acridine Orange, or left unstained for use on a control chip. For
the control chip, proceed to step 5.
[0743] The cells were incubated 10 min at room temperature.
[0744] The cells were pelleted and washed twice in PBS.
[0745] The cells were resuspended in 1 mL PBS.
[0746] The chip was preparing by washing the microfluidic network
with deionized water, and then was mounted on an inverted
fluorescent microscope. The microscope's 63.times. oil-immersion
lens was used to maximize fluorescent signal.
[0747] The cells were loaded onto the chip, positioned, and
retained.
[0748] PBS containing peroxide was loaded into a reagent-well of
the chip.
[0749] Exposure of the chip to light from the UV lamp was
minimized, to minimize photobleaching.
[0750] The UV shutter was opened to expose stained cells to
fluorescent light.
[0751] PBS containing peroxide was pumped over the cells for 2 min
or until lysis or photobleaching occurred.
[0752] Cells were then exposed sequentially to PBS/2% TWEEN-20,
WINDEX, and finally water.
[0753] Results
[0754] The conditions of peroxide, TWEEN, and WINDEX did not lyse
the cells on the first attempt of this experiment. Subsequently,
water was used successfully to demonstrate cell lysis. Lysis
probably occurred under the other conditions, but was not as
obvious. Jurkat cells are fairly robust and may not be a good model
cell line for this experiment.
Example 20
Inducing and Detecting Cell Apoptosis in a Microfluidic
Environment
[0755] This example describes induction and detection of cell
apoptosis in a microfluidic system; see FIG. 76.
[0756] Background
[0757] Apoptosis, also termed programmed cell death, is a carefully
regulated process of cell death that occurs as a normal part of
development. Inappropriately regulated apoptosis is implicated in
disease states, such as Alzheimer's disease and cancer. Apoptosis
is distinguished from necrosis, or accidental cell death, by
characteristic morphological and biochemical changes, including
compaction and fragmentation of the nuclear chromatin, shrinkage of
the cytoplasm, and loss of membrane asymmetry..sup.1-5
[0758] Phosphatidylserine (PS) distribution also can act as a
marker for apoptosis. In normal viable cells, phosphatidylserine is
located on the cytoplasmic side of the cell membrane. However, in
apoptotic cells, PS is translocated from the inner to the outer
leaflet of the plasma membrane, thus exposing PS to the cell
exterior..sup.6 In leukocyte apoptosis, PS on the outer surface of
the cell marks the cell for recognition and phagocytosis by
macrophages..sup.7,8 The human anticoagulant, annexin V, is a 35-36
kD Ca.sup.+2-dependent phospholipid-binding protein that has a high
affinity for PS..sup.9 Annexin V can identify apoptotic cells by
binding to PS exposed on the outer leaflet..sup.10 Bound annexin V
may be detected through a dye, a specific binding member conjugated
to annexin V, an anti-annexin-V antibody, and/or the like.
[0759] Hydrogen peroxide has been shown to induce markers of
apoptosis, such as PS translocation, in cultured cells. The
cellular toxicity of hydrogen peroxide (H.sub.2O.sub.2) is
initiated by oxidative stress, resulting in rapid modification of
cytoplasmic constituents, depletion of intracellular glutathione
(GSH) and ATP, a decrease in NAD.sup.+ level, an increase in free
cytosolic Ca.sup.2+, and lipid peroxidation..sup.11 H.sub.2O.sub.2
also activates the mitochondria permeability transition pore and
the release of cytochrome c..sup.12 In the cytoplasm, cytochrome c,
in combination with Apaf-1, activates caspase-9, leading to the
activation of caspase-3 and subsequent apoptosis.sup.13-15.
[0760] Method
[0761] This example demonstrates induction and detection of cell
apoptosis in a microfluidic system. Jurkat cells are positioned and
retained in a microfluidic system, and then programmed cell death
is initiated by exposure of these cells to hydrogen peroxide.
Translocation of PS to the outer membrane leaflet is monitored with
annexin V, to measure apoptosis. At the same time, cells are
exposed to propidium iodide, which stains cells with disrupted
membranes, an indicator of necrosis rather than apoptosis.
[0762] Materials used were as follows. Microfluidic chips were
constructed based on system 250 of Example 2. Jurkat T-cells were
cultured in RPMI. The VYBRANT Apoptosis Assay Kit #2 was obtained
from Molecular Probes, Eugene, Oreg. This kit includes
fluorophore-conjugated annexin V (green) and propidium iodide
(red). Data were collected on an inverted fluorescent microscope
equipped with a video camera.
[0763] The analysis was conducted according to the following
numbered steps.
[0764] The video camera was turned on.
[0765] Cells were trapped in the retention chamber of the chip.
[0766] Annexin-V-conjugate was loaded into reagent well #1 of the
chip.
[0767] Propidium iodide was loaded into reagent well #2 of the
chip.
[0768] Binding Buffer (BB) was loaded into the shield buffer well
of the chip.
[0769] The cells were perfused with BB for 5 min.
[0770] The cells were perfused with annexin-V-conjugate for 5
min.
[0771] Cells were checked for staining. (Note: This is a negative
control. No staining occurred at this stage because the cells had
not apoptosed.)
[0772] The valves regulating flow of the shield buffer and reagent
wells were each closed.
[0773] The BB was replaced with 800 .mu.M H.sub.2O.sub.2 in
PBS.
[0774] The cells were exposed to the H.sub.2O.sub.2/PBS by opening
the valve regulating flow from of the shield buffer.
[0775] Cells were observed under light microscopy during induction
of apoptosis.
[0776] After 15 min, the valve regulating flow of the shield buffer
was closed. The well was washed with BB, and then replaced with
BB.
[0777] The cells were then perfused with BB for 5 min.
[0778] The valve for the annexin-V-conjugate was opened, and the
shielding buffer valve was closed.
[0779] The cells were exposed to the annexin-V-conjugate for 5
min.
[0780] The valve controlling the annexin-V-conjuagate was closed,
and the BB valve was opened to wash the cells.
[0781] The cells were exposed to excitation light by opening the
microscope shutter. Green fluorescence indicated a positive
reaction for phosphatidylserine.
[0782] The valve that regulates flow of propidium iodide ("the PI
valve") was opened, while the valve that regulates BB ("the BB
valve") was closed.
[0783] After 2 min, the BB valve was reopened, and the PI valve was
closed.
[0784] After washing for 5 min, the fluorescent shutter was opened
while using the red filter set on the microscope.
[0785] Finally, the BB was replaced with water, and the cells were
lysed and then re-exposed to the PI.
[0786] Results
[0787] FIG. 76 shows selected video frames from this analysis. In
panel A, cells 1590 have been trapped in chamber 270 and are
visible under bright field illumination. Panels B and C compare
labeling of cells with the annexin-V-conjugate before (B) and after
(C) exposure to hydrogen peroxide. Cells 1590 do not label with the
annexin-conjugate before exposure to hydrogen peroxide (panel B),
but a weak annexin-conjugate signal is detectable after hydrogen
peroxide exposure (panel C), demonstrating that at least some of
the cells have initiated apoptosis. Panels D-F compare propidium
iodide staining of cells 1590 at different times during the
analysis. Panels D and E show no propidium iodide staining, either
before or after induction of apoptosis by exposure to hydrogen
peroxide. In contrast, panel F reveals detectable propidium-iodide
staining after exposure of cells to water, which renders the cells
necrotic.
References
[0788] Immunol. Cell Biol. 76, 1 (1998).
[0789] Cytometry 27, 1 (1997).
[0790] J. Pharmacol Toxicol. Methods 37, 215 (1997).
[0791] FASEB J. 9, 1277 (1995).
[0792] Am J. Pathol. 146, 3 (1995).
[0793] Cytometry 31, 1 (1998).
[0794] J. Immunol. 148, 2207 (1992).
[0795] J. Immunol. 151, 4274 (1993).
[0796] J. Biol. Chem. 265, 4923 (1990).
[0797] Blood 84, 1415 (1994).
[0798] Am. J. Physiol. 273, G7 (1997).
[0799] Free Radic. Biol. Med. 24, 624 (1998).
[0800] FEBS Lett. 447, 274 (1999).
[0801] Cell 91, 479 (1997).
[0802] Annu. Rev. Cell Dev. Biol. 15, 269 (1999).
Example 21
Analysis of Aquatic Microorganisms in a Microfluidic System
[0803] This example describes the capture and visualization of
aquatic microorganisms, such as plankton, using a microfluidic
system.
[0804] Background
[0805] Plankton are a very diverse group of marine and fresh water
organisms that spend some or all of their lives drifting in water.
Plankton represent both the animal and plant kingdoms and include a
range of sizes from submicron to over a centimeter. These seemingly
listless organisms play critical roles, both positive and negative,
in the health of not only other aquatic organisms but also in the
composition of the earth's atmosphere. For example, these organisms
are thought to produce a large fraction of the earth's oxygen. In
addition, they play a critical role in global carbon dioxide
exchange, removing much of the excess carbon dioxide produced by
burning fossil fuels and sending this carbon dioxide to the ocean
floor. In contrast, some plankton are infamous for their negative
impact on the economy. For example, explosive population growth of
dinoflagellate plankton produce a toxic "red tide" that poisons
fish and shellfish. However, occurrences of red tides are difficult
to predict and/or prevent, resulting in extensive fish-kills and
beach closures, which have a large economic impact. Therefore,
systems are needed to manipulate, treat, and analyze plankton,
including laboratory or natural populations that benefit or harm
the environment.
[0806] Method and Results
[0807] This example provides a microfluidic system capable of
manipulating and detecting small plankton, particularly
picoplankton (0-2 .mu.m), ultraplankton (2-5 .mu.m), and/or
nannoplankton (5-60 .mu.m). Plankton may be retained, treated,
and/or detected in an integrated microfluidic environment.
[0808] Plankton were manipulated and detected in a microfluidic
system as follows. A sample of seawater was collected from San
Francisco Bay and centrifuged to concentrate organisms in the
sample. A 20 .mu.L aliquot of the concentrated sample was loaded
into the input reservoir of microfluidic system 250, described in
Example 2 above. Naturally-fluorescent plankton were retained in
chamber 270 and detected successfully by fluorescent microscopy
(not shown).
[0809] This method of this example may be modified by changing any
suitable parameters. For example, plankton may be collected from
freshwater sources or cultured, an aqueous plankton sample may be
loaded directly into a microfluidic environment without
concentration, and/or retained plankton may be exposed to any
suitable reagents. Alternatively, or in addition, microfluidic
systems may be used that sort a heterogeneous population of
plankton according to a physical property (such as size or density,
among others) or a measured property/characteristic (such as
labeling with a dye and/or specific binding member).
Example 22
Analysis of Membrane Trafficking in a Microfluidic System using
Membrane Dyes
[0810] This example describes microfluidic analysis of membrane
trafficking pathways in cells treated with membrane-labeling
dyes.
[0811] Background
[0812] Studies of vesicle trafficking often rely on optically
detectable dyes that label membranes. Brief exposure of cells to
such a dye results in labeling of the surface-membrane of these
cells. Subsequent dye movement to interior membranes, such as
endosomes, Golgi apparatuses, lysosomes, and/or endoplasmic
reticulum, tracks corresponding transit of surface membranes,
receptors, and/or ligands, among others, through intracellular
vesicle trafficking pathways. Using this approach, cell endocytic,
recycling, degradative, and/or secretory pathways may be monitored
and analyzed.
[0813] Some "FM" dyes available from Molecular Probes bind to cell
membranes. Thus these FM membrane dyes may be used as
general-purpose probes for endocytosis, because they are generally
nontoxic. FM membrane dyes are virtually non-fluorescent in aqueous
solution, but become intensely fluorescent upon association with a
membrane.
[0814] Goals and Method
[0815] The goals of this analysis included the following. I) Define
the staining conditions for two FM membrane dyes, FM 1-43 and FM
4-64, using Jurkat cells. FIGS. 77 and 78 show the structure and
excitation/emission spectra of these dyes. These two FM dyes have
substantially nonoverlapping emission spectra. II) Test the
affinity of FM dyes for microfluidic chips formed with PDMS, to
define a background level of staining. III) Trap a Jurkat cell in a
microfluidic chip and perform two-color staining of the cell using
the two FM membrane dyes.
[0816] Materials used for this analysis included the following. FM
1-43 and FM 4-64 were obtained from Molecular Probes. Microfluidic
chips were produced based on system 250 of Example 2. Results were
collected and recording using an inverted fluorescent microscope
equipped with a video camera.
[0817] Conditions for labeling Jurkat cells with FM membrane dyes
were determined with the following labeling protocol.
[0818] Cultured Jurkat cells (5 mL of cells/media) were pelleted by
centrifugation at 1000 rpm for 5 min.
[0819] The cell pellet was washed twice-with PBS.
[0820] The cell pellet was resuspended in 2 mL PBS.
[0821] Aliquots (500 .mu.L) of the resulting cell suspension were
dispensed into four microcentrifuge tubes.
[0822] Dye was added to each of the four tubes as follows: no dye
was added to tube #1, FM 1-43 was added to tube #2, FM 4-64 was
added to tube #3, and both FM 1-43 and FM4-64 were added to tube
#4. The final dye concentration for each dye was 2 .mu.M.
[0823] The cells were observed with the fluorescent microscope.
[0824] Each staining condition was documented by saving digital
image files.
[0825] Labeling of the microfluidic chip with the FM membrane dyes
to determine background signal was carried out as follows.
[0826] Each dye was diluted to a final concentration of 2 .mu.M in
PBS.
[0827] FM 1-43 (5 .mu.L) was introduced into a first chip.
[0828] FM 4-64 (5 .mu.L) was introduced into a second available
chip.
[0829] A mixture of the FM 1-43 and 4-64 dyes (1:1) was introduced
into a third chip.
[0830] Each dye-loaded chip was observed using a fluorescent
microscope.
[0831] The level of background staining was determined relative to
fluorescence intensity of the cells stained with FM dyes in part A
above.
[0832] Cells were labeled with FM dyes in a microfluidic system as
follows.
[0833] Unlabeled Jurkat cells were loaded and captured in a
microfluidic chip using PBS as a carrier buffer.
[0834] Each FM membrane dye (5 .mu.L) was placed in one of the two
reagent wells on the chip.
[0835] Chip features and cells were visualized using minimal
incandescent light.
[0836] The video camera was turned on, and the 100.times.
oil-immersion objective on the fluorescent scope was used.
[0837] The first FM membrane dye (1-43) was delivered to the
cells.
[0838] The fluorescent signal was observed.
[0839] The second FM membrane dye (4-64) was delivered to the
cells.
[0840] The fluorescent signal was observed.
[0841] Steps 5-8 were repeated as necessary until the signal
intensity was maximized.
[0842] Results
[0843] The results of the three protocols are as follows.
[0844] Protocol A produced significant labeling of Jurkat cells
with the dyes after a 5-minute incubation at room temperature. Each
dye stained the cells with sufficient intensity to visualize using
the fluorescent microscope. For example, FIG. 79 shows Jurkat cells
stained with FM 1-43. However, the emission profile of each dye was
not distinguishable as a discrete color using the green/red filter
set on a Leica microscope. Properly selected filter sets may allow
a two-color assay using these dyes.
[0845] Protocol B produced significant background labeling of
microfluidic chips formed with PDMS, using either dye. The PDMS may
be surface-modified to minimize binding of these dyes to the
chip.
[0846] Protocol C was foiled by the high background produced by dye
binding to PDMS. After trapping a single cell in the chip, FM 1-43
bound to the chip more efficiently than to the membrane of the
trapped cell.
Example 23
Capturing Cells in Single-cell or Multi-cell Microfluidic
Chambers
[0847] This example describes capture of a single cell or a cell
population in a microfluidic system; see FIGS. 80-82.
[0848] FIG. 80 shows a single cell captured at a retention site
using a chip fabricated generally according to system 850 of
Example 7. In FIG. 80A, cells 1610 follow a divided flow path
extending in opposite directions above retention site 1612. In FIG.
80B, a trapped cell 1614 is positioned at the retention site.
[0849] Multiple cells were captured in a larger retention chamber
formed by a chip fabricated generally according to system 250 of
Example 2. FIGS. 81A, 81B, and 81C show empty chamber 270, the
chamber with two cells, and with six cells, respectively. FIG. 82
shows a similar capture of cells, but here the cells are prelabeled
with a fluorescent dye so that the cells are easily visible as
bright green using fluorescent microscopy. FIGS. 82A and 82B show a
chamber with only three cells and during the entry of a fourth
cell, respectively.
Example 24
Fixing and Staining Cells in a Microfluidic System
[0850] This example describes the use of a microfluidic system to
fix a cell with an organic solvent, methanol, and label the cell
with acridine orange; see FIG. 83.
[0851] All cell manipulations and treatments were as described in
Example 2. FIG. 83A shows a single cell 1630 retained at the bottom
of retention site 1632. The cell is barely visible due to the low
level of light used. The cell was perfused with methanol to fix the
cell, and visible cell-shrinkage was evident (not shown). FIG. 83B
shows that the cell exhibits no fluorescence. However, after the
cell was perfused with a solution of acridine orange, the cell
fluoresces brightly (see FIG. 83C).
Example 25
Microfluidic Mechanism for Measuring Cell Secretion
[0852] This example describes the structure and use of a soft
lithography-based, microfluidic system for measuring secretion of
molecules, complexes, and/or small particles from cells.
[0853] Many cell analyses measure release, and/or secretion of
materials from cells. In some cases, the cells secrete material
naturally. For example, neurons are analyzed for their ability to
secrete neurotransmitters at neural synapses; endocrine cells for
secretion of endocrine hormones, such as insulin, growth hormone,
prolactin, steroid hormones, etc.; and a broad range of cell types
for secretion of cytokines. In other cases, cells are lysed to
define an aspect of their internal contents. However, in any of
these cases, a secreted or released material of interest may no
longer be held in a fixed position by the cells, and thus may be
free to diffuse into the ambient solution. Accordingly, such
secreted or released materials may be difficult to analyze without
concentrating them and/or without using immobilized, high-affinity
binding partners, for example, in ELISA.
[0854] Microfluidic systems may ameliorate some of the difficulties
associated with measuring material released from cells, but may
introduce additional considerations. In microfluidic systems, cells
may be grown in isolated chambers having small volumes, as
described above in Example 10. The chambers may maintain released
materials in the small volumes, promoting subsequent analysis.
However, to maintain the released materials in a concentrated form,
the chambers may be isolated from other portions of the
microfluidic network. Such isolated chambers do not promote ready
analysis of the released materials, since the materials may be
isolated from analytical reagents and may be difficult to collect
without substantially diluting the released materials. Therefore, a
microfluidic mechanism is needed that allows material released from
cells to be collected and/or analyzed in a distinct fluidic
compartment that is not part of a primary fluidic layer of a
microfluidic system.
[0855] This example provides a microfluidic system having a cell
chamber and a separate material collection compartment that
communicate fluidically through a semi-permeable membrane. The
semi-permeable membrane permits movement of material that is
secreted/released from cells, but prevents movement of cells
themselves. The membrane may be form a portion of a fluid layer, or
interface with a fluid layer above and/or below the fluid layer.
When disposed below, the membrane may form some or all of the
substrate for the fluid layer. Accordingly, secreted/released
material may pass through the membrane for collection and/or
analysis in another compartment of the fluid layer, a compartment
above the fluid layer, and/or below the substrate. For example, the
microfluidic system may include a layer similar to the base layer
of Example 11.
Example 26
Microfluidic Analysis of a Heterogeneous Particle Population--Part
II
[0856] This example describes microfluidic systems for sorting and
analyzing heterogeneous populations of particles, such as blood
samples, based on differences in particle size; see FIGS. 84-88.
Example 26 expands upon aspects of Example 15 above.
[0857] Description
[0858] This example provides a microfluidic system 1650 that
selectively retains and analyzes larger particles from a mixture of
larger and smaller particles; see FIGS. 84 and 85. System 1650
includes an input mechanism 1652, a positioning mechanism 1654, a
filtration mechanism 1656, a retention mechanism 1658, a perfusion
mechanism 1660, a release mechanism 1662, and a flow-based
detection mechanism 1664, among others. These mechanisms may be
grouped into a first set for inputting sample and size-selecting
the sample, and a second set for retaining, treating, measuring,
and outputting the size-selected sample.
[0859] The first set of mechanisms may functionally interconnect as
follows. Input mechanism 1652 introduces particles from a particle
sample placed in particle input-reservoir 5 1666, into microfluidic
network 1668 of system 1650. Particles are moved by positioning
mechanism 1654 to filtration mechanism 1656 by flow along inlet
channel 1670. Filtration mechanism 1656 may act as a size-dependent
and regulatable retention mechanism, or prefilter, that removes
smaller particles from the inputted particles, while retaining
larger particles. After suitable filtration, the larger particles
may be released from filtration mechanism 1656 and moved by
positioning mechanism 1654 toward retention mechanism 1658.
[0860] The second set of mechanisms may functionally interconnect
as follows. Positioning mechanism 1654 may use a first focusing
mechanism 1672 to focus and direct particles toward retention
mechanism 1658. Particles retained by retention mechanism 1658 may
be perfused with desired reagents from perfusion mechanism 1660,
then released by release mechanism 1662. Released cells may be
moved by positioning mechanism 1654 toward flow-based detection
mechanism 1664. During positioning, cells may be focused into a
single stream of particles by a second focusing mechanism 1674.
Finally, detected cells may be passed to output mechanism 1676.
[0861] System 1650 may include a plurality of regulators, or
valves, that may regulate various aspects of the mechanisms
described above; see FIG. 85. Valve V1 may regulate input mechanism
1652. Valve V2 may regulate alternative input mechanism 1678.
Alternative input mechanism 1678 may provide an alternative source
of input fluid, and may be used to supply particle-free fluid for
washing filtration mechanism 1658, for carrying particles from
filtration mechanism to first focusing mechanism 1672 and on to
retention mechanism 1658, and/or the like. Valve V3 may regulate
input from first reagent reservoir 1680. Valve V4 may regulate
input from second reagent reservoir 1682. Valve V5 may regulate
flow of a shield buffer to space reagents from retained particles
until the desired moment for beginning treatment. V6 may regulate
flow through a first waste channel 1684. V7 may regulate release
mechanism 1662, V8 may regulate flow through a second waste channel
1686. V9 may regulate flow toward detection mechanism 1664.
Finally, V10 may regulate a filter-release mechanism 1688 that
regulates release of particles from regulatable retention mechanism
1656.
[0862] Further aspects of input mechanism 1652, positioning
mechanism 1654, retention mechanism 1658, perfusion mechanism 1660,
release mechanism 1662, and output mechanism 1676 elsewhere in
Section XIII.
[0863] Applications
[0864] The description that follows exemplifies use of system 1650
for separation and analysis of white blood cells from a sample of
whole blood. However, system 1650 may be suitable for use with any
heterogeneous (or homogeneous) population of particles.
[0865] System 1650 first separates white blood cells from smaller
red blood cells and platelets. These separated white blood cells
are directed to a retention site, retained, and then processed by
the perfusion mechanism to stain the retained white blood cells.
These stained cells are then released from the retention site and
then positioned to a separate flow-based detection site. The
detection site then detects a characteristic of the stained cells,
based on the staining method/reagents used.
[0866] A chip fabricated according to system 1650 may be readied
for use as follows. First, the chip may be loaded with water. Next,
when all the channels are filled, the water may be replaced with a
buffer solution. At this point, the following valves generally are
closed: V1, V2, V3, V4, V5, V9, and V10. By contrast, the following
valves generally are open: V6, V7, and V8. All input reservoirs may
be loaded with their respective buffers/reagents. However, particle
input-reservoir 1666 typically is not loaded yet. Each waste
reservoir 1692, 1694, 1696, and 1698 may be emptied (or is already
empty).
[0867] A sample of whole blood may be loaded and filtered as
follows. An aliquot of blood is loaded into particle
input-reservoir 1666. Valve V1 may be opened and the blood allowed
to flow into filtration mechanism 1656. FIG. 86 shows the operation
of filtration mechanism 1656 in greater detail. A first set of
particle-selective channels 1700, for example, channels that are
about 7 .mu.m wide and 5 .mu.m high, may be disposed along the
walls of inlet channel 1702. A second set of particle-selective
channels or chamber channels 1704 also may be disposed around the
perimeter of capture chamber 1706. Accordingly, red blood cells may
travel to flow-through chambers 1708 and then waste reservoirs
1692, 1694, along a substantial area formed by inlet channel 1702
and chamber 1706. In particular, travel of red blood cells through
particle-selective channels 1700 from inlet channel 1702 may avoid
clogging chamber channels 1704. However, the white blood cells may
be retained in chamber 1704, because they cannot pass through
channels 1700 and may not travel past chamber 1704 because
filter-release mechanism 1688 (valve V10) is closed.
[0868] White blood cells retained in capture chamber 1706 may be
washed as follows. After a suitable number of white blood cells
have entered chamber 1706, valve V1 may be closed so that no more
whole blood enters inlet channel 1702 and chamber 1706. Then, valve
V2 may be opened to allow the carrying buffer provided by
alternative input mechanism 1678 to wash residual red blood cells
out of chamber 1706. At this point, waste reservoirs 1692, 1694 may
be emptied to avoid reverse flow of the red blood cells back into
chamber 1706.
[0869] Filtered white blood cells may be retained by retention
mechanism 1658 as follows; see FIGS. 85-87. Valve V10 may be opened
to allow the filtered white blood cells from chamber 1706 to be
released. The released cells may be focused by first focusing
mechanism 1672 and carried toward retention site 1710 (see FIG.
87). Flow of carrying buffer from alternative input mechanism 1678
may act during this process to reposition the white blood. cells
from chamber 1706 to retention site 1710.
[0870] Retained white blood cells may be stained with reagents as
follows. Valve V11 may be closed to prevent additional white blood
cells from leaving chamber 1706 and entering retention site 1710.
Next, valve V6 may be closed to facilitate directing reagents along
a flow path toward the retained white blood cells by perfusion
mechanism 1660. Next, white blood cells may be stained or otherwise
treated/processed using perfusion mechanism 1660, as described
elsewhere in Section XIII, particularly Example 2. Pump P1 may be
used by perfusion mechanism 1660 to actively move reagents, buffer,
and/or fluid during particle treatment (see FIG. 85). At this
point, the valves may be in the following configuration. Valves V1,
V3, V4, V5, V6, V9, are V10 closed. Valves V2, V7, and V8 are open.
After cell treatment has been completed, pump P1 may be turned off,
and valves V3, V4, and V5 may be closed to terminate action of
perfusion mechanism 1660.
[0871] Treated/processed cells may be released and detected as
follows; see FIGS. 84, 85, 87, and 88. Pump P2 may be turned on.
This pump may be used to pull fluid, particles, and/or reaction
products toward detection mechanism 1664 and waste (output)
reservoir 1698. Next, valve V8 may be closed and valve V9 opened
(see FIG. 87). With this valve configuration, fluid and particle
may be directed toward waste reservoir 1698 instead of waste
reservoir 1696 (see FIG. 85). At this point, each focusing
reservoir 1712, 1714, 1716, 1718 may be refilled with buffer and
waste reservoir 1698 may be emptied. Then, partial or complete
closure of valve V7 may be used to release white blood cells from
retention mechanism 1658. During release, buffer flowing from
reservoirs 1712, 1714, or alternative input mechanism 1678, may be
used to carry the released white blood cells toward detection
mechanism 1664. Buffer flowing from reservoirs 1716, 1718 may act
in second focusing mechanism 1674, to position (focus) the released
cells to a desired cross-sectional portion of outlet channel 1720,
generally a central portion (see FIG. 88). After cell focusing,
outlet channel 1720 may constrict to a narrowed channel 1722, which
may facilitate positioning the cells in single file, that is,
one-by-one at detection site 1724, rather than in groups.
[0872] System 1650 may be used to measure any suitable aspect of a
blood sample or other inputted particle population, including
samples from patients, research subjects, volunteers, forensic
studies, cadavers, etc. Suitable aspects may include analysis of
leukemias, anemias, blood abnormalities, blood health, genetic
diseases, infections, ratios of specific blood cell types, presence
of nonblood cells, and/or the like. Exemplary leukemias may include
acute lymphoblastic leukemias, chronic myelogenous leukemias, acute
myelogenous leukemias, acute lymphoid leukemias, chronic
lymphocystic leukemias, and/or juvenile myelolymphocystic
leukemias, among others. Exemplary anemias and/or genetic diseases
may include aplastic anemias, Faconi anemias, sickle-cell anemias,
and/or the like. Other aspects or characteristics of blood cells
(or other heterogeneous particle populations) that may be suitable
for analysis are described above in Sections VIII and XII.
[0873] FIG. 89 is a top plan view of a perfusion device for
exposing particles to an array of different reagents or different
reagent concentrations. Here, microfluidic passage device 2000
provides a plurality of growth/perfusion chambers 2030 for loading
particles, such as cells, through loading passage 2010 which is
controlled by valving line 2020 which is in operable communication
with control input 2070, and which, when actuated, isolates each
chamber 2030 from one another. Particles may then be flushed out
the chambers 2030 by opening valving line 2020 and pushing fluid
from loading passage 2010 through each chamber 2030 towards exit
passage 2080. Once each chamber 2030 is loaded with particles, such
as cells, and isolated, valve line 2040, which is in operable
communication with control input 2140, then opens to permit flow of
reagent and diluent, such as media or a fluid that dilutes the
reagent, through flow lines 2120, which originate from a diluent
reservoir 2110, and optionally, reagent reservoir 2100, which may
hold a reagent for exposure to the particles. The ratio of diluent
to reagent may be controlled by valving, or, preferably, by
controlling the bore of the lines connecting the diluent reservoir
2110 to flow line 2120 and reagent reservoir 2100 to flow line
2120. Diluent and reagent are then fed into chambers 2030 by
pumping action caused by, for example, a peristaltic pump 2090,
which is actuated by pump input lines 2150a-c, thus particles are
perfused with reagent/diluent. Diluent, in the case of cells, may
be cell culture media. Effluent from chambers 2030 may be collected
into waste reservoir 2050.
[0874] FIGS. 90 through 94 depict a top plan view of a device being
used to measure the response of cells to a chemo-attractant.
Microfluidic passage device 2200 provides reagent loading chamber
2230, wherein reagent is metered into reagent chamber 2300 by the
opening of valve 2210 and blind filling reagent into reagent
chamber 2300. Once reagent chamber 2230 is filled, particles 2320,
such as cells, which were previously introduced into particle
chamber 2300 are then exposed to a gradient of reagent upon the
opening of valve 2220, valve 2210, preferably, remains closed
during the formation of the gradient. FIG. 91 shows reagent
entering into gradient forming mechanism 2250, which has channels
2270 for limiting reagent flow into particle chamber 2320. FIG. 92
depicts the advancement of reagent towards particle chamber 2300.
FIGS. 93 and 94 depict the movement of particles 2320 toward
channels 2270 where the chemo-attractant reagent is emanating from.
2230 is filled, particles 2320, such as cells, which were
previously introduced into particle chamber 2300 are then exposed
to a gradient of reagent upon the opening of valve 2220, valve
2210, preferably, remains closed during the formation of the
gradient. FIG. 91 shows reagent entering into gradient forming
mechanism 2250, which has channels 2270 for limiting reagent flow
into particle chamber 2320. FIG. 92 depicts the advancement of
reagent towards particle chamber 2300. FIGS. 93 and 94 depict the
movement of particles 2320 toward channels 2270 where the
chemo-attractant reagent is emanating from.
[0875] FIG. 95 is a close-up top plan view of a perfusion chamber
with associated valving system. Particles, such as cells, can be
loaded into a series of particle chambers 2450 by opening isolation
valve line 2430 which, when closed, isolates each chamber 2450 from
each other. Particles do not enter flow line 2460 since they are
retained in chamber 2450 by screen or comb 2490, which each
obstruction is spaced-apart from the other at a distance less than
that of the particle, so as to retain the particle on one side of
the screen or comb 2490. In use, particles are introduced into
chamber 2450 by the opening of isolation valve line 2430 which
allows the particles to flow through and fill each chamber 2450.
Once filled with the desired amount of particles, isolation valves
2430 are closed to isolate each chamber 2450 from each other, and
then flow valves 2440 are opened to allow for flow of reagent
through chamber 2450 to perfuse the particles with reagent. Once an
experiment is complete, flow valves 2440 may then be closed,
isolation valves 2430 may than be opened to flush out particles. If
the particles are adherent cells, such cells can be liberated if
attached by exposing such adhered cells to a cell dislodging
reagent such as trypsin. Once liberated, the cells can be flushed
out of the system, and the system reused.
[0876] FIGS. 96a through 96c depict a preferred embodiment of a
perfusion chamber device wherein a plurality of different compounds
from a plurality of different compound sources can be perfused
through a plurality of cell chambers such that each different
compound is perfused through a different chamber, and then a common
detection reagent can be perfused through each different cell
chamber from a common detection reagent source. In some
embodiments, the different compounds are perfused in one direction
through each cell chamber and the common detection agent is
perfused in an different direction through the detection chamber.
In some embodiments, the common detection reagent source is in
fluid communication, preferably selectively, with a waste
receptacle. In some embodiments, the different compound sources may
also serve as a second waste receptacle after the different
compounds have been delivered to the different cell chambers.
[0877] For example, FIG. 96a shows a device 2400 wherein cells are
introduced through cell inlet 2480 and flowed through until line
2410 is filled with cell containing solution. During cell loading,
chamber valves 2440 are closed, and, preferably, detection reagent
valve 2448 and waste valve 2447 remain closed. Once chambers 2450
have been loaded with cells, chamber isolation valves 2430 close to
isolate each chamber from the other, as shown in FIG. 96b. As shown
in FIG. 96c, with isolation valves 2430 remaining closed, cells
contained within chambers 2450 are then exposed different compounds
supplied by opening valves 2440, which permits flow of the
different compounds through channels 2460 and 2500 into chambers
2450, perfusing across through chamber 2450, and out through to
waste 2441. After a selected period of perfusion, waste valve 2447
may be closed and detection reagent valve 2448 may be opened
wherein a reagents, for example, media, wash solution, detection
agent(s), and/or developing reagents may be flowed back through
chambers 2450 out through channels 2460 and 2500. Alternatively, a
single common compound may be first flowed to each of chambers 2450
from detection reagent input 2448, and then different detection
reagents may be flowed through each chamber from channels 2460 and
2500.
[0878] Perfusion type devices, such as those described above, may
be useful for conducting toxicological assays. The invention
provides in one aspect for devices and methods for conducting cell
toxicity assays wherein cells are exposed, preferably transiently
for a selected period of time, to a compound which may be toxic to
the cells, or becomes toxic to the cells through further
processing. For example, liver cells, previously loaded into
chambers, may be introduced into the chambers, and then a drug
candidate, or a plurality of drug candidates may be presented to
the cells within the chambers for a selected period of time and/or
at selected concentrations. The drug candidates may then be flushed
out by a wash step, which is then followed, after a selected period
of time, by a detection treatment, where the drug exposed cells are
treated with a reagent to detect a change in state caused by the
drug candidate. In some embodiments, no detection reagent is used.
Instead, a change in some physical property of the cells is
observed or measured, such as impedance, resistivity, conductivity,
cell morphology, proliferation, and lysis. Changes in state may
include, for example, apoptosis, proliferation, senescence, changes
in membrane chemistry, and changes in nuclear or organelle
structure. Detection reagents may include, annexin V type assays,
apoptosis detection reagents, vital dye reagents, Quinn2 dyes, LDH
Assays: (lactase dehydrogenase enzyme leakage from plasma
membrane), ATP measurements (cell proliferation/cytotocicity
assessment), and MTT salt assay, WST-1 type assays.
[0879] Cell assays may include: trypan blue; eosin Y nigrosine;
propidium iodide; ethidium bromide, wherein dead and viable cells
are discriminated by differential staining and counted using a
light or fluorescence microscope. These methods do not allow the
processing of large sample numbers and do not account for dead
cells which may have lysed. Thus, the rate of cell death in long
term cultures can be underestimated. In other embodiments,
fluorescent dyes: [51Cr]; [3H]-thymidine; [3H]-proline;
[75Se]-methionine; [125J]-5-iodo-2-deoxyuridine;
bis-carboxyethyl-carboxy- fluorescein (BCECF); calcein-AM from
prelabeled target cells.
[0880] Yet another embodiment includes assays based on the
measurement of cytoplasmic enzyme activity released by damaged
cells. The amount of enzyme activity detected in the culture
supernatant correlates to the proportion of lysed cells. Enzyme
release assays have been described for alkaline and acid
phosphatase; glutamate-oxalacetate transaminase; glutamate pyruvate
transaminase; arginosuccinate lyase.
[0881] The disclosure set forth above may encompass one or more
distinct inventions, with independent utility. Each of these
inventions has been disclosed in its preferred form(s). These
preferred forms, including the specific embodiments thereof as
disclosed and illustrated herein, are not intended to be considered
in a limiting sense, because numerous variations are possible. The
subject matter of the inventions includes all novel and nonobvious
combinations and subcombinations of the various elements, features,
functions, and/or properties disclosed herein.
* * * * *