U.S. patent application number 12/087713 was filed with the patent office on 2010-06-03 for microfluidic devices and methods of use in the formation and control of nanoreactors.
Invention is credited to Laurent Boitard, Jeffrey Branciforte, Yves Charles, Gilbert Feke, Wolfgang Hinz, Darren R. Link, John Q. Lu, David Marran, Jonathan M. Rothberg, Ahmadali Tabatabai, Michael Weiner.
Application Number | 20100137163 12/087713 |
Document ID | / |
Family ID | 36940391 |
Filed Date | 2010-06-03 |
United States Patent
Application |
20100137163 |
Kind Code |
A1 |
Link; Darren R. ; et
al. |
June 3, 2010 |
Microfluidic Devices and Methods of Use in The Formation and
Control of Nanoreactors
Abstract
The present invention provides novel microfluidic devices and
methods that are useful for performing high-throughput screening
assays and combinatorial chemistry. Such methods can include
labeling a library of compounds by emulsifying aqueous solutions of
the compounds and aqueous solutions of unique liquid labels on a
microfluidic device, which includes a plurality of electrically
addressable, channel bearing fluidic modules integrally arranged on
a microfabricated substrate such that a continuous channel is
provided for flow of immiscible fluids, whereby each compound is
labeled with a unique liquid label, pooling the labeled emulsions,
coalescing the labeled emulsions with emulsions containing a
specific cell or enzyme, thereby forming a nanoreactor, screening
the nanoreactors for a desirable reaction between the contents of
the nanoreactor, and decoding the liquid label, thereby identifying
a single compound from a library of compounds.
Inventors: |
Link; Darren R.; (Lexington,
MA) ; Boitard; Laurent; (Paris, FR) ;
Branciforte; Jeffrey; (Cromwell, CT) ; Charles;
Yves; (Waterbury, CT) ; Feke; Gilbert;
(Durham, CT) ; Lu; John Q.; (Plano, TX) ;
Marran; David; (Durham, CT) ; Tabatabai;
Ahmadali; (Branford, CT) ; Weiner; Michael;
(Guilford, CT) ; Hinz; Wolfgang; (Guilford,
CT) ; Rothberg; Jonathan M.; (Guilford, CT) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY AND POPEO, P.C
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
36940391 |
Appl. No.: |
12/087713 |
Filed: |
June 1, 2006 |
PCT Filed: |
June 1, 2006 |
PCT NO: |
PCT/US2006/021280 |
371 Date: |
September 28, 2009 |
Current U.S.
Class: |
506/16 ; 506/30;
506/37; 506/40 |
Current CPC
Class: |
B01L 3/50273 20130101;
B01L 3/502792 20130101; B82Y 5/00 20130101; C12Q 1/6869 20130101;
B01F 13/0076 20130101; C12Q 1/6816 20130101; C12Q 1/6874 20130101;
C40B 60/08 20130101; B01J 2219/00459 20130101; B01L 2200/0673
20130101; C40B 70/00 20130101; C40B 50/08 20130101; B01J 2219/0072
20130101; C12Q 2565/629 20130101; B01J 2219/00743 20130101; B01L
2300/0896 20130101; B01L 2400/0424 20130101; C12Q 2563/159
20130101; C12Q 2525/161 20130101; B01F 13/0071 20130101; B01J
2219/00479 20130101; B01L 3/502746 20130101; G01N 21/6458 20130101;
C12Q 1/6834 20130101; C40B 30/04 20130101; G01N 33/543 20130101;
B01L 2200/0647 20130101; B01F 3/0807 20130101; B01J 2219/00576
20130101; B01L 3/502715 20130101; B01L 2300/0816 20130101; C12Q
2563/103 20130101; B01L 7/52 20130101; B01L 2200/0621 20130101;
B01L 2400/0415 20130101; B01J 2219/00286 20130101; B01L 3/5027
20130101; B01L 3/565 20130101; B01L 2200/027 20130101; B01L
2200/0652 20130101; C12N 15/1075 20130101; C12Q 2565/537 20130101;
G01N 33/542 20130101; C07K 1/047 20130101; C40B 40/04 20130101;
B01J 2219/005 20130101; B01L 2400/084 20130101; C12Q 2561/119
20130101; G01N 21/6428 20130101; B01L 3/502761 20130101; B01L
2400/0487 20130101; B01J 2219/00466 20130101; B01L 2300/0867
20130101; G01N 33/6854 20130101; B01J 19/0046 20130101; G01N
21/6445 20130101; C12Q 1/6876 20130101; C12Q 2563/107 20130101;
C40B 60/12 20130101; B01J 2219/00599 20130101; B01L 2300/0864
20130101; B82Y 30/00 20130101; G01N 2500/10 20130101; B01J
2219/00702 20130101; C12Q 1/686 20130101; G01N 33/536 20130101;
G01N 33/6845 20130101; B01L 3/502784 20130101; C12Q 1/6804
20130101; C12Q 1/6825 20130101; C12Q 2600/16 20130101; B01J
2219/00468 20130101; C12Q 1/686 20130101; C12Q 2563/159 20130101;
C12Q 2565/629 20130101 |
Class at
Publication: |
506/16 ; 506/40;
506/37; 506/30 |
International
Class: |
C40B 40/06 20060101
C40B040/06; C40B 60/14 20060101 C40B060/14; C40B 60/08 20060101
C40B060/08; C40B 50/14 20060101 C40B050/14 |
Claims
1-29. (canceled)
30. A microfluidic device comprising a microfabricated substrate,
said substrate further comprising: a) one or more inlet modules
having at least one fluorinated inlet channel adapted to carry at
least one dispersed phase fluid comprising an aqueous solution; b)
at least one fluorinated main channel adapted to carry at least one
continuous phase fluid comprising a fluorinated oil, wherein said
inlet channel is in fluid communication with said main channel such
that said dispersed phase fluid is immiscible with said continuous
phase fluid and forms a plurality of monodisperse droplets in said
continuous phase fluid; and c) a coalescence module downstream from
and in fluid communication with said one or more inlet modules via
the main channel, wherein two or more droplets passing there
through are coalesced to form a nanoreactor, wherein the
nanoreactor is about 25 .mu.m in diameter.
31. The device of claim 30, wherein the fluorinated oil further
comprises a surfactant.
32. The device of claim 31, wherein the surfactant is
sorbitan-based carboxylic acid esters, perfluorinated polyethers,
non-ionic surfactants or ionic surfactants.
33. The device of claim 30, wherein said channels are coated with
fluorosilane, an amorphous soluble perfluoropolymer, BSA,
PEG-silane, a silica primer layer followed by a
perfluoroalkylalkylsilane compound, Teflon or fluorinated oils.
34. The device of claim 33, wherein the fluorinated oils are
octadecafluoroctane, Fluorinert or fluorononane.
35. The device of claim 34, wherein the fluorinated oil further
comprises a surfactant.
36. The device of claim 30, wherein said dispersed phase comprises
a library of droplets.
37. The device of claim 36, wherein said library of droplets
comprises a biological/chemical material, wherein said
biological/chemical material is selected from the group consisting
of tissues, cells, particles, proteins, antibodies, amino acids,
nucleotides, small molecules, and pharmaceuticals.
38. The device of claim 30, wherein said inlet channel is further
connected to a means for introducing a sample to said device.
39. The device of claim 30 further comprising a means for
simultaneously loading multiple samples onto the device.
40. The device of claim 30, further comprising one or more
electrodes that generate an electric field.
41. The device of claim 40, wherein the electrodes comprise metal
alloy components and the metal alloy components are integrally
contained in one or more channels and are isolated from the inlet
and main channels.
42. The device of claim 30, wherein said substrate further
comprises at least one detection module downstream from and in
fluid communication with said coalescence module, said detection
module comprising a detection apparatus for evaluating the contents
and/or characteristics of the nanoreactor.
43. The device of claim 42, wherein said detection apparatus
comprises an optical detector.
44. The device of claim 30, wherein said substrate further
comprises a sorting module downstream from and in fluid
communication with said detection module, said sorting module
comprising a sorting apparatus adapted to direct said nanoreactor
into or away from a collection module in response to the contents
or characterization of the nanoreactor.
45. The device of claim 30, wherein said substrate further
comprises a mixing module in fluid communication with the main
channel, wherein the mixing module is downstream of the coalescence
module and upstream of the detection module.
46. The device of claim 30, wherein said substrate further
comprises a delay module in fluid communication with the main
channel, wherein the delay module is downstream of the coalescence
module and upstream of the detection module.
47. The device of claim 30, wherein said substrate further
comprises a UV-release module in fluid communication with the main
channel, wherein the UV-release module is downstream of the inlet
module and upstream of the coalescence module.
48. The device of claim 30, wherein said substrate further
comprises a collection module connected to a means for storing a
sample from said device.
49. The device of claim 30, wherein said substrate further
comprises a waste module connected to a means for collecting a
sample discarded from said device.
50. A method of creating a nanoreactor, the method comprising: a)
providing a microfabricated substrate comprising a plurality of
electrically addressable channel bearing microfluidic modules
integrally arranged on said substrate so as to be in fluid
communication with each other, thereby forming at least one
fluorinated main channel adapted to carry at least one continuous
phase fluid comprising a fluorinated oil; b) flowing a first
dispersed phase fluid comprising an aqueous solution through a
first fluorinated inlet channel into the main channel such that one
or more monodisperse droplets are formed in said continuous phase
fluid flowing therein; c) flowing a second dispersed phase fluid
comprising an aqueous solution through a second inlet channel into
the main channel such that one or more monodisperse droplets are
formed in said continuous phase fluid flowing therein; and d)
coalescing at least one droplet formed in step (b) with at least
one droplet formed in step (c) as the droplets pass through a
coalescence module of the microfabricated substrate, thereby
producing a nanoreactor, wherein the nanoreactor is about 25 .mu.m
in diameter.
51. An emulsion library comprising individual specific primer-pairs
to different exons and a plurality of monodisperse droplets,
wherein each droplet is about 25 .mu.m in diameter and wherein on
average one specific primer-pair is comprised within each droplet,
and wherein each droplet is an water-in-oil emulsion formed using a
continuous phase fluid comprising a fluorocarbon oil and a
surfactant and a discontinuous phase fluid comprising an aqueous
fluid.
52. A method of forming an emulsion library comprising: (a)
providing individual specific primer-pairs to different exons; (b)
separately emulsifying the individual primer-pairs of step (a) in a
monodisperse droplet, wherein each droplet contains on average one
primer-pair; and (c) pooling the droplets of step (b) to form a
plurality of monodisperse droplets, wherein each droplet is about
25 .mu.m in diameter; wherein each droplet is an water-in-oil
emulsion formed using a continuous phase fluid comprising a
fluorocarbon oil and a surfactant and a discontinuous phase fluid
comprising an aqueous fluid.
Description
FIELD OF INVENTION
[0001] The present invention generally relates to systems and
methods for the formation and/or control of fluidic species, and
articles produced by such systems and methods. More particularly,
the present invention relates to the development of high throughput
microfluidic devices for precision fluid handling and use of such
systems in various biological, chemical, or diagnostic assays.
BACKGROUND
[0002] High throughput molecular screening (HTS) is the automated,
rapid testing of thousands of distinct small molecules or probes in
cellular models of biological mechanisms or disease, or in
biochemical or pharmacological assays. Active compounds identified
through HTS can provide powerful research tools to elucidate
biological processes through chemical genetic approaches, or can
form the basis of therapeutics or imaging agent development
programs. HTS has experienced revolutionary changes in technology
since the advent of molecular biology and combinatorial chemistry,
and the incorporation of modern information management systems.
Current HTS instrumentation allows screening of hundreds of
thousands of compounds in a single day at a rate orders of
magnitude greater than was possible a decade ago. However, there
are still bottlenecks which currently limit HTS capacity, such as
(a) compound collection maintenance, tracking, and disbursement,
and (b) rapidity, accuracy, and content of assay
instrumentation.
[0003] The manipulation of fluids to form fluid streams of desired
configuration, discontinuous fluid streams, droplets, particles,
dispersions, etc., for purposes of fluid delivery, product
manufacture, analysis, and the like, is a relatively well-studied
art. For example, highly monodisperse gas bubbles, less than 100
microns in diameter, have been produced using a technique referred
to as capillary flow focusing. In this technique, gas is forced out
of a capillary tube into a bath of liquid, where the tube is
positioned above a small orifice, and the contraction flow of the
external liquid through this orifice focuses the gas into a thin
jet which subsequently breaks into equal-sized bubbles via a
capillary instability. A similar arrangement can be used to produce
liquid droplets in air.
[0004] Microfluidic systems have been described in a variety of
contexts, typically in the context of miniaturized laboratory
(e.g., clinical) analysis. Other uses have been described as well.
For example, International Patent Application Publication No. WO
01/89788 describes multi-level microfluidic systems that can be
used to provide patterns of materials, such as biological materials
and cells, on surfaces. Other publications describe microfluidic
systems including valves, switches, and other components.
[0005] Precision manipulation of streams of fluids with
microfluidic devices is revolutionizing many fluid-based
technologies. Networks of small channels are a flexible platform
for the precision manipulation of small amounts of fluids. The
utility of such microfluidic devices depends critically on enabling
technologies such as the microfluidic peristaltic pump,
electrokinetic pumping, dielectrophoretic pump or electrowetting
driven flow. The assembly of such modules into complete systems
provides a convenient and robust way to construct microfluidic
devices. However, virtually all microfluidic devices are based on
flows of streams of fluids; this sets a limit on the smallest
volume of reagent that can effectively be used because of the
contaminating effects of diffusion and surface adsorption. As the
dimensions of small volumes shrink, diffusion becomes the dominant
mechanism for mixing leading to dispersion of reactants; moreover,
surface adsorption of reactants, while small, can be highly
detrimental when the concentrations are low and volumes are small.
As a result, current microfluidic technologies cannot be reliably
used for applications involving minute quantities of reagent; for
example, bioassays on single cells or library searches involving
single beads are not easily performed. An alternate approach that
overcomes these limitations is the use of aqueous droplets in an
immiscible carrier fluid; these provide a well defined,
encapsulated microenvironment that eliminates cross contamination
or changes in concentration due to diffusion or surface
interactions. Droplets provide the ideal microcapsule that can
isolate reactive materials, cells, or small particles for further
manipulation and study. However, essentially all enabling
technology for microfluidic systems developed thus far has focused
on single phase fluid flow and there are few equivalent active
means to manipulate droplets requiring the development of droplet
handling technology. While significant advances have been made in
dynamics at the macro- or microfluidic scale, improved techniques
and the results of these techniques are still needed. For example,
as the scale of these reactors shrinks, contamination effects due
to surface adsorption and diffusion limit the smallest quantities
that can be used. Confinement of reagents in droplets in an
immiscible carrier fluid overcomes these limitations, but demands
new fluid-handling technology.
[0006] Furthermore, the underlying physics of the influence of
electric fields on fluids is well known. The attractive and
repulsive forces produced by an electric field on positive or
negative charges give rise to the forces on charged fluid elements,
the polarization of non-polar molecules, and the torque on polar
molecules which aligns them with the field. In a non-uniform field,
because the force on the positively charged portion of the
distribution is different than the force on the negatively charged
portion, polar molecules will also experience a net force toward
the region of higher field intensity. In the continuum limit, the
result is a pondermotive force in the fluid. In the limit of high
droplet surface tension, it is useful to describe the net
pondermotive force on a droplet as if it were a rigid sphere:
F=qE+2.pi.(.di-elect cons..sub.m)r.sup.3(k).gradient.E.sup.2.
where the first term is the electrophoretic force on the droplet (q
is the net droplet charge and E is the electric field), and the
second term is the dielectrophoretic force (r is the radius of the
sphere, (K) is the real part of the Clausius-Mossotti factor
K=(.di-elect cons.*.sub.p-.di-elect cons.*.sub.m)/(.di-elect
cons.*.sub.p+2.di-elect cons.*.sub.m),
and .di-elect cons.*.sub.p and .di-elect cons.*.sub.m are the
complex permittivities of the droplet and carrier fluid).
[0007] Although utility of electrophoretic control of droplets is
great, it does have significant limitations. First, the charging of
droplets is only effectively accomplished at the nozzle. Second,
the discharge path required to eliminate screening effects also
discharges the droplets. Third, finite conductivity of the carrier
fluid, however small, will eventually discharge the droplets.
Therefore, once the droplet is formed, there is essentially only
one opportunity to perform any pondermotive function which relies
on the droplet's charge density (such as coalescing oppositely
charged droplets through their mutual Coulombic attraction, or
electrophoretically sorting a droplet), and that function can only
be performed as long as sufficient charge has not leaked off of the
droplet.
[0008] Thus, it would be desirable to develop an electrically
addressable emulsification system that combines
compartmentalization and electrical manipulation, which allows for
multi-step chemical processing, including analysis and sorting, to
be initiated in confinement with exquisite timing and metering
precision, for use in a variety of chemical, biological, and
screening assays, in which the cost and time to perform such assays
would be drastically reduced. It would also be desirable to develop
a device using dielectrophoretic force (which does not rely on
charge density) to manipulate droplets so that more than one
electrical pondermotive function can be carried out following a
significantly long delay from droplet formation.
SUMMARY OF THE INVENTION
[0009] The present invention provides devices having individual
fluid handling modules that can be combined into fluid processing
systems so as to perform multi-step processing of isolated
components, which is essential for searching through molecular
libraries for rare interactions with cells, nucleic acids, enzymes,
coded microbeads, and other biomaterials. Using principles based on
the electrostatic and dielectrophoretic manipulation of charged and
neutral droplets 20 to 100 microns in diameter, the microfluidic
devices as described herein can inexpensively encapsulate reagents,
combine same, analyze, and sort in the range of 1.times.10.sup.9
droplets per day. The present invention provides a microfluidic
device that includes a microfabricated substrate. The substrate can
include a plurality of electrically addressable channel bearing
microfluidic modules integrally arranged with each other so as to
be in fluid communication. The microfabricated substrate can have,
for example, (i) one or more inlet modules that have at least one
inlet channel adapted to carry a dispersed phase fluid, (ii) at
least one main channel adapted to carry a continuous phase fluid,
wherein the inlet channel is in fluid communication with the main
channel such that the dispersed phase fluid is immiscible with the
continuous phase fluid and forms a plurality of droplets in the
continuous phase fluid, and (iii) a coalescence module downstream
from and in fluid communication with the inlet modules via the main
channel, wherein two or more droplets passing there through are
coalesced to form a nanoreactor. The microfluidic device of the
present invention can further include a sorting module, mixing
module, delay module, UV-release module, detection module,
collection module, waste module and/or acoustic actuator, and or
combinations thereof, in any order. These modules are in fluid
communication with the main channel. The flow of the dispersed
phase and continuous phase can be pressure driven, for example.
[0010] The present invention also provides methods of creating a
nanoreactor. The method includes, for example, a) providing a
microfabricated substrate having a plurality of electrically
addressable channel bearing microfluidic modules integrally
arranged on the substrate so as to be in fluid communication with
each other, thereby forming at least one main channel adapted to
carry at least one continuous phase fluid; b) flowing a first
dispersed phase fluid through a first inlet channel into the main
channel such that one or more droplets is formed in the continuous
phase fluid flowing therein; c) flowing a second dispersed phase
fluid through a second inlet channel into the main channel such
that one or more droplets is formed in the continuous phase fluid
flowing therein; and d) coalescing at least one droplet formed in
step (b) with at least one droplet formed in step (c) as the
droplets pass through a coalescence module of the microfabricated
substrate, thereby producing a nanoreactor. The coalescing step can
be achieved by an electric field or passively. The first and second
dispersed phase fluids can include a biological or chemical
material, which can include, for example, tissues, cells,
particles, proteins, antibodies, amino acids, nucleotides, small
molecules, and pharmaceuticals. The nanoreactor can further be
incubated within a delay module, and then interrogated for a
predetermined characteristic within a detection module.
[0011] The present invention also includes methods of synthesizing
a compound from two or more reactive substructures. In a particular
aspect the method includes a) labeling the reactive substructures
with a label unique to the substructure; b) emulsifying aqueous
solutions of the labeled reactive substructures on a microfluidic
device to form droplets; and c) randomly combining the droplets on
the microfluidic device to form a compound. The one embodiment, the
method further includes d) screening the compound formed in step
(c) based on a desirable chemical or biological property exhibited
by the compound; and e) identifying the structure of the compound
by decoding the label. In another embodiment, steps (a) and (b) are
alternatively performed by introducing a preformed labeled
emulsion.
[0012] In another aspect, the present invention provides methods
for identifying a single compound from a library on a microfluidic
device. The method can include a) labeling a library of compounds
by emulsifying aqueous solutions of the compounds and aqueous
solutions of unique liquid labels, whereby each compound is labeled
with a unique liquid label; b) pooling the labeled emulsions
resulting from step (a); c) coalescing the labeled emulsions with
emulsions containing a specific cell or enzyme, thereby forming a
nanoreactor; d) screening the nanoreactors for a desirable reaction
between the contents of the nanoreactor; and e) decoding the liquid
label, thereby identifying a single compound from a library of
compounds. In various embodiments of the method, the contents of
the nanoreactor can be incubated prior to screening. The screening
step can be performed by fluorescent polarization, for example. The
liquid label can be a quantum dot (q-dot) or a dye.
[0013] In yet another aspect, the present invention includes
methods for controlling the quality of a library of emulsified
compounds. The method can include, for example, a) providing a
library of emulsified compounds; b) emulsifying a q-dot encoded
aqueous buffer in an inert fluorocarbon medium, thereby forming
droplets; c) incubating the q-dot encoded droplet with the library
of emulsified compounds; d) sorting the q-dot encoded droplet away
from the library; e) analyzing the q-dot encoded droplet for the
presence of any of the compounds emulsified in the library; and f)
eliminating the compounds identified in step (e) from the library
of emulsified compounds, wherein one or more of steps (a)-(f) are
performed on a microfluidic device. In one embodiment, the
analyzing step is performed by mass spectroscopy.
[0014] In still a further aspect, the present invention provides
methods for sorting cells. The method can include a) fusing an
affinity-reagent to an enzyme; b) mixing the fusion product of step
(a) with a cell population; c) isolating cells attached to the
fusion product; d) emulsifying the cells of step (c) in an inert
fluorocarbon medium; e) coalescing the cell emulsion of step (d)
with an emulsion comprising a substrate corresponding to the enzyme
of step (a), thereby forming a nanoreactor; and f) screening the
nanoreactor for a desirable reaction between the contents of the
nanoreactor, wherein one or more steps of (a)-(f) are performed on
a microfluidic device. In one embodiment, the affinity-reagent can
be an antibody that is specific for a cell-surface cancer marker.
The enzyme can include alk/phos, .beta.-galactosidase, or
horseradish peroxidase. The affinity-reagent can be fused to
multiple enzymes, and multiple substrates can be emulsified and
coalesced with the cell emulsions.
[0015] Another aspect of the present invention provides methods for
sequencing individual exons from individual chromosomes. The method
can include, for example, a) emulsifying specific primer-pairs to
an exon with beads that can bind to said primer-pairs; b) pooling
the emulsions of step (a) to create a library emulsion; c)
providing a separate chromosomal DNA emulsion; d) coalescing the
library emulsion of step (b) with the chromosomal emulsion of step
(c), thereby forming a nanoreactor; e) amplifying the DNA in the
nanoreactor; f) isolating the beads; g) screening for beads
containing DNA; and h) sequencing the beads containing DNA, wherein
one or more steps of (a)-(h) are performed on a microfluidic
device.
BRIEF DESCRIPTION OF DRAWINGS
[0016] Non-limiting embodiments of the present invention will be
described by way of example with reference to the accompanying
drawings, which are schematic and are not intended to be drawn to
scale. In the drawings, each identical or nearly identical
component illustrated is typically represented by a single numeral.
For the purposes of clarity, not every component is labeled in
every drawing, nor is every component of each embodiment of the
invention shown where illustration is not necessary to allow those
of ordinary skill in the art to understand the invention. In the
drawings:
[0017] FIG. 1 is an schematic illustrating the interacting modules
of a microfluidic device of the present invention.
[0018] FIG. 2 is a photograph and accompanying graphs showing the
flow cytometric-cell-based assay for human and bacterial cells.
[0019] FIG. 3 is a schematic and graph showing simultaneous two
color fluorescence detection.
[0020] FIG. 4 is a schematic illustrating chemical library
screening using a nanoreactor of the present invention.
[0021] FIG. 5 is a schematic illustrating the physical basis of
fluorescence polarization assays.
[0022] FIG. 6 is several graphs showing the absorption and emission
spectra of q-dots and organic dye.
[0023] FIG. 7 is a schematic showing the extension to the
fluorescence test station required to perform polarization
fluorescence measurements simultaneously with q-dot readout.
[0024] FIG. 8 is a schematic and accompany graphs showing a
fluorescence polarization based kinase assay.
[0025] FIG. 9 is a schematic illustrating the retrosynthetic
analysis of Gleevec.
[0026] FIG. 10 is a schematic illustrating the synthesis of a
fluorescent and non-fluorescent product from the same
precursor.
[0027] FIG. 11 is schematic showing the use of nucleic acids for
chemical encoding and decoding tagging of chemical reactions.
[0028] FIG. 12 is a schematic illustrating a taqman assay and
molecular beacon probes.
[0029] FIG. 13 is a graph showing polarized fluorescence
signals.
[0030] FIG. 14 is a graph showing a time trace and histogram of
fluorescence polarization calculated from analyzed droplets.
[0031] FIG. 15 (A) is a schematic illustrating dielectrophoretic
stopping of droplet A allowing droplet B to contact A and coalesce.
The dielectrophoretic force is not strong enough to stop the
combined A+B and they move off in the stream. This is shown in the
photomicrograph, FIG. 15 (B).
[0032] FIG. 16 is a schematic (a) and photographs (b, e) showing
droplets made on-chip (A) being interdigitated with library
droplets (B). The droplets are of different size and the smaller
droplets B move at a higher velocity than droplets A until they
catch up after which they move together. An electric field causes
the droplets to coalesce, FIG. 16 (c). Droplets A and B may both
come from libraries (made off-chip) FIG. 16 (d), or be made
on-chip.
[0033] FIG. 17 (A-D) shows alternate ways to achieve
interdigitation of droplets of different type.
[0034] FIG. 18A-D highlight observed passive coalescence of coupled
droplet pairs--(A) Tee 0.25 mM FC-1% E5-5% PVP 70-FF 1% E5-5%
PVP-0.1 mM FC-PBS 70-11172005-nozzles-3.cin; (B) Tee 0.25 mM FC-1%
E5-5% PVP 90-FF 1% E5-5% PVP-0.1 mM FC-PBS
70-11172005-nozzles-5.cin; (C) Tee 0.25 mM FC-1% E5-5% PVP 90-FF 1%
E5-5% PVP-0.1 mM FC-PBS 70-11172005-nozzles-prior to
coalesce-8.cin; (D) 1% E5-5% PVP-0.25 mM FC-50-1% E5-5% PVP-0.1 mM
FC-PBS 50-11172005-overview-1.cin.
[0035] FIG. 19 shows a schematic diagram of the assembly of modules
used for sequencing exons of individual chromosomes. (A) Individual
specific primer-pairs to different exons along with a primer-bound
bead are each separately emulsified and then pooled to create a
library emulsion (a set of 96 exon primer pairs are shown for
illustrative purposes); (B) Individual modules are strung together
in a sequence of droplet operations. A chromosomal DNA solution is
diluted such that a 30 micron drop contains, on average, slightly
less than a half-genome's concentration of DNA. Droplets from the
pooled emulsion library set of exon-specific primers are combined
with droplets containing the diluted solution of chromosomal DNA
and used in a bead-based DNA amplification reaction (i.e., PCR);
(C) The DNA-containing beads will be randomly placed into a
picotiter plate and sequenced using a 454 Corp.'s Life Sciences DNA
sequencing instrument.
[0036] FIG. 20 describes sample preparation and DNA sequencing on
the 454 Instrument. A) Genomic DNA is isolated, fragmented, ligated
to adapters and separated into single strands (top left). Fragments
are bound to beads under conditions that favor one fragment per
bead, the beads are captured in the droplets of a
PCR-reaction-mixture-in-oil emulsion and PCR amplification occurs
within each droplet, resulting in beads each carrying ten million
copies of a unique DNA template (top, second from the left). The
emulsion is broken, the DNA strands are denatured, and beads
carrying single-stranded DNA clones are deposited into wells of a
fiber-optic slide (bottom left). Smaller beads carrying immobilized
enzymes required for pyrophosphate sequencing are deposited into
each well (bottom, second from the left); B) Microscope photograph
of emulsion showing droplets containing a bead and empty droplets.
The thin arrow points to a 28-mm bead; the thick arrow points to an
approximately 100-mm droplet; C) Scanning electron micrograph of a
portion of a fiber-optic slide, showing fiber-optic cladding and
wells before bead deposition; D) The sequencing instrument consists
of the following major subsystems: a fluidic assembly; E) a flow
chamber that includes the well-containing fiber-optic slide; F) a
CCD camera-based imaging assembly; G) and a computer that provides
the necessary user interface and instrument control.
[0037] FIG. 21 shows measurement of DNA hybridization using
fluorescence polarization (FP) on the microfluidic device described
herein. (A) FP can be used to measure the binding of 2 DNA
molecules on the microfluidic device. (B) Oligonucleotide 102 is
complement to both 101 and 103. The addition of either
oligonucleotide to the labeled 102 shifts the mP value, indicative
of binding. Addition of 102 to non-complementing oligos does not
change its mP (data not shown). Oligonucleotides; #101: 5'
Biotin-ATCCGCCCCAGCAGCTGCCAGGCACAGCCCCTAAACTCCTGATTTATGCTGCATCCATTTTGC
3'; #102: 5' Fluorescein-GCAAAATGGATGCAGCATAAATCAGGAGTTTAG 3';
#103: 5' Fluorescein-CTAAACTCCTGATTTA TGCTGCATCCATTTTGC-3'.
DETAILED DESCRIPTION
[0038] The microfluidic devices and methods of use described herein
are based on the creation and electrical manipulation of aqueous
phase droplets completely encapsulated by an inert fluorocarbon oil
stream. This combination enables electrically addressable droplet
generation, highly efficient droplet coalescence, precision droplet
breaking and recharging, and controllable single droplet sorting.
Additional passive modules include multi-stream droplet
formulations, mixing modules, and precision break-up modules. The
integration of these modules is an essential enabling technology
for a droplet based, high-throughput microfluidic reactor system.
The microfluidic devices of the present invention can use a
flow-focusing geometry to form the droplets. For example, a water
stream can be infused from one channel through a narrow
constriction; counter propagating oil streams (preferably
fluorinated oil) hydrodynamically focus the water stream and
stabilize its breakup into micron size droplets as it passes
through the constriction. In order to form droplets, the viscous
forces applied by the oil to the water stream must overcome the
water surface tension. The generation rate, spacing and size of the
water droplets is controlled by the relative flow rates of the oil
and the water streams and nozzle geometry. While this
emulsification technology is extremely robust, droplet size and
rate are tightly coupled to the fluid flow rates and channel
dimensions. Moreover, the timing and phase of the droplet
production cannot be controlled. To overcome these limitations, the
microfluidic devices of the present invention can incorporate
integrated electric fields, thereby creating an electrically
addressable emulsification system. In one embodiment, this can be
achieved by applying high voltage to the aqueous stream and charge
the oil water interface. The water stream behaves as a conductor
while the oil is an insulator; electrochemical reactions charge the
fluid interface like a capacitor. At snap-off, charge on the
interface remains on the droplet. The droplet size decreases with
increasing field strength. At low applied voltages the electric
field has a negligible effect, and droplet formation is driven
exclusively by the competition between surface tension and viscous
flow, as described above. The microfluidic, droplet-based
reaction-confinement system of the present invention can further
include a mixer which combines two or more reagents to initiate a
chemical reaction. Multi-component droplets can easily be generated
by bringing together streams of materials at the point where
droplets are made. However, all but the simplest reactions require
multiple steps where new reagents are added during each step. In
droplet-based microfluidic devices, this can be best accomplished
by combining (i.e. coalescing) different droplets, each containing
individual reactants. However, this is particularly difficult to
achieve in a microfluidic device because surface tension,
surfactant stabilization, and drainage forces all hinder droplet
coalescence; moreover, the droplets must cross the stream lines
that define their respective flows and must be perfectly
synchronized to arrive at a precise location for coalescence. The
microfluidic devices of the present invention overcome these
difficulties by making use of electrostatic charge, placing charges
of opposite sign on each droplet, and applying an electric field to
force them to coalesce. By way of non-limiting example, a device
according to the present invention can include two separate nozzles
that generate droplets with different compositions and opposite
charges. The droplets are brought together at the confluence of the
two streams. The electrodes used to charge the droplets upon
formation also provide the electric field to force the droplets
across the streamlines, leading to coalescence. In the absence of
an electric field, droplets in the two streams do not in general
arrive at the point of confluence at exactly the same time. When
they do arrive synchronously the oil layer separating the droplets
cannot drain quickly enough to facilitate coalescence and as a
result the droplets do not coalesce. In contrast, upon application
of an electric field, droplet formation becomes exactly
synchronized, ensuring that droplets each reach the point of
confluence simultaneously (i.e., paired droplets).
[0039] Moreover, since the droplets are oppositely charged they are
attracted to one another, which forces them to traverse the fluid
stream lines and contact each other, thereby causing them to
coalesce. The remarkable synchronization of the droplet formation
results from coupling of the break-off of each of the pair of
droplets as mediated by the electric field. The use of oppositely
charged droplets and an electric field to combine and mix reagents
is extremely robust, and 100% of the droplets coalesce with their
partner from the opposite stream.
[0040] Other embodiments of the microfluidic devices of the present
invention can include a droplet sorter. The contents of individual
droplets must be probed, and selected droplets sorted into discreet
streams. In one embodiment, such sorting in microfluidic devices
can be accomplished through the use of mechanical valves. In
another embodiment of the present invention, the use of
electrostatic charging of droplets provides an alternate means that
can be precisely controlled, can be switched at high frequencies,
and requires no moving parts. Electrostatic charge on the droplets
enables drop-by-drop sorting based on the linear coupling of charge
to an external electric field. As an example, a T-junction
bifurcation that splits the flow of carrier fluid equally will also
randomly split the droplet population equally into the two streams.
However, a small electric field applied at the bifurcation
precisely dictates which channel the drops enter. Varying the
direction of the field varies the direction of the sorted droplets.
The large forces that can be imparted on the droplets and the short
time required to switch the field make this a fast and robust
sorting engine with no moving parts; thus the processing rate is
limited only by the rate of droplet generation and electric field
switching time, and can easily exceed 20,000 per second.
[0041] Accordingly, in one embodiment the present invention
provides a microfluidic device comprising a microfabricated
substrate comprising at least one inlet channel adapted to carry at
least one dispersed phase fluid and at least one main channel
adapted to carry a continuous phase fluid, where inlet channel is
in fluid communication with the main channel at one or more inlet
modules such that the dispersed phase fluid is immiscible with the
continuous phase fluid and forms a plurality of droplets in the
continuous phase fluid; a coalescence module, where an electric
field is applied to cause two or more droplets to coalesce; and c)
a detection module including a detection apparatus for evaluating
the contents and/or characteristics of the coalesced droplets
produced in the coalescence module. The microfabricated substrate
can further comprise one or more sorting modules, collection
modules, waste modules, branch channels, delay modules, mixing
modules and/or UV release modules, or any combinations thereof in
any order. FIG. 1.
[0042] The present invention also provides methods of creating a
nanoreactor. The method includes a) providing a microfabricated
substrate comprising at least one inlet channel adapted to carry at
least one dispersed phase fluid and at least one main channel
adapted to carry a continuous phase fluid, where the inlet channel
is in fluid communication with the main channel at one or more
inlet modules, and where the dispersed phase fluid is immiscible
with the continuous phase fluid; b) flowing a first dispersed phase
fluid through a first inlet channel such that the first dispersed
phase fluid forms one or more droplets in the continuous phase
fluid; c) flowing at least a second dispersed phase fluid through
an at least second inlet channel such that the second dispersed
phase fluid forms one or more droplets in the continuous phase
fluid; and d) coalescing at least one droplet formed in step (b)
with at least one droplet formed in step (c) under the influence of
an electric field, thereby producing a nanoreactor.
[0043] The present invention also provides a method for
manipulating a nanoreactor. The method includes providing a
nanoreactor as described herein; providing a plurality of
electrically addressable channel bearing microfluidic modules
integrally arranged with each other on a microfabricated substrate
so as to be in fluid communication and providing a control system
for manipulating the nanoreactor.
[0044] The present invention also provides methods of manipulating
biological/chemical material. The method includes a) providing a
microfabricated substrate comprising at least one inlet channel
adapted to carry at least one dispersed phase fluid and at least
one main channel adapted to carry a continuous phase fluid, where
the inlet channel is in fluid communication with the main channel
at one or more inlet modules, and where the dispersed phase fluid
is immiscible with the continuous phase fluid; b) flowing a first
dispersed phase fluid comprising a first biological/chemical
material through a first inlet channel such that the first
dispersed phase fluid resides as one or more droplets in the
continuous phase fluid; c) flowing at least a second dispersed
phase fluid comprising a second biological/chemical material
through a second inlet channel such that the second dispersed phase
fluid resides as one or more droplets in the continuous phase
fluid; d) slowing or stopping at least one droplet formed in step
(b) by exerting a dielectrophoretic force onto the droplet; e)
coalescing at least one droplet formed in step (c) with the droplet
slowed or stopped in step (d) under the influence of an electric
field within a coalescence module, thereby producing a nanoreactor,
f) incubating the nanoreactor within a delay module; and g)
interrogating the nanoreactor for a predetermined characteristic
within a detection module. Slowing or stopping the droplets from
step (b) allows pairing of the droplets from step (c) before they
move to the location (e) where they are driven to coalesce by an
electric field, or passively by passing through a narrowing of the
channel.
[0045] In step (d), the pairing of droplets from (b) and (c) may be
achieved in one of three ways: (i) using the dielectrophoretic
force produced by the electric field gradient; (ii) using droplets
of two different sizes, which works best when one droplet is
comparable to the channel width and one droplet is smaller than the
channel width, so that the smaller droplet catches up to the larger
droplet; and (iii) the droplet in steps (b) and (c) have different
viscosities, and thus, move at different velocities. Preferably,
the droplets are of different sizes, and more preferably, the
larger droplet has enough volume so that it would have a diameter
greater than the channel width if it were spherical.
[0046] Methods of sorting biological/chemical material, although
frequently desired, is not necessary in order to use the devices or
practice the methods of the present invention. In particular, the
devices and methods of the invention also include embodiments
wherein the biological/chemical material is analyzed and/or
identified, but is not sorted. The generation of nanoreactors
through the coalescence of two droplets, although frequently
desired, is not necessary in order to use the devices or practice
the methods of the present invention. In particular, the devices
and methods of the invention also include embodiments wherein the
biological/chemical material is sorted without a coalescence
event.
Substrates
[0047] The present invention also provides methods of producing a
microfluidic device. The method of producing a microfluidic device
comprises one or more of the following steps in any combination: 1)
hard lithography, 2) soft lithography, 3) extraction and/or punch
though, 4) bonding, 5) channel coating, 6) interconnect assembly,
7) electrode injection and 8) waveguide injection and fiber
installation. The foregoing steps are described in more detail
herein.
[0048] An "analysis unit" is a microfabricated substrate, e.g., a
microfabricated chip, having at least one inlet channel, at least
one main channel, at least one coalescence module, and at least one
detection module. The analysis unit can further contain one or more
sorting module. The sorting module can be in fluid communication
with branch channels in communication with one or more outlet
modules (collection module or waste module). For sorting, at least
one detection module cooperates with at least one sorting module to
divert flow via a detector-originated signal. It shall be
appreciated that the "modules" and "channels" are in fluid
communication with each other and therefore may overlap; i.e.,
there may be no clear boundary where a module or channel begins or
ends. A device according to the invention may comprise a plurality
of analysis units.
[0049] A variety of channels for sample flow and mixing can be
microfabricated on a single chip and can be positioned at any
location on the chip as the detection or sorting modules, e.g., for
kinetic studies. A plurality of analysis units of the invention may
be combined in one device. Microfabrication applied according to
the invention eliminates the dead time occurring in conventional
gel electrophoresis or flow cytometric kinetic studies, and
achieves a better time-resolution. Furthermore, linear arrays of
channels on a single chip, i.e., a multiplex system, can
simultaneously detect and sort a sample by using an array of photo
multiplier tubes (PMT) for parallel analysis of different channels.
This arrangement can be used to improve throughput or for
successive sample enrichment, and can be adapted to provide a very
high throughput to the microfluidic devices that exceeds the
capacity permitted by conventional flow sorters. Circulation
systems can be used in cooperation with these and other features of
the invention. Positive displacement pressure driven flow is a
preferred way of controlling fluid flow and electric fields and
electric field gradients are a preferred way of manipulating
droplets within that flow.
[0050] Microfabrication permits other technologies to be integrated
or combined with flow cytometry on a single chip, such as PCR,
moving cells using optical tweezer/cell trapping, transformation of
cells by electroporation, .mu.TAS, and DNA hybridization. Detectors
and/or light filters that are used to detect cellular
characteristics of the reporters can also be fabricated directly on
the chip. Preferably, detectors are off-chip free space optics or
off-chip electronics with on-chip leads.
[0051] A device of the invention can be microfabricated with a
sample solution reservoir or well or other apparatus for
introducing a sample to the device, at the inlet module, which is
typically in fluid communication with an inlet channel. A reservoir
may facilitate introduction of molecules or cells into the device
and into the sample inlet channel of each analysis unit. An inlet
module may have an opening such as in the floor of the
microfabricated chip, to permit entry of the sample into the
device. The inlet module may also contain a connector adapted to
receive a suitable piece of tubing, such as liquid chromatography
or HPLC tubing, through which a sample may be supplied. Such an
arrangement facilitates introducing the sample solution under
positive pressure in order to achieve a desired infusion rate at
the inlet module.
[0052] A microfabricated device of the invention is preferably
fabricated from a silicon microchip or silicon elastomer. The
dimensions of the chip are those of typical microchips, ranging
between about 0.5 cm to about 7.5 cm per side and about 1 micron to
about 1 cm in thickness. A microfabricated device can be
transparent and can be covered with a material having transparent
properties, such as a glass coverslip, to permit detection of a
reporter, for example, by an optical device such as an optical
microscope.
[0053] The device of the present invention can comprise inlet and
outlet interconnects. The interconnections, including tubes, must
be extremely clean and make excellent bonding with the PDMS surface
in order to allow proper operation of the device. The difficulty in
making a fluidic connection to a microfluidic device is primarily
due to the difficulty in transitioning from a macroscopic fluid
line into the device while minimizing dead volume. Development of a
commercial microfluidic platform requires a simple, reliable
fluidic interconnect in order to reduce the chance of operator and
error leaks. The curing and manufacturing of the PDMS slab with the
tubes already placed on the silicon wafer accomplish these
goals.
[0054] The template process can include, but is not limited to, the
following features. In order to minimize contamination and leakage,
process operations that allow for greater reproducibility and
reliability are improved. Tubes and interconnects for the PDMS slab
can be cured in place. The tubes and interconnects can be placed in
position by applying a UV-cured adhesive to allow for holding the
tubes in place on the silicone wafer. Once the tubes are placed in
position, PDMS can be poured over the wafer and cured. The cured
PDMS, along with the tubes in place, can be peeled off of the
silicone wafer easily. This process can be applied to fluidics
channels as well as other connection channels. Once the adhesive is
applied onto the wafer, the process will allow for quick templating
of PDMS slabs with exact reproducibility of channel locations and
cleanliness. Tubes of any size can be implemented for this process.
This process allows for less stress on the interconnection joints
and smaller interconnection footprints in the device.
[0055] In one embodiment, small interconnects based on creating a
face seal between the tubing and the device are used. A grommet may
be placed into either a tapered hole or a hole with perpendicular
walls. In one embodiment, the raised contact surface between the
two sides is formed on the tubing side instead of the device side.
In another embodiment, the sealing feature can be molded into the
device. In yet another embodiment, a possible interconnect can be
molded and bonded on a glass substrate directly from PDMS. In this
embodiment, a thin film of PDMS can be simultaneously formed and
bonded to the top of the glass slide and permits the use of
isolated patterned electrodes and heating elements beneath the
fluid channels. If not required, the seals could be made without
the top skin. The raised contact surface could also be built into
the tubing side. The sealing surface on the tubing side of the
connection can be formed directly into the face of the tubing,
although a separate piece secured to the tubing assembly/retaining
nut may also be used.
[0056] The tubing side of the interconnect can be mounted into a
retaining block that provides precise registration of the tubing,
while the microfluidic device can be positioned accurately in a
carrier that the retaining block would align and clamp to. The
total dead volume associated with these designs would be critically
dependent on how accurately the two mating surfaces could be
positioned relative to each other. the maximum force required to
maintain the seal would be limited by the exact shape and
composition of the sealing materials as well as the rigidity and
strength of the device itself. The shapes of the mating surfaces
can be tailored to the minimal leakage potential, sealing force
required, and potential for mis-alignment. By way of non-limiting
example, the single ring used in the fluidic interconnects can be
replaced with a series of rings of appropriate cross-sectional
shape.
[0057] The device of the present invention can comprise a layer,
such as a glass slide, which is perforated for functional
interconnects, such as fluidic, electrical, and/or optical
interconnects, and sealed to the back interface of the device so
that the junction of the interconnects to the device is leak-proof.
Such a device can allow for application of high pressure to fluid
channels without leaking.
[0058] A silicon substrate containing the microfabricated flow
channels and other components is preferably covered and sealed,
most preferably with a transparent cover, e.g., thin glass or
quartz, although other clear or opaque cover materials may be used.
When external radiation sources or detectors are employed, the
detection module is covered with a clear cover material to allow
optical access to the cells. For example, anodic bonding to a
"PYREX" cover slip can be accomplished by washing both components
in an aqueous H.sub.2SO.sub.4/H.sub.2O.sub.2 bath, rinsing in
water, and then, for example, heating to about 350.degree. C. while
applying a voltage of 450 V.
[0059] The present invention provides improved methods of bonding
PDMS to incompatible media. Normal methods of bonding various
materials (plastic, metals, etc) directly to materials such as
PDMS, silicone, Teflon, and PEEK using traditional bonding
practices (adhesives, epoxies, etc) do not work well due to the
poor adhesion of the bonding agent to materials such as PDMS.
Normal surface preparation by commercially available surface
activators has not worked well in microfluidic device
manufacturing. This problem is eliminated by treating the PDMS
surface to be bonded with high intensity oxygen or air plasma. The
process converts the top layer of PDMS to glass which bonds
extremely well with normal adhesives. Tests using this method to
bond external fluid lines to PDMS using a UV-cure adhesive (Loctite
352, 363, and others) resulted in a bond that is stronger than the
PDMS substrate, resulting in fracture of the PDMS prior to failure
of the bond. The present method combines high radiant flux,
wavelength selection, and cure exposure time to significantly
enhance the bond strength of the adhesive.
Channels
[0060] The invention provides microfluidic devices having channels
that form the boundary for a fluid. The channels of the
device-carry a mixture of incompatible or immiscible fluids, such
as an oil-water mixture. Droplets of aqueous solution containing a
biological/chemical material are dispersed within the oil or other
incompatible solvent. Each droplet of this multi-phase mixture can
encapsulate one or more molecules, particles, or cells. The
droplets are trapped and their boundaries are defined by channel
walls, and therefore they do not diffuse and/or mix. Individual
particles or molecules can be separately compartmentalized inside
individual droplets. These droplets can be analyzed, combined with
other droplets (e.g. to react droplet contents) and analyzed, and
then sorted. Thus, the invention also provides methods for
analyzing, combining, detecting and/or sorting of
biological/chemical materials.
[0061] The channels present in the device can be made with micron
dimensions and the volume of the detection module is precisely
controlled. The planar geometry of the device allows the use of
high numerical aperture optics, thereby increasing the sensitivity
of the system. Because the system is entirely self-contained, there
is no aerosol formation, allowing for much safer sorting of
biohazardous materials. Materials sorted in the device are
compartmentalized within individual droplets of an aqueous solution
traveling in a flow of a second, incompatible or immiscible
solution. Thus, there is no problem with the material diffusing or
exchanging positions, even when sorting or analyzing extremely
small particles, molecules, or reagents. In a preferred embodiment,
water droplets are extruded into a flow of oil, but any fluid phase
may be used as a droplet phase and any other incompatible or
immiscible fluid or phase may be used as a barrier phase.
[0062] A "channel," as used herein, means a feature on or in a
device (e.g., a substrate) that at least partially directs the flow
of a fluid. In some cases, the channel may be formed, at least in
part, by a single component, e.g., an etched substrate or molded
unit. The channel can have any cross-sectional shape, for example,
circular, oval, triangular, irregular, square or rectangular
(having any aspect ratio), or the like, and can be covered or
uncovered (i.e., open to the external environment surrounding the
channel). In embodiments where the channel is completely covered,
at least one portion of the channel can have a cross-section that
is completely enclosed, and/or the entire channel may be completely
enclosed along its entire length with the exception of its inlet
and outlet.
[0063] A channel may have an aspect ratio (length to average
cross-sectional dimension) of at least 2:1, more typically at least
3:1, 5:1, or 10:1. As used herein, a "cross-sectional dimension,"
in reference to a fluidic or microfluidic channel, is measured in a
direction generally perpendicular to fluid flow within the channel.
An open channel generally will include characteristics that
facilitate control over fluid transport, e.g., structural
characteristics (an elongated indentation) and/or physical or
chemical characteristics (hydrophobicity vs. hydrophilicity) and/or
other characteristics that can exert a force (e.g., a containing
force) on a fluid. The fluid within the channel may partially or
completely fill the channel. In some cases the fluid may be held or
confined within the channel or a portion of the channel in some
fashion, for example, using surface tension (e.g., such that the
fluid is held within the channel within a meniscus, such as a
concave or convex meniscus). In an article or substrate, some (or
all) of the channels may be of a particular size or less, for
example, having a largest dimension perpendicular to fluid flow of
less than about 5 mm, less than about 2 mm, less than about 1 mm,
less than about 500 microns, less than about 200 microns, less than
about 100 microns, less than about 60 microns, less than about 50
microns, less than about 40 microns, less than about 30 microns,
less than about 25 microns, less than about 10 microns, less than
about 3 microns, less than about 1 micron, less than about 300 nm,
less than about 100 nm, less than about 30 nm, or less than about
10 nm or less in some cases. In one embodiment, the channel is a
capillary. Of course, in some cases, larger channels, tubes, etc.
can be used to store fluids in bulk and/or deliver a fluid to the
channel.
[0064] In some embodiments, the dimensions of the channel may be
chosen such that fluid is able to freely flow through the channel,
for example, if the fluid contains cells. The dimensions of the
channel may also be chosen, for example, to allow a certain
volumetric or linear flow rate of fluid in the channel. Of course,
the number of channels and the shape of the channels can be varied
by any method known to those of ordinary skill in the art. In some
cases, more than one channel or capillary may be used. For example,
two or more channels may be used, where they are positioned inside
each other, positioned adjacent to each other, etc.
[0065] A "main channel" is a channel of the device of the invention
which permits the flow of molecules, cells, small molecules or
particles past a coalescence module for coalescing one or more
droplets, a detection module for detection (identification) or
measurement or a droplet and a sorting module, if present, for
sorting a droplet based on the detection in the detection module.
The coalescence, detection and/or sorting modules can be placed or
fabricated into the main channel. The main channel is typically in
fluid communication with an inlet channel or inlet module. An
"inlet channel" permits the flow of molecules, cells, small
molecules or particles into the main channel. One or more inlet
channels communicate with one or more means for introducing a
sample into the device of the present invention. The inlet channel
communicates with the main channel at an inlet module. The main
channel is also typically in fluid communication with an outlet
module and optionally with branch channels, each of which may have
a collection module or waste module. These channels permit the flow
of cells out of the main channel.
[0066] Channels of the device of the present invention can be
formed from silicon elastomer (e.g. RTV), urethane compositions, of
from silicon-urethane composites such as those available from
Polymer Technology Group (Berkeley, Calif.), e.g. PurSil.TM. and
CarboSil.TM.. The channels may also be coated with additives or
agents, such as surfactants, TEFLON, or fluorinated oils such as
octadecafluoroctane (98%, Aldrich), Fluorinert (FC-3283; 3M), or
fluorononane, any of which can be modified to contain a
fluorosurfactant. Fluorinated oils have favorable properties
including chemical inertness, high gas permeability, and
biocompatibility, which are desirable in microfluidic applications.
TEFLON is particularly suitable for silicon elastomer (RTV)
channels, which are hydrophobic and advantageously do not absorb
water, but they may tend to swell when exposed to an oil phase.
Swelling may alter channel dimensions and shape, and may even close
off channels, or may affect the integrity of the chip, for example
by stressing the seal between the elastomer and a coverslip.
Urethane substrates do not tend to swell in oil but are
hydrophillic, they may undesirably absorb water, and tend to use
higher operating pressures. Hydrophobic coatings may be used to
reduce or eliminate water absorption. Absorption or swelling issues
may also be addressed by altering or optimizing pressure or droplet
frequency (e.g. increasing periodicity to reduce absorption).
RTV-urethane hybrids may be used to combine the hydrophobic
properties of silicon with the hydrophilic properties of
urethane.
[0067] The channels of the invention are microfabricated, for
example by etching a silicon chip using conventional
photolithography techniques, or using a micromachining technology
called "soft lithography" as described by Whitesides and Xia,
Angewandte Chemie International Edition 37, 550 (1998). These and
other microfabrication methods may be used to provide inexpensive
miniaturized devices, and in the case of soft lithography, can
provide robust devices having beneficial properties such as
improved flexibility, stability, and mechanical strength. When
optical detection is employed, the invention also provides minimal
light scatter from molecule, cell, small molecule or particle
suspension and chamber material. Devices according to the invention
are relatively inexpensive and easy to set up. They can also be
disposable, which greatly relieves many of the concerns of gel
electrophoresis (for molecules), and of sterilization and permanent
adsorption of particles into the flow chambers and channels of
conventional FACS machines.
[0068] The channels of the device of the present invention can be
of any geometry as described. However, the channels of the device
can comprise a specific geometry such that the contents of the
channel are manipulated, e.g., sorted, mixed, prevent clogging,
etc.
[0069] For particles (e.g., cells) or molecules that are in
droplets (i.e., deposited by the inlet module) within the flow of
the main channel, the channels of the device are preferably
rounded, with a diameter between about 2 and 100 microns,
preferably about 60 microns, and more preferably about 30 microns
at the cross-flow area or droplet extrusion region. This geometry
facilitates an orderly flow of droplets in the channels. Similarly,
the volume of the detection module in an analysis device is
typically in the range of between about 10 femtoliters (fl) and
5000 fl, preferably about 40 or 50 fl to about 1000 or 2000 fl,
most preferably on the order of about 200 fl. In preferred
embodiments, the channels of the device, and particularly the
channels of the inlet connecting to a droplet extrusion region, are
between about 2 and 50 microns, most preferably about 30
microns.
[0070] A microfluidic device can include a bifurcation geometry
designed in such a manner as to minimize fluidic shear forces on
droplets during sorting. Known devices describe bifurcation
geometries in which significant shear forces affect droplets during
sorting. Specifically droplets may experience shear forces when
moving under the influence of the sorting force across the width of
the input channel prior to encountering the bifurcation, and
droplets may experience shear forces at the bifurcation point which
are applied in such a manner as to elongate or even tear the
droplet apart.
[0071] A microfluidic device comprising channels having a
bifurcation geometry can minimize these shear forces by (i)
including a necked-down segment of the input channel upstream of
the bifurcation where the droplet is diagnosed to make the sorting
decision, and/or by (ii) including a flaired-out segment of the
input channel immediately prior to the bifurcation, and/or by (iii)
including a fork on the far wall of the bifurcation. The shear
forces are minimized by component (i) because the sorting field is
applied while the droplet is in the necked-down segment. Therefore,
when the droplet exits the necked-down segment, the droplet is
placed on fluid streamlines, which will carry it out the desired
branch of the bifurcation. Furthermore, the droplet does not
significantly encounter fluid streamlines, which follow the
undesired branch of the bifurcation. The shear forces are minimized
by component (ii) because the droplet does not significantly impact
the far wall of the bifurcation at a point where it would
experience fluid streamlines, which follow the undesired branch of
the bifurcation. The shear forces are minimized by component (iii)
because the fork serves to focus the two sets of fluid streamlines
(i.e., the one set which follows one branch of the bifurcation, and
the other set which follows the other branch of the bifurcation)
away from each other.
[0072] A microfluidic device can include a specific geometry
designed in such a manner as to prevent the aggregation of
biological/chemical material and keep the biological/chemical
material separated from each other prior to encapsulation in
droplets. The geometry of channel dimension can be changed to
disturb the aggregates and break them apart by various methods,
that can include, but is not limited to, geometric pinching (to
force cells through a (or a series of) narrow region(s), whose
dimension is smaller or comparable to the dimension of a single
cell) or a barricade (place a series of barricades on the way of
the moving cells to disturb the movement and break up the
aggregates of cells).
[0073] Channel design can force biological/chemical material moving
along the center streamline through flow focus, e.g., using two
dilution channels at the entrance of the channel to prevent
attachment to the channel surface. This can also be used to prevent
the surface attachment by cells.
[0074] Droplets at these dimensions tend to conform to the size and
shape of the channels, while maintaining their respective volumes.
Thus, as droplets move from a wider channel to a narrower channel
they become longer and thinner, and vice versa. Droplets can be at
least about four times as long as they are wide. This droplet
configuration, which can be envisioned as a lozenge shape, flows
smoothly and well through the channels. Longer droplets, produced
in narrower channels, provides a higher shear, meaning that
droplets can more easily be sheared or broken off from a flow, i.e.
using less force. Droplets can also tend to adhere to channel
surfaces, which can slow or block the flow, or produce turbulence.
Droplet adherence is overcome when the droplet is massive enough in
relation to the channel size to break free. Thus, droplets of
varying size, if present, can combine to form uniform droplets
having a so-called critical mass or volume that results in smooth
or laminar droplet flow. Droplets that are longer than they are
wide, preferably about four times longer than they are wide,
generally have the ability to overcome channel adherence and move
freely through the microfluidic device. Thus, in an exemplary
embodiment with 60 micron channels, a typical free-flowing droplet
is about 60 microns wide and 240 microns long. Droplet dimensions
and flow characteristics can be influenced as desired, in part by
changing the channel dimensions, e.g. the channel width.
[0075] The microfabricated devices of this invention most
preferably generate round, monodisperse droplets. The droplets can
have a diameter that is smaller than the diameter of the
microchannel; i.e., preferably 40 to 100 .mu.m when cells are used
or 5 to 40 .mu.m when reagents are used. Monodisperse droplets may
be particularly preferably, e.g., in high throughput devices and
other embodiments where it is desirable to generate droplets at
high frequency and of high uniformity.
[0076] To prevent material (e.g., cells and other particles or
molecules) from adhering to the sides of the channels, the channels
(and coverslip, if used) may have a coating which minimizes
adhesion. Such a coating may be intrinsic to the material from
which the device is manufactured, or it may be applied after the
structural aspects of the channels have been microfabricated.
"TEFLON" is an example of a coating that has suitable surface
properties.
[0077] The surface of the channels of the microfluidic device can
be coated with any anti-wetting or blocking agent for the dispersed
phase. The channel can be coated with any protein to prevent
adhesion of the biological/chemical sample. For example, in one
embodiment the channels are coated with BSA, PEG-silane and/or
fluorosilane. For example, 5 mg/ml BSA is sufficient to prevent
attachment and prevent clogging. In another embodiment, the
channels can be coated with a cyclized transparent optical polymer
obtained by copolymerization of perfluoro (alkenyl vinyl ethers),
such as the type sold by Asahi Glass Co. under the trademark Cytop.
In such an embodiment, the coating is applied from a 0.1-0.5 wt %
solution of Cytop CTL-809M in CT-Solv 180. This solution can be
injected into the channels of a microfluidic device via a plastic
syringe. The device can then be heated to about 90.degree. C. for 2
hours, followed by heating at 200.degree. C. for an additional 2
hours. In another embodiment, the channels can be coated with a
hydrophobic coating of the type sold by PPG Industries, Inc. under
the trademark Aquapel (e.g., perfluoroalkylalkylsilane surface
treatment of plastic and coated plastic substrate surfaces in
conjunction with the use of a silica primer layer) and disclosed in
U.S. Pat. No. 5,523,162, which patent is hereby incorporated by
reference. By fluorinating the surfaces of the channels, the
continuous phase preferentially wets the channels and allows for
the stable generation and movement of droplets through the device.
The low surface tension of the channel walls thereby minimizes the
accumulation of channel clogging particulates.
[0078] The surface of the channels in the microfluidic device can
be also fluorinated to prevent undesired wetting behaviors. For
example, a microfluidic device can be placed in a polycarbonate
dessicator with an open bottle of
(tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane. The
dessicator is evacuated for 5 minutes, and then sealed for 20-40
minutes. The dessicator is then backfilled with air and removed.
This approach uses a simple diffusion mechanism to enable facile
infiltration of channels of the microfluidic device with the
fluorosilane and can be readily scaled up for simultaneous device
fluorination.
[0079] The microfluidic device can include a syringe (or other
glass container) that is treated with a vapor or solution of an
appropriate PEG-silane to effect the surface PEG functionalization.
The purpose for treating the walls of glass containers (e.g.,
syringes) with a PEG functionality is to prevent biological
adhesion to the inner walls of the container, which frustrates the
proper transfer of biological/chemical materials into the
microfluidic device of the present invention.
[0080] The device of the present invention can comprise one or more
fluid channels to inject or remove fluid in between droplets in a
droplet stream for the purpose of changing the spacing between
droplets.
[0081] The invention provides methods of cell manipulation by
channel geometry. Most cells, especially mammalian cells intend to
attach each other in suspension. The purpose of changing channel
geometry is to detach the cell from aggregates and keep them
separated from each other before they are encapsulated in the
drops. The geometry of channel dimension can be changed to disturb
the aggregates and break them apart by various methods, that can
include, for example, geometric pinching and/or barricades. With
geometric pinching, cells are forced through one or more narrow
regions, whose dimension is smaller or comparable to the dimension
of a single cell. With a barricade, a series of
obstacles/impediments (barricades) are placed in the way of the
moving cells to disturb the movement and break up the aggregates of
cells.
[0082] The present invention provides methods to prevent channel
clogging including methods of fluid pinching and surface coating.
Some cells and polystyrene beads tend to attach to the PDMS/Glass
surface. This is an undesired result as the accumulated beads can
clog the channel, especially the narrow region (i.e. nozzle).
Channel design and blocking reagent can be used in some embodiments
to prevent the beads' attachment to the channel surface and to each
other. Non-limiting examples include coating reagents and channel
design. A coating reagent, such as BSA (or any other protein), is
added to the bead buffer to coat the channel surface as well as the
beads' surface. 5 mg/ml BSA has shown to be sufficient to prevent
the beads' attachment. No clogging is observed in an experiment
with 10 um diameter beads in a 30 um wide and 25 um deep nozzle
device. With channel design, cells/beads are forced to move along
the center streamline through flow focus--Using two dilution
channels at the entrance of the beads' channel to prevent beads'
attachment to the channel surface. This can also used to prevent
the surface attachment by cells.
[0083] A typical analysis unit of the invention comprises a main
inlet channel that is part of and feeds or communicates directly
with a main channel, along with one or more sample inlet channels
in communication with the main channel at a inlet module situated
downstream from the main inlet. In one embodiment, each different
sample inlet channel preferably communicates with the main channel
at a different inlet module. In another embodiment, different
sample inlet channels can communication with the main channel at
the same inlet module. The inlet channel is further connected to a
means for introducing a sample to said device. The means can be a
well or reservoir. The well or reservoir further include an
acoustic actuator. The means can be temperature controlled. The
main channel is further connected to a means for collecting a
sample from said device. The means can be a well or reservoir. The
means can be temperature controlled.
[0084] The inlet module generally comprises a junction between the
sample inlet channel and the main channel such that a solution of a
sample (i.e., a fluid containing a sample such as molecules, cells,
small molecules (organic or inorganic) or particles) is introduced
to the main channel and forms a plurality of droplets. The sample
solution can be pressurized. The sample inlet channel can intersect
the main channel such that the sample solution is introduced into
the main channel at an angle perpendicular to a stream of fluid
passing through the main channel. For example, the sample inlet
channel and main channel intercept at a T-shaped junction; i.e.,
such that the sample inlet channel is perpendicular (90 degrees) to
the main channel. However, the sample inlet channel can intercept
the main channel at any angle, and need not introduce the sample
fluid to the main channel at an angle that is perpendicular to that
flow. The angle between intersecting channels is in the range of
from about 60 to about 120 degrees. Particular exemplary angles are
45, 60, 90, and 120 degrees.
[0085] The main channel in turn can communicate with two or more
branch channels at the sorting module or "branch point", if
present, forming, for example, a T-shape or a Y-shape. Other shapes
and channel geometries may be used as desired.
[0086] The device of the present invention can comprise one more
means for chromatographically sorting the sample prior to droplet
formation. The means can be in fluid communication with the inlet
channel and/or the inlet module. Preferably, the means is a
channel. The sample can be sorted by size, charge, hydrophobicity,
atomic mass, etc. The separating can be done isocratic or by
generating a gradient chemically, (for example using salt or
hydrophobicity), electrically, by pressure, or etc. For size
exclusion, the channel can be preloaded with Sepharose. The sample
is then loaded at one end, and the droplets are formed at an
opposing end. The sample separates by size prior to becoming
incorporated within a droplet.
Fluids
[0087] The term "flow" means any movement of liquid or solid
through a device or in a method of the invention, and encompasses
without limitation any fluid stream, and any material moving with,
within or against the stream, whether or not the material is
carried by the stream. For example, the movement of molecules,
beads, cells or virions through a device or in a method of the
invention, e.g. through channels of a microfluidic chip of the
invention, comprises a flow. This is so, according to the
invention, whether or not the molecules, beads, cells or virions
are carried by a stream of fluid also comprising a flow, or whether
the molecules, cells or virions are caused to move by some other
direct or indirect force or motivation, and whether or not the
nature of any motivating force is known or understood. The
application of any force may be used to provide a flow, including
without limitation, pressure, capillary action, electro-osmosis,
electrophoresis, dielectrophoresis, optical tweezers, and
combinations thereof, without regard for any particular theory or
mechanism of action, so long as molecules, cells or virions are
directed for detection, measurement or sorting according to the
invention.
[0088] The flow stream in the main channel is typically, but not
necessarily, continuous and may be stopped and started, reversed or
changed in speed. Prior to sorting, a liquid that does not contain
sample molecules, cells or particles can be introduced into a
sample inlet well or channel and directed through the inlet module,
e.g., by capillary action, to hydrate and prepare the device for
use. Likewise, buffer or oil can also be introduced into a main
inlet region that communicates directly with the main channel to
purge the device (e.g., or "dead" air) and prepare it for use. If
desired, the pressure can be adjusted or equalized, for example, by
adding buffer or oil to an outlet module.
[0089] The pressure at the inlet module can also be regulated by
adjusting the pressure on the main and sample inlet channels, for
example, with pressurized syringes feeding into those inlet
channels. By controlling the pressure difference between the oil
and water sources at the inlet module, the size and periodicity of
the droplets generated may be regulated. Alternatively, a valve may
be placed at or coincident to either the inlet module or the sample
inlet channel connected thereto to control the flow of solution
into the inlet module, thereby controlling the size and periodicity
of the droplets. Periodicity and droplet volume may also depend on
channel diameter, the viscosity of the fluids, and shear
pressure.
[0090] As used herein, the term "fluid stream" or "fluidic stream"
refers to the flow of a fluid, typically generally in a specific
direction. The fluidic stream may be continuous and/or
discontinuous. A "continuous" fluidic stream is a fluidic stream
that is produced as a single entity, e.g., if a continuous fluidic
stream is produced from a channel, the fluidic stream, after
production, appears to be contiguous with the channel outlet. The
continuous fluidic stream is also referred to as a continuous phase
fluid or carrier fluid. The continuous fluidic stream may be
laminar, or turbulent in some cases. The continuous fluidic stream
may be, e.g., solid or hollow (i.e., containing a second fluid
internally, for example, as in a hollow tube). It is to be
understood that wherever "tube" is used herein, the structure can
be a hollow, a solid or filled (i.e., not hollow) stream, a stream
that includes a central core and a surrounding layer or layers, any
of which can be selectively reacted with any others, or solidified,
or the like. In some cases, the central core is hollow, and/or
fluid may be removed from a hardened surrounding fluid to produce a
hollow tube. The continuous phase fluid can be a non-polar solvent.
The continuous phase fluid can be a fluorocarbon oil.
[0091] Similarly, a "discontinuous" fluidic stream is a fluidic
stream that is not produced as a single entity. The discontinuous
fluidic stream is also referred to as the dispersed phase fluid or
sample fluid. A discontinuous fluidic stream may have the
appearance of individual droplets, optionally surrounded by a
second fluid. A "droplet," as used herein, is an isolated portion
of a first fluid that completely surrounded by a second fluid. In
some cases, the droplets may be spherical or substantially
spherical; however, in other cases, the droplets may be
non-spherical, for example, the droplets may have the appearance of
"blobs" or other irregular shapes, for instance, depending on the
external environment. As used herein, a first entity is
"surrounded" by a second entity if a closed loop can be drawn or
idealized around the first entity through only the second entity.
The dispersed phase fluid can include a biological/chemical
material. The biological/chemical material can be tissues, cells,
particles, proteins, antibodies, amino acids, nucleotides, small
molecules, and pharmaceuticals. The biological/chemical material
can include one or more labels. The label can be a DNA tag, dyes or
quantum dot, or combinations thereof.
[0092] The term "emulsion" refers to a preparation of one liquid
distributed in small globules (also referred to herein as drops,
droplets or NanoReactors) in the body of a second liquid. The first
and second fluids are immiscible with each other. For example, the
discontinuous phase can be an aqueous solution and the continuous
phase can a hydrophobic fluid such as an oil. This is termed a
water in oil emulsion. Alternatively, the emulsion may be a oil in
water emulsion. In that example, the first liquid, which is
dispersed in globules, is referred to as the discontinuous phase,
whereas the second liquid is referred to as the continuous phase or
the dispersion medium. The continuous phase can be an aqueous
solution and the discontinuous phase is a hydrophobic fluid, such
as an oil (e.g., decane, tetradecane, or hexadecane). The droplets
or globules of oil in an oil in water emulsion are also referred to
herein as "micelles", whereas globules of water in a water in oil
emulsion may be referred to as "reverse micelles".
[0093] As used herein, the term "NanoReactor" and its plural
encompass the terms "droplet", "microdrop" or "microdroplet" as
defined herein, as well as an integrated system for the
manipulation and probing of droplets, as described in detail
herein. Nanoreactors as described herein can be 0-100 .mu.m (e.g.,
5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100)
[0094] The droplet forming liquid is typically an aqueous buffer
solution, such as ultrapure water (e.g., 18 mega-ohm resistivity,
obtained, for example by column chromatography), 10 mM Tris HCl and
1 mM EDTA (TE) buffer, phosphate buffer saline (PBS) or acetate
buffer. Any liquid or buffer that is physiologically compatible
with the population of molecules, cells or particles to be analyzed
and/or sorted can be used. The fluid passing through the main
channel and in which the droplets are formed is one that is
immiscible with the droplet forming fluid. The fluid passing
through the main channel can be a non-polar solvent, most
preferably decane (e.g., tetradecane or hexadecane), fluorocarbon
oil or another oil (for example, mineral oil).
[0095] The dispersed phase fluid may also contain
biological/chemical material (e.g., molecules, cells, or other
particles) for combination, analysis and/or sorting in the device.
The droplets of the dispersed phase fluid can contain more than one
particle or can contain no more than one particle. For example,
where the biological material comprises cells, each droplet
preferably contains, on average, no more than one cell. The
droplets can be detected and/or sorted according to their
contents.
[0096] The fluids used in the invention may contain one or more
additives, such as agents which reduce surface tensions
(surfactants). Surfactants can include Tween, Span,
fluorosurfactants, and other agents that are soluble in oil
relative to water. Surfactants can aid in controlling or optimizing
droplet size, flow and uniformity, for example by reducing the
shear force needed to extrude or inject droplets into an
intersecting channel. This can affect droplet volume and
periodicity, or the rate or frequency at which droplets break off
into an intersecting channel. Furthermore, the surfactant can serve
to stabilize aqueous emulsions in fluorinated oils from
coalescing.
[0097] The droplets may be coated with a surfactant. Preferred
surfactants that may be added to the continuous phase fluid
include, but are not limited to, surfactants such as sorbitan-based
carboxylic acid esters (e.g., the "Span" surfactants, Fluka
Chemika), including sorbitan monolaurate (Span 20), sorbitan
monopalmitate (Span 40), sorbitan monostearate (Span 60) and
sorbitan monooleate (Span 80), and perfluorinated polyethers (e.g.,
DuPont Krytox 157 FSL, FSM, and/or FSH). Other non-limiting
examples of non-ionic surfactants which may be used include
polyoxyethylenated alkylphenols (for example, nonyl-, p-dodecyl-,
and dinonylphenols), polyoxyethylenated straight chain alcohols,
polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated
mercaptans, long chain carboxylic acid esters (for example,
glyceryl and polyglycerl esters of natural fatty acids, propylene
glycol, sorbitol, polyoxyethylenated sorbitol esters,
polyoxyethylene glycol esters, etc.) and alkanolamines (e.g.,
diethanolamine-fatty acid condensates and isopropanolamine-fatty
acid condensates). In addition, ionic surfactants such as sodium
dodecyl sulfate (SDS) may also be used. However, such surfactants
are generally less preferably for many embodiments of the
invention. For instance, in those embodiments where aqueous
droplets are used as nanoreactors for chemical reactions (including
biochemical reactions) or are used to analyze and/or sort
biomaterials, a water soluble surfactant such as SDS may denature
or inactivate the contents of the droplet.
[0098] The carrier fluid can be an oil (e.g., decane, tetradecane
or hexadecane) or fluorocarbon oil that contains a surfactant
(e.g., a non-ionic surfactant such as a Span surfactant) as an
additive (preferably between about 0.2 and 5% by volume, more
preferably about 2%). A user can preferably cause the carrier fluid
to flow through channels of the microfluidic device so that the
surfactant in the carrier fluid coats the channel walls.
[0099] In one embodiment, the fluorosurfactant can be prepared by
reacting the perflourinated polyether DuPont Krytox 157 FSL, FSM,
or FSH with aqueous ammonium hydroxide in a volatile fluorinated
solvent. The solvent and residual water and ammonia can be removed
with a rotary evaporator. The surfactant can then be dissolved
(e.g., 2.5 wt %) in a fluorinated oil (e.g., Flourinert (3M)),
which then serves as the continuous phase of the emulsion.
[0100] The invention can use pressure drive flow control, e.g.,
utilizing valves and pumps, to manipulate the flow of cells,
particles, molecules, enzymes or reagents in one or more directions
and/or into one or more channels of a microfluidic device. However,
other methods may also be used, alone or in combination with pumps
and valves, such as electro-osmotic flow control, electrophoresis
and dielectrophoresis (Fulwyer, Science 156, 910 (1974); Li and
Harrison, Analytical Chemistry 69, 1564 (1997); Fiedler, et al.
Analytical Chemistry 70, 1909-1915 (1998); U.S. Pat. No.
5,656,155). Application of these techniques according to the
invention provides more rapid and accurate devices and methods for
analysis or sorting, for example, because the sorting occurs at or
in a sorting module that can be placed at or immediately after a
detection module. This provides a shorter distance for molecules or
cells to travel, they can move more rapidly and with less
turbulence, and can more readily be moved, examined, and sorted in
single file, i.e., one at a time.
[0101] Without being bound by any theory, electro-osmosis is
believed to produce motion in a stream containing ions e.g. a
liquid such as a buffer, by application of a voltage differential
or charge gradient between two or more electrodes. Neutral
(uncharged) molecules or cells can be carried by the stream.
Electro-osmosis is particularly suitable for rapidly changing the
course, direction or speed of flow. Electrophoresis is believed to
produce movement of charged objects in a fluid toward one or more
electrodes of opposite charge, and away from one on or more
electrodes of like charge. Where an aqueous phase is combined with
an oil phase, aqueous droplets are encapsulated or separated from
each other by oil. Typically, the oil phase is not an electrical
conductor and may insulate the droplets from the electro-osmotic
field. In this example, electro-osmosis may be used to drive the
flow of droplets if the oil is modified to carry or react to an
electrical field, or if the oil is substituted for another phase
that is immiscible in water but which does not insulate the water
phase from electrical fields.
[0102] Dielectrophoresis is believed to produce movement of
dielectric objects, which have no net charge, but have regions that
are positively or negatively charged in relation to each other.
Alternating, non-homogeneous electric fields in the presence of
droplets and/or particles, such as cells or molecules, cause the
droplets and/or particles to become electrically polarized and thus
to experience dielectrophoretic forces. Depending on the dielectric
polarizability of the particles and the suspending medium,
dielectric particles will move either toward the regions of high
field strength or low field strength. For example, the
polarizability of living cells depends on their composition,
morphology, and phenotype and is highly dependent on the frequency
of the applied electrical field. Thus, cells of different types and
in different physiological states generally possess distinctly
different dielectric properties, which may provide a basis for cell
separation, e.g., by differential dielectrophoretic forces.
Likewise, the polarizability of droplets also depends upon their
size, shape and composition. For example, droplets that contain
salts can be polarized. According to formulas provided in Fiedler,
et al. Analytical Chemistry 70, 1909-1915 (1998), individual
manipulation of single droplets requires field differences
(inhomogeneities) with dimensions close to the droplets.
[0103] Manipulation is also dependent on permittivity (a dielectric
property) of the droplets and/or particles with the suspending
medium. Thus, polymer particles, living cells show negative
dielectrophoresis at high-field frequencies in water. For example,
dielectrophoretic forces experienced by a latex sphere in a 0.5
MV/m field (10 V for a 20 micron electrode gap) in water are
predicted to be about 0.2 piconewtons (pN) for a 3.4 micron latex
sphere to 15 pN for a 15 micron latex sphere (Fiedler, et al.
Analytical Chemistry 70, 1909-1915 (1998)). These values are mostly
greater than the hydrodynamic forces experienced by the sphere in a
stream (about 0.3 pN for a 3.4 micron sphere and 1.5 pN for a 15
micron sphere). Therefore, manipulation of individual cells or
particles can be accomplished in a streaming fluid, such as in a
cell sorter device, using dielectrophoresis. Using conventional
semiconductor technologies, electrodes can be microfabricated onto
a substrate to control the force fields in a microfabricated
sorting device of the invention. Dielectrophoresis is particularly
suitable for moving objects that are electrical conductors. The use
of AC current is preferred, to prevent permanent alignment of ions.
Megahertz frequencies are suitable to provide a net alignment,
attractive force, and motion over relatively long distances. See
U.S. Pat. No. 5,454,472.
[0104] Radiation pressure can also be used in the invention to
deflect and move objects, e.g. droplets and particles (molecules,
cells, particles, etc.) contained therein, with focused beams of
light such as lasers. Flow can also be obtained and controlled by
providing a pressure differential or gradient between one or more
channels of a device or in a method of the invention.
[0105] Molecules, cells or particles (or droplets containing
molecules, cells or particles) can be moved by direct mechanical
switching, e.g., with on-off valves or by squeezing the channels.
Pressure control may also be used, for example, by raising or
lowering an output well to change the pressure inside the channels
on the chip. See, e.g., the devices and methods described U.S. Pat.
No. 6,540,895. These methods and devices can further be used in
combination with the methods and devices described in pending U.S.
Patent Application Publication No. 20010029983 and 20050226742. The
"pump and valve" drive systems are particularly preferred. They are
rapid, efficient, economical, and relatively easy to fabricate and
control. Additionally, they do not rely on electrical fields or
electrical charges, which may be harder to control and in some
cases may potentially affect the droplet contents. Different
switching and flow control mechanisms can be combined on one chip
or in one device and can work independently or together as
desired.
[0106] The device can exchange constituents within a droplet
through the use of fluid flow in such a way that the droplet, while
in a first immiscible fluid, is exposed to a second immiscible
fluid such that constituents within the droplet that are immiscible
in the first immiscible fluid are soluble in the second immiscible
fluid. In one example, an aqueous droplet containing a chemical
reaction produces by-products that are soluble in a lipid solvent.
The chemical reaction is performed in a water-environment in a
silicon-based solvent. After the chemical reaction occurs, the
droplet is exposed to an organic-oil based solvent where the
chemical byproducts are allowed to diffuse out of the droplet. The
resulting droplet is then assayed for cell-killing activity by
combining the droplet with live cells. Alternatively, the change in
the non-aqueous fluid flow is used to add a particular constituent
from the second immerscible fluid to diffuse into the aqueous drop
before the droplet is returned to the 100% first immiscible fluid
flow.
[0107] The concentration (i.e., number) of molecules, cells or
particles in a droplet can influence sorting efficiently and
therefore is preferably optimized. In particular, the sample
concentration should be dilute enough that most of the droplets
contain no more than a single molecule, cell or particle, with only
a small statistical chance that a droplet will contain two or more
molecules, cells or particles. This is to ensure that for the large
majority of measurements, the level of reporter measured in each
droplet as it passes through the detection module corresponds to a
single molecule, cell or particle and not to two or more molecules,
cells or particles.
[0108] The parameters which govern this relationship are the volume
of the droplets and the concentration of molecules, cells or
particles in the sample solution. The probability that a droplet
will contain two or more molecules, cells or particles
(P.sub..ltoreq.2) can be expressed as
P.sub..ltoreq.2=1-{1+[cell].times.V}.times.e.sup.-[cell].times.V
[0109] where "[cell]" is the concentration of molecules, cells or
particles in units of number of molecules, cells or particles per
cubic micron (.mu.m.sup.3), and V is the volume of the droplet in
units of .mu.m.sup.3.
[0110] It will be appreciated that P.sub..ltoreq.2 can be minimized
by decreasing the concentration of molecules, cells or particles in
the sample solution. However, decreasing the concentration of
molecules, cells or particles in the sample solution also results
in an increased volume of solution processed through the device and
can result in longer run times. Accordingly, it is desirable to
minimize to presence of multiple molecules, cells or particles in
the droplets (thereby increasing the accuracy of the sorting) and
to reduce the volume of sample, thereby permitting a sorted sample
in a reasonable time in a reasonable volume containing an
acceptable concentration of molecules, cells or particles.
[0111] The maximum tolerable P.sub..ltoreq.2 depends on the desired
"purity" of the sorted sample. The "purity" in this case refers to
the fraction of sorted molecules, cells or particles that posses a
desired characteristic (e.g., display a particular antigen, are in
a specified size range or are a particular type of molecule, cell
or particle). The purity of the sorted sample is inversely
proportional to P.sub..ltoreq.2. For example, in applications where
high purity is not needed or desired a relatively high
P.sub..ltoreq.2 (e.g., P.sub..ltoreq.2=0.2) may be acceptable. For
most applications, maintaining P.sub..ltoreq.2 at or below about
0.1, preferably at or below about 0.01, provides satisfactory
results.
[0112] A sample solution containing a mixture or population of
molecule, cells or particles in a suitable fluid (such as a liquid
or buffer described above) is supplied to the sample inlet channel,
and droplets of the sample solution are introduced, at the inlet
module, into the flow passing through the main channel. The force
and direction of flow can be controlled by any desired method for
controlling flow, for example, by a pressure differential, by valve
action or by electro-osmotic flow (e.g., produced by one or more
electrodes or patterned electrically conductive layers at inlet
and/or outlet modules). This permits the movement of the cells into
one or more desired branch channels or outlet modules.
[0113] Both the fluid comprising the droplets and the fluid
carrying the droplets (i.e., the aqueous and non-polar fluids)
have, preferably, a relatively low Reynolds Number, for example
10.sup.-2. The Reynolds Number represents an inverse relationship
between the density and velocity of a fluid and its viscosity in a
channel of given length. More viscous, less dense, slower moving
fluids over a shorter distance will have a lower Reynolds Number,
and are easier to divert, stop, start, or reverse without
turbulence. Because of the small sizes and slow velocities,
microfabricated fluid systems are often in a low Reynolds number
regime (Re<<1). In this regime, inertial effects, which cause
turbulence and secondary flows, are negligible; viscous effects
dominate the dynamics. These conditions are advantageous for
sorting, and are provided by microfabricated devices of the
invention. Accordingly the microfabricated devices of the invention
are preferably if not exclusively operated at a low or very low
Reynold's number.
[0114] The device of the present invention can be used to generate
droplets whose composition may vary from one droplet to the next
droplet due to any number of reasons (chemical reaction, sample
preparation, etc). Within the same device, the droplets can be
passed through a measurement volume in which the contents can be
interrogated using various means (optical or electrical). The
result of the measurement can be used to decide which flow path the
droplets should take. The means of changing the flow path can be
accomplished through mechanical, electrical, optical, or other
technique as described herein or well known in the art.
[0115] The present invention provides methods for the determination
of droplet size and rate information without the need for optical
measurements on a microfluidic device. The need to control the
timing between multiple events requires the determination of the
exact time when a droplet passes a given point. It is also
essential to know which channel a droplet enters. This method can
significantly reduce the cost and complexity of such
measurements.
[0116] The fluids used to generate droplets in microfluidic devices
are typically immiscible liquids such as oil and water. These two
materials generally have very different dielectric constants
associated with them. These differences can be exploited to
determine droplet rate and size for every drop passing through a
small section of a microfluidic device. One method to directly
monitor this variation in the dielectric constant measures the
change in capacitance over time between a pair of closely spaced
electrodes. This change in capacitance can be detected by the
change in current measured in these electrodes:
i = V C t ##EQU00001##
Where i is the current, V is the voltage applied across the
electrodes, and dC/dt is the change in capacitance with time.
Alternatively, the capacitance can be measured directly if a time
varying voltage is applied to these same electrodes: i=CdV/dt Where
C is the measured capacitance, and dV/dt is the change in voltage
with time. As a first approximation, the electrode pair can be
determined as a parallel plate capacitor:
C = 0 k A d ##EQU00002##
Where .di-elect cons..sub.0 is the permittivity of free space, k is
the effective dielectric constant (this changes every time a
droplet passes through), A is the area of the capacitor and d is
the electrode separation. The current measured in the device is
then plotted as a function of time.
Inlet Module
[0117] An "inlet module" is an area of a microfabricated device
that receives molecules, cells, small molecules or particles for
coalescence, detection and/or sorting. The inlet module can contain
one or more inlet channels, wells or reservoirs, openings, and
other features which facilitate the entry of molecules, cells,
small molecules or particles into the device. A chip may contain
more than one inlet module if desired. The inlet module is in fluid
communication with the main channel. The inlet module can include a
junction between an inlet channel and the main channel of a device
of the invention. The junction can permit the introduction of a
pressurized fluid to the main channel. The inlet channel can be at
an angle perpendicular to the flow of fluid in the main channel.
The fluid introduced to the main channel through the inlet module
is "incompatible" (i.e., immiscible) with the fluid in the main
channel so that droplets of the fluid introduced through the inlet
module are formed in the stream of continuous fluid in the main
channel.
[0118] Embodiments of the invention are also provided in which
there are two or more inlet modules introducing droplets of samples
into the main channel. For example, a first inlet module may
introduce droplets of a first sample into a flow of fluid (e.g.,
oil) in the main channel and a second inlet module may introduce
droplets of a second sample into the flow of fluid in main channel,
and so forth. The second inlet module is preferably downstream from
the first inlet module (e.g., about 30 .mu.m). The fluids
introduced into the two or more different inlet modules can
comprise the same fluid or the same type of fluid (e.g., different
aqueous solutions). For example, droplets of an aqueous solution
containing an enzyme are introduced into the main channel at the
first inlet module and droplets of aqueous solution containing a
substrate for the enzyme are introduced into the main channel at
the second inlet module. Alternatively, the droplets introduced at
the different inlet modules may be droplets of different fluids
which may be compatible or incompatible. For example, the different
droplets may be different aqueous solutions, or droplets introduced
at a first inlet module may be droplets of one fluid (e.g., an
aqueous solution) whereas droplets introduced at a second inlet
module may be another fluid (e.g., alcohol or oil).
[0119] To obtain one droplet comprising a single element of a
specific biological/chemical material (e.g., a cell), separation of
biological/chemical material, and uniformity of the number density
of biological/chemical materials in a microfluidic channel is
desirable. Accordingly, the microfluidic device can include an
acoustic actuator. The loaded sample (biological/chemical material)
can be well mixed and separated in a small chamber by acoustic wave
before sending out to the nozzle region for encapsulation. The
frequency of the acoustic wave should be fine tuned so as not to
cause any damage to the cells. The biological effects of acoustic
mixing have been well studied (e.g., in the ink-jet industry) and
many published literatures also showed that piezoelectric
microfluidic device can deliver intact biological payloads such as
live microorganisms and DNA.
[0120] The design of the acoustic resonant can use a Piezoelectric
bimorph flat plate located on the side of the carved resonant in
the PDMS slab. The resonant inlet can connect to the cell flow
input channel and the outlet can connect to the cell flow pinching
channel. The piezoelectric driving waveform can be carefully
optimized to select the critical frequencies that can separate
cells in fluids. There are five parameters to optimize beyond the
frequency parameter and Lab electronics can be used to optimize the
piezoelectric driving waveform. Afterwards, a low cost circuit can
be designed to generate only the optimized waveform in a preferred
microfluidic device.
Coalescence Module
[0121] The device of the invention also comprises one or more
coalescence modules. A "coalescence module" is within or coincident
with at least a portion of the main channel at or downstream of the
inlet module where molecules, cells, small molecules or particles
comprised within droplets are brought within proximity of other
droplets comprising molecules, cells, small molecules or particles
and where the droplets in proximity coalesce or combine their
contents. The coalescence module can also include an apparatus,
preferably one or more electrodes or patterned electrically
conductive layers for generating a dielectrophoretic force. The
dielectrophoretic force generated by one or more electrodes or
patterned electrically conductive layers can slow or stop the
droplets within the main channel thereby facilitating their
proximity and resulting coalescence or combination.
[0122] Two or more precursor droplets in one or more droplet
streams can be coalesced into a larger droplet by applying a
voltage to produce an electric field. The voltage can be
alternating. The electric field can be an AC electric field, or a
DC electric field.
[0123] The coalescing influence can create a dielectrophoretic
force that slows or stops a first precursor droplet relative to the
velocity of the stream that carries the droplet. The first
precursor droplet will remain slow or stopped until a second (or
more) precursor droplet arrives and coalesces with the first
precursor droplet due to interactions between the field induced
dipoles in the droplets. The new droplet of increased volume is
then too large to be held by the dielectric field and moves off
under the influence of the flow of the continuous phase fluid. See
FIGS. 15-17. No change in the applied voltage is required and the
electric field remains constant. Both the trapping (slowing or
stopping) of the precursor droplets and the release of the
coalesced droplet can be passive. Once the new droplet of increased
volume moves off, the next precursor droplet is then trapped in the
field and the process repeated. The voltage can be tuned such that
more than one droplet is coalesced with a trapped droplet. An
advantage of coalescing more than one droplet with another is that
it allows for pairwise combinations. Alternatively, a variation on
this geometry will allow precise control of the droplet phase by
temporarily shifting droplets to low velocity streamlines in the
flow.
[0124] The precursor droplets can come at different times in the
same fluid stream and subsequently coalesce. Alternatively, the
precursor droplets can arrive together in different (e.g., two or
more) fluid streams so that the droplets are in a substantially
adjacent position with respect to each other when they come under
the influence of the dielectrophoretic force and then subsequently
coalesce due to the interactions between the field induced dipoles
in the droplets. The different fluid streams can be substantially
parallel.
[0125] The electric field gradient can be stronger for precursor
droplets in a first parallel fluid stream than for precursor
droplets in a second (or more) parallel fluid stream. Accordingly,
it is only the precursor droplets from the first stream that are
trapped, thereby preventing coalescence of precursor droplets in
the other fluid stream(s) among each other in the case where the
frequency of precursor droplets in the other stream(s) is greater
than the frequency of precursor droplets in the first stream. In
this manner the electric field can be changed to cause coalescence
of only correct pairs of precursor droplets. Thus, in some
embodiments the trapping and release of the droplets can be
non-passive (i.e., based on whether the electric field is on or
off).
[0126] The device can include channels for use in fluid control and
other channels filled with a metal alloy for casting integrated
metal alloy components (i.e., electrodes). Alternatively, the
electrodes can be manufactured using other technologies (e.g.,
lithographically patterned electrodes made from indium tin oxide or
a metal such as platinum). The microfluidic device can include
metal alloy components useful for performing electrical functions
on fluids, including but not limited to, coalescing droplets,
charging droplets, sorting droplets, detecting droplets and shaking
droplets to mix the contents of coalesced droplets. The device can
contain more than one of the above mentioned components for more
than one of the above mentioned functions.
[0127] The present invention also provides methods of manipulating
biological/chemical material. In one embodiment, the first and
second droplets can be brought into proximity prior to coalescence
by slowing or stopping at least one droplet comprising a first
biological/chemical material by exerting a dielectrophoretic force
onto the droplet produced by an electric field gradient. In another
embodiment, the first and second droplets can be brought into
proximity prior to coalescence where the first and second droplet
are of different size. In some embodiments, one of the first and
second droplets can be the size of the channel width and the other
droplet can be smaller than the channel width. In other
embodiments, the larger droplet has enough volume so that it would
have a diameter greater than the channel width if it were
spherical. In a further embodiment, the first and second droplets
can be brought into proximity prior to coalescence where the first
and second droplet are of different viscosities and thus move at
different velocities. Viscosity of a droplet can be changed by
changing the content of the droplet. For example, glycerol can be
added to a droplet to give it an increased viscosity.
[0128] In one embodiment, the method of manipulating biological and
chemical material, further comprises coalescing at least one
droplet with a droplet slowed or stopped under the influence of a
dielectrophoretic force from an electric field gradient created
within a coalescence module, thereby producing a nanoreactor.
[0129] The droplet size can be controlled such that the droplet
formed from flowing a first dispersed phase fluid in a continuous
phase fluid moves at a different velocity with respect to a droplet
formed from flowing a second dispersed phase fluid in a continuous
phase fluid, such that droplets arrive in pairs at a region where
an electric field induces them to coalesce, thereby producing a
nanoreactor. In some embodiments, greater than 50% of the droplets
are paired. In other embodiments, greater than 75% of the droplets
are paired.
[0130] The droplet viscosity can be controlled such that the
droplet formed from flowing a first dispersed phase fluid in a
continuous phase fluid moves at a different velocity with respect
the droplet formed from flowing a second dispersed phase fluid in a
continuous phase fluid, such that droplets arrive in pairs at a
region where an electric field induces them to coalesce, thereby
producing a nanoreactor. In some embodiments, greater than 50% of
the droplets are paired. In other embodiments, greater than 75% of
the droplets are paired.
[0131] The electrodes comprising metal alloy components may either
terminate at fluid channels or be isolated from fluid channels. The
electrodes can be constructed by filling the appropriate channels
with metal alloy. One way this can be accomplished to use positive
pressure injection of the metal alloy in a melted state, such as
with a syringe, into the channels, and then cool the metal alloy to
a solid form. Another example is to use negative pressure to suck
the metal alloy in a melted state into the channels, and then cool
the metal alloy to a solid form. This can be accomplished for
example by use of capillary forces. Another method of construction
can use any of the above mentioned embodiments, and then flush out
the metal alloy in a melted state with another liquid to define the
geometry of the metal alloy components. Another example is to use
any of the above mentioned embodiments, and then use a localized
cold probe to define a solid termination point for the metal alloy,
and then cool the remaining metal alloy to a solid form. A further
example is to use another material, such as microscopic solder
spheres or UV curable conductive ink, to form a barrier between
fluid and metal alloy channels, to define the geometry of the metal
alloy components.
[0132] The device can include a combination of both integrated
metal alloy components and a patterned electrically conductive
layer. The patterned electrically conductive layer can have
features patterned such that their boundaries are within a
leak-proof seal. The device can have a patterned electrically
conductive feature as one of two charging electrodes and one
integrated metal alloy component as the other of two charging
electrodes. Alternatively, the device can have metal alloy
components as the two halves of a bowtie antenna and patterned
electrically conductive features as the two halves of a pickup
antenna for dielectric droplet detection.
[0133] The device can include a plurality of electrodes that are
insulated from the fluid present in the device, and the method of
operation including appropriate application of dielectrical signals
and appropriate fluids. In known devices, the electrodes are
typically in contact with the fluids in order to allow discharge of
species that would otherwise screen the applied dielectric field.
Whereas, in devices where the electrodes have been insulated from
the fluid, this screening effect typically arises so quickly that
the device is not useful for any significantly extended period of
time. The drawbacks of electrodes in contact with the fluids vs.
insulated electrodes are (a) degraded reliability against leaking
(since the interface between the electrodes and the other
components of the device may be more difficult to effect a
leak-proof seal), and (b) degraded reliability against electrode
corrosion (whose failure mode effects include failure of
application of dielectric fields, and fluid channel
contamination).
[0134] The device of the present invention comprising a plurality
of electrodes that are insulated from the fluid present in the
device counteracts this screening effect by extending the screening
rise time and including a polarity switch for all of the different
dielectric fields applied in the device. The screening rise time is
extended by using fluids with dielectrical properties. A polarity
switch for all of the different dielectric fields applied in the
device is achieved by using an algorithm for dielectrical control,
which switches the polarity of the dielectrical fields at a
frequency sufficiently high to maintain proper dielectrical
function of the device. This dielectrical control algorithm may
also switch the polarity for the dielectric fields in a cascading,
time controlled manner starting at the fluid origin point and
progressing downstream, so that given fluid components experience
one polarity at every point along their course. The device of the
present invention can be used with metal alloy electrodes or using
a combination of metal alloy electrodes and patterned conductive
film electrodes.
[0135] In one embodiment, the invention provides a microfluidic
device using injected electrodes. The interface between the
microscopic electrode (typically 25 .mu.m thick) and the
macroscopic interconnect can easily fail if the joint between the
two is flexed. The flexing of the joint can be eliminated by
securing a firm material that serves to fasten, support, and
reinforce the joint (i.e., a grommet) into the interface. In order
to prevent flexing, the mating surface of the device can be
manufactured from a hard material such as glass or plastic. The
electrical connection with the external system can be made by
securing the device such that it connects to a spring loaded
contact, which is either offset from the grommet (thereby
minimizing the force applied to the solder region), or centered on
the grommet (as long as the contact does not touch the solder).
[0136] The metal alloy components are also useful for performing
optical functions on fluids, including but not limited to, optical
detection of droplets in a geometry which may include a mirror.
[0137] To prevent leakage of fluid out of electrodes placed within
microfluidic channels, the microfluidic device can include a layer
patterned with channels for fluid control, and another layer with
patterned electrically conductive features, where the features are
patterned such that their boundaries are within a leak-proof seal.
The leak-proof seal can be achieved at the interface between the
unpatterned areas of the fluid control layer and the unpatterned
areas of the electrically conductive layer. The leak-proof seal can
also be achieved by a third interfacial layer between the fluid
control layer and the unpatterned areas of the electrically
conductive layer. The third interfacial layer can or can not be
perforated at specific locations to allow contact between the fluid
and the electrically conductive layer. Electrical access ports can
also be patterned in the fluid control layer.
[0138] The electrodes and patterned electrically conductive layers
as described can be associated with any module of the device (inlet
module, coalescence module, mixing module, delay module, detection
module and sorting module) to generate dielectric or electric
fields to manipulate and control the droplets and their
contents.
[0139] The microfluidic device can combine dielectric or electric
fields with droplet fission to separate ionic species during
droplet breakup.
[0140] The present invention provides methods of controlling
droplets using fringing fields. Effective control of uncharged
droplets within microfluidic devices can require the generation of
extremely strong dielectric field gradients. The fringe fields from
the edges of a parallel plate capacitor can provide an excellent
topology to form these gradients. The microfluidic device according
to the present invention can include placing a fluidic channel
between two parallel electrodes, which can result in a steep
electric field gradient at the entrance to the electrodes due to
edge effects at the ends of the electrode pair. Placing these pairs
of electrodes at a symmetric channel split can allow precise
bi-directional control of droplet within a device. Using the same
principle, only with asymmetric splits, can allow single ended
control of the droplet direction in the same manner. Alternatively,
a variation on this geometry will allow precise control of the
droplet phase by shifting.
[0141] A device of the invention can be used for the application of
an electric field at a junction between two immiscible fluids. The
electric field created charged droplets and large forces necessary
for emulsification, while the junction stabilized droplet
production even at high fields, when a Taylor cone was present.
Applications of this technology include, but are not limited to,
the generation of charged droplets with a narrow distribution in
radius down to submicron sizes and controlled droplet coalescence
by oppositely charged droplets.
[0142] The device of this embodiment can be created by patterning
PDMS on a glass substrate having electrodes formed from indium tin
oxide ("ITO"). A voltage difference can be applied to the
electrodes to create an applied dielectric field. The device can
include a two-fluid injection system where a conductive fluid can
be injected into a non-conductive fluid in the presence of the
electric field to generate droplets of the conductive fluid
dispersed in the non-conductive fluid. Droplets can be created
having diameters of less than about 1 micron to about 100 microns.
These droplets can remain charged with the sign of the charge
dependent on the sign of the dielectric field with respect to the
direction of flow.
[0143] In the absence of an electric field, large droplets can be
generated, while in the presence an electric field (E=2 V/micron),
a Taylor cone can be stabilized with uniform submicron droplets
being emitted from the tip. The droplets may also be discharged on
a ground electrode located further downstream. Such a device can
have many applications, for example, in generating well controlled
nanoemulsions.
[0144] Oppositely oriented devices can also be used to generate
droplets having opposite sign of charge. Using this charge, the
droplets can coalesce at a precise or generally predetermined
location. If there is no electric field applied, the droplets
cannot coalesce. The electrostatic attraction can cause the drops
to coalesce. The electric field, in some cases, can be used to
control the phase between when the droplets are generated to ensure
simultaneous arrival at a central location and subsequent
coalescence, for example, through an auto feedback mechanism or a
using an AC dither. The surface of the droplets can be deformed and
electrostatic forces may overcome surface tension to produce a
fluid bridge to coalesce and/or neutralize the droplets.
Interdigitation and Coalescence of Droplets
[0145] Particular design embodiments of the microfluidic device
described herein allow for a more reproducible and controllable
interdigitation of droplets of specific liquids followed by
pair-wise coalescence of these droplets. The droplet pairs can
contain liquids of different compositions and/or volumes, which
would then combine to allow for a specific reaction to be
investigated. The pair of droplets can come from any of the
following: (i) two continuous aqueous streams and an oil stream;
(ii) a continuous stream, an emulsion stream, and an oil stream, or
(iii) two emulsion streams and an oil stream. FIG. 17 A-D.
[0146] The nozzle design enhances the interdigitation of droplets
and further improves coalescence of droplets due to the better
control of the interdigitation and smaller distance between pairs
of droplets. The greater control over interdigitation allows for a
perfect control over the frequency of either of the droplets.
Coalescence can be accomplished by localized electric field
application, as described above. Coalescence may also be
accomplished by passive coalescence of droplets without application
of any external effects for the appropriate mix). Passive
coalescence significantly simplifies the device operation and
control, which is critical as the same procedure is repeated
multiple times in a given process. To obtain the optimum operation,
the spacing between droplets and coupling of the droplets can be
adjusted by adjusting flow of any of the streams, viscosity of the
streams, nozzle design (including orifice diameter, the channel
angle, and post-orifice neck of the nozzle).
[0147] In one embodiment, passive coalescence of paired droplets
can be achieved by passing the droplets through a narrowing of a
channel (or a neck-down or a pinch). FIG. 18A-D. In such an
embodiment, droplets passing through the pinch are touching while
being elongated as they are passing through the channel. Due to the
elongation and redistribution of surface activities at the
elongated ends, the droplet pair coalesces spontaneously and
passively.
Detection Module
[0148] A "detection module" is a location within the device,
typically within the main channel where molecules, cells, small
molecules or particles are to be detected, identified, measured or
interrogated on the basis of at least one predetermined
characteristic. The molecules, cells, small molecules or particles
can be examined one at a time, and the characteristic is detected
or measured optically, for example, by testing for the presence or
amount of a reporter. For example, the detection module is in
communication with one or more detection apparatuses. The detection
apparatuses can be optical or electrical detectors or combinations
thereof. Examples of suitable detection apparatuses include optical
waveguides, microscopes, diodes, light stimulating devices, (e.g.,
lasers), photo multiplier tubes, and processors (e.g., computers
and software), and combinations thereof, which cooperate to detect
a signal representative of a characteristic, marker, or reporter,
and to determine and direct the measurement or the sorting action
at the sorting module.
[0149] A detection module is within, communicating or coincident
with a portion of the main channel at or downstream of the inlet
module and, in sorting embodiments, at, proximate to, or upstream
of, the sorting module or branch point. Precise boundaries for the
detection module are not required, but are preferred. The sorting
module may be located immediately downstream of the detection
module or it may be separated by a suitable distance consistent
with the size of the molecules, the channel dimensions and the
detection system. It will be appreciated that the channels may have
any suitable shape or cross-section (for example, tubular or
grooved), and can be arranged in any suitable manner so long as
flow can be directed from inlet to outlet and from one channel into
another.
[0150] The detection module can have features to detect the
droplets, including but not limited to, integrated metal alloy
components and/or features patterned in an electrically conductive
layer, to broadcast a signal around a droplet and pick up an
electrical signal in proximity to the droplet.
[0151] As each droplet passes into the detection module, it is
examined for a predetermined characteristic (i.e., using the
detector) and a corresponding signal is produced, for example
indicating that "yes" the characteristic is present, or "no" it is
not. The signal may correspond to a characteristic qualitatively or
quantitatively. That is, the amount of the signal can be measured
and can correspond to the degree to which a characteristic is
present. For example, the strength of the signal may indicate the
size of a molecule, or the potency or amount of an enzyme expressed
by a cell, or a positive or negative reaction such as binding or
hybridization of one molecule to another, or a chemical reaction of
a substrate catalyzed by an enzyme. In response to the signal, data
can be collected and/or a control system in the sorting module, if
present, can be activated to divert a droplet into one branch
channel or another for delivery to the collection module or waste
module. Thus, in sorting embodiments, molecules or cells within a
droplet at a sorting module can be sorted into an appropriate
branch channel according to a signal produced by the corresponding
examination at a detection module. The detection can be optical
detection of molecular, cellular or other characteristics, for
example directly or by use of a reporter associated with a
characteristic chosen for sorting. However, other detection
techniques can also be employed.
[0152] The device can be used to generate droplets whose
composition may vary from one to the next due to any number of
reasons (chemical reaction, sample preparation, etc). Within the
same device, the droplets are passed through a measurement volume
in which the contents are interrogated using various means (optical
or electrical). The result of the measurement is used to decide
which flow path the droplets should take. The means of changing the
flow path can be accomplished through mechanical, electrical,
optical, or some other technique as described herein.
[0153] The device can provide an accurate means of precisely
aligning optical waveguides and their associated optical elements
(lenses, prisms, mirrors, interconnects, etc.) to the fluidic
channels contained within the microfluidic device. Such waveguides
can be used to provide well defined optical access to the fluidic
channels to permit optical scattering, absorption, fluorescence, or
any other optical measurement technique.
[0154] Channels within the device are typically made using
semiconductor lithographic processes. In order to create the
waveguides, a separate series of channels and useful shapes
(lenses, mirrors, etc) can be created either simultaneously (i.e.
in the same processing step) or in successive steps. The reusable
master created in this way can then used to fabricate the waveguide
components and fluid channels without the need for special
fixturing or careful alignment in subsequent steps. The extra
channels or shapes can then filled with a high index of refraction
liquid (for waveguides) or reflective material (for mirrors)
through injection into the channel or void. The liquid can either
remain as a fluid or be allowed to solidify. UV cure epoxies used
by the telecommunications industry are excellent choices for the
waveguide materials. Possible waveguide geometry can include a
focusing lens and a back-reflecting mirror.
[0155] The device of the present invention also comprises the use
of beads and methods for analyzing and sorting beads (i.e, bead
reader device). The device can read and either sort or not sort
droplets containing one or more of a set of two or more beads. Each
bead can be differentiated from each other bead within a set. Beads
can be separated by several tags including, but not limited to,
quantum dyes, fluorescent dyes, ratios of fluorescent dyes,
radioactivity, radio-tags, etc. For example, a set of beads
containing a ratio of two dyes in discrete amounts with an
apparatus for detecting and differentiating beads containing one
discrete ratio from the other beads in this set having a different
ratio of the two dyes. The microfluidic device can include a
paramagnetic beads. The paramagnetic beads can introduce and remove
chemical components from droplets using droplet coalescence and
breakup events. The paramagnetic beads can also be used for sorting
droplets.
[0156] The present invention provides methods of screening
molecular libraries on beads through limited-dilusion-loading and
then chemical or optical release inside of droplets. Provided are
methods for chemical synthesis on a bead and releasing said
chemical attached to the bead using a releasing means (chemical, UV
light, heat, etc) within a droplet, and then combining a second
droplet to the first droplet for further manipulation. For example,
tea-bag synthesis of chemicals on a bead simultaneously with a
means for identifying said bead (using, for example, a mass spec
tag). Using the resulting mixed-chemistry beads in a droplet within
a fluid flow, and exposing the beads to UV light to release the
chemical synthesized from the bead into the droplet environment.
Combining the droplet containing the released chemical with a
droplet containing a cell, and performing a cell-based assay.
Sorting droplets having the desired characteristics (for example,
turn on of a reporter gene), and then analyzing the sorted beads
using mass spectroscopy.
[0157] The device of the present invention can comprise column
separation prior to bead sorting. A device containing a channel
loaded with a separating means for chromatographically sorting the
sample prior to droplet formation. Such separating means could
include size, charge, hydrophobicity, atomic mass, etc. The
separating can be done isocratic or by use of a means for
generating a gradient chemically, (for example using salt or
hydrophobicity), electrically, by pressure, or etc. For example, a
channel is preloaded with Sepharose size exclusion media. A sample
is loaded at one end, and the droplets are formed at an opposing
end. The sample separates by size prior to becoming incorporated
within a droplet.
[0158] The detector can be any device or method for interrogating a
molecule, a cell or particle as it passes through the detection
module. Typically, molecules, cells or particles (or droplets
containing molecules, cells or particles) are to be analyzed or
sorted according to a predetermined characteristic that is directly
or indirectly detectable, and the detector is selected or adapted
to detect that characteristic. A preferred detector is an optical
detector, such as a microscope, which may be coupled with a
computer and/or other image processing or enhancement devices to
process images or information produced by the microscope using
known techniques. For example, molecules can be analyzed and/or
sorted by size or molecular weight. Enzymes can be analyzed and/or
sorted by the extent to which they catalyze chemical reaction of a
substrate (conversely, substrate can be analyzed and/or sorted by
the level of chemical reactivity catalyzed by an enzyme). Cells can
be sorted according to whether they contain or produce a particular
protein, by using an optical detector to examine each cell for an
optical indication of the presence or amount of that protein. The
protein may itself be detectable, for example by a characteristic
fluorescence, or it may be labeled or associated with a reporter
that produces a detectable signal when the desired protein is
present, or is present in at least a threshold amount. There is no
limit to the kind or number of characteristics that can be
identified or measured using the techniques of the invention, which
include without limitation surface characteristics of the cell and
intracellular characteristics, provided only that the
characteristic or characteristics of interest for sorting can be
sufficiently identified and detected or measured to distinguish
cells having the desired characteristic(s) from those which do not.
For example, any label or reporter as described herein can be used
as the basis for analyzing and/or sorting molecules or cells, i.e.
detecting molecules or cells to be collected.
[0159] The molecules or cells or particles (or droplets containing
them) are analyzed and/or separated based on the intensity of a
signal from an optically-detectable reporter bound to or associated
with them as they pass through a detection module in the device.
Molecules or cells or particles having an amount or level of the
reporter at a selected threshold or within a selected range are
diverted into a predetermined outlet or branch channel of the
device. The reporter signal may be collected by a microscope and
measured by a photo multiplier tube (PMT). A computer digitizes the
PMT signal and controls the flow via valve action or
electro-osmotic potentials. Alternatively, the signal can be
recorded or quantified as a measure of the reporter and/or its
corresponding characteristic or marker, e.g., for the purpose of
evaluation and without necessarily proceeding to sort the molecules
or cells.
[0160] The chip can be mounted on an inverted optical microscope.
Fluorescence produced by a reporter is excited using a laser beam
focused on molecules (e.g., DNA, protein, enzyme or substrate) or
cells passing through a detection region. Fluorescent reporters can
include, but are not limited to, rhodamine, fluorescein, Texas red,
Cy 3, Cy 5, phycobiliprotein (e.g., phycoerythrin), green
fluorescent protein (GFP), YOYO-1 and PicoGreen. In molecular
fingerprinting applications, the reporter labels can be
fluorescently labeled single nucleotides, such as fluorescein-dNTP,
rhodamine-dNTP, Cy3-dNTP, etc.; where dNTP represents dATP, dTTP,
dUTP or dCTP. The reporter can also be chemically-modified single
nucleotides, such as biotin-dNTP. The reporter can be fluorescently
or chemically labeled amino acids or antibodies (which bind to a
particular antigen, or fragment thereof, when expressed or
displayed by a cell or virus).
[0161] The device can analyze and/or sort cells based on the level
of expression of selected cell markers, such as cell surface
markers, which have a detectable reporter bound thereto, in a
manner similar to that currently employed using
fluorescence-activated cell sorting (SACS) machines. Proteins or
other characteristics within a cell, and which do not necessarily
appear on the cell surface, can also be identified and used as a
basis for sorting. The device can also determine the size or
molecular weight of molecules such as polynucleotides or
polypeptides (including enzymes and other proteins) or fragments
thereof passing through the detection module. Alternatively, the
device can determine the presence or degree of some other
characteristic indicated by a reporter. If desired, the cells,
particles or molecules can be sorted based on this analysis. The
sorted cells, particles or molecules can be collected from the
outlet channels in collection modules (or discarded in wasted
modules) and used as needed. The collected cells, particles or
molecules can be removed from the device or reintroduced to the
device for additional coalescence, analysis and sorting.
[0162] To detect a reporter or determine whether a molecule, cell
or particle has a desired characteristic, the detection module may
include an apparatus for stimulating a reporter for that
characteristic to emit measurable light energy, e.g., a light
source such as a laser, laser diode, light emitting diode (LED),
high-intensity lamp, (e.g., mercury lamp), and the like. Where a
lamp is used, the channels are preferably shielded from light in
all regions except the detection module. Where a laser is used, the
laser can be set to scan across a set of detection modules from
different analysis units. In addition, laser diodes or LED's may be
microfabricated into the same chip that contains the analysis
units. Alternatively, laser diodes or LED's may be incorporated
into a second chip (i.e., a laser diode chip) that is placed
adjacent to the microfabricated analysis or sorter chip such that
the laser light from the diodes shines on the detection
module(s).
[0163] An integrated semiconductor laser and/or an integrated
photodiode detector can be included on the silicon wafer in the
vicinity of the detection module. This design provides the
advantages of compactness and a shorter optical path for exciting
and/or emitted radiation, thus minimizing distortion and
losses.
[0164] The present invention provides methods of droplet detection
using electrical signal broadcasting. The device of the present
invention can comprise features, such as integrated metal alloy
components and/or features patterned in an electrically conductive
layer, for detecting droplets by broadcasting a signal around a
droplet and picking up an electrical signal in proximity to the
droplet.
[0165] The present invention provides self-aligning optical
waveguides and optical elements for detection and control of
droplets. The device of the present invention can comprise an
accurate means of precisely aligning optical waveguides and their
associated optical elements (lenses, prisms, mirrors,
interconnects, etc.) to the fluidic channels contained within the
device. Such waveguides can be used to provide well defined optical
access to the fluidic channels to permit optical scattering,
absorption, fluorescence, or any other optical measurement
technique.
[0166] Fluidic channels within a microfluidic device are typically
made using semiconductor lithographic processes. In order to create
the waveguides, a separate series of channels and useful shapes
(lenses, minors, etc) can be created either simultaneously (i.e. in
the same processing step) or in successive steps. The reusable
master created in this way can then used to fabricate the waveguide
components and fluid channels without the need for special
fixturing or careful alignment in subsequent steps. The extra
channels or shapes can then filled with a high index of refraction
liquid (for waveguides) or reflective material (for mirrors)
through injection into the channel or void. The liquid can either
remain as a fluid or be allowed to solidify. UV cure epoxies used
by the telecommunications industry are excellent choices for the
waveguide materials. Possible waveguide geometries can include a
focusing lens and a back-reflecting minor.
[0167] The dimensions of the detection module are influenced by the
nature of the sample under study and, in particular, by the size of
the droplets, beads, particles, molecules or cells (including
virions) under study. For example, mammalian cells can have a
diameter of about 1 to 50 microns, more typically 10 to 30 microns,
although some mammalian cells (e.g., fat cells) can be larger than
120 microns. Plant cells are generally 10 to 100 microns. However,
other molecules or particles can be smaller with a diameter from
about 20 nm to about 500 nm.
[0168] Detection modules used for detecting molecules and cells
have a cross-sectional area large enough to allow a desired
molecule, cells, bead, or particles to pass through without being
substantially slowed down relative to the flow carrying it.
[0169] In another embodiment, the droplet content detection can be
achieved by simultaneous detection of contents of multiple droplets
in parallel using spectroscopic fluorescence imaging with
sensitivity as high as single-molecule limit. In this embodiment,
one can spatially distribute droplets containing fluorescent
entities such as Fluorophore biological markers and/or quantum dots
in a two-dimensional sheet in a microscopic field-of-view. The
field-of-view of those droplets can then be illuminated by a
fluorescence excitation source and the resulting fluorescence can
be spectroscopically imaged. Therefore, for a given fluorescence
detection sensitivity, the throughput of fluorescence detection
compared to a single-drop fluorescence detection method can be
increased by a factor of a/b for a given sensitivity, where a is
the number of droplets that can be imaged within a given
field-of-view, and b is the ratio of the fluorescence sensitivity
of a single-drop fluorescence detector compared to that of the
multiple drop fluorescence detector. Furthermore, unlike the prior
art single-drop fluorescent detection method where the drops are
flowed through a detection volume so that their residence time in
the detection volume, and hence the signal integration time and
sensitivity, is limited, the residence time of the droplet in the
field-of-view can be unlimited, thereby allowing sensitivity as
high as the single-molecule limit.
Sorting Module
[0170] The device of the present invention can further include one
or more sorting modules. A "sorting module" is a junction of a
channel where the flow of molecules, cells, small molecules or
particles can change direction to enter one or more other channels,
e.g., a branch channel for delivery to an outlet module (i.e.,
collection or waste module), depending on a signal received in
connection with an examination in the detection module. Typically,
a sorting module is monitored and/or under the control of a
detection module, and therefore a sorting module may "correspond"
to such detection module. The sorting region is in communication
with and is influenced by one or more sorting apparatuses. A
sorting apparatus comprises techniques or control systems, e.g.,
dielectric, electric, electro-osmotic, (micro-) valve, etc. A
control system can employ a variety of sorting techniques to change
or direct the flow of molecules, cells, small molecules or
particles into a predetermined branch channel. A "branch channel"
is a channel which is in communication with a sorting region and a
main channel. Typically, a branch channel receives molecules,
cells, small molecules or particles depending on the molecule,
cells, small molecules or particles characteristic of interest as
detected by the detection module and sorted at the sorting module.
A branch channel can have an outlet module and/or terminate with a
well or reservoir to allow collection or disposal (collection
module or waste module, respectively) of the molecules, cells,
small molecules or particles. Alternatively, a branch channel may
be in communication with other channels to permit additional
sorting.
[0171] The device of the present invention can further include one
or more outlet modules. An "outlet module" is an area of a
microfabricated device that collects or dispenses molecules, cells,
small molecules or particles after coalescence, detection and/or
sorting. The outlet module can include a collection module and/or a
waste module. The collection module can be connected to a means for
storing a sample. The collection module can be a well or reservoir
for collecting and containing droplets detected to have a specific
predetermined characteristic in the detection module. The
collection module can be temperature controlled. The waste module
can be connected to a means for discarding a sample. The waste
module can be a well or reservoir for collecting and containing
droplets detected to not have a specific predetermined
characteristic in the detection module. The outlet module is
downstream from a sorting module, if present, or downstream from
the detection module if a sorting module is not present. The outlet
module may contain branch channels or outlet channels for
connection to a collection module or waste module. A device can
contain more than one outlet module.
Mixing Module
[0172] Although coalescence of one or more droplets in one or more
coalescence modules can be sufficient to mix the contents of the
coalesced droplets (e.g., through rotating vortexes existing within
the droplet), the device of the present invention can further
include one or more mixing modules. A "mixing module" can comprise
features for shaking or otherwise manipulate droplets so as to mix
their contents. The mixing module is preferably downstream from the
coalescing module and upstream from the detection module. The
mixing module can include, but is not limited to, the use of metal
alloy component electrodes or electrically conductive patterned
electrodes to mix the contents of droplets and to reduce mixing
times for fluids combined into a single droplet in the microfluidic
device.
[0173] The device of the present invention can comprise features,
such as, acoustic actuators, metal alloy component electrodes or
electrically conductive patterned electrodes, for shaking droplets
to reduce mixing times for fluids combined into a single
droplet.
[0174] For acoustic manipulation, the frequency of the acoustic
wave should be fine tuned so as not to cause any damage to the
cells. The biological effects of acoustic mixing have been well
studied (e.g., in the ink-jet industry) and many published
literatures also showed that piezoelectric microfluidic device can
deliver intact biological payloads such as live microorganisms and
DNA. In an exemplary embodiment, the design of the acoustic
resonant uses a Piezoelectric bimorph flat plate located on the
side of the carved resonant in the PDMS slab. The piezoelectric
driving waveform is carefully optimized to select the critical
frequencies that can separate cells in fluids. There are five
parameters to optimize beyond the frequency parameter. Lab
electronics is used to optimize the piezoelectric driving waveform.
Afterwards, a low cost circuit can be designed to generate only the
optimized waveform in a preferred microfluidic device.
Delay Module
[0175] The device of the present invention can further include one
or more delay modules. The "delay module" can be a delay line. The
operation of a microfluidics device where a reaction within a
droplet is allowed to occur for a non-trivial length of time
requires a delay line to increase the residence time within the
device. For reactions demanding extensive residence time, longer or
larger delay lines are required. Accordingly, the invention
provides methods to increase residence times within microfluidic
devices.
[0176] The delay module is in fluid communication with the main
channel. The delay module can be located downstream of the
coalescence module and upstream of the detection module. The delay
module can be a serpentine channel or a buoyant hourglass. The
delay module can further comprise heating and cooling regions. The
heating and cooling regions can be used for performing on-chip,
flow-through PCR with the devices described herein.
[0177] The channel dimensions and configurations can be designed to
accommodate the required residence time with minimum pressure drops
across the device. For example, to accommodate very long delay
lines within the microfluidic device, the device can comprise a
multilayered PDMS slab which is composed of several patterned PDMS
slabs.
[0178] The channel dimensions can also be designed so as to allow
for required flow, residence time and pressure drop. Some channels
may be required to be very large in width and height. In order to
avoid collapse of the channels, the device includes support posts
within the channel design. In order to reduce dead volume behind
posts and further improve droplet stability, the support posts are
designed to optimize a streamlined flow within the channel. These
designs can include curved features as opposed to sharp edges.
[0179] To allow for longer period of device operation, delay lines
can also be extended to the outside of the chip. The off-chip delay
lines can be tubes within micron-sized internal diameter.
[0180] In order to allow more efficient use of available space and
faster operation, in methods where droplets are charged, after
charging, asymmetric splitting of oil and drops can be accommodated
by siphoning off oil from channels after droplets are charged. The
delay lines can be in the form of a tower a structure which is
vertical with respect to the ambient gravitational field) as to
allow buoyant forces to assist controlled droplet transport. Known
delay lines involve transporting droplets by emulsifying them in a
carrier fluid flowing in a channel and/or tube. Because the
velocity profile of the carrier fluid through the cross-section of
the channel and/or tube is not uniform, the velocity distribution
of the droplets will not be narrow, which causes the delay time
distribution of the droplets to not be narrow (i.e., some droplets
will be delayed more or less than others).
[0181] The devices of the present invention can also include
buoyancy-assisted microfluidic delay lines. In buoyancy-assisted
microfluidic delay lines, buoyant forces act on droplets emulsified
in a fluid in one or more towers. This can include allowing the
tower to fill for the desired delay time, and then releasing the
droplets. The tower can or can not continue to fill and release
droplets as needed. In this example, one may desire to have a
cylindrical tower section that is capped by a pyramidal funnel
section. The tower can effectively functions as an hourglass.
Droplets that have a density less than their carrier fluid are fed
into the base of the tower, buoyantly rise to the top of the tower
with a substantially uniform velocity distribution, and are
funneled into a functional component of the microfluidic device
(such as a y-branch). Carrier fluid is exhausted at the base of the
tower at the same rate as it is introduced at the apex so that the
net flow of carrier fluid through the delay line is zero. The tower
and funnel sections can have any cross-sectional shape, such as
circular, elliptical, or polygonal. The microfluidic device can
include a tower with adjustable length.
[0182] The device can also include a switching network of twenty
towers to guarantee a delay time dispersion of 5% (because
1/20=0.05). The capacity of each tower is 0.05*T, where T is the
delay time. The concept includes, for example: (a) upon device
start-lip, filling the first tower for 0.05*T, but stop-cock its
exhaust, and also have the other nineteen towers closed; (b) after
0.05*T, closing the first tower and filling the second between
0.05*T and 0.10*T; (c) repeating step (b) for the remaining
eighteen towers; (d) at time T, allowing the first tower to
exhaust; (e) at time 1.05*T, stop-cocking the exhaust of the first
tower, allowing the second tower to exhaust, and allowing the first
tower to fill; (f) at time 1.10*T, stop-cocking the exhaust of the
second tower, allowing the third tower to exhaust, closing the
first tower, and allowing the second tower to fill, and (g)
repeating step (f) ad infinitum. More than twenty towers may
provide an even tighter control over the width of the delay time
dispersion. This scheme may require a valve network. This network
of towers can be outside the microfluidic device.
UV-Release Module
[0183] The device of the present invention can further include one
or more UV-release modules. The "UV-release module" is in fluid
communication with the main channel. The UV-release module is
located downstream of the inlet module and upstream of the
coalescence module. The UV-module can be a used in bead assays.
Compounds from encapsulated beads can be cleaved in a UV-releasing
module using UV light. Photolabile linkers can be broken down on
demand after a single bead has been encapsulated thus releasing
multiple copies of a single compound into solution. In the cell
based assay disclosed herein the chemical compound assayed is
desired to be in solution in order to penetrate the cell membrane.
Furthermore, to ensure compartmentalization of a single compound
with a cell the cleavage of the compound from the solid support can
only be done after the bead has been encapsulated. Photocleavable
linkers can be utilized to cleave the compounds of the bead after
drop formation by passing the drop through a UV-release module
(i.e., laser of the appropriate wavelength).
[0184] The present invention also provides methods for chemical
synthesis on a bead and releasing said chemical attached to the
bead using a releasing means (chemical, UV light, heat, etc) within
a droplet, and then combining a second droplet to the first droplet
for further manipulation. Preferably, the releasing means is a
UV-module. For example, tea-bag synthesis of chemicals on a bead
simultaneously with a means for identifying said bead (using, for
example, a mass spec tag). Using the resulting mixed-chemistry
beads in a droplet within a fluid flow, and exposing the beads to
UV light to release the chemical synthesized from the bead into the
droplet environment. Combining the droplet containing the released
chemical with a droplet containing a cell, and performing a
cell-based assay. Sorting droplets having the desired
characteristics (for example, turn on of a reporter gene), and then
analyzing the sorted beads using mass spectroscopy.
Kits
[0185] As a matter of convenience, predetermined amounts of the
reagents, compound libraries, and/or emulsions described herein and
employed in the present invention can be optionally provided in a
kit in packaged combination to facilitate the application of the
various assays and methods described herein. Such kits also
typically include instructions for carrying out the subject assay,
and may optionally include the fluid receptacle, e.g., the cuvette,
multiwell plate, microfluidic device, etc. in which the reaction is
to be carried out.
[0186] Typically, reagents included within the kit are uniquely
labeled emulsions containing tissues, cells, particles, proteins,
antibodies, amino acids, nucleotides, small molecules, substrates,
and/or pharmaceuticals. These reagents may be provided in
pre-measured container (e.g., vials or ampoules) which are
co-packaged in a single box, pouch or the like that is ready for
use. The container holding the reagents can be configured so as to
readily attach to the fluid receptacle of the device in which the
reaction is to be carried out (e.g., the inlet module of the
microfluidic device as described herein). In one embodiment, the
kit can include an RNAi kit. In another embodiment, the kit can
include a chemical synthesis kit. It will be appreciated by persons
of ordinary skill in the art that these embodiments are merely
illustrative and that other kits are also within the scope of the
present invention.
DEFINITIONS
[0187] The terms used in this specification generally have their
ordinary meanings in the art, within the context of this invention
and in the specific context where each term is used. Certain terms
are discussed below, or elsewhere in the specification, to provide
additional guidance to the practitioner in describing the devices
and methods of the invention and how to make and use them. It will
be appreciated that the same thing can typically be described in
more than one way. Consequently, alternative language and synonyms
may be used for any one or more of the terms discussed herein.
Synonyms for certain terms are provided. However, a recital of one
or more synonyms does not exclude the use of other synonyms, nor is
any special significance to be placed upon whether or not a term is
elaborated or discussed herein. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference. In the case of conflict, the present
specification, including definitions, will control. In addition,
the materials, methods, and examples are illustrative only and are
not intended to be limiting.
[0188] The invention is also described by means of particular
examples. However, the use of such examples anywhere in the
specification, including examples of any terms discussed herein, is
illustrative only and in no way limits the scope and meaning of the
invention or of any exemplified term. Likewise, the invention is
not limited to any particular preferred embodiments described
herein. Indeed, many modifications and variations of the invention
will be apparent to those skilled in the art upon reading this
specification and can be made without departing from its spirit and
scope. The invention is therefore to be limited only by the terms
of the appended claims along with the full scope of equivalents to
which the claims are entitled.
[0189] As used herein; "about" or "approximately" shall generally
mean within 20 percent, preferably within 10 percent, and more
preferably within 5 percent of a given value or range.
[0190] The term "molecule" means any distinct or distinguishable
structural unit of matter comprising one or more atoms, and
includes for example polypeptides and polynucleotides.
[0191] The term "polymer" means any substance or compound that is
composed of two or more building blocks (`mers`) that are
repetitively linked to each other. For example, a "dimer" is a
compound in which two building blocks have been joined
together.
[0192] The term "polynucleotide" as used herein refers to a
polymeric molecule having a backbone that supports bases capable of
hydrogen bonding to typical polynucleotides, where the polymer
backbone presents the bases in a manner to permit such hydrogen
bonding in a sequence specific fashion between the polymeric
molecule and a typical polynucleotide (e.g., single-stranded DNA).
Such bases are typically inosine, adenosine, guanosine, cytosine,
uracil and thymidine. Polymeric molecules include double and single
stranded RNA and DNA, and backbone modifications thereof, for
example, methylphosphonate linkages.
[0193] Thus, a "polynucleotide" or "nucleotide sequence" is a
series of nucleotide bases (also called "nucleotides") generally in
DNA and RNA, and means any chain of two or more nucleotides. A
nucleotide sequence typically carries genetic information,
including the information used by cellular machinery to make
proteins and enzymes. These terms include double or single stranded
genomic and cDNA, RNA, any synthetic and genetically manipulated
polynucleotide, and both sense and anti-sense polynucleotide
(although only sense stands are being represented herein). This
includes single- and double-stranded molecules, i.e., DNA-DNA,
DNA-RNA and RNA-RNA hybrids, as well as "protein nucleic acids"
(PNA) formed by conjugating bases to an amino acid backbone. This
also includes nucleic acids containing modified bases, for example
thio-uracil, thio-guanine and fluoro-uracil.
[0194] The polynucleotides herein may be flanked by natural
regulatory sequences, or may be associated with heterologous
sequences, including promoters, enhancers, response elements,
signal sequences, polyadenylation sequences, introns, 5'- and
3'-non-coding regions, and the like. The nucleic acids may also be
modified by many means known in the art. Non-limiting examples of
such modifications include methylation, "caps", substitution of one
or more of the naturally occurring nucleotides with an analog, and
internucleotide modifications such as, for example, those with
uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoroamidates, carbamates, etc.) and with charged linkages
(e.g., phosphorothioates, phosphorodithioates, etc.).
Polynucleotides may contain one or more additional covalently
linked moieties, such as, for example, proteins (e.g., nucleases,
toxins, antibodies, signal peptides, poly-L-lysine, etc.),
intercalators (e.g., acridine, psoralen, etc.), chelators (e.g.,
metals, radioactive metals, iron, oxidative metals, etc.), and
alkylators. The polynucleotides may be derivatized by formation of
a methyl or ethyl phosphotriester or an alkyl phosphoramidate
linkage. Furthermore, the polynucleotides herein may also be
modified with a label capable of providing a detectable signal,
either directly or indirectly. Exemplary labels include
radioisotopes, fluorescent molecules, biotin, and the like.
[0195] The term "interdigitation" as used herein means pairing of
droplets from separate aqueous streams, or from two separate inlet
nozzles, for eventual coalescence.
[0196] The term "dielectrophoretic force gradient" means a
dielectrophoretic force is exerted on an object in an electric
field provided that the object has a different dielectric constant
than the surrounding media. This force can either pull the object
into the region of larger field or push it out of the region of
larger field. The force is attractive or repulsive depending
respectively on whether the object or the surrounding media has the
larger dielectric constant.
[0197] "DNA" (deoxyribonucleic acid) means any chain or sequence of
the chemical building blocks adenine (A), guanine (G), cytosine (C)
and thymine (T), called nucleotide bases, that are linked together
on a deoxyribose sugar backbone. DNA can have one strand of
nucleotide bases, or two complimentary strands which may form a
double helix structure. "RNA" (ribonucleic acid) means any chain or
sequence of the chemical building blocks adenine (A), guanine (G),
cytosine (C) and uracil (U), called nucleotide bases, that are
linked together on a ribose sugar backbone. RNA typically has one
strand of nucleotide bases.
[0198] A "polypeptide" (one or more peptides) is a chain of
chemical building blocks called amino acids that are linked
together by chemical bonds called peptide bonds. A "protein" is a
polypeptide produced by a living organism. A protein or polypeptide
may be "native" or "wild-type", meaning that it occurs in nature;
or it may be a "mutant", "variant" or "modified", meaning that it
has been made, altered, derived, or is in some way different or
changed from a native protein, or from another mutant.
[0199] An "enzyme" is a polypeptide molecule, usually a protein
produced by a living organism, that catalyzes chemical reactions of
other substances. The enzyme is not itself altered or destroyed
upon completion of the reaction, and can therefore be used
repeatedly to catalyze reactions. A "substrate" refers to any
substance upon which an enzyme acts.
[0200] As used herein, "particles" means any substance that may be
encapsulated within a droplet for analysis, reaction, sorting, or
any operation according to the invention. Particles are not only
objects such as microscopic beads (e.g., chromatographic and
fluorescent beads), latex, glass, silica or paramagnetic beads, but
also includes other encapsulating porous and/or biomaterials such
as liposomes, vesicles and other emulsions. Beads ranging in size
from 0.1 micron to 1 mm can be used in the devices and methods of
the invention and are therefore encompassed with the term
"particle" as used herein. The term particle also encompasses
biological cells, as well as beads and other microscopic objects of
similar size (e.g., from about 0.1 to 120 microns, and typically
from about 1 to 50 microns) or smaller (e.g., from about 0.1 to 150
nm). The devices and methods of the invention are also directed to
sorting and/or analyzing molecules of any kind, including
polynucleotides, polypeptides and proteins (including enzymes) and
their substrates and small molecules (organic or inorganic). Thus,
the term particle further encompasses these materials.
[0201] The particles (including, e.g., cells and molecules) are
sorted and/or analyzed by encapsulating the particles into
individual droplets (e.g., droplets of aqueous solution in oil),
and these droplets are then sorted, combined and/or analyzed in a
microfabricated device. Accordingly, the term "droplet" generally
includes anything that is or can be contained within a droplet.
[0202] A "small molecule" as used herein, is meant to refer to a
composition that has a molecular weight of less than about 5 kD and
most preferably less than about 4 kD. Small molecules can be, e.g.,
nucleic acids, peptides, polypeptides, peptidomimetics,
carbohydrates, lipids or other organic or inorganic molecules.
Libraries of chemical and/or biological mixtures, such as fungal,
bacterial, or algal extracts, are known in the art.
[0203] As used herein, "cell" means any cell or cells, as well as
viruses or any other particles having a microscopic size, e.g. a
size that is similar to or smaller than that of a biological cell,
and includes any prokaryotic or eukaryotic cell, e.g., bacteria,
fungi, plant and animal cells. Cells are typically spherical, but
can also be elongated, flattened, deformable and asymmetrical,
i.e., non-spherical. The size or diameter of a cell typically
ranges from about 0.1 to 120 microns, and typically is from about 1
to 50 microns. A cell may be living or dead. Since the
microfabricated device of the invention is directed to sorting
materials having a size similar to a biological cell (e.g. about
0.1 to 120 microns) or smaller (e.g., about 0.1 to 150 nm) any
material having a size similar to or smaller than a biological cell
can be characterized and sorted using the microfabricated device of
the invention. Thus, the term cell shall further include
microscopic beads (such as chromatographic and fluorescent beads),
liposomes, emulsions, or any other encapsulating biomaterials and
porous materials. Non-limiting examples include latex, glass, or
paramagnetic beads; and vesicles such as emulsions and liposomes,
and other porous materials such as silica beads. Beads ranging in
size from 0.1 micron to 1 mm can also be used, for example in
sorting a library of compounds produced by combinatorial chemistry.
As used herein, a cell may be charged or uncharged. For example,
charged beads may be used to facilitate flow or detection, or as a
reporter. Biological cells, living or dead, may be charged for
example by using a surfactant, such as SDS (sodium dodecyl
sulfate). The term cell further encompasses "virions", whether or
not virions are expressly mentioned.
[0204] A "virion", "virus particle" is the complete particle of a
virus. Viruses typically comprise a nucleic acid core (comprising
DNA or RNA) and, in certain viruses, a protein coat or "capsid".
Certain viruses may have an outer protein covering called an
"envelope". A virion may be either living (i.e., "viable") or dead
(i.e., "non-viable"). A living or "viable" virus is one capable of
infecting a living cell. Viruses are generally smaller than
biological cells and typically range in size from about 20-25 nm
diameter or less (parvoviridae, picornoviridae) to approximately
200-450 nm (poxviridae). However, some filamentous viruses may
reach lengths of 2000 nm (closterviruses) and are therefore larger
than some bacterial cells. Since the microfabricated device of the
invention is particularly suited for sorting materials having a
size similar to a virus (i.e., about 0.1 to 150 nm), any material
having a size similar to a virion can be characterized and sorted
using the microfabricated device of the invention. Non-limiting
examples include latex, glass or paramagnetic beads; vesicles such
as emulsions and liposomes; and other porous materials such as
silica beads. Beads ranging in size from 0.1 to 150 nm can also be
used, for example, in sorting a library of compounds produced by
combinatorial chemistry. As used herein, a virion may be charged or
uncharged. For example, charged beads may be used to facilitate
flow or detection, or as a reporter. Biological viruses, whether
viable or non-viable, may be charged, for example, by using a
surfactant, such as SDS.
[0205] A "reporter" is any molecule, or a portion thereof, that is
detectable, or measurable, for example, by optical detection. In
addition, the reporter associates with a molecule, cell or virion
or with a particular marker or characteristic of the molecule, cell
or virion, or is itself detectable to permit identification of the
molecule, cell or virion's, or the presence or absence of a
characteristic of the molecule, cell or virion. In the case of
molecules such as polynucleotides such characteristics include
size, molecular weight, the presence or absence of particular
constituents or moieties (such as particular nucleotide sequences
or restrictions sites). In the case of cells, characteristics which
may be marked by a reporter includes antibodies, proteins and sugar
moieties, receptors, polynucleotides, and fragments thereof. The
term "label" can be used interchangeably with "reporter". The
reporter is typically a dye, fluorescent, ultraviolet, or
chemiluminescent agent, chromophore, or radio-label, any of which
may be detected with or without some kind of stimulatory event,
e.g., fluoresce with or without a reagent. In one embodiment, the
reporter is a protein that is optically detectable without a
device, e.g. a laser, to stimulate the reporter, such as
horseradish peroxidase (HRP). A protein reporter can be expressed
in the cell that is to be detected, and such expression may be
indicative of the presence of the protein or it can indicate the
presence of another protein that may or may not be coexpressed with
the reporter. A reporter may also include any substance on or in a
cell that causes a detectable reaction, for example by acting as a
starting material, reactant or a catalyst for a reaction which
produces a detectable product. Cells may be sorted, for example,
based on the presence of the substance, or on the ability of the
cell to produce the detectable product when the reporter substance
is provided.
[0206] A "marker" is a characteristic of a molecule, cell or virion
that is detectable or is made detectable by a reporter, or which
may be coexpressed with a reporter. For molecules. a marker can be
particular constituents or moieties, such as restrictions sites or
particular nucleic acid sequences in the case of polynucleotides.
For cells and virions, characteristics may include a protein,
including enzyme, receptor and ligand proteins, saccharides,
polynucleotides, and combinations thereof, or any biological
material associated with a cell or virion. The product of an
enzymatic reaction may also be used as a marker. The marker may be
directly or indirectly associated with the reporter or can itself
be a reporter. Thus, a marker is generally a distinguishing feature
of a molecule, cell or virion, and a reporter is generally an agent
which directly or indirectly identifies or permits measurement of a
marker. These terms may, however, be used interchangeably.
[0207] The invention is further described below, by way of the
following examples. The examples include descriptions of
particular, exemplary embodiments of the devices and methods of the
present invention, including particular embodiments of channel
architectures, valves, switching and flow control devices and
methods which may be implemented as part of the devices and methods
of the invention. The examples are provided for illustrative
purposes only and are not limiting of the above-described invention
in any way. For example, many of these specific embodiments are
described and discussed primarily in terms of detecting and sorting
cells suspended directly in the fluid that flows through a main
channel of the device. Nevertheless, it will be appreciated by
persons of ordinary skill in the art that these preferred
embodiments are merely illustrative and that the invention may be
practiced in a variety of embodiments that share the same inventive
concept. In particular, the devices and methods described in this
example (including the channel architectures, valves, switching and
flow control devices and methods) may be readily adapted to a
multi-phased device so that droplets which contain, e.g.,
molecules, cells or virions may be analyzed and/or sorted as
desired by a user.
EXAMPLES
Example 1
[0208] The device of the present invention can be used for
Live/Dead Cell Based Assays. In one example, the assay uses two
fluorophores; one is permeable across cell membranes, and a second
dye binds DNA and can enter the cell only if the membrane is
compromised. Similar Live/Dead assays exist for bacteria and yeast.
Tagged chemical libraries and photocleavable linkers can be used in
such assays. Combinatorial one-bead-one-compound libraries obtained
through split-bead synthesis require a tag which describes their
synthetic history in order to identify the compound reliably.
Several encoding technologies for microcarriers such as beads, rods
and crowns have been developed over the last decade to address this
need. A simple and effective method relies on spectrometric
chemical tags which are generated in parallel to the chemical
entity of interested utilizing orthogonal chemistry. Alternatives
include the use of nucleic acids such as DNA, followed by the use
of the polymerase chain reaction (PCR) to decode the encoded
beads.
[0209] In the cell based assay disclosed herein the chemical
compound assayed is desired to be in solution in order to penetrate
the cell membrane. Furthermore, to ensure compartmentalization of a
single compound with a cell the cleavage of the compound from the
solid support can only be done after the bead has been
encapsulated. Photocleavable linkers can be utilized to cleave the
compounds of the bead after drop formation by passing the drop
through a UV-release module (i.e., laser of the appropriate
wavelength).
[0210] To evaluate the effect of individual components contained in
the molecular library, a two color fluorescence detection for
standard cytotoxicity assays can be used [available from Invitrogen
(Carlsbad, Calif.) or Cell Technology]. While any cells can be
used, for illustrative purposes, the Invitrogen LIVE/DEAD
Viability/Cytotoxicity Kit #L3224 for animal cells are used here.
This kit contains two probes that measure two recognized parameters
of cell viability: intracellular esterase activity and plasma
membrane integrity. Live cells are identified by the presence of
intracellular esterase activity, detected by the enzymatic
conversion of the almost nonfluorescent cell-permeant calcein AM to
the extremely fluorescent calcein. The calcein is retained within
live cells, producing an intense uniform green fluorescence. EthD-1
enters cells with damaged membranes and undergoes a 40-fold
enhancement of fluorescence upon binding to nucleic acids, thereby
producing a bright red fluorescence in dead cells. EthD-1 is
excluded by the intact plasma membrane of live cells. The
determination of cell viability depends on these physical and
biochemical properties of cells. Background fluorescence levels are
inherently low with this assay technique because the dyes are
essentially non-fluorescent before interacting with cells.
[0211] The spectral absorption and emission characteristics for
both the calcein and EthD-1 are presented in FIG. 3, while FIG. 2
plots results presented by Molecular probes when a 50/50 mix of
live and dead cells are run through a flow cytometer. The
absorption characteristics of both dyes makes it possible to excite
fluorescence using the existing 488 nm excitation source. FIG. 2,
left panel, shows a mixture of live and ethanol-killed bovine
pulmonary artery epithelial cells stained with the reagents in
Molecular Probes Live/Dead Cell Viability/Cytotoxicity Assay Kit
(L3224). Live cells fluoresce bright green, whereas dead cells with
compromised membranes fluoresce red-orange. (Molecular Probes). The
middle panel shows a viability assay using Molecular Probes'
LIVE/DEAD Viability/Cytotoxicity Kit on a flow cytometer. A 1:1
mixture of live and ethanol-fixed human B cells was stained with
calcein AM and ethidium homodimer-1, flow cytometric analysis was
carried out with excitation at 488 nm. The right panel shows
analysis of bacterial cultures using the Live/Dead BacLight
Bacterial Viability and Counting Kit available from www.molecular
probes.com. As shown in FIG. 3, left panel, the present invention
further provides a fluorescence detection system comprising a
fluorescence detection stand capable of measuring green
fluorophores within microfluidic channels while simultaneously
permitting visual monitoring via a high speed video microscope. The
optical components of this system are commercially available. The
modular layout of this system permits straightforward modification
of the excitation and detection wavelengths. This modularity also
makes it possible to upgrade the system to multi-wavelength
excitation, multiwavelength detection, and detection of orthogonal
polarization states. Currently, the 488 nm transition of a
multiline Argon-Ion Laser is used as the excitation source for
Fluorescein (FITC). The laser provides between 3 and 20 milliwatts
of power and is focused to a spot approximately 17 microns in
diameter (full width half maximum, FWHM). When the stand is
configured to use a photomultiplier tube, it is able to detect less
than 10,000 FITC molecules at a 10 kHz droplet rate. The
sensitivity of this system is limited by fluorescence interference
generated by the microfluidic device itself. The right panel of
FIG. 3 shows excitation and emission spectra for calcein AM and
EthD-1 dyes. Normal cytometry protocol excites both at 488 nm. FIG.
3 indicated the changes required to convert the single fluorophore
station to a two color fluorescence station. The calcein
fluorescence can be collected using filters designed for
fluorescein detection, while the EthD-1 can be monitored using
filters designed for propidium iodide or Texas Red.
[0212] Using water droplets doped with fluorescein and propidium
iodide over a range of concentrations from 1.times.10.sup.-3M to
1.times.10.sup.-8M, both dyes have similar absorption and
fluorescence properties to calcein and Ethd-1. In seventeen micron
droplets, this corresponds to a range of 1.5.times.10.sup.9 to
1.5.times.10.sup.4 molecules within the measurement volume. Once
the baseline performance has been verified, tests can begin on
droplets containing live cells, dead cells, and mixtures of the
two. This establishes selectivity and detection limits on the two
types of cells.
[0213] The dyes selected have been used extensively in flow
cytometry and are commonly used in most cell-based assays. They are
designed not to overlap significantly with each other and can be
evaluated both independently and together to assess the cross-talk.
The status of (potentially) many cells within one drop can thus be
determined. The use of inexpensive optics on our instrument will be
more than compensated for by the theoretical increase of dye
molecules in the nanoreactor. Optics with higher efficiencies can
be used.
[0214] Compounds from encapsulated beads can be cleaved in a
UV-releasing module using UV light. Photolabile linkers can be
broken down on demand after a single bead has been encapsulated
thus releasing multiple copies of a single compound into
solution.
[0215] Synthetic chemistry relies on the differential activity of
chemical groups in order to control bond breaking and forming
processes. Photolabile protecting groups form a fourth orthogonal
type of functionality which survive reaction conditions capable of
cleaving protective groups of the other types. Several of these
photolabile protecting groups have been used to link organic
molecules to solid support and their use as linkers has been
reviewed. This allows the synthesis of solid supported molecules
with the option of releasing the final product by irradiation with
the appropriate wavelength. The repertoire of chemical groups for
which photocleavable protecting groups have been devised is
extensive, which allows the synthesis of diverse combinatorial
libraries.
[0216] In lieu of the high sample rate a triazene-based photolabile
linker, which is cleaved by irradiation with a 355 nm 3 w Nd-YAG
laser, can be used. This linker is stable under a wide range of
reaction conditions with the exception of strong acids lending
itself to solid supported split-bead synthesis.
[0217] If the residence time of the bead inside the UV laser is
insufficient to cleave all of the compound off the substrate bead,
the residence time can be increased by slowing down the flow of the
bead containing drops by widening the channel. Alternatively the
intensity of laser beam can be increased to ensure complete
cleavage.
[0218] As previously discussed, long incubation times are desirable
for cytotoxicity studies, but cannot be easily achieved in known
microfluidic channel layouts. Accordingly, one embodiment of the
device used in the live/dead cell-based assay disclosed herein uses
a passive means to achieve uniform droplet residency times
exceeding one hour in a delay line module located directly before
the sorting module. It is possible to achieve a delay time of one
hour between droplet generation and detection without stopping
droplet generation.
[0219] In one example, a "buoyancy hourglass" delay line can be
used, wherein, similar to sands in an hourglass which depend on
gravity, the droplets will rise from a large reservoir to an
exhaust port due to their density mismatch with the carrier oil.
Microfluidic modules (e.g., inlet module, UV-releasing module,
coalescence module, and mixing module) which are utilized before
the delay module can be patterned at the bottom of the stack, and
microfluidic modules which are utilized after the delay module
(e.g., detection module and sorting module) can be patterned at the
top of the stack. Upon start-up, the hourglass will be stop-cocked
to allow droplets to fill until the desired delay time is reached
and then droplet will be removed from the device at the same rate
that they enter, thereby ensuring essentially the same residency
time for all droplets. Spontaneous droplet coalescence in the
hourglass can be prevented by using one or more surfactants to
stabilize the droplets.
[0220] The shape and timing of electric field gradients through the
use of computer modeling can be optimized by tailoring the geometry
of the electrodes and the fluid channels and the synchronization of
the applied voltages to the droplets.
[0221] The FEMLAB (COMSOL, Inc.) partial differential equation
solver software can be used to model the combination of fluid
dynamics and electrostatics. The model can include "still-frame
captures" of the trajectory of droplets through bifurcations, and
can optimize the electrode geometry, the fluid channel geometry,
and the distribution of applied voltages as a function of the
incremental droplet trajectory. Further, a high-speed digital
camera and driving electronics can be used to acquire "still-frame
captures" of the actual droplet trajectories and comparing those
captures to those produced by the model. The model and the
electrode and fluid channel geometries can be iteratively optimized
using inexpensive rapid prototyping capability (24 hours from
design to test-results). Finally, the electric field gradients can
be satisfactorily optimized when bidirectional sorting at rates of
1000 droplets/second or greater, without breaking the droplets and
with an acceptably low error rate for the given application, is
achieved. If the electromechanical relay network is not fast enough
to operate with the optimized timing parameters, a solid-state
relay network (e.g., using Behlke electronic relays) can be used to
increase the speed of the driving electronics.
[0222] Droplets containing beads can be sorted using
dielectrophoretic and electrostrictive forces based on a
fluorescence probe at rates of 1000 droplets/s or greater. A
fluorescence detection system and Electrical Control System can be
used to trigger the optimal "pulse" (i.e., distribution of applied
voltages as a function of time) to sort neutral droplets based on
the fluorescence probe. Dielectrophoretic/electrostrictive sorting
of droplets containing fluorescent dye can be performed, wherein
the sorting is triggered by the droplet number (e.g., every nth
droplet is sorted in one direction, or every nth or mth droplet is
sorted in one direction, etc.). Fluorescent dye can be used to
perform dielectrophoretic sorting of droplets because it is
convenient and inexpensive; the trigger signal for the
dielectrophoretic/electrostrictive sorting can be exactly the same
as was used for electrophoretic sorting. This process is the direct
logical consequence of optimizing the electric field gradients.
[0223] Additionally, dielectrophoretic/electrostrictive sorting of
droplets containing fluorescent beads can be performed. This step
is intermediate between droplets containing fluorescent dye and
droplets containing cells and beads laden with chemical libraries.
Finally, dielectrophoretic/electrostrictive sorting of droplets
containing fluorescent cells can be performed. This step is
intermediate between droplets containing fluorescent dye and
droplets containing cells and beads laden with chemical
libraries.
[0224] In the event that the solutions containing the beads or
cells have dielectric properties so different than the solution
containing fluorescent dye that the dielectric field gradients are
not optimal, the dielectric field gradients can be optimized
separately for each solution. The fluorescent dye solution can be
modified to better resemble the bead or cell solutions in order to
continue to take advantage of the convenience of the fluorescent
dye for the development of the sorting parameters.
[0225] Droplets sorted based on a particular phenotype (for
example, dead cells) will be decoded (by using a decoding scheme)
to identify the compound added in that droplet.
[0226] In some embodiments, the assay can be based on a
nucleic-acid based encoded bead system. Two types of beads can be
used for example--one contains a cytotoxic compound and
oligonucleotide tag, and a second bead contains only a different
oligonucleotide tag. The two types of beads may (optionally) also
be encoded by a different fluorescent tag (i.e., other than the
ones being used for the cell-based assays, as an example, two
different Q-dots) so that the beads can be examined under a
fluorescent microscope after sorting to determine the sorting
efficiency.
[0227] The sorted beads from dead-cell containing nanoreactors can
then be taken, and using the polymerase chain reaction (PCR), the
tags on the beads can be amplified using PCR primers. These tags
can be `hard-copied` by cloning them into a plasmid vector,
transforming them into E. coli, and the tag sequence of 100
different E. coli transformants determined by DNA sequencing.
[0228] Additionally, more complex libraries using T-bag synthesis
on beads, can be constructed, wherein oligonucleotide tags are
specific for each round of synthesis for a monomer. The same
monomer used in two different rounds can have two separate tags. As
a non-limiting example, if 30 monomers in a bead-based T-bag
synthesis were used for 5 rounds, 5.times.30, or 150 different tags
will be required. The complexity of a library of 30 monomers after
5 rounds is 30.sup.5, or nearly 25 million compounds. The beads in
a specific T-bag after each round of monomer synthesis can have a
specific oligonucleotide tag ligated, using T4 DNA ligase, onto the
beads. These tags, from sorted beads, can be amplified, cloned and
sequenced. By knowing what tags were used in which round of
synthesis, an internal check of validation of the bead that was
positive in that droplet is achieved. The sequencing reaction can
be eliminated by using a hybridization chip containing the 150
tags.
Example 2
[0229] The present invention provides methods for performing
polymerase chain reaction in nanoreactors of the present invention
as described. PCR can be performed on a drop-by-drop basis in a
microfluidic device according to the present invention. A
monolithic chip can be provided wherein the heating and cooling
lines are built into the chip and a sorting means is provided.
Advantages of performing PCR in droplets on such a chip are that
the chip is disposable and the reaction can be repeated without
cleaning the device between reactions. Furthermore, the chip
provides a convenient way of getting all the components to perform
PCR in the droplets in the right concentration. Additionally, the
PCR is more efficient because the heat transfer is more efficient
due to the small volume. This provides for shorter
incubation/residence times. Droplets containing the nucleic acids,
all PCR primers, and, if present, beads are generated one at a time
at rates between 100 and 20,000 droplets per second. The droplets
can then be sent through a serpentine path between heating and
cooling lines to amplify the genetic material inside the droplets.
Upon exiting the device the droplets may be sent for further
on-chip or off-chip processing, directed into another chip, or the
emulsion may be broken to release the PCR product. If present,
beads may be harvested by passing the emulsion through a filtration
device, sedimentation, or centrifugation.
[0230] The width and depth of the channel can be adjusted to set
the residence time at each temperature, which can be controlled to
anywhere between less than a second and minutes. At a typical rate
of 1000 drops per second, 1 million strands of DNA can be amplified
in approximately 20 minutes on one device. A typical flow rate of
250 .mu.L/hour would correspond to 1000 drops of 50 microns in
diameter being generated every second. Flow rates and droplet sizes
can be adjusted as needed by controlling the nozzle geometry.
[0231] In an example bead based application, the purpose is to
amplify at most one DNA fragment in a droplet containing a single
micro-bead (1 to 100 microns in diameter) and then separate and
collect only the beads coated with DNA. This is achieved by
starting with a dilute mixture of DNA fragments and beads in a
solution containing the appropriate PCR primers. Droplets are then
made in the limited dilution regime where most of the droplets are
empty, but some droplets have a DNA strand in them and some
droplets have beads in them. The target droplets have both a single
DNA fragment and a single bead. After PCR amplification of the DNA
on the surface of the beads a fluorescence activated sorting module
(NanoFACS) can be added to the end of the device to separate the
droplets into two populations, one containing amplified DNA and one
without amplified DNA. The beads are then removed from the emulsion
where the droplets all contain DNA to achieve a collection of beads
where essentially all beads are coated with only one type of DNA
fragment.
[0232] The quality of the collection of beads where each fragment
is amplified in the presence of only one bead can be enhanced by
ensuring that each droplet contains at most one bead. Droplets
containing more than one bead can be removed using a
fluorescence-based sorting step.
[0233] Along with PCR, nucleic acid based signal methods such as
tyramide assays using an appropriate enzyme reaction,
oligonucleotides decorated with two or more detecting groups, or
other amplification means, for example, rolling circle
amplification, ligase chain reaction, and NASBA can be used to
increase the signal within a droplet.
Example 3
[0234] The device of the present invention can be used to screen
chemical libraries composed of at least 10.sup.6 molecules against
an established cell line. In this manner, positive and negative
nanoreactors can be tracked and sorted using either a nucleic-acid
based, or multi-colored bead-based encoding scheme For example, a
control library with known hits can be screened against a human
cancer cell line.
[0235] In one embodiment, a chemical library can be screened using
a nanoreactor as described in detail herein. The power of the
present invention comes from a combination of compartmentalization
and electrical manipulation that enables multi-step chemical
processing, including analysis and sorting, to be initiated in
confinement with exquisite timing and metering precision. This
multi-step processing of isolated components is essential for
searching through molecular libraries for rare interactions with
cells, nucleic acids, enzymes, coded microbeads, and other
biomaterials. For example, a set of encoding nucleic acids, (i.e.,
DNA tags) can be combined into solutions of unique chemical
compounds such that the DNA tags and chemicals are emulsified
together. In one embodiment, the DNA tag acts as a surrogate
identifier to track the associated chemical compound in droplets
sorted by a nanoreactor described herein. After sorting, the
emulsion can be broken and the nucleic acids can be decoded (FIG.
4). As shown in FIG. 4 (Left panel), (A) An individual compound
from a library of compounds will each be combined (B) with a
unique, differentiatable set of q-dots. The combined mixture will
each (C) be separately emulsified off-line using a flow-focusing
microfluidics emulsifier to synthesize individual droplets
containing both a specific compound and a unique set of q-dots. As
shown in FIG. 4 (Right panel), the set of individually-emulsified
encoded compounds will be (D) pooled together and injected, along
with either cells or enzymes, into the RDT instrument and (E) the
two droplets combined to form individual NanoReactors. Depending on
the reaction being monitored, a separate combining (not shown in
the figure) of these nanoreactors with droplets containing assay
components may be needed. In addition, a delay loop may be placed
between these combinings and the detector (F) to allow sufficient
time to occur in the droplet as to allow any potential
chemical/cellular/enzymatic reaction to occur. The nanoreactors are
next sent past a detector to both monitor the reaction and decode
the q-dots contained within it. The nanoreactors can be further
sorted (G) if necessary. In designing the device to be placed on
the Instrument, individual modules are strung together in a
sequence of droplet operations. Operations can be used to
encapsulate cells or enzyme, inject the labeled pre-formed compound
library emulsion, coalesce pairs of droplets, mix the contents of
droplets, incubate reactions over time, detect fluorescence, decode
the liquid label, sort (if needed) based on the detected signal,
and transport droplets to collection and waste streams. The
individual modules operate independently, much like resisters and
capacitors in an electrical circuit, to collectively perform
complex fluid processing operations. Several methods encompassing
various chemical library screening embodiments of the invention as
described. In one example, a kinase enzyme assay is used as an
enzyme model, three different quantum dots (q-dots) for the liquid
label, and a set of 96 different chemical compounds (in which 1-2
will be preferred kinase substrates) as the library. Fluorescence
polarization is preferred since the argument can be made that it
can be adapted to many different types of assays. In one example,
water-soluble q-dots that emit at 620 nm, 650 nm and 680 nm are
used. These emission bands are well outside of spectral region
where the target enzyme assays emit (below 580 nm), so these q-dots
are an excellent choice for In another example, near-IR q-dots are
used to enhance their water solubility for the purpose of expanding
the non-overlapping spectral region of the target assays. Moving
the q-dot readout to this "unused" wavelength band can permit
virtually any fluorescence assay of interest to be adapted to the
nanoreactors of the present invention without modification,
tremendously expanding the application space immediately
available.
[0236] The nucleic acid can be a linear molecule wherein the ends
can be used as priming sites for PCR, and the middle sequence is
unique to each chemical compound; it is this middle sequence that
is used as the encode. The nucleic acid and chemical compound are
together combined into one droplet by pre-emulsifying the nucleic
acid and chemical together and then adding them to a microfluidic
device as described herein, as a pre-made, compound droplet. The
compound droplet can be combined with a another droplet on the
instrument. This other droplet can contain an item under
investigation (including for example, but not limited to, a cell or
enzyme), which, when combined with the compound droplet forms an
`assay` droplet. The assay droplets having a desired detected
property (for example, inhibition of enzyme activity through the
use of a fluorescent substrate added to the compound microdrop) can
then be sorted. The sorted assay droplets can be collected, the
emulsion broken, and the nucleic acid sequence can be decoded. The
decoding can be performed by emulsion PCR (as described in U.S.
Application Publication No 2005-0227264) and sequencing on a
sequencing instrument. Alternatively, the decoding can be performed
by cloning the PCR product into an appropriate host (for example,
E. coli), and the resultant clones subjected to DNA sequencing.
[0237] The nucleic acid can be a linear molecule having a region of
uniqueness, and the decoding can be performed by cloning and
subsequently transforming the DNA obtained from sorted assay
droplets into an appropriate host (e.g., E. coli). The resultant
clones can then be subjected to decoding by hybridizing a PCR
product containing the unique identifier to a complementary strand
of nucleic acid fixed to a solid support (for example a chip,
wafer, or bead).
[0238] The nucleic acid can be a plasmid having a region of
uniqueness, and the decoding can be performed by transforming the
DNA obtained from sorted assay droplets into an appropriate host
(e.g., E. coli). The resultant clones can then be subjected to DNA
sequencing to identify the encoded sequence.
[0239] The nucleic acid can be a plasmid having a region of
uniqueness, and the decoding can be performed by transforming the
DNA obtained from sorted assay droplets into an appropriate host
(e.g., E. coli). The resultant clones can then be subjected to
decoding by hybridizing a labeled-PCR product containing the unique
identifier to a complementary strand of nucleic acid fixed to a
solid support (for example a chip, wafer, or bead).
[0240] The nucleic acid can be either a plasmid or linear fragment
having a region of uniqueness, and the decoding can be performed by
transforming the DNA obtained from sorted assay droplets into an
appropriate host (e.g., E. coli). The resultant clones can then be
subjected to decoding by hybridizing a labeled-PCR product
containing the unique identifier to a complementary strand of
nucleic acid fixed to a solid support (for example a chip, wafer,
or bead). Preferably, the bead can be encoded with dyes or Qdots,
and the decoding can be performed on a microfluidic device
according to the present invention, or on a Qdot or Luminex
instrument.
[0241] A set of unique nucleic acids can be added to a set of
unique chemical entities, wherein each combined set is separately
emulsified. The separately emulsified combined set can be further
combined to generate an emulsified mixed solution of droplets,
wherein each droplet can contain both a nucleic acid and a unique
chemical entity. This combined mixed solution can be injected into
a microfluidic device according to the present invention for use in
various assays contemplated by one of ordinary skill in the
art.
[0242] The nucleic acid containing unique identifiers can be
generated by PCR of an antibiotic resistance or other selectable
gene with a set of the forward and reverse PCR primers each
containing a 5' nucleotide sequence common to each other, forward
and downstream primers, respectively, a unique sequence 3' to the
common sequence, and a region of the antibiotic or other selectable
gene. Said primers can be used in a PCR reaction to generate an
antibiotic resistance or other selectable gene bracketed by unique
identifiers which in turn can be bracketed by either a forward or
reverse common sequence. The PCR product can then be cloned into a
vector having a second antibiotic resistance or other selectable
gene, and the vector can be cloned into an appropriate host (e.g.,
E. coli), thereby selecting for antibiotic resistance and another
selectable gene simultaneously.
[0243] The label can also be a solution containing a dye such as an
organic dye (for example cy3, cy5, flourescein) or inorganic label
such as a quantum dot. The dot can be further coated or
encapsulated by hydrophobic residues. More than one dye can be
added to a solution prior to emulsification and the ratio of one or
more dyes can be used to decode the droplet.
[0244] Additionally, many bead-encoded assays have already been
developed for microspheres that should be directly ported to the
devices and systems disclosed herein. Such assays include, for
example: allergy testing, disease markers (including, autoimmune,
cancer and cardiac), cytokine, genotyping, gene expression,
infectious disease, kinase/phosphorylated proteins, metabolic
markers, tissue typing, transcription factors/nuclear receptors and
others.
[0245] The present invention also provides methods of using a
drop-washer for combinatorial chemistry/biology. A device of the
present invention capable of exchanging constituents within a
droplet through the use of fluid flow in such a way that the
microdrop, while in a first immiscible fluid, is exposed to a
second immiscible fluid such that constituents within the droplet
that are immiscible in the first immiscible fluid are soluble in
the second immiscible fluid.
[0246] For example, an aqueous droplet containing a chemical
reaction produces by-products that are soluble in a lipid solvent.
The chemical reaction is performed in a water-environment in a
silicon-based solvent. After the chemical reaction occurs, the
droplet is exposed to an organic-oil based solvent where the
chemical byproducts are allowed to diffuse out of the droplet. The
resulting droplet is then assayed for cell-killing activity by
combining the droplet with live cells.
[0247] Similar to the preceding example, but the change in the
non-aqueous fluid flow is used to add a particular constituent from
the second immiscible fluid to diffuse into the aqueous drop before
the droplet is returned to the 100% first immiscible fluid
flow.
Example 4
[0248] The present invention also provides methods of performing
biological assays in nanoreactors using fluorescence polarization
(FP). Fluorescence polarization technology has been used in basic
research and commercial diagnostic assays for many decades, but has
begun to be widely used in drug discovery only in the past six
years. Originally, FP assays for drug discovery were developed for
single-tube analytical instruments, but the technology was rapidly
converted to high-throughput screening assays when commercial plate
readers with equivalent sensitivity became available. These assays
include such well-known pharmaceutical targets such as kinases,
phosphatases, proteases, G-protein coupled receptors, and nuclear
receptors.
[0249] Nuclear Receptors; FP has been used to develop high
throughput screening (HTS) assays for nuclear receptor-ligand
displacement (Parker G J, et al., Development of high throughput
screening assays using fluorescence polarization: nuclear
receptor-ligand-binding and kinase/phosphatase assays). The
FP-based estrogen receptor (ER) assay is based on the competition
of fluorescein-labeled estradiol and estrogen-like compounds for
binding to ER. In a screen of 50 lead compounds from a
transcriptional activation screen, 21 compounds had IC50 values
below 10 microM, with one exhibiting roughly a 100-fold higher
affinity for ERbeta over ERalpha. An FP-based competitive binding
assay can be used to screen diverse compounds with a broad range of
binding affinities for ERs.
[0250] Phosphatases and Kinases; A nonradioactive, simple,
sensitive fluorescence polarization assay has been developed to
assay protein tyrosine kinase activity (Seethala R.; Menzel R. A
Homogeneous, Fluorescence Polarization Assay for Src-Family
Tyrosine Kinases. Analytical Biochemistry, November 1997, vol. 253,
no. 2, pp. 210-218(9)). This assay involves incubation of a
fluorescenylated peptide substrate with the kinase, ATP, and
anti-phosphotyrosine antibody. The phosphorylated peptide product
is immunocomplexed with the anti-phosphotyrosine antibody resulting
in an increase in the polarization signal as measured in a
fluorescence polarization analyzer. These results show that the
fluorescence polarization assay can detect inhibitors and is
comparable to the .sup.32PO.sub.4 transfer assay. The fluorescence
polarization method is advantageous compared to the .sup.32PO.sub.4
transfer assay or ELISA or DELFIA because it is a one-step assay
that does not involve several washings, liquid transfer, and sample
preparation steps. It has the added advantage of using nonisotopic
substrates. The fluorescence polarization assay thus is
environmentally safe and minimizes handling problems.
[0251] G-protein coupled receptors; High-throughput fluorescence
polarization (FP) assays offer a nonradioactive, homogeneous, and
low-cost alternative to radioligand binding assays for cell surface
receptors (G protein-coupled receptors and ligand-gated ion
channels) (Allen M, Reeves J, Mellor G. High throughput
fluorescence polarization: a homogeneous alternative to radioligand
binding for cell surface receptors. J Biomol Screen. 2000 April;
5(2):63-9). FP assays were shown to work across a range of both
peptide (vasopressin V1a and delta-opioid) and nonpeptide (beta
1-adrenoceptor, 5-hydroxytryptamine-3) receptors. Assays could be
run in 384-well plates with little loss of signal window or
sensitivity compared to 96-well plate assays. New advances in FP
measurement have therefore enabled FP to offer a high throughput
alternative to radioligand binding for cell surface receptors.
[0252] GTPases; A 30,000-member compound library was screened using
filter binding [FB (.sup.33P)] and FP detection systems, and
compounds that were active in either assay were retested in 5-point
curve confirmation assays (C. L. Hubert et al. Data Concordance
from a Comparison between Filter Binding and Fluorescence
Polarization Assay Formats for Identification of ROCK-II
Inhibitors). Analysis of these data showed an approximate 95%
agreement of compounds identified as active in both assay formats.
Also, compound potency determinations from FB and FP had a high
degree of correlation and were considered equivalent. These data
suggest that the assay methodology has little impact on the quality
and productivity of the screen, provided that the assays are
developed to standardize kinetic conditions.
[0253] Diagnostics using Antibodies: The control of equine
infectious anemia virus (EIAV) infections of horses has been over
the past 20 years based primarily on the identification and
elimination of seropositive horses, predominantly by a standardized
agar gel immunodiffusion (AGID) assay in centralized reference
laboratories. Peptides derived from antigenic regions of EIAV core
and envelope proteins were initially screened for their utility as
probes in an FP assay to select the bestpeptide antigen candidates
(S. B. Tencza, et al. Development of a Fluorescence
Polarization-Based Diagnostic Assay for Equine Infectious Anemia
Virus. Journal of Clinical Microbiology, May 2000, p. 1854-1859,
Vol. 38, No. 5). The FP assay was optimized to detect the presence
of EIAV-specific antibodies by a change in the FP of a
fluorescein-labeled immunoreactive peptide diagnostic antigen. The
most sensitive and specific peptide probe was a peptide
corresponding to the immunodominant region of the EIAV
transmembrane protein, gp45. This probe was tested for its
reactivity in the optimized FP assay with 151 AGID-positive horse
sera and 106 AGID-negative serum samples. The results of these
studies demonstrated that the FP assay reactivity correlated with
reported AGID results in 106 of 106 negative serum samples (100%
specificity) and in 135 of 151 positive serum samples (89.4%
sensitivity). The FP assay was also found to have a very low
background reactivity and to readily detect antibodies produced
early in infection (<3 weeks postinfection).
[0254] FP is a homogeneous technology with very rapid reactions;
seconds to minutes suffice to reach equilibrium. As the reagents
are stable, large highly reproducible batches may be prepared.
Because of these properties, FP has proven to be highly
automatable, often performed with a single incubation with a
single, premixed, tracer-receptor reagent. The fact that there are
no washing steps increases the precision and speed over
heterogeneous technologies and dramatically reduces waste.
[0255] Other homogeneous technologies based on fluorescence
intensity have been developed. These include energy transfer,
quenching, and enhancement assays. FP offers several advantages
over these. The assays are usually easier to construct, since the
tracers do not have to respond to binding by intensity changes. In
addition, only one tracer is required and crude receptor
preparations may be utilized. Furthermore, since FP is independent
of intensity, it is relatively immune to colored solutions and
cloudy suspensions. FP offers several advantages in the area of
instrumentation. Because FP is a fundamental property of the
molecule, and the reagents are stable, little or no standardization
is required. FP is relatively insensitive to drift in detector gain
settings and laser power.
[0256] The concept of molecular movement and rotation is the basis
of fluorescence polarization. By using a fluorescent dye to label a
small molecule, its binding to another molecule of equal or greater
size can be monitored through its speed of rotation. As shown in
FIG. 5, dye molecules with their absorption transition vectors
(arrows) aligned parallel to the electric vector of linearly
polarized light (along the vertical page axis) are selectively
excited. For dyes attached to small, rapidly rotating molecules,
the initially photoselected orientational distribution becomes
randomized prior to emission, resulting in low fluorescence
polarization. Conversely, binding of the low molecular weight
tracer to a large, slowly rotating molecule results in high
fluorescence polarization. Fluorescence polarization therefore
provides a direct readout of the extent of tracer binding to
proteins, nucleic acids and other biopolymers.
[0257] Fluorescence polarization, first described in 1926 by
Perrin, has a long history. FP theory and the first instrument for
measuring was developed by Weber. This work was expanded to
biological systems, such as antigen-antibody reactions and
hormone-receptor interactions by Dandliker. The first commercial
systems, aimed at monitoring drugs in body fluids come from Jolley
and co-workers.
[0258] Fluorescence polarization is defined by the following
equation: P=(V-H)/(V+H) where P is the polarization unit, V is the
intensity of the vertical component of the emitted light, and H is
the intensity of horizontal component of the emitted light of a
fluorophore excited by vertical plane polarized light. The
"polarization unit" P is a dimensionless entity and is not
dependent on the intensity of the emitted light or on the
concentration of the fluorophore. This is the fundamental power of
FP. The term "mP" is now in general use, where 1 mP equals one
thousandth of a P.
[0259] The excitation dipole is the direction in which the molecule
prefers to absorb light. The emission dipole is the direction in
which a molecule prefers to emit light. This assumed (for the sake
of simplicity) that these directions are parallel. In one
experiment, if the fluorescent molecules are fixed so that all
excitation dipoles are aligned in the vertical plane and assume
there is only fluoresces with a polarization along the emission
dipole then a maximum polarization unit of 1000 mP is observed. If,
however, the excitation dipoles were randomly oriented this maximum
polarization unit is reduced to 500 mP. In another experiment, if
the requirement that the dipoles are fixed was removed and they are
allowed to reorient between the time when they are excited and the
time when they fluoresce the polarization unit falls below 500
mP.
[0260] In another experiment, a collection of randomly oriented
transition moments are free to rotate. In this case, the
polarization unit is between 0 and 500 mP and is dependent on how
far the molecule has rotated during the fluorescence lifetime of
the excited state. The smaller the molecule, the faster it rotates,
and so the lower the FP will be.
[0261] The rate of rotation of a molecule is described by the
Stokes equation: .rho.=(3.eta.V)/(RT) where .rho. is the rotational
relaxation time (the time required to rotate through an angle whose
cosine is 1/e, or approximately 68.5.degree.), .eta. is the
viscosity of the medium, V is the molecular volume of the molecule,
R is the gas constant, and T is the temperature in degrees Kelvin.
From the previous equations we can see that the higher the
molecular weight of a molecule, the higher the rotational
relaxation time will be; V=vM where M (Perrin equation) is the
molecular weight of the molecule in Daltons and v is its partial
specific volume (cm.sup.3 g.sup.-1). The Perrin equation was first
described in 1926, and describes the relationship between the
observed FP, the limiting polarization, the fluorescence lifetime
of the fluorophore (.tau.), and its rotational relaxation time.
((1/P)-(1/3))=((1/P.sub.0)-(1/3)).times.((1+(3.tau./P)).
[0262] The shorter the fluorescence lifetime, the higher the FP
will be. Conversely, the shorter the rotational relaxation time,
the smaller the FP will be. Combining the Stokes equation and the
Perrin equation, and substituting M for V and rearranging, we get
the relationship between the molecular weight of a molecule and its
FP (1/P is proportional to 1/M);
(1/P)=(1/P.sub.0)+((1/P.sub.0)-(1/3)).times.(RT/vM).times.(.tau./.eta.).
From this equation we can see that P equals P.sub.0 in the limiting
cases of high molecular weight, high viscosity, and short lifetime.
In fact, P.sub.0 can be determined by measuring FP at various
viscosities, plotting P against 1/.eta., and determining the
intercept on the ordinate.
[0263] Organic fluorophores have characteristics, such as narrow
excitation bands and broad red-tailing emission band. FIG. 6 (Left,
Center Panel) shows the absorption and emission spectra of q-dot
535 nanocrystals and fluorescein, respectively. FIG. 6 (Right
panel) shows the emission spectra of a several sizes of CdSe--ZnS
quantum dots, with excitation of ZnSe at 290 nm, all others at 365
nm. nm in all cases. These bands often limit their effectiveness.
This makes concurrent resolution of multiple light-emitting probes
problematic due to spectral overlap. Also, many organic dyes
exhibit low resistance to photodegradation.
[0264] Luminescent colloidal semiconductor nanocrystals called
quantum dots or q-dots (QD) are inorganic fluorophores that have
the potential to circumvent some of the functional limitations
encountered by organic dyes. In particular, CdSe--ZnS core-shell
QDs exhibit size-dependent tunable photoluminescence (PL) with
narrow emission bandwidths (FWHM .about.30 to 45 nm) that span the
visible spectrum and broad absorption bands. These allow
simultaneous excitation of several particle sizes (colors) at a
common wavelength. This, in turn, allows simultaneous resolution of
several colors using standard instrumentation (FIG. 6, right
panel). CdSe--ZnS QDs also have high quantum yields, are resistant
to photodegradation, and can be detected optically at
concentrations comparable to organic dyes.
[0265] Quantum dots are nano-scale semiconductors typically
consisting of materials such as crystalline cadmium selenide. The
term `q-dot` emphasizes the quantum confinement effect of these
materials, and typically refers to fluorescent nanocrystals in the
quantum confined size range. Quantum confinement refers to the
light emission from bulk (macroscopic) semiconductors such as LEDs
which results from exciting the semiconductor either electrically
or by shining light on it, creating electron-hole pairs which, when
they recombine, emit light. The energy, and therefore the
wavelength, of the emitted light is governed by the composition of
the semiconductor material. If, however, the physical size of the
semiconductor is considerably reduced to be much smaller than the
natural radius of the electron-hole pair (Bohr radius), additional
energy is required to "confine" this excitation within the
nanoscopic semiconductor structure leading to a shift in the
emission to shorter wavelengths.
[0266] Fluorescence polarization assays can be used in a
microfluidics device to monitor the activity of kinase enzymes,
phosphatases, proteases, ligand-ligand binding, and others.
Extension of the existing fluorescence detection system to perform
fluorescence polarization measurements requires the incorporation
of a linearly polarized laser and polarizing optics into the
design. As shown in FIG. 7, linearly polarized laser and polarizing
optics is incorporated into the design. A linearly polarized
frequency doubled diode laser operating at 488 nm passes through a
1/2 waveplate and linear polarizer (Meadowlark Optics, >2000:1
contrast ratio). This makes it possible to orient and lock the
exciting laser polarization as required for FP. The laser is
reflected and focused into the sample using a dichroic beamsplitter
and anti-reflection coated lenses. Fluorescence from the sample is
transmitted back through the lenses and dichroic beamsplitter and
isolated using the emission filter. This fluorescence signal is
then split into orthogonal polarizations using a polarizing
beamsplitter (Meadowlark Optics polarizing cube beamsplitter,
contrast ratio >500:1 transmitted, >20:1 reflected). Contrast
is further enhanced with linear polarizers (Meadowlark Optics,
>2000:1 contrast ratio). Finally, each polarization signal is
measured using a pair of photomultiplier tubes (Hamamastsu H5789),
digitized and analyzed by computer. A linearly polarized
(>200:1) frequency doubled diode laser operating at 488 nm from
Picarro is used for this purpose. As seen in the figure, the laser
passes through a 1/2 waveplate and linear polarizer (Meadowlark
Optics, >2000:1 contrast ratio). This makes it possible to
orient and lock the exciting laser polarization as required for FP.
As with the standard station, the laser is reflected and focused
into the sample using a dichroic beamsplitter and anti-reflection
coated lenses. Fluorescence from the sample is transmitted back
through the lenses and dichroic beamsplitter and isolated using the
emission filter. This fluorescence signal is then split into
orthogonal polarizations using a polarizing beamsplitter
(Meadowlark Optics polarizing cube beamsplitter, contrast ratio
>500:1 transmitted, >20:1 reflected) and contrast is further
enhanced with linear polarizers (Meadowlark Optics, >2000:1
contrast ratio). Finally, each polarization signal is measured
using a pair of photomultiplier tubes (Hamamastsu H5789), digitized
and analyzed on the computer. It is expected that these optics will
permit better than mP sensitivity.
[0267] These fluorescence polarization systems were tested using a
model enzyme system; Src-family tyrosine kinase. A nonradioactive,
simple, sensitive fluorescence polarization assay has been
developed to assay protein tyrosine kinase activity (Seethala R.;
Menzel R. A Homogeneous, Fluorescence Polarization Assay for
Src-Family Tyrosine Kinases. Analytical Biochemistry, November
1997, vol. 253, no. 2, pp. 210-218(9)). This assay involves
incubation of a fluorescenylated peptide substrate with the kinase,
ATP, and anti-phosphotyrosine antibody. As shown in FIG. 8, the
phosphorylated peptide product is immunocomplexed with the
anti-phosphotyrosine antibody resulting in an increase in the
polarization signal as measured in a fluorescence polarization
analyzer. FIG. 8, left panel, shows the IMAP principle of
operation. When a fluorescent substrate is phosphorylated by a
kinase, it can bind to the IMAP binding reagent, whose molecular
size is large relative to the substrate. This gives a large
increase in the polarization of the fluorescence. FIG. 8, middle
panel, shows the IMAP assay of MAPKAP-K2, a serine/threonine
kinase. MAPKAP-K2, from Upstate, was assayed in a volume of 20
.mu.L using the amounts of enzyme indicated. Concentrations of ATP
and substrate were 5.0 and 0.5 .mu.M, respectively. Incubation was
60 minutes at room temperature, followed by the addition of 60
.mu.L IMAP binding reagent. FP was read on an Analyst system 30
minutes later. FIG. 8, right panel, shows the IMAP quantification
of kinase inhibition. MAPKAP-K2 (0.25 units/mL) was incubated using
the amounts of enzyme shown for 15 minutes. The activity of the
enzyme was then assessed as described in above. These results show
that the fluorescence polarization assay can detect inhibitors and
is comparable to the .sup.32PO.sub.4 transfer assay. The
fluorescence polarization method is advantageous compared to the
.sup.32PO.sub.4 transfer assay or ELISA or DELFIA because it is a
one-step assay that does not involve several washings, liquid
transfer, and sample preparation steps. It has the added advantage
of using nonisotopic substrates. The fluorescence polarization
assay thus is environmentally safe and minimizes handling
problems.
[0268] The dyes that are chosen are used extensively in flow
cytometry and in our instrument will be determining the status of
(potentially) many dyes within one drop. The use of inexpensive
optics on our instrument will be more than compensated for by the
theoretical increase of dye molecules in the nanoreactor.
[0269] FP assays have been shown to tolerate up to 5% DMSO with no
loss in sensitivity or signal window. From a random set of 1,280
compounds, Allen et al found that 1.9% significantly interfere with
FP measurement (J Biomol Screen. 2000 April, 5(2):63-9. High
throughput fluorescence polarization: a homogeneous alternative to
radioligand binding for cell surface receptors. Allen M, Reeves J,
Mellor G. Receptor & Enzyme Screening Technologies, Glaxo
Wellcome Medicines Research Centre, Stevenage, Herts, UK.). If
fluorescent or quenching compounds were eliminated (3% of all
compounds), less than 0.4% of compounds were found to interfere
with FP measurement. Compounds are assayed a priori and those that
have these undesirable characteristics are eliminated.
[0270] In some enzymatic assays, a delay module (i.e., delay line)
will be utilized. This is less true for enzyme reaction mechanisms
in a small volume. And even many cell-based assays can be measured
within 5 minutes. Longer assay times can be accomplished by
collecting the droplets, incubating them for an appropriate amount
of time, and then re-injecting them into the device.
[0271] Three different q-dots in several concentrations each can be
placed in a microdroplet, and can then be used with the device of
the present invention to decode what is in the drop. In one
experiment, the initial labeling scheme used three colors of q-dots
having emission wavelengths of 620 nm (CdSe/ZnS), 650 nm
(InGaP/ZnS), and 680 nm (InGaP/ZnS) (excitation at 488 nm is
appropriate for all). In one specific example, one q-dot was
maintained at a constant concentration and varying the second and
third q-dots at least 10 different concentrations giving 100
different encodes (1.times.10.times.10). Decoding will be computed
by referencing the intensity of the second and third q-dots
relative to the first q-dot. Other labeling schemes can be used
during the course of these experiments.
[0272] The Q-dot readout extension to the fluorescence station is
described herein and is easily incorporated into the design due to
the modular layout developed. As seen, a series of dichroic
beamsplitters, emission filters, and detectors are stacked onto the
system, allowing measurement of the required five emission channels
(two fluorescence polarization signals and three q-dot bands).
Dichroic beamsplitters and emission filters capable of separating
the q-dot wavelength bands from each other are readily available,
so it is a straightforward process to configure the station
appropriately.
[0273] The residence time can be increased by slowing down the flow
of drops by widening the channel. Alternatively the intensity of
the laser beam can be increased to compensate or increase the
concentration of the q-dots within the droplet.
[0274] As described herein, the dyes chosen for FP are commonly
used in most cell- and enzyme-based assays and are designed not to
overlap significantly with the q-dots. The dyes are evaluated both
independently and together with the q-dots (at first
off-instrument) to assess the cross-talk. Preferably, the liquid
q-dot labels are read outside a spectral wavelength band currently
used in FACS analysis and sorting (i.e., the dyes flourescein, Cy3,
Cy5, etc). This permits the use of currently-available assays
(dependent on these dyes). Using specific q-dots, crosstalk is
minimized. Several commercial entities sell q-dots that can be read
by the optics being designed. The three colors of q-dots used
currently are the non-functionalized T2 EviTags having emission
wavelengths of 620 nm (CdSe/ZnS), 650 nm (InGaP/ZnS), and 680 nm
(InGaP/ZnS) (excitation at 488 nm is appropriate for all).
[0275] It is possible to generate 96 types of droplets, each
droplet containing both a unique set of q-dot labels and a chemical
compound, and as the droplet flows through the device of the
present invention kinase enzyme activity can be analyzed using FP
and the q-dot label can be decoded. This method allows for scaling
to more complex and interesting libraries.
[0276] FP assays have been shown to tolerate up to 5% DMSO with no
loss in sensitivity or signal window. From a random set of 1,280
compounds, Allen et al. found that 1.9% significantly interfere
with FP measurement (J Biomol Screen. 2000 April, 5(2):63-9.
High-throughput fluorescence polarization: a homogeneous
alternative to radioligand binding for cell surface receptors.
Allen M, et al. Receptor & Enzyme Screening Technologies, Glaxo
Wellcome Medicines Research Centre, Stevenage, Herts, UK.). If
fluorescent or quenching compounds are eliminated (3% of all
compounds) then less than 0.4% of compounds are found to interfere
with FP measurements. Compounds are assayed a priori and those that
quench FP are eliminated.
[0277] The three colors of q-dots we will use are the
non-functionalized T2 EviTags having emission wavelengths of 620
nm, 650 nm, and 680 nm; excitation at 488 nm is appropriate for
all. The >620 nm liquid labeling emission band was chosen not to
interfere with the FP assay band found between 488 and 620 nm.
These q-dots are commercially-available, stable in some buffers and
remain suspended in aqueous solution.
[0278] A mixture of two types of droplets, buffer-only and
fluorescein-containing, are stable for at least 1 month without any
detectable diffusion of the organic dye into the buffer-only
droplets. Other surfactants may be substituted for different kinds
of compounds. For other compound testing, i) similar mixtures of
compound-containing and buffer-only droplets can be created, ii)
they can be sorted based on their q-dot labels, and iii) Mass
Spectrometry can be used on the buffer-only droplets to
quantitatively detect the presence of other chemicals
compounds.
[0279] In some embodiments, a delay module (i.e., delay line) can
be utilized. This will be true for enzyme reaction mechanisms in a
small volume. But even many cell-based assays can be measured
within 5 minutes. Droplets can also be taken off-line and stored
for at least a month a month before re-injection into the device of
the present invention with no apparent change in the droplets.
Longer delay times can be achieved by taking mixed droplets
off-line, and then re-injected them.
Example 5
[0280] The present invention provides methods for performing
condensation chemistry in nanoreactors of the present invention as
described to synthesize libraries of drug-like molecules in a
highly convergent manner.
[0281] All life processes can be reduced to chemical reactions that
take place in aqueous media. Hence water is considered to be the
universal ultimate solvent and inevitably biological experiments
are performed in aqueous media. Furthermore, organic Solvents of
all types are detrimental to biochemical reactions limiting their
use to small percentages of water miscible solvents such as
dimethyl sulfoxide and ethylene glycol. On chip synthesis in our
system therefore requires that all chemical reactions are performed
in biologically compatible aqueous media. In contrast, conventional
synthetic organic chemistry relies on highly activated substrates
and highly reactive reagents to conduct bond forming processes.
Typically these substrates and/or reagents are unstable in the
presence of and react with water rendering them useless. An ever
increasing effort to reduce cost, enhance safety and to address
environmental concerns w.r.t. solvent choice has driven the
development of synthetic methods that utilize water as the primary
and in many cases the only solvent (Li). In the case in which water
is not the only solvent, water miscible organic solvents are used
to aid the dissolution of substrates.
[0282] While material can be removed from NanoReactors by breaking
them in a controlled way, it is preferable to avoid having to do
so. To eliminate the requirements of removing material from
doplests, we have identified 5 reaction types, which can be
performed in aqueous media, that can be used to "stitch" drug-like
molecules together from a highly diverse library of sub-structural
components. These reactions generate commonly occurring functional
groups in drug-like molecules and include: i) N-Acylation ii)
N-Sulfonylation iii) Cycloaddittions iv) Reductive alkylation of
amines and v) SNAr reactions (Morgan). Random combination of
sub-structures will yield a library of all possible combinations.
The reactions are sufficiently orthogonal to perform multi-step
reactions. Furthermore, simple protection and deprotection schemes
can be used to increase the number of condensations. A sufficient
number excess of component nanoreactors will yield a library of all
possible combinations with multiple copies of each combination for
testing in a biomolecular assay. This redundancy is required to
reduce the impact of false positive sorting events.
[0283] This technology is based on the assembly of drug-like
chemical entities by two or more step convergent syntheses from
diverse sub-structural components. A two step process would
assemble the final chemical species from four substructures. An
example of such a synthesis is the construction of the kinase
inhibitor Gleevec from relatively simple building blocks. Gleevec
is the first of a class of kinase inhibitors which targets the
chimeric tyrosine kinase bcr-abl. Bcr-abl is constitutively active
causing a rare life-threatening form of cancer called chronic
myeloid leukemia (CIVIL). Gleevec was given FDA approval in a
record breaking three months in May of 2001. Analogues to a sphere,
the three dimensional structure can be broken down into hemispheres
and quadrant by the appropriate disconnections. FIG. 9 shows that
Quadrants A and B are combined utilizing a reductive alkylation.
Quadrant C and D are combined utilizing a 1,3 dipolar
cycloaddition. The Hemisphere AB and CD are combined utilizing an
N-acylation.
[0284] Chemical Diversity is achieved by varying the sub-structural
units (Quadrants) and randomly combining to achieve all possible
combinations. Table 1 shows the theoretical diversity for number of
each quadrant (e.g. 4 different Quadrants A, with 4 different
Quadrants B, with 4 different Quadrants C and 4 different Quadrants
D yield 256 unique products after two steps as described above.
TABLE-US-00001 TABLE 1 Diversity of quadrants (equal Number of
number of diverse members for Unique each quadrant) solutions 1 1 2
16 3 81 4 256 5 625 6 1,295 7 2,401 8 4,096 9 6,561 10 10,000
[0285] Sufficiently diverse condensation chemistry in aqueous media
exists to synthesize diverse libraries of drug-like molecules in a
highly convergent manner.
[0286] The majority of synthetic reactions routinely used in the
synthesis of complex drug-like molecules rely on reagents and
substrates which are sensitive to hydrolysis in the presence of
water and hence require stringent exclusion of water from the
reaction vessel. Furthermore, condensation reaction often require
the equivalent of a dehydration to proceed, with one or both of the
condensation substrates being sensitive to degradation by water.
The pursuit of "greener" chemistry, using benign and
environmentally friendly solvents such as water, has resulted in
the development of several condensation reactions which take place
in the presence of water. Aqueous organic chemistry has been
extensively reviewed in the primary literature and books.
[0287] The encapsulation technology of the present invention which
relies on the addition of reagents and/or substrates to
nanoreactors without the option for reaction work-up (i.e.
purification of product). This precludes the use of reactions which
have side products which could potentially interfere with
subsequent steps or the biological assay intended to be performed
on the final product. Furthermore, in multistep reactions, the two
steps will have to be orthogonal with respect to their coupling
chemistry, i.e. the functional groups for consecutive reactions may
not interfere with each other. Five reaction types have been
identified which can be performed in aqueous media and which do not
require purification of the product prior to the next synthetic
step or testing in biological assays. These reactions include: i)
N-Acylation ii) N-Sulfonylation iii) Cycloadditions iv) reductive
alkylation of amines and v) SNAr reactions.
[0288] The present invention provides methods of performing these
condensation reactions in a highly convergent, "one pot" synthesis
to stitch together complex drug-like molecules from at least 2-16
substructures.
[0289] The solubility of organic compounds in aqueous media is
strongly dependent on their structure. To enhance solubility of the
library compounds in aqueous media, it is common in the
biomolecular screening community to dissolve library compounds in
DMSO and subsequently dilute the DMSO solution with water. DMSO is
compatible with the nanoreactors described herein.
[0290] A fluorescent product resulting from the condensation of two
suitable fragments can be distinguished from droplets that have
components which did not react to form the fluorescent product thus
enabling the optical readout to distinguish between the two cases
and sort the droplets accordingly. The components able to form the
fluorescent product would contain a different tag from the
components which are not able to form a fluorescent product. Hence
this system can be used to test the tagging strategy chosen to
identify the composition of the final product.
[0291] Traditional combinatorial chemistry relies on complex
deconvolution methods to determine the structural identity of the
final product once it has been determined to be active in any
particular biomolecular assay. Massively parallel synthetic
approaches use encoding technologies to infer the structure of any
particular product from the tag associated with it which typically
identifies the reaction history of that particular compound. In one
example, the reagent droplets are encoded with nucleic acids tag
which will provide a unique PCR signature for the final product
from which the reactant composition and hence structure can be
inferred.
[0292] The synthesis of a fluorescent molecule which will be the
product of one particular component with a common reaction partner
will be used to test the tagging technology. FIG. 10 shows three
tags denoted A, B and C label one of each of the following unique
components: A is a fragment which if combined with C will yield a
fluorescent molecule. B is a fragment which if combined with C will
yield a non-fluorescent molecule. The fluorescence detector will be
able to distinguish between drops that contain A, B, C or the
mixture BC and between the drops which contain the mixture AC (if
the reaction has taken place and the fluorescent product is
formed). The fluorescence based sorting will yield a population of
AC tags which are completely devoid of B. The second (waste)
population of drops may contain B, C and A tag if not every drop
containing the tag A has been fused to a drop containing C.
[0293] Multistep convergent syntheses of drug-like compounds can be
performed in nano-reactors by the selective fusion of droplets.
These compounds can then be tested in a biomolecular screen
immediately after being synthesized on chip.
[0294] The synthesis of the bcr-abl kinase inhibitor Gleevec has
been described herein. The synthesis of this inhibitor from four
sub-structural units followed by an assay determining its ability
to inhibit the bcr-abl kinase in a fluorescence polarization based
assay would serve as proof of principle for this technology.
[0295] Two different sub-structures will be used for each quadrant
such that at least 16 possible products can be formed of which one
is Gleevec. Although some of the other products will have
sub-structural elements of Gleevec, the "alternative" quadrants
will be considerably different to ensure that a completely
non-active product will be amongst the possible combinations. Each
unique quadrant will be tagged with a suitable nuclei acid
oligomer.
[0296] The products will be tested in a fluorescence polarization
based kinase assay with the expectation that Gleevec will strongly
inhibit the activity of bcr-abl. Based on the assay readout, the
drops containing Gleevec will be sorted and collected separately.
Analysis of the nucleic acid tags of those drops can reveal the
composition of the hit compound.
[0297] The present invention also provides methods of using nucleic
acids for chemical encoding and decoding tagging of chemical
reactions Current technology exists for the tagging of beads with
chemical tags which "record" the synthetic history of any
particular bead thereby allowing the deconvolution of the active
small molecule's structure. The encapsulation of the reagents used
to assemble the library members enables the use of homogeneous
nucleic acid based tags to determine the structure of any
particular quaternary reaction combination. Positive hits from the
biomolecular screen would be cloned into E. coli and decoded using
polymerase chain reaction (PCR) to determine the composition of
quadrants used to assemble the bio-active molecule. FIG. 11 (left
panel) shows four groups of DNA tags. One of sixteen
double-stranded oligodeoxynucleotide `surrogate` tags will be added
to each of four `groups` of the sixteen different chemicals being
used for chemical synthesis (see text for details). Each group of
tags will have unique overlapping 5' and 3' ends that are the same
for each member of the group, but complementary between adjacent
groups. The tags within the groups are designed with asymmetric 5'
overhangs such that they can ligate once with a member of an
adjacent group. The first and fourth groups will additionally
contain 5' and 3' sequences (respectively) that can be used as
priming sites to PCR up final products containing all four groups.
The top-strand in groups 2, 3 and 4 will contain a 5' phosphate
needed for DNA ligation. FIG. 11 (right panel) shows the tags in
each reaction are sorted based on the enzyme assay. In the example
shown, chemical synthesis is allowed to occur (see text for
details) and droplets (in this example) containing tags 2, 7, 11
and 14 have within them a synthesized compound that reacts
positively in an enzyme/cell-based assay. The positive droplets are
then subjected to a polymerase chain reaction (PCR) using primers
complementary to the ends of groups 1 and 4. The resulting PCR
product will next be cloned into an appropriate DNA vector.
Finally, colonies of transformed E. coli containing the catenated
tags will be DNA-sequenced to decode the synthesis history of the
compound associated with positively-sorted droplets.
[0298] Alternatively soluble quantum dot dyes can be used to encode
the input emulsions which can identify the chemical composition of
a positive hit by measuring relative fluorescence signals of
multicolored quantum dots eliminating the need for sorting. An
assay point with appropriate signal from a fluorescent marker (in
the case of the kinase assay proposed here we would measure changes
in fluorescence polarization) the synthetic history of the molecule
responsible for this signal would be read out by determining
relative levels of dyes. This tagging technology is limited by the
number of unique combinations that can be discerned with
appropriate confidence and hence would be applied to smaller, more
focused libraries typically used to explore a sub-set of chemical
space surrounding an early lead.
Example 6
[0299] The present invention provides methods of isolating
self-antigens. A first sample droplet set consisting of a tumor
obtained from a multicellular organism treated in such a way as to
create single cells that are then each separately or multiply
contained within said first droplet set are combined with a second
set of droplets consisting of one or more t-cells isolated from the
organism, and the resulting combined droplets are analyzed for
t-cell killing of the tumor cells contained within the combined
droplets using a detecting means. The detecting means can include
analysis for cytoplasmic enzymes that would be released to the
droplet environment upon cell lysis. The droplets can be either
sorted or not sorted and then further analyzed for identification
of tumor cell epitopes recognized by the t-cell.
Example 7
[0300] The present invention provides methods of matrix screening
using a phased-drop approach or derivatives thereof. A device
composed of a multitude of samples each separately contained within
sample wells connected to one or more inlet channels such that that
can be operated in such a way that each sample can be encapsulated
within a droplet within a fluid-flow and be both sequentially and
separately combined with each of the other samples by varying the
phase of the combining of the separate, sequential droplets.
[0301] For example, by changing the phase of the combining of the
drops it is possible to have, for example, with five separate
samples each combine with the other samples, in this example in
pairs, to yield drops containing a mixture of all possible pairs of
compounds 1+2, 2+3, 3+4, 4+5, 5+1, . . . 1+4. The phasing can be by
one of several means, including channel length, valves, pressure,
etc.
[0302] In another example, a matrix of 100 chemical compounds are
loaded into 100 separate wells and are each combined in separate
pairs to yield 100.sup.2 different pairwise combinations. These
10.sup.3 combinations are each separately used in a cell-based
assay to determine their combined effects on cell survival.
[0303] The devices and systems disclosed herein have several
distinct advantages over current devices and methods for analyzing
samples. These advantages include, for example: reliability and
reproducibility, flexibility (the ability to `swap out`), the
greatly reduced cost of an assay, speed and handling, reduced
skill-level required needed to perform the an analysis, scalability
of assays from one to many nanoreactors, automatable with current
liquid-handling robotics, multiple sort capability and previously
unachievable assay architecture enabled by NanoRector confinement
and manipulation
[0304] The enhanced functionality that electrostatic charge brings
to droplets in microfluidic devices has the potential to enable an
expansive list of microfluidics applications. This toolkit of
techniques for manipulating droplets described herein can enable
modular integration of systems for transporting and reacting small
numbers of molecules. High throughput screening, combinatorial
chemistry, and the search for rare biological function in libraries
of biomolecules all benefit from electrostatic manipulation of
droplets in microchannels. Droplet-based microfluidic technology
can also be used to develop a chip-scale fluorescence activated
cell sorter (FACS) with enhanced activation functionality that goes
beyond fluorescence to include multiple reagent-based assays
between the droplet formation and sorting steps. Moreover by using
femtoliter droplets, which are a few microns in diameter, even a
single biomolecule represents concentrations of >>1 nM,
sufficient for efficient chemical reactivity and single-molecule
assays.
[0305] Many of the potential uses of droplet-based microfluidic
devices are driven by a need to encapsulate a varied population or
library of molecules, cells or particles into microreactors,
perform an assay on the contents, perhaps through the addition of
reagents, and then, finally, to selectively remove specific
microreactors from the collection in a search for rare events. This
requires a processing rates of 10.sup.3 per second to sort through
the smallest libraries in a reasonable time while rates on the
order 10.sup.5 per second are desirable for larger libraries. These
rates are feasible using the charged droplet paradigm. Moreover,
because the microfluidic devices are stamped, parallel flow streams
can be fabricated, further enhancing the total throughput.
Combined, the advantages of droplets and high throughput
manipulation provide significant opportunity for widespread
application. The inventions presented and described in detail
herein will facilitate the application of droplet-based
microfluidic technology.
Example 8
[0306] The present invention also provides adaptations of known
assays for use on the microfluidic device according to the present
invention. For example, fluorescence polarization, molecular
beacons, and taqman assays can be adapted for use in SNP, DGE, and
nucleic acid identification. In a high-throughput mode the
individual droplets can be labeled with either organic or inorganic
dyes, or colored beads. A distinct advantage is that beads are not
required and the entire assay can be performed in solution. Some
exemplary assays are described.
[0307] The present invention can be used to identify CDRs in a
pre-defined CDR library. In one example, there can be 100
pre-defined CDRs for each of the 6 CDRs in an scFv (i.e., 3 in VH,
and 3 in VL). 600 molecular beacons can be created, each beacon
separately emulsified with a different (for example, q-dot)
LiquidLabel. The 600 separate emulsions can be pooled to create one
emulsion library mixture (composed of 600 different types of
droplets, and as stated each droplet containing both a molecular
beacon specific to a specific CDR, and a LiquidLabel specific to
droplets containing that molecular beacon). scFv Ab genes from
antigen-interacting antibodies isolated by either phage display or
yeast two-hybrid can be amplified by PCR using 5' and 3' flanking
primers. The PCR product of the Ab gene can be either emulsified on
the RDT Instrument prior to combining with the library mixture, or,
in a separate example, combined with it's own unique LiquidLabel
off-line, and mixed with several (other) amplified Ab fragments,
thereby allowing several PCR fragments to be analyzed
simultaneously. The amplified fragment will then be combined with
the library mixture, and run past the detector. The detector will
identify the molecular beacon within the droplet using the
LiquidLabel and further detect whether hybridization has occurred
by examining the status of the fluorophore relative to the
probe-containing quencher.
[0308] An oligonucleotide assay can be used to generate a product
against which an fluorescence polarization (FP) organic-dye type
tag, molecular beacon or taqman oligonucleotide can be used in an
assay as described above. Other assays are also possible.
[0309] The present invention can also be utilized in differential
gene expression. Taqman or molecular beacons can be used in a
modification of the methods as described herein.
[0310] The TaqMan system requires the use of a polymerase with 5'
to 3' nuclease activity, such as Taq DNA polymerase, and a short
oligonucleotide probe labeled with a reporter-dye and a quencher
dye that anneals to the target downstream from one of the primers
(See, FIG. 12, left panel). If the probe is hybridized to the
target, the polymerase cleaves the hybridized probe, separating the
reporter from the quencher, which results in a higher fluorescent
signal. The fluorescent signal increases proportionally to the
number of amplicons generated during the log-linear phase of
amplification. It is important that the probe hybridizes before the
primers so the polymerase can cleave the probe and release the
reporter dye as primers are extended. Otherwise, amplification
occurs but is not monitored because the probe is not cleaved.
[0311] Molecular beacon probes are hairpin-shaped oligonucleotide
molecules that have a fluorophore and a non-fluorescent quencher
dye attached to the 5' and 3' ends (See, FIG. 12, right panel).
Generally, DABCYL is the non-fluorescent universal quencher and the
other dye is a reporter fluorophore such as, FAM, TET, TAMRA or
ROX. The molecular beacon is in a hairpin configuration when it is
not hybridized to the target site. It is designed to have two
"arms" with complementary sequences that form a very stable hybrid
or stem. The close proximity of the quencher and reporter
suppresses reporter fluorescence when the beacon is in a hairpin
configuration. When the beacon hybridizes to the target during the
annealing step the reporter dye is separated from the quencher,
which allows the reporter to fluoresce. In order for the beacon to
anneal to the target sequence, it must form a hybrid that is even
more stable than the hairpin to remain in the hybridized
conformation. Therefore, the probe is less likely to form a hybrid
with the target if there are mismatched base pairs.
[0312] In addition to molecular beacons and Taqman, the devices of
the present invention can be used to carry out fluorescence
polarization as described herein. Most SNP assays can be adapted
for both mini-sequencing and gene expression analysis.
A series of fluorescence polarization measurements have been made
inside a microfluidic device according to the present invention
while looking at droplets containing Fluorescein, Fluorescein bound
to biotin, and Fluorescein bound to biotin+Steptaviden. The
fluorescence signal was split into two orthogonal polarizations:
one parallel to the laser excitation polarization, and one
perpendicular to the polarization. These signal were collected and
analyzed to determine the change in polarization of the
fluorescence for each of these binding conditions.
[0313] The Polarization is calculated from:
P = ( V - H ) ( V + H ) ##EQU00003##
Where V=fluorescence signal polarized parallel to laser excitation
polarization, and H=fluorescence signal polarized perpendicular. A
mP ("milli-P") is 1000*P. The Polarization is equal to zero when
the fluorescence is completely depolarized, and has a maximum of
500 mP when the fluorescent molecule is "frozen" (i.e. bound to a
large molecule that does not rotate between excitation and
emission).
[0314] The fluorescence station was modified to include cleanup
polarizer for the laser and a polarizing beamsplitter with cleanup
polarizers for collection. The two resulting fluorescence channels
collect light with orthogonal polarizations ("Vertical" is parallel
to the laser polarization, "Horizontal" is perpendicular to the
laser polarization). The device used to generate alternating
droplets is built from RDT Master #257 (50 um deep channels). Table
2 lists the fluids used for these tests.
TABLE-US-00002 TABLE 2 Fluids ran through the double nozzle device.
Fluid Name Composition Oil FC3283 + 10% "Avocado" BTFC 1 .times.
10.sup.-6 Molar Biotinylated Fluorescein in 10 mM Borate pH 9 BTFC
+ SA 1 .times. 10.sup.-6 Molar Biotinylated Fluorescein in 10 mM
Borate pH 9 + 0.5 .times. 10.sup.-6 Molar Steptaviden (4 binding
sites per molecule)
[0315] During these experiments, the fluid flow rates were 200
ul/hr for FC, BTFC, and BTFC+SA, and 600 ul/hr for the oil
(FC3283+10% "Avacado"). Droplets ranged in size from 65 to 75 um in
diameter, depending on which water solution was used. Time between
droplets varied from 1300 us to 2200 us, again depending on which
fluid was used. Fluorescence polarization measurements were made by
injecting BTFC in one nozzle, and BTFC+SA in the other. Once these
measurements were completed, the BTFC+SA was replaced with FC and a
second series of data was collected. FIG. 13 plots typical
fluorescence data collected from both these runs, as well as the
calculated Polarization for each of the droplets shown. FIG. 13
(top panel) shows raw polarized fluorescence signals collected on
the test station when measuring BTFC and BTFC+SA, then BTFC and FC
(the data is normalized so P(Fluorescein)=0.0). FIG. 13 (bottom
panel) shows polarization calculated from each droplet in the top
plot. The transition at t=1.5 sec is a mathematical artifact where
the data collected for the two conditions were concatenated in
software (the data after t=1.5 was collected approximately 30
minutes after the data collected before t=1.5). In this data,
Vertical is fluorescence parallel to laser polarization, Horizontal
is polarization perpendicular. In this data, the "horizontal"
polarization has been normalized such that the Polarization is
equal to zero for Fluorescein. The Polarization was calculated by
integrating the fluorescence signal across the droplet for each
polarization, then plugging the results into Equation 1. FIG. 14
plots the Polarization measured for a longer number of droplets,
along with the histogram created from this data. FIG. 14 (top
panel) shows polarization calculated from each droplet for a longer
time period than in FIG. 3. FIG. 14 (bottom panel) shows histogram
generated from the data in the top figure. The data after t=1.5 was
collected approximately 30 minutes after the data collected before
t=1.5.
[0316] As seen in the histograms of the Polarization, the
polarization clusters tightly around three different mean values.
The zero-centered mean (.sigma.=1.7) corresponds to Fluorescein,
while the 18.4 mP (.sigma.=1.6) grouping corresponds to
Biotinalated Fluorescein and the 96 mP (.sigma.=3.46) grouping
corresponds to Biotinalated Fluorescein bound to Steptaviden.
Example 9
[0317] Fluorosurfactants are synthesized by reacting Krytox 157
FSL, FSM, or FSH with aqueous ammonium hydroxide in a volatile
fluorinated solvent. The solvent and residual water and ammonia are
removed with a rotary evaporator. The surfactant can then be
dissolved in a fluorinated oil (e.g., FC-3283 from 3M), which can
then be used as the continuous phase of the emulsion. A typical
concentration is 2.5 wt % of surfactant dissolved in the oil.
[0318] The channels of the microfluidic device are also coated with
a fluorinated surface product. For example, the coating is applied
from a 0.1-0.5 wt % solution of Cytop CTL-809M in CT-Sols 180. This
solution is injected into the channels of a microfluidic device via
a plastic syringe. The device is then heated to 90.degree. C. for
two hours, followed by 200.degree. C. for an additional two hours.
These surfactants in the fluorinated oil stabilize the aqueous
droplets from spontaneously coalescing. By fluorinating the channel
surfaces, the oil phase preferentially wets the channels and allows
for the stable generation and movement of droplets through the
device. the low surface tension of the channel walls minimizes the
accumulation of channel clogging particulates.
Example 10
[0319] The quality of libraries of emulsified compounds can also be
controlled so as to eliminate from the library those compounds that
cross between droplets. By emulsifying a q-dot encoded, buffer-only
droplet and allowing it to incubate with 1-1000 separately
emulsified compounds quality control of libraries can be achieved.
After incubation, the uniquely encoded q-dot droplet is sorted away
from the compound-containing droplets and analyzed (e.g., by mass
spec) for the presence of any of the compounds emulsified in the
other 1-1000 types of droplets. Compounds that cross between
droplets are identified and eliminated from the library.
Example 11
[0320] The present invention also enables the user to sort cells
based on binding of an affinity-reagent attached to a means for
signal amplification. As a non-limiting example, an antibody fused
to an enzyme (e.g., alk/phos, .beta.-gal, horseradish peroxidase,
etc.) is added to a mix of cells and incubated. The antibody can be
against a particular cell-surface marker, for example, such as a
cancer marker. The cell suspension can be washed or unwashed (if
the antibody is in low concentration, i.e., less than 1 antibody
per droplet, and the antibody has good binding properties).
[0321] The cells that have antibodies attached to them are then
emulsified into droplets and an appropriate enzyme-substrate is
added. The presence of a fluorigenic substrate product is amplified
from one to many copies by the enzyme turning-over the substrate.
Multiple enzymes and multiple substrates can be used to allow
analysis of multiple samples with multiple fluorophores at the same
time or sequentially. The affinity-reagent can be a protein,
nucleic acid, or other molecule to which an enzyme (or portion
thereof that when brought together becomes active) can be attached
either covalently or through a reasonably strong interaction.
Example 12
[0322] The device of the present invention can also be used to
sequence individual exons from individual chromosomes or tumor
cells. A schematic diagram for performing this method is provided
in FIG. 19. Individual specific primer-pairs to different exons
(e.g., epidermal growth factor receptor (EGFR) exon-specific primer
pairs) along with a primer-bound bead (e.g., a Dynal strepavidin
bead) are each emulsified and then pooled to create a library
emulsion (in FIG. 19 a set of 96 exon primer pairs are shown for
illustrative purposes). Separately, a chromosomal DNA solution is
diluted in an aqueous buffer such that upon emulsification on a
microfluidic device described herein, a 30-50 micron droplet
contains, on average, slightly less than a half-genome's
concentration of DNA. Droplets from the pooled emulsion library set
of exon-specific primers are coalesced with droplets containing the
diluted solution of chromosomal DNA on a microfluidic device as
described herein, and used in a bead-based DNA amplification
reaction (i.e., PCR). The microfluidic device as described herein
collects 1.times.10.sup.9 of these droplets in 24 hours, which
results in an emulsion of droplets, some of which contain beads
with amplified exon-DNA attached. After PCR, the emulsions are
broken by centrifugation, the beads are isolated, washed, and then
enriched for DNA-containing beads on a microfluidic device as
described herein. The exon- and chromosome-specific DNA-containing
beads are randomly placed into a picotiter plate (454 Corp.) and
sequenced using a Life Sciences DNA sequencing instrument (as
provided by 454 Corp. and described in any of U.S. application Ser.
No. 09/814,338, filed Mar. 21, 2001; U.S. application Ser. No.
10/104,280, filed Mar. 21, 2002; U.S. application Ser. No.
10/767,899, filed Jan. 28, 2004; U.S. application Ser. No.
11/045,678, filed Jan. 28, 2005; or U.S. application Ser. No.
11/195,254, filed Aug. 1, 2005, each of which are incorporated
herein by reference in their entirety). A summary of this process
is provided by FIG. 20. The emulsion PCR amplification reaction can
be performed off-chip using control chromosomal DNA as template and
a single set of exon-specific primers, or on-chip (i.e., on the
microfluidic devices of the present invention as described
herein).
[0323] In a more specific example, the microfluidic devices and
methods of the present invention have been used to develop
individual exon-, and chromosome-specific sequencing methods with
off-line emulsion PCR using chromosomal DNA as template and a
single set of exon-specific primers.
[0324] The ability to combine (i.e., coalesce) two droplets
together can be used to amplify an exon from an individual
chromosome.
[0325] a. PCR amplification of DNA within a droplet. 454 Life
Sciences has previously demonstrated emulsion solid-phase PCR in
droplets of a size range anticipated for the microfluidic devices
according to the present invention. Successful DNA amplification
using an emulsion of a PCR with the perfluorocarbon oils and
surfactants used to generate and manipulate droplets on
microfluidic devices of the present invention have also been
demonstrated. Several polymerases (notably those from archea, e.g.,
Thermal Ace DNA Pol; Pfu Turbo Pol; Advantage 2-CG Taq Pol; and
Advantage Taq Pol) work well in the buffers and oils used in the
devices described herein.
[0326] b. Setting up the exon-specific PCR reactions. In the first
set of experiments, robust bulk conditions for the droplet-based
exon amplification are developed. Primers within an exon in the
EGFR gene can be used. Approximately 10% of patients with
non-small-cell lung cancer (NSCLC) show responsiveness to targeted
tyrosine kinase inhibitor (TKI) chemotherapy regimens. Response in
patients has been strongly associated with somatic heterozygous
mutations in the ATP cleft of the EGFR gene.
[0327] Wild-type and mutant chromosomal DNAs containing an 18 base
pair (bp) deletion in an EGFR exon encoding this ATP cleft are
used. Initial experiments can be performed in a bulk (i.e.,
off-instrument) solution using the perfluorocarbon oils and
surfactants as described herein. Once conditions have been
established with limiting chromosomal DNA these amplification
experiments in mono-dispersed droplets formulated on-instrument are
repeated. These droplets are collected and the DNA contained within
amplified. The emulsion containing the amplified droplets are
broken, and the aqueous phase analyzed by gel electrophoresis.
[0328] c. Measurement of solid phase amplification. Once the exon
PCR reactions are working in solution from droplets formed on the
instrument, these experiments will be repeated with droplets
containing both primers and beads. The beads are emulsified with
the exon primer pairs. One of the primers is attached to the bead
using standard oligonucleotide coupling chemistry. Both primers
will also be in solution (those skilled in the art will appreciate
that the goal of solid-phase amplification is to generate enough
amplified product in solution such that some of it is driven to the
oligonucleotide primer attached to the bead).
[0329] A serial dilution of chromosomal DNA in several trials is
then added to the primer-bead solution. Droplets are formulated at
a concentration of less than one bead per droplet. The
DNA/primer/bead solution is gently shaken to keep the beads in
suspension as the droplets are being formed on the microfluidic
devices of the present invention. The droplets are collected from
the instrument and the DNA within them is amplified by PCR
off-line.
[0330] d. Measurement of single-chromosome PCR. On-bead
hybridization of two separately-labeled cy3- and cy5-containing
oligonucleotide probes is used to measure amplified-DNA attachment
efficiency. A cy3-labeled probe is synthesized complementary to the
sequence within the 18 by deletion region, and a second cy5-labeled
oligonucleotide probe is synthesized that will span this deletion
(with complementarity to both 5' and 3' sides). The probes are
designed such that at 30.degree. C. they do not cross-hybridize.
The quantitation and ratio of cy3:cy5 dyes on the bead is a measure
of the amount of each specific allele of the DNA present on the
bead.
[0331] As a control, a fluorescein-labeled oligonucleotide
complementary to the oligonucleotide attached to the bead is used.
This control oligonucleotide is used to estimate the maximum amount
of cy3 or cy5-labeled probe that can attach to the bead. The
hybridized beads are washed to remove un-hybridized probe and the
amount of fluorescein still attached to individual beads is
compared to fluorescein-standard concentrations. Other methods and
controls for estimating attached DNA (such as fluorescence
polarization) can be used in conjunction with the microfluidic
devices according to the present invention. FIG. 21. The
chromosomal DNAs are diluted and added to exon-primer containing
droplets on the microfluidic devices of the present invention using
conditions established in the bulk emulsions. After PCR
amplification, the beads are isolated, washed, and hybridized in
solution to the cy3-, cy5-labeled probes. The hybridized beads are
washed to remove un-hybridized and non-specifically-bound labeled
nucleotide and the amount of dye still attached to individual beads
is determined using a fluorescent microscope. The % synthesized is
estimated from the maximum estimated to be able to be synthesized.
The 454 instrument requires 1.times.10.sup.7 copies of DNA per bead
for accurate reads. Using the methods described herein, more than
1.times.10.sup.7 DNA molecules per bead can be attached.
[0332] PCR is a typical temperature-controlled and enzyme catalyzed
biochemical reaction that consists of the periodical repetition of
three different temperatures (melting, annealing and extension
temperature). Alternatively, two temperatures can be applied by
combining the annealing and extension temperatures, thus further
reducing the complexity of the thermal cycling profile and
increasing the speed and efficiency of the PCR reaction. Because of
the temperature-sensitivity of the PCR system a minor temperature
difference may significantly affect the efficiency of DNA
amplification, especially in emulsion PCR microfluidic systems.
[0333] Accordingly, the effects of temperature on enzyme kinetics,
heating and temperature-measuring methods in emulsion PCR
microfluidics are critical in order to gain a better understanding
of PCR kinetics in microfluidics.
[0334] Several thermostable polymerases that work well in oils used
in the microfluidic devices have been identified herein. A syringe
pump attached to a microfluidic device of the present invention
with appropriate sensors and heating elements (described below) can
be used to model the ability of the polymerases to generate PCR
product off-instrument.
[0335] The choice of a heating method for PCR microfluidics is of
importance for achieving faster temperature ramping rates. In one
embodiment, a contact-heating method (e.g., the use of hot air) can
be used. Contact-heating methods utilize electrothermal conversion
to heat the PCR solution, in which the thermal components embedding
the heating element are in direct contact with the components of
the PCR amplification. To date, along with the thin film heaters,
metallic heating blocks and Peltier-effect-based thermo-electric
ceramic heating blocks have been widely applied in temperature
control of PCR.
[0336] In one embodiment, 2 Kaptan Thermofoil heaters from Minco
and a two-step PCR cycling method can be used. Thermofoil heaters
are thin, flexible heating elements consisting of an etched foil
resistive element laminated between layers of flexible insulation.
Thermofoil heaters are applied to the surface of the part to be
heated. Their thin profile gives close thermal coupling between the
heater and heat sink. The flat foil element of thermofoil heaters
transfers heat more efficiently and over a larger surface area than
round wire. Thermofoil heaters, therefore, develop less thermal
gradient between the resistive element and heat sink.
[0337] Methods of temperature measurement for PCR microfluidics. In
emulsion PCR microfluidics, it is critical to select methods for
temperature measurement to accurately control temperature during
PCR cycling. Temperature measurement methods are usually divided
into two categories: contact and non-contact temperature
measurement. The former includes thin-film type temperature sensing
and non-thin-film-type temperature sensing.
[0338] In one embodiment, temperature measurement can be performed
by using the Minco Non-Invasive Sensors Design Kit. This kit comes
with thermal-ribbon, thermal-tab, and bolt-on resistance
temperature detectors that will allow us to accurately sense
temperature without having to drill or tap into the chip. The
detectors are accurate to +/-0.25.degree. C. In another embodiment,
temperature measurements using a temperature dependent fluorescent
dye (e.g., a dilute fluorophore such as rhodamine B or rhodamine
3B) can constitute a second technique for measuring temperature in
microfluidic structures.
[0339] The methods discussed above can be repeated with a second
primer set, which consists of 96 different exons. Primers can be
designed and tested on an MJ Research PCR instrument a priori to
establish suitability to the two-step PCR conditions to be used
on-chip.
[0340] All exons to be amplified are first sequenced by traditional
Sanger methods to establish a base-line read. Where possible, exons
with known polymorphisms within the to-be-sequenced DNA (i.e., the
individual will be polymorphic at a site within the exon) are
selected. We expect 50:50 for each polymorphism, this analysis will
enable us to gather information about the bias in sequencing during
the on-chip amplification reactions. The controls for attachment
are the same as that described above.
[0341] A pool of primer sets are tested on a diluted genomic DNA
solution whereby the genomic DNA is at several concentrations.
[0342] Once the ideal amount and quality of DNA attached to the
beads is achieved, the 454 instrument will be used to sequence the
beads. DNA-containing beads are enriched for on microfluidic
devices according to the present invention either using a DNA
staining agent (ex, Syber green) or by hybridization to a
fluorescent oligonucleotide probe. Appropriate controls are used to
estimate the number of exon copies per bead.
Sequence CWU 1
1
3163DNAArtificial SequenceOligonucleotide 1atccgcccca gcagctgcca
ggcacagccc ctaaactcct gatttatgct gcatccattt 60tgc
63233DNAArtificial SequenceOligonucleotide 2gcaaaatgga tgcagcataa
atcaggagtt tag 33333DNAArtificial SequenceOligonucleotide
3ctaaactcct gatttatgct gcatccattt tgc 33
* * * * *
References