U.S. patent application number 16/733132 was filed with the patent office on 2020-07-16 for enzyme quantification.
The applicant listed for this patent is Bio-Rad Laboratories, Inc.. Invention is credited to Darren R. Link, Michael L. Samuels.
Application Number | 20200225232 16/733132 |
Document ID | / |
Family ID | 47260399 |
Filed Date | 2020-07-16 |
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United States Patent
Application |
20200225232 |
Kind Code |
A1 |
Link; Darren R. ; et
al. |
July 16, 2020 |
ENZYME QUANTIFICATION
Abstract
The invention generally relates to methods for quantifying an
amount of enzyme molecules. Systems and methods of the invention
are provided for measuring an amount of target by forming a
plurality of fluid partitions, a subset of which include the
target, performing an enzyme-catalyzed reaction in the subset, and
detecting the number of partitions in the subset. The amount of
target can be determined based on the detected number.
Inventors: |
Link; Darren R.; (Lexington,
MA) ; Samuels; Michael L.; (Windham, NH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bio-Rad Laboratories, Inc. |
Hercules |
CA |
US |
|
|
Family ID: |
47260399 |
Appl. No.: |
16/733132 |
Filed: |
January 2, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13487030 |
Jun 1, 2012 |
10533998 |
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16733132 |
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12504764 |
Jul 17, 2009 |
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13487030 |
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61081930 |
Jul 18, 2008 |
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61492602 |
Jun 2, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/542 20130101;
B01J 2219/00702 20130101; C12Q 1/25 20130101; B01J 2219/0072
20130101; B01L 2300/0864 20130101; B01L 2300/0867 20130101; C12Q
1/6827 20130101; B01J 2219/00418 20130101; B01L 2400/0487 20130101;
G01N 33/5008 20130101; B01F 5/0646 20130101; B01J 2219/00722
20130101; B01L 2300/0681 20130101; C12Q 1/6804 20130101; B01L
2300/0654 20130101; B01F 13/0062 20130101; C40B 40/04 20130101;
B01J 2219/00585 20130101; B01J 2219/00664 20130101; B01J 2219/00576
20130101; B01L 7/52 20130101; C12Q 1/6818 20130101; B01L 2400/0415
20130101; G01N 33/582 20130101; C40B 60/10 20130101; G01N 21/6445
20130101; B01J 2219/00743 20130101; B01J 2219/00479 20130101; B01J
2219/00351 20130101; B01L 2200/027 20130101; B01L 2300/0636
20130101; G01N 33/573 20130101; B01F 5/0653 20130101; B01J
2219/0059 20130101; B01J 19/0046 20130101; B01J 2219/00592
20130101; B01J 2219/00599 20130101; B01J 2219/00286 20130101; B01L
2300/0645 20130101; C40B 50/08 20130101; G01N 21/6428 20130101;
B01J 2219/00657 20130101; B01L 3/502761 20130101; G01N 2500/00
20130101; B01J 2219/0065 20130101; B01F 3/0807 20130101; B01J
2219/0074 20130101 |
International
Class: |
G01N 33/573 20060101
G01N033/573; B01F 5/06 20060101 B01F005/06; B01J 19/00 20060101
B01J019/00; B01F 13/00 20060101 B01F013/00; B01L 3/00 20060101
B01L003/00; B01F 3/08 20060101 B01F003/08; C40B 40/04 20060101
C40B040/04; C40B 50/08 20060101 C40B050/08; C40B 60/10 20060101
C40B060/10; G01N 33/50 20060101 G01N033/50; C12Q 1/25 20060101
C12Q001/25; C12Q 1/6804 20060101 C12Q001/6804; C12Q 1/6818 20060101
C12Q001/6818; C12Q 1/6827 20060101 C12Q001/6827; G01N 33/542
20060101 G01N033/542; G01N 33/58 20060101 G01N033/58 |
Claims
1-20. (canceled)
21. A method for detecting a condition in a human, the method
comprising: forming fluid partitions comprising components of a
chemical reaction; conducting said chemical reaction; determining a
distribution of at least one product of said chemical reaction;
comparing the distribution to an expected distribution of said
product; and identifying the presence of said condition if said
distribution is statistically-significantly different than said
expected distribution.
22. The method of claim 21, wherein said product is a protein.
23. The method of claim 22, wherein said protein is beta amyloid
protein.
24. The method of claim 23, wherein said distribution is measured
as an aggregate of said beta amyloid protein.
25. The method of claim 21, wherein at least one of the components
of the chemical reaction comprises a detectable label that is acted
on by the chemical reaction.
26. The method of claim 25, further comprising the step of
identifying fluid partitions that contain released detectable
label.
27. The method of claim 25, wherein the components comprise an
enzyme and at least one substrate of the enzyme.
28. The method of claim 27, wherein the enzyme catalyzes a reaction
that results in release of a detectable label from the
substrate.
29. The method of claim 28, wherein the determining step comprises
quantifying an amount of enzyme in the fluid partitions.
30. The method of claim 29, further comprising determining a number
of enzyme molecules within each partition based upon signal
strength of the detectable label.
31. The method of claim 25, wherein the determining step is based
upon a localized concentration of the detectable label.
32. The method of claim 31, wherein the localized concentration is
detected within a fluid partition.
33. The method of claim 21, wherein the fluid partitions are
droplets.
34. The method of claim 33, wherein the droplets are surrounded by
an immiscible carrier fluid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 13/487,030, filed Jun. 1, 2012, which is a
continuation-in-part of U.S. patent application Ser. No.
12/504,764, filed Jul. 17, 2009, which claims priority to, and the
benefit of, U.S. Provisional Application No. 61/081,930, filed Jul.
18, 2008, the contents of each of which are hereby incorporated by
reference in their entirety. In addition, U.S. patent application
Ser. No. 13/487,030 also claims priority to, and the benefit of,
U.S. Provisional Patent Application No. 61/492,602, filed on Jun.
2, 2011, the contents of which are hereby incorporated by reference
in their entirety.
FIELD OF INVENTION
[0002] The present invention generally relates to droplet libraries
and to systems and methods for the formation of libraries of
droplets. The present invention also relates to methods utilizing
these droplet libraries in various biological, chemical, or
diagnostic assays.
BACKGROUND
[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. 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 Nos. WO
01/89788; WO 2006/040551; WO 2006/040554; WO 2004/002627; WO
2008/063227; WO 2004/091763; WO 2005/021151; WO 2006/096571; WO
2007/089541; WO 2007/081385 and WO 2008/063227.
[0004] 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. 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.
[0005] 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] The present invention overcomes the current limitations in
the field by providing precise, well-defined, droplet libraries
which can be utilized alone, or within microfluidic channels and
devices, to perform various biological and chemical assays
efficiently and effectively, especially at high speeds.
SUMMARY
[0007] This invention provides methods to identify and quantify the
presence, type, and amount of reactants and products of chemical
reactions. The invention takes advantage of the ability to form
discrete droplets that contain the components of a chemical
reaction. Because measurements can be performed on individual
droplets and collections of individual droplet, it is possible to
identify and quantify chemical reaction components in the droplets
according to methods described herein. Methods of the invention are
useful to detect and/or quantify any component of a chemical
reaction. In one preferred embodiment, enzyme molecules are
quantified based on their activity inside individual droplets. In
order to identify and quantitate enzyme activity, droplets are
identified as "negative" and/or "positive" droplets for the
reaction catalyzed by the target enzyme, and the number of enzyme
molecules within positive droplets (e.g., based on the quantized
signal strength) is determined. Digital counting of enzyme
molecules provides an extremely wide dynamic range of detection,
with a lower limit of detection dependent on the number of
molecules available to count and the total number of droplets read
(e.g. 1 in 10.sup.7, in one hour using a droplet flow rate of
10.sup.7 per hour) and the upper limit for single molecule counting
determined by the number of droplets but also includes a further
range where multiple or average numbers of molecules are present in
droplets.
[0008] In general, the invention involves incorporating components
of a chemical reaction in a droplet and allowing the chemical
reaction to occur in the droplet. One or more of the components of
the reaction is detectably labeled (e.g., with a reporter molecule)
such that label is detectable as a result of the reaction (e.g.,
release of a reporter). Detection and quantification of the label
allows detection and quantification of the reaction components. The
reporter moiety may be any detectable moiety that can be used as an
indicator of reaction components (e.g., enzyme activity). Any
reporter system known in the art may be used with methods of the
invention. In certain embodiments, the reporter moiety is a
fluorescent moiety.
[0009] In a preferred embodiment, a reporter is attached to one or
more substrate(s) of a chemical reaction in a droplet, which label
is released upon enzymatic catalysis. The number of droplets
containing quantified enzyme molecules are then determined based
upon the presence and/or signal strength of the reporter. Reporter
(and therefore enzyme) can be quantified based upon these
measurements as well. Methods of the invention involve forming a
sample droplet. Any technique known in the art for forming sample
droplets may be used with methods of the invention. An exemplary
method involves flowing a stream of sample fluid so that the sample
stream intersects one or more opposing streams of flowing carrier
fluid. The carrier fluid is immiscible with the sample fluid.
Intersection of the sample fluid with the two opposing streams of
flowing carrier fluid results in partitioning of the sample fluid
into individual sample droplets. The carrier fluid may be any fluid
that is immiscible with the sample fluid. An exemplary carrier
fluid is oil, which may in some cases be fluorinated. In certain
embodiments, the carrier fluid includes a surfactant, such as a
fluorosurfactant.
[0010] In some aspects, the invention provides methods for digital
distribution assays that allow, for example, for detection of a
physiological condition in a human. Detectable physiological
conditions include conditions associated with aggregation of
proteins or other targets. Methods include forming fluid partitions
that include components of a detectable chemical reaction and
conducting the reaction. A distribution of at least one of the
components is determined based on detecting the detectable
reaction. A statistically expected distribution can be computed and
compared to the determined distribution or comparisons of
distributions from known or typical samples. Based on these
comparisons, the presence and/or the severity of the potential
condition can be determined. In certain embodiments, the condition
involves protein aggregation. The protein can be a protein from a
sample from a patient. In some embodiments, methods assay for
Alzheimer's disease, Parkinson's disease, Huntington's disease,
Type II diabeties mellitus, prion-associated diseases, or other
conditions.
[0011] Another droplet formation method includes merging at least
two droplets, in which each droplet includes different material.
Another droplet formation method includes forming a droplet from a
sample, and contacting the droplet with a fluid stream, in which a
portion of the fluid stream integrates with the droplet to form a
droplet. An electric field may be applied to the droplet and the
fluid stream. The electric field assists in rupturing the interface
separating the two fluids. In particular embodiments, the electric
field is a high-frequency electric field.
[0012] Methods of the invention may be conducted in microfluidic
channels. As such, in certain embodiments, methods of the invention
may further involve flowing the droplet channels and under
microfluidic control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] 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:
[0014] FIG. 1 is an schematic illustrating the interacting modules
of a microfluidic device of the present invention.
[0015] FIG. 2 is a schematic illustrating a one emulsion
library.
[0016] FIG. 3 is a schematic illustrating an antibody pair library
for ELISA Application.
[0017] FIG. 4 Panel A is a schematic illustrating that the cell in
the Protein-Fragment Complementation Assay is not secreting any
antigen hence the fluorogenic substrate is not converted into a
fluorescent product. Panel B is a schematic showing the conversion
to a fluorescent product.
[0018] FIG. 5 Panel A is a photograph showing droplets containing
Ammonium Carboxylate Salt of Krytox 157 FSH 2 Wt % in FC 3283
without PEG amine salt. Panel B is a photograph showing droplets
containing PEG 600 Diammonium Carboxylate Salt of Krytox 157 FSH at
4.0% by volume.
[0019] FIG. 6 is a schematic illustrating a primer library
generation.
[0020] FIG. 7 is a schematic illustrating enzyme amplified flow
cytometry.
[0021] FIGS. 8A-8G show droplet formation and detection of reaction
positive droplets.
[0022] FIGS. 9A-9D show readouts of time traces at different enzyme
concentrations. Time traces show digital reactions in droplets at
low enzyme concentrations.
[0023] FIGS. 10A-10D show readouts of histograms. Increasing enzyme
concentrations shifts the distribution from quantized to average
regime.
[0024] FIGS. 11A and 11B illustrate concentration determination
using digital droplet data. Digital counting measurement of enzyme
concentration matches known starting amount.
[0025] FIG. 12 is a schematic showing sandwich formation for
digital droplet ELISA.
[0026] FIGS. 13A-13D illustrates different digital droplet ELISA
readout counting modes.
[0027] FIGS. 14A and 14B show embodiments for multiplexing digital
assay.
[0028] FIGS. 15A and 15B show fluorescent polarization as another
mode for readout.
[0029] FIGS. 16A and 16B illustrate localized florescence as
another mode for readout.
[0030] FIG. 17 is illustrates a digital competitive allele specific
enzyme (CASE) assay.
[0031] FIGS. 18A-18C show multiplexing embodiments.
[0032] FIGS. 19A and 19B show a workflow for a localized
fluorescence binding assay.
[0033] FIG. 20 shows monocyte detection according to certain
embodiments.
[0034] FIGS. 21A-21C illustrate single droplet traces including
optical labels.
[0035] FIGS. 22A-22C give single droplet traces with a scatter plot
and histogram.
[0036] FIGS. 23A-23C diagram adjusting a dynamic range of a
localized fluorescence assay.
[0037] FIG. 24 is a drawing showing a device for droplet
formation.
[0038] FIG. 25 is a drawing showing a device for droplet
formation.
[0039] FIG. 26 is a diagram of results of a digital distribution
assay.
DETAILED DESCRIPTION
[0040] Droplet Libraries
[0041] Droplet libraries are useful to perform large numbers of
assays while consuming only limited amounts of reagents. 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.
[0042] In general, a droplet library is made up of a number of
library elements that are pooled together in a single collection.
Libraries may vary in complexity from a single library element to
10.sup.15 library elements or more. Each library element is one or
more given components at a fixed concentration. The element may be,
but is not limited to, cells, virus, bacteria, yeast, beads, amino
acids, proteins, polypeptides, nucleic acids, polynucleotides or
small molecule chemical compounds. The element may contain an
identifier such as a label. The terms "droplet library" or "droplet
libraries" are also referred to herein as an "emulsion library" or
"emulsion libraries." These terms are used interchangeably
throughout the specification.
[0043] A cell library element can include, but is not limited to,
hybridomas, B-cells, primary cells, cultured cell lines, cancer
cells, stem cells, or any other cell type. Cellular library
elements are prepared by encapsulating a number of cells from one
to tens of thousands in individual droplets. The number of cells
encapsulated is usually given by Poisson statistics from the number
density of cells and volume of the droplet. However, in some cases
the number deviates from Poisson statistics as described in Edd et
al., "Controlled encapsulation of single-cells into monodisperse
picolitre drops" Lab Chip, 8(8):1262-1264, 2008. The discreet
nature of cells allows for libraries to be prepared in mass with a
plurality of cellular variants all present in a single starting
media and then that media is broken up into individual droplet
capsules that contain at most one cell. These individual droplets
capsules are then combined or pooled to form a library consisting
of unique library elements. Cell division subsequent to, or in some
embodiments following, encapsulation produces a clonal library
element.
[0044] A bead based library element contains one or more beads, of
a given type and may also contain other reagents, such as
antibodies, enzymes or other proteins. In the case where all
library elements contain different types of beads, but the same
surrounding media, the library elements can all be prepared from a
single starting fluid or have a variety of starting fluids. In the
case of cellular libraries prepared in mass from a collection of
variants, such as genomically modified, yeast or bacteria cells,
the library elements will be prepared from a variety of starting
fluids.
[0045] Often it is desirable to have exactly one cell per droplet
with only a few droplets containing more than one cell when
starting with a plurality of cells or yeast or bacteria, engineered
to produce variants on a protein. In some cases, variations from
Poisson statistics can be achieved to provide an enhanced loading
of droplets such that there are more droplets with exactly one cell
per droplet and few exceptions of empty droplets or droplets
containing more than one cell.
[0046] Examples of droplet libraries are collections of droplets
that have different contents, ranging from beads, cells, small
molecules, DNA, primers, antibodies. The droplets range in size
from roughly 0.5 micron to 500 micron in diameter, which
corresponds to about 1 pico liter to 1 nano liter. However,
droplets can be as small as 5 microns and as large as 500 microns.
Preferably, the droplets are at less than 100 microns, about 1
micron to about 100 microns in diameter. The most preferred size is
about 20 to 40 microns in diameter (10 to 100 picoliters). The
preferred properties examined of droplet libraries include osmotic
pressure balance, uniform size, and size ranges.
[0047] The droplets comprised within the droplet library provided
by the instant invention are uniform in size. That is, the diameter
of any droplet within the library will vary less than 5%, 4%, 3%,
2%, 1% or 0.5% when compared to the diameter of other droplets
within the same library. The uniform size of the droplets in the
library is critical to maintain the stability and integrity of the
droplets and is also essential for the subsequent use of the
droplets within the library for the various biological and chemical
assays described herein.
[0048] The droplets comprised within the emulsion libraries of the
present invention are contained within an immiscible fluorocarbon
oil comprising at least one fluorosurfactant. In some embodiments,
the fluorosurfactant comprised within immiscible fluorocarbon oil
is a block copolymer consisting of one or more perfluorinated
polyether (PFPE) blocks and one or more polyethylene glycol (PEG)
blocks. In other embodiments, the fluorosurfactant is a triblock
copolymer consisting of a PEG center block covalently bound to two
PFPE blocks by amide linking groups. The presence of the
fluorosurfactant (similar to uniform size of the droplets in the
library) is critical to maintain the stability and integrity of the
droplets and is also essential for the subsequent use of the
droplets within the library for the various biological and chemical
assays described herein. Fluids (e.g., aqueous fluids, immiscible
oils, etc.) and other surfactants that can be utilized in the
droplet libraries of the present invention are described in greater
detail herein.
[0049] The droplet libraries of the present invention are very
stable and are capable of long-term storage. The droplet libraries
are determined to be stable if the droplets comprised within the
libraries maintain their structural integrity, that is the droplets
do not rupture and elements do not diffuse from the droplets. The
droplets libraries are also determined to be stable if the droplets
comprised within the libraries do not coalesce spontaneously
(without additional energy input, such as electrical fields
described in detail herein). Stability can be measured at any
temperature. For example, the droplets are very stable and are
capable of long-term storage at any temperature; for example, e.g.,
-70.degree. C., 0.degree. C., 4.degree. C., 37.degree. C., room
temperature, 75.degree. C. and 95.degree. C. Specifically, the
droplet libraries of the present invention are stable for at least
30 days. More preferably, the droplets are stable for at least 60
days. Most preferably, the droplets are stable for at least 90
days.
[0050] The present invention provides an emulsion library
comprising a plurality of aqueous droplets within an immiscible
fluorocarbon oil comprising at least one fluorosurfactant, wherein
each droplet is uniform in size and comprises the same aqueous
fluid and comprises a different library element. The present
invention also provides a method for forming the emulsion library
comprising providing a single aqueous fluid comprising different
library elements, encapsulating each library element into an
aqueous droplet within an immiscible fluorocarbon oil comprising at
least one fluorosurfactant, wherein each droplet is uniform in size
and comprises the same aqueous fluid and comprises a different
library element, and pooling the aqueous droplets within an
immiscible fluorocarbon oil comprising at least one
fluorosurfactant, thereby forming an emulsion library.
[0051] For example, in one type of emulsion library, all different
types of elements (e.g., cells or beads), are pooled in a single
source contained in the same medium. After the initial pooling, the
cells or beads are then encapsulated in droplets to generate a
library of droplets wherein each droplet with a different type of
bead or cell is a different library element. The dilution of the
initial solution enables the encapsulation process. In some
embodiments, the droplets formed will either contain a single cell
or bead or will not contain anything, i.e., be empty. In other
embodiments, the droplets formed will contain multiple copies of a
library element. The cells or beads being encapsulated are
generally variants on the same type of cell or bead. In one
example, the cells can comprise cancer cells of a tissue biopsy,
and each cell type is encapsulated to be screened for genomic data
or against different drug therapies. Another example is that
10.sup.11 or 10.sup.15 different type of bacteria; each having a
different plasmid spliced therein, are encapsulated. One example is
a bacterial library where each library element grows into a clonal
population that secretes a variant on an enzyme.
[0052] In another example, the emulsion library comprises a
plurality of aqueous droplets within an immiscible fluorocarbon
oil, wherein a single molecule is encapsulated, such that there is
a single molecule contained within a droplet for every 20-60
droplets produced (e.g., 20, 25, 30, 35, 40, 45, 50, 55, 60
droplets, or any integer in between). Single molecules are
encapsulated by diluting the solution containing the molecules to
such a low concentration that the encapsulation of single molecules
is enabled. In one specific example, a LacZ plasmid DNA was
encapsulated at a concentration of 20 fM after two hours of
incubation such that there was about one gene in 40 droplets, where
10 .mu.m droplets were made at 10 kHz per second. Formation of
these libraries rely on limiting dilutions.
[0053] The present invention also provides an emulsion library
comprising at least a first aqueous droplet and at least a second
aqueous droplet within a fluorocarbon oil comprising at least one
fluorosurfactant, wherein the at least first and the at least
second droplets are uniform in size and comprise a different
aqueous fluid and a different library element. The present
invention also provides a method for forming the emulsion library
comprising providing at least a first aqueous fluid comprising at
least a first library of elements, providing at least a second
aqueous fluid comprising at least a second library of elements,
encapsulating each element of said at least first library into at
least a first aqueous droplet within an immiscible fluorocarbon oil
comprising at least one fluorosurfactant, encapsulating each
element of said at least second library into at least a second
aqueous droplet within an immiscible fluorocarbon oil comprising at
least one fluorosurfactant, wherein the at least first and the at
least second droplets are uniform in size and comprise a different
aqueous fluid and a different library element, and pooling the at
least first aqueous droplet and the at least second aqueous droplet
within an immiscible fluorocarbon oil comprising at least one
fluorosurfactant thereby forming an emulsion library.
[0054] For example, in one type of emulsion library, there are
library elements that have different particles, i.e., cells or
beads in a different medium and are encapsulated prior to pooling.
As exemplified in FIG. 2, a specified number of library of
elements, i.e., n number of different cells or beads, are contained
within different mediums. Each of the library elements are
separately emulsified and pooled, at which point each of the n
number of pooled different library elements are combined and pooled
into a single pool. The resultant pool contains a plurality of
water-in-oil emulsion droplets each containing a different type of
particle.
[0055] In some embodiments, the droplets formed will either contain
a single library element or will not contain anything, i.e., be
empty. In other embodiments, the droplets formed will contain
multiple copies of a library element. The contents of the beads
follow a Poisson distribution, where there is a discrete
probability distribution that expresses the probability of a number
of events occurring in a fixed period of time if these events occur
with a known average rate and independently of the time since the
last event. The oils and surfactants used to create the libraries
prevents the exchange of the contents of the library between
droplets.
[0056] Examples of assays that utilize these emulsion libraries are
ELISA assays. The present invention provides another emulsion
library comprising a plurality of aqueous droplets within an
immiscible fluorocarbon oil comprising at least one
fluorosurfactant, wherein each droplet is uniform in size and
comprises at least a first antibody, and a single element linked to
at least a second antibody, wherein said first and second
antibodies are different. In one example, each library element
comprises a different bead, wherein each bead is attached to a
number of antibodies and the bead is encapsulated within a droplet
that contains a different antibody in solution. These antibodies
can then be allowed to form "ELISA sandwiches," which can be washed
and prepared for a ELISA assay. Further, these contents of the
droplets can be altered to be specific for the antibody contained
therein to maximize the results of the assay. A specific example of
an ELSA assay is shown in Example 5 and in FIG. 3.
[0057] The present invention also provides another emulsion library
comprising a plurality of aqueous droplets within an immiscible
fluorocarbon oil comprising at least one fluorosurfactant, wherein
each droplet is uniform in size and comprises at least a first
element linked to at least a first antibody, and at least a second
element linked to at least a second antibody, wherein said first
and second antibodies are different. In one example of a
Protein-Fragment Complementation Assay (PCA), library droplets are
prepared to contain a mixture of two different antibodies. Wherein
the two antibodies bind with strong affinity to different epitopes
of the antigen molecule that is to be detected. Detection is
achieved by tethering protein fragments to the each of the
antibodies such that when held in proximity the two fragments
create an active enzyme capable of turning over a fluorogenic
substrate, only in the presence of the antigen. For example, as
shown in FIG. 4 Panel A, the cell is not secreting any antigen
hence the fluorogenic substrate is not converted into a fluorescent
product. By contrast, in Panel B, the cell is secreting the
antigen. Hence when the antibodies bind to it the two protein
fragments are held in close enough proximity to form an active
enzyme. The intensity of the fluorescence signal generated from
these sandwiches is indicative of the antigen concentration in the
droplet.
[0058] In another example, of the emulsion library, the library
begins with a certain number of library elements, which may contain
proteins, enzymes, small molecules and PCR primers, among other
reagents. However, there is no Poisson distribution in these
droplet libraries, Rather, each library is added to a droplet in a
specific concentration. In these droplet libraries, there are a
large number of the reagent contained within the droplets. With
small molecule chemical compounds, each library element can be the
same small molecule chemical compound at different concentrations
or be completely different small molecule chemical compound per
element. When encapsulating PCR primers there are any number of a
single type of primer pairs contained within each droplet.
[0059] Labels can be used for identification of the library
elements of the various types of droplet libraries. Libraries can
be labeled for unique identification of each library element by any
means known in the art. The label can be an optical label, an
enzymatic label or a radioactive label. The label can be any
detectable label, e.g., a protein, a DNA tag, a dye, a quantum dot
or a radio frequency identification tag, or combinations thereof.
Preferably the label is an optical label. The label can be detected
by any means known in the art. Preferably, the label is detected by
fluorescence polarization, fluorescence intensity, fluorescence
lifetime, fluorescence energy transfer, pH, ionic content,
temperature or combinations thereof. Various labels and means for
detection are described in greater detail herein.
[0060] Specifically, after a label is added to each of the various
library elements, the elements are then encapsulated and each of
the droplets contains a unique label so that the library elements
may be identified. In one example, by using various combinations of
labels and detection methods, it is possible to use two different
colors with different intensities or to use a single color at a
different intensity and different florescence anisotropy.
[0061] Optical labels are also utilized in quality control in order
to ensure that the droplet libraries are well controlled, and that
equal number of each library elements are contained within uniform
volumes in each droplet library. After 120 minutes of mixing, using
8-labels in a 96-member library, the average number of droplets is
13,883 for each of the library elements. As Table 1 shows below,
there is very little variation between the number of droplets for
each library element, i.e., between -0.8% to +1.1%. The slight
variation in the number of droplets allows the pooled droplet
libraries to be used in any number of assays.
TABLE-US-00001 TABLE 1 Element G1 G2 G3 G4 G5 G6 G7 G8 # drops
13913 13898 14036 13898 13834 13927 13788 13769 % variation +0.2%
0% +1.1% 0% -0.4% +0.3% -0.7% -0.8%
[0062] In some quality control examples, 384-member libraries were
prepared with eight optical labels; typically 5 to 20 micro-liters
of each library element are emulsified into approximately 10
picoliter volume droplets so there are about 1 million droplets of
each library element and 384 million droplets in the library.
[0063] The eight optical labels are a dye at concentrations that
increase by a factor of c (where c ranges from about 1.2 to 1.4)
from one optical label to the next so that the nth optical label
has (c)(n-1) the dye concentration of the lowest concentration.
Optical labels are used with concentrations between 10 nM and1 uM.
Typically, the range of optical label concentrations for one series
of labels is 1 order of magnitude (e.g., 10 nM to 100 nM with a
multiplier of 1.43 for each increasing label concentration). A
larger range of droplet label concentrations can also be used.
Further, multiplexed two-color labels can be used as well.
[0064] Plates are prepared with 384 separate library elements in
separate wells of the 384-well plates; 8 of which have optical
labels. The library elements are made into droplets, collected in a
vial, (also known as a creaming tower) and the collection is mixed
on the mixer for several hours. The mixer works by flipping the
vial over about once every 30 seconds and then allowing the
droplets to rise. Multiple plates can be emulsified and pooled or
collected sequentially into the same vial.
[0065] A small fraction of the droplets are taken out of the vial
to verify 1) that the droplets are present in the correct
predetermined ratio and 2) that the droplets are of uniform size.
Typically, 1,000 to 10,000 droplets of each library element (0.384
to 3.84 million QC-droplets) are removed from the vial through a
PEEK line in the center opening in the vial cap by positive
displacement with a drive oil infused through the side opening in
vial cap. The PEEK line takes the droplets into a port on a
microfluidic chip at a rate of several thousand droplets/second;
for 10 picoliter droplets at a rate of 3000 droplets/s corresponds
to a typical infusion rate of roughly 110 micro-liters/hr. Once on
chip the droplets are spaced out by adding oil before they are
imaged and pass one droplet at a time through a laser excitation
spot. Maximum fluorescence intensity data from individual droplets
is collected for all of the QC-droplets and histograms are built to
show the number of droplets within a given fluorescence intensity
range. As expected, if eight of the library elements have optical
labels, then there are eight peaks in the histograms. The
increasing concentration factor c=1.38 results in uniformly
separated peaks across one decade when plotted on a log scale. The
relative number of droplets in each peak is used as a quality
metric to validate that the libraries were prepared with the
expected relative representation. In this example, the percent
variation is determined to be only 2.7% demonstrating that all
library elements have uniform representation.
[0066] Image analysis can be utilized to determine and monitor
osmotic pressure within the droplets. Osmotic pressure (e.g., two
member library prepared with a small difference in buffer
concentration) can effect droplets. Specifically, droplets with a
lower salt concentration shrink over time and droplets with higher
salt concentration grow over time, until uniform salt
concentrations are achieved. Thus it.
[0067] Image analysis can also be utilizes for quality control of
the library reformatting process. After the various library
elements are generated, pooled and mixed, optical labels can be
used to verify uniform representation of all library elements.
Additionally, image analysis is used to verify uniform volume for
all droplets.
[0068] Further, image analysis can be used for shelf life testing
by quantifying the materials performance. Droplets are stored in
vials under a variety of conditions to test droplets stability
against droplet-droplet coalescence events. Conditions tested
include temperature, vibration, presence of air in vials,
surfactant type, and surfactant concentration. A Quality Score of
percent coalescence is calculated by image analysis. Shelf-life for
the droplet libraries of the present invention exceed 90 days.
[0069] Microfluidic Systems
[0070] Reagents can be reformatted as droplet libraries utilizing
automated devices. Specifically, the library elements can be placed
onto plates containing any number of wells, i.e. 96, 384, etc. The
plates can then be placed in an Automated Droplet Library
Production machine (or other such automated device known in the
art), which forms the droplets and puts them into a vial or other
such container, containing the ready to use droplet library. In
general, the process aspirates each of the library elements from
the well plates through tubing connected to a microfluidic device
(described in greater detail herein) which can be used to form the
droplets. The tubing that aspirates the library elements can be
rinsed at a wash station and then the process can be repeated for
the next library element.
[0071] A collection vial can be used to contain the droplets made
using the Automated Droplet Library Production. In one example, the
collection vial has two holes, a first hole in the center of the
vial cap and a second hole part way to the edge of the vial cap.
The vial is first filled with oil through the second hole to purge
air out first hole, the emulsion is then introduced to the vial
through the first hole, typically this is done sequentially one
library element at a time at low volume fraction, and oil is
displaced and goes out through the second hole. The collected
droplets can be stored in the vial for periods of time in excess of
3 months. To remove the emulsion for use or to make smaller
aliquots, oil is introduced through the second opening to displace
the emulsion and drive out the first opening.
[0072] The droplet libraries of the present invention are
preferably formed by utilizing microfluidic devices and are
preferably utilized to perform various biological and chemical
assays on microfluidic devices, as described in detail herein. The
present invention also provides embedded microfluidic nozzles. In
order to create a monodisperse (<1.5% polydispersity) emulsion
directly from a library well, a nozzle can be formed directly into
the fitting used to connect the storage well/reservoir (e.g.
syringe) to a syringe tip (e.g. capillary tubing). Examples of
suitable nozzles to create the droplet libraries of the instant
invention are described in WO 2007/081385 and WO 2008/063227.
[0073] Since the flow is three dimensional, under this design
surface wetting effects are minimized. The nozzle can be made from
one or two oil lines providing constant flow of oil into the
nozzle, a connection to the capillary tubing, and a connection to
the storage well/reservoir (e.g. syringe). The high resolution part
of the nozzle can be made out of a small bore tubing or a small,
simple part molded or stamped from an appropriate material (TEFLON,
plastic, metal, etc). If necessary, the nozzle itself could be
formed into the tip of the ferrule using post mold processing such
as laser ablation or drilling.
[0074] This nozzle design eliminates the surface wetting issues
surrounding the quasi-2D flow associated with typical microfluidic
nozzles made using soft lithography or other standard microfluidic
chip manufacturing techniques. This is because the nozzle design is
fully 3-dimensional, resulting is a complete isolation of the
nozzle section from the continuous aqueous phase. This same design
can also be used for generation of emulsions required for immediate
use, where the aqueous line would be attached directly to a syringe
and the outlet of the nozzle would be used to transport the
emulsion to the point of use (e.g. into a microfluidic PCR chip,
delay line, etc).
[0075] In another embodiment, the present invention provides
compositions and methods to directly emulsify library elements from
standard library storage geometries (e.g. 96 well plates, etc). In
order to create a monodisperse emulsion from fluids contained in a
library well plate, this invention would include microfluidic based
nozzles manufactured simultaneously with an appropriately designed
fluidic interconnect or well.
[0076] Specifically, the microfluidic devices and methods described
herein are based on the creation and electrical manipulation of
aqueous phase droplets (e.g., droplet libraries) completely
encapsulated by an inert immiscible oil stream. This combination
enables precise droplet generation, highly efficient, electrically
addressable, droplet coalescence, and controllable, electrically
addressable single droplet sorting. The microfluidic devices
include one or more channels and modules. A schematic illustrating
one example of interacting modules of a microfluidic substrate is
shown in FIG. 1. The integration of these modules is an essential
enabling technology for a droplet based, high-throughput
microfluidic reactor system and provides an ideal system for
utilizing the droplet libraries provided herein for numerous
biological, chemical, or diagnostic applications.
[0077] Substrates
[0078] The microfluidic device of the present invention includes
one or more analysis units. An "analysis unit" is a microsubstrate,
e.g., a microchip. The terms microsubstrate, substrate, microchip,
and chip are used interchangeably herein. The analysis unit
includes at least one inlet channel, at least one main channel and
at least one inlet module. The analysis unit can further include at
least one coalescence module. at least one detection module and one
or more sorting modules. The sorting module can be in fluid
communication with branch channels which are in fluid communication
with one or more outlet modules (collection module or waste
module). For sorting applications, 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 plurality of analysis
units of the invention may be combined in one device. The
dimensions of the substrate are those of typical microchips,
ranging between about 0.5 cm to about 15 cm per side and about 1
micron to about 1 cm in thickness. The analysis unit and specific
modules are described in further detail in WO 2006/040551; WO
2006/040554; WO 2004/002627; WO 2004/091763; WO 2005/021151; WO
2006/096571; WO 2007/089541; WO 2007/081385 and WO 2008/063227. A
variety of materials and methods can be used to form any of the
described components of the systems and devices of the invention.
For example, various components of the invention can be formed from
solid materials, in which the channels can be formed via molding,
micromachining, film deposition processes such as spin coating and
chemical vapor deposition, laser fabrication, photolithographic
techniques, etching methods including wet chemical or plasma
processes, and the like. See, for example, Angell, et al.,
Scientific American, 248:44-55, 1983. At least a portion of the
fluidic system can be formed of silicone by molding a silicone
chip. Technologies for precise and efficient formation of various
fluidic systems and devices of the invention from silicone are
known. Various components of the systems and devices of the
invention can also be formed of a polymer, for example, an
elastomeric polymer such as polydimethylsiloxane ("PDMS"),
polytetrafluoroethylene ("PTFE") or TEFLON, or the like.
[0079] Silicone polymers are preferred, for example, the silicone
elastomer polydimethylsiloxane. Non-limiting examples of PDMS
polymers include those sold under the trademark Sylgard by Dow
Chemical Co., Midland, Mich., and particularly Sylgard 182, Sylgard
184, and Sylgard 186. Silicone polymers including PDMS have several
beneficial properties simplifying formation of the microfluidic
structures of the invention. For instance, such materials are
inexpensive, readily available, and can be solidified from a
prepolymeric liquid via curing with heat. For example, PDMSs are
typically curable by exposure of the prepolymeric liquid to
temperatures of about, for example, about 65 .degree. C. to about
75 .degree. C. for exposure times of, for example, about an hour.
Also, silicone polymers, such as PDMS, can be elastomeric and thus
may be useful for forming very small features with relatively high
aspect ratios, necessary in certain embodiments of the invention.
Flexible (e.g., elastomeric) molds or masters can be advantageous
in this regard.
[0080] One advantage of forming structures such as microfluidic
structures of the invention from silicone polymers, such as PDMS,
is the ability of such polymers to be oxidized, for example by
exposure to an oxygen-containing plasma such as an air plasma, so
that the oxidized structures contain, at their surface, chemical
groups capable of cross-linking to other oxidized silicone polymer
surfaces or to the oxidized surfaces of a variety of other
polymeric and non-polymeric materials. Thus, components can be
formed and then oxidized and essentially irreversibly sealed to
other silicone polymer surfaces, or to the surfaces of other
substrates reactive with the oxidized silicone polymer surfaces,
without the need for separate adhesives or other sealing means. In
most cases, sealing can be completed simply by contacting an
oxidized silicone surface to another surface without the need to
apply auxiliary pressure to form the seal. That is, the
pre-oxidized silicone surface acts as a contact adhesive against
suitable mating surfaces. Specifically, in addition to being
irreversibly sealable to itself, oxidized silicone such as oxidized
PDMS can also be sealed irreversibly to a range of oxidized
materials other than itself including, for example, glass, silicon,
silicon oxide, quartz, silicon nitride, polyethylene, polystyrene,
glassy carbon, and epoxy polymers, which have been oxidized in a
similar fashion to the PDMS surface (for example, via exposure to
an oxygen-containing plasma). Oxidation and sealing methods useful
in the context of the present invention, as well as overall molding
techniques, are described in the art, for example, in Duffy et al.,
"Rapid Prototyping of Microfluidic Systems and
Polydimethylsiloxane," Anal. Chem., 70:474-480, 1998.
Another advantage to forming microfluidic structures of the
invention (or interior, fluid-contacting surfaces) from oxidized
silicone polymers is that these surfaces can be much more
hydrophilic than the surfaces of typical elastomeric polymers
(where a hydrophilic interior surface is desired). Such hydrophilic
channel surfaces can thus be more easily filled and wetted with
aqueous solutions than can structures comprised of typical,
unoxidized elastomeric polymers or other hydrophobic materials.
Channels
[0081] The microfluidic substrates of the present invention include
channels that form the boundary for a fluid. A "channel," as used
herein, means a feature on or in 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.
[0082] The channels of the invention can be formed, 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).
[0083] 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 100nm, less than about 30 nm, or less than about 10
nm or less in some cases.
[0084] 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, and, if present, a detection module for detection
(identification) or measurement of a droplet and a sorting module
for sorting a droplet based on the detection in the detection
module. The main channel is typically in fluid communication with
the coalescence, detection and/or sorting modules, as well as, an
inlet channel of the 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 molecules, cells, small molecules or particles out of the main
channel. 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.
[0085] The microfluidic substrate can also 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.
[0086] 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.
[0087] A microfluidic substrate can also 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).
[0088] 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. 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. Channels can be
coated by any means known in the art. For example, the channels are
coated with TEFLON, BSA, PEG-silane and/or fluorosilane in an
amount sufficient to prevent attachment and prevent clogging. In
another example, 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 example, 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. 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.
[0089] The surface of the channels in the microfluidic device can
be also fluorinated by any means known in the art 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.
[0090] Fluids
[0091] The fluids described herein are related to the fluids within
the droplet libraries and to the fluids within a microfluidic
device.
[0092] The microfluidic device of the present invention is capable
of controlling the direction and flow of fluids and entities within
the device. 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. Specific flow forces are described in further detail
herein.
[0093] The flow stream in the main channel is typically, but not
necessarily, continuous and may be stopped and started, reversed or
changed in speed. 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. 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.
[0094] 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. 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 known
in the art. The label can be an optical label, an enzymatic label
or a radioactive label. The label can be any detectable label,
e.g., a protein, a DNA tag, a dye, a quantum dot or a radio
frequency identification tag, or combinations thereof. Preferably
the label is an optical label. The label can be detected by any
means known in the art. Preferably, the label is detected by
fluorescence polarization, fluorescence intensity, fluorescence
lifetime, fluorescence energy transfer, pH, ionic content,
temperature or combinations thereof. Various labels and means for
detection are described in greater detail herein.
[0095] 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".
[0096] The fluidic droplets may each be substantially the same
shape and/or size. The droplets may be uniform in size. The shape
and/or size can be determined, for example, by measuring the
average diameter or other characteristic dimension of the droplets.
The "average diameter" of a plurality or series of droplets is the
arithmetic average of the average diameters of each of the
droplets. Those of ordinary skill in the art will be able to
determine the average diameter (or other characteristic dimension)
of a plurality or series of droplets, for example, using laser
light scattering, microscopic examination, or other known
techniques. The diameter of a droplet, in a non-spherical droplet,
is the mathematically-defined average diameter of the droplet,
integrated across the entire surface. The average diameter of a
droplet (and/or of a plurality or series of droplets) may be, for
example, less than about 1 mm, less than about 500 micrometers,
less than about 200 micrometers, less than about 100 micrometers,
less than about 75 micrometers, less than about 50 micrometers,
less than about 25 micrometers, less than about 10 micrometers, or
less than about 5 micrometers in some cases. The average diameter
may also be at least about 1 micrometer, at least about 2
micrometers, at least about 3 micrometers, at least about 5
micrometers, at least about 10 micrometers, at least about 15
micrometers, or at least about 20 micrometers in certain cases.
[0097] As used herein, the term "NanoReactor" and its plural
encompass the terms "droplet", "nanodrop", "nanodroplet",
"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
0.1-1000 .mu.m (e.g., 0.1, 0.2 . . . 5, 10, 15, 20, 25, 30, 35, 40,
45, 50 . . . 1000), or any size within this range. 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.
[0098] The microfluidic substrate of this invention most preferably
generate round, highly uniform, monodisperse droplets (<1.5%
polydispersity). Droplets and methods of forming monodisperse
droplets in microfluidic channels is described in WO 2006/040551;
WO 2006/040554; WO 2004/002627; WO 2004/091763; WO 2005/021151; WO
2006/096571; WO 2007/089541; WO 2007/081385 and WO 2008/063227.
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, decane (e.g., tetradecane or
hexadecane), fluorocarbon oil, silicone oil or another oil (for
example, mineral oil).
[0099] The droplet 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.
[0100] Droplets of a sample fluid can be formed within the inlet
module on the microfluidic device or droplets (or droplet
libraries) can be formed before the sample fluid is introduced to
the microfluidic device ("off chip" droplet formation). To permit
effective interdigitation, coalescence and detection, the droplets
comprising each sample to be analyzed must be monodisperse. As
described in more detail herein, in many applications, different
samples to be analyzed are contained within droplets of different
sizes. Droplet size must be highly controlled to ensure that
droplets containing the correct contents for analysis and coalesced
properly. As such, the present invention provides devices and
methods for forming droplets and droplet libraries.
[0101] Surfactants
[0102] 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. In some applications, performance is improved by
adding a second surfactant to the aqueous phase. 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. The present invention provides compositions and methods
to stabilize aqueous droplets in a fluorinated oil and minimize the
transport of positively charged reagents (particularly, fluorescent
dyes) from the aqueous phase to the oil phase.
[0103] 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.
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.
[0104] Fluorocarbon oil continuous phases are well-suited as the
continuous phase for aqueous droplet libraries for a number of
reasons. Fluorous oils are both hydrophobic and lipophobic.
Therefore, they have low solubility for components of the aqueous
phase and they limit molecular diffusion between droplets. Also,
fluorous oils present an inert interface for chemistry and biology
within droplets. In contrast to hydrocarbon or silicone oils,
fluorous oils do not swell PDMS materials, which is a convenient
material for constructing microfluidic channels. Finally,
fluorocarbon oils have good solubility for gases, which is
necessary for the viability of encapsulated cells.
[0105] Combinations of surfactant(s) and oils must be developed to
facilitate generation, storage, and manipulation of droplets to
maintain the unique chemical/biochemical/biological environment
within each droplet of a diverse library. Therefore, the surfactant
and oil combination must (1) stabilize droplets against
uncontrolled coalescence during the drop forming process and
subsequent collection and storage, (2) minimize transport of any
droplet contents to the oil phase and/or between droplets, and (3)
maintain chemical and biological inertness with contents of each
droplet (e.g., no adsorption or reaction of encapsulated contents
at the oil-water interface, and no adverse effects on biological or
chemical constituents in the droplets). In addition to the
requirements on the droplet library function and stability, the
surfactant-in-oil solution must be coupled with the fluid physics
and materials associated with the platform. Specifically, the oil
solution must not swell, dissolve, or degrade the materials used to
construct the microfluidic chip, and the physical properties of the
oil (e.g., viscosity, boiling point, etc.) must be suited for the
flow and operating conditions of the platform.
[0106] Droplets formed in oil without surfactant are not stable to
permit coalescence, so surfactants must be dissolved in the
fluorous oil that is used as the continuous phase for the emulsion
library. Surfactant molecules are amphiphilic--part of the molecule
is oil soluble, and part of the molecule is water soluble. When a
water-oil interface is formed at the nozzle of a microfluidic chip
for example in the inlet module described herein, surfactant
molecules that are dissolved in the oil phase adsorb to the
interface. The hydrophilic portion of the molecule resides inside
the droplet and the fluorophilic portion of the molecule decorates
the exterior of the droplet. The surface tension of a droplet is
reduced when the interface is populated with surfactant, so the
stability of an emulsion is improved. In addition to stabilizing
the droplets against coalescence, the surfactant should be inert to
the contents of each droplet and the surfactant should not promote
transport of encapsulated components to the oil or other
droplets.
[0107] A very large body of fundamental research and commercial
application development exists for non-fluorous surfactants and
emulsions ranging from sub-micron droplets to very large,
macroscopic emulsions. In contrast, fundamental knowledge and
commercial practice with fluorinated oils and surfactants is much
less common. More specifically, testing and development of
fluorosurfactants and fluorous oil formulations for the application
of creating large libraries of micron-scale droplets with unique
composition is limited to only a few groups throughout the world.
Only a few commercially-available fluorosurfactants that stabilize
water-in-fluorocarbon oil emulsions exist. For instance,
surfactants with short fluorotelomer-tails (typically
perfluorinated C.sub.6 to C.sub.10) are available, but they do not
provide sufficient long-term emulsion stability. Fluorosurfactants
with longer fluorocarbon tails, such as perfluoropolyether (PFPE),
are limited to molecules with ionic headgroups.
[0108] Classes of oils are available from wide variety of
fluorinated oils and are available from commercial sources. The
requirements for the oil are (1) immiscibility with the aqueous
phase, (2) solubility of emulsion stabilizing surfactants in the
oil, and (3) compatibility and/or insolubility of reagents from the
droplet phase. Oils include hydrofluoroethers, which are
fluorinated alkyl chains coupled with hydrocarbon chemistry through
and ether bond. One supplier of this type of oil is 3M. The
products are marketed as Novec Engineered Fluids or HFE-series
oils. This oils include but are not limited to, HFE-7500, which is
a preferred embodiment as it provides superior droplet stability
seems to be higher. Other oils include HFE-7100, -7200, -7600,
which are examples of other HFEs available from 3M. These can be
used as stand-alone oils or components of oil mixtures to optimize
emulsion properties and performance. Other hydrofluoroethers are
also available from other producers, distributors, or resellers may
offer hydrofluoroethers.
[0109] Another class of oil is perfluoroalkylamines, which are
perfluorinated oils based on perfluoroalkyl amine structures. 3M
produces these oils as Fluorinert Electronic Liquids (FC-oils).
Fluorinert products differ by variations in alkyl chain length,
branch structure, and combinations of different structures or pure
oils. Many of them offer the potential for stand-alone oils or
components of oil mixtures to optimize emulsion properties and
performance. Specific examples are Fluorinert FC-3283, Fluorinert
FC-40, which are a preferred embodiments. Higher viscosity and
boiling point useful for applications requiring elevated
temperature (e.g., thermocyling for PCR). Other Fluorinert series
can be used for stand-alone oils or components of oil mixtures to
optimize emulsion properties and performance. Again, other
perfluoroalkylamines are available from other producers,
distributors, or resellers may offer perfluoroalkylamines.
[0110] Fluorinated organics/solvents offer a number of
perfluorinated or partially fluorinated solvents are available from
a variety of producers, distributors, and/or resellers. Many of
them offer the potential for stand-alone oils or components of oil
mixtures to optimize emulsion properties and performance. Examples
of fluorinated organic reagents utilized, included (but not limited
to) trifluoroacetic acid and hexafluoroisopropanol, to improve
droplet stability in other fluorinated oil systems. Addtionally,
fluoropolymers may also be used within a microfluidic system.
Examples of fluoropolymers include, Krytox GPL oils, Solvay Galden
oils, and other liquid fluoropolymers. Other fluorinated materials
find widespread use in a variety of industries, but they are
generally not well-known in the disciplines of interfacial,
colloidal, physical, or synthetic organic chemistry. Therefore, a
number of other candidates for oils exist in specialty and niche
market applications. As such, new oils have been targeted partially
that are per-fluorinated materials, which are not widely
recognized.
[0111] The properties of oils selected are based upon their
chemical properties, such as, among others molecular structure,
fluorine content and solvating strength. Physical properties of
oils examined include viscosity, boiling point, thermal expansion
coefficient, oil-in-water solubility, water-in-oil solubility,
dielectric constant, polarity, and oil-in-water surface
tension.
[0112] Classes of surfactants include fluorosurfactants that can be
categorized by the type of fluorophilic portion of the molecule,
the type of hydrophilic, or polar, portion, and the chemistry used
to link the different parts of the molecule. Materials developed
are capable of stabilizing an emulsion or droplet library. The
preferred embodiment is the EA surfactant. Specifically, the EA
surfactant is a Krytox-PEG-Krytox. The EA surfactant is a nonionic
tri-block copolymer surfactant was developed to avoid issues that
the ionic surfactants (e.g., RR, see below) which result from the
use of some other ionic surfactant. Specifically, ionic surfactants
interact with charged species in the droplets and can sequester
ions (e.g., magnesium required for the PCR reaction) or other
reagents to the oil phase. The structure of the EA surfactant
comprises a PEG--approximately 600 Da with amine end functionality,
PFPE--Mn is .about.5000-8000 from a Krytox FSH starting material
and the linker is an amide coupling. Another surfactant includes
the RR surfactant, which is a Krytox ammonium carboxylate.
[0113] Alternative materials are alternative fluorophilic portion,
i.e., PFPE (Solvay or Demnum), Poly(fluoroalkylacrylate) and other
non-polymeric and partially fluorinated materials. Alternative
head-group chemistry for the hydrophilic portion includes,
non-ionic head groups like PEG (Mw, Mw/Mn (PDI)) and functionality
(i.e., diblock, triblock and dendritic). Others include morpholino.
Ionic head groups for the hydrophilic portion include anionic, such
as elemental and amine salts and further cationic head portions.
Other head group chemistries include zwitterionic, hybrid (e.g.,
PEG-ionic and zonyl FSO/FSN), lipophilic (e.g, lipophilic to
promote bilayer and lipophilic spacer to hydrophile). Another
alternative is alternative coupling chemistry such as,
phosphoryl/Friedel-Crafts, spacer to organic handle and others.
[0114] Characteristics of surfactants are their molecular
structure, determined by NMR, chromatography (e.g., HPLC, GPC/SEC),
FTIR, mass spectrometry, and titrations. Purity of surfactants is
another characteristic examined in addition to the
fluorophile-hydrophile balance.
[0115] A preferred embodiment includes oil-surfactant formulation
for the application of library emulsions is R-oil (HFE-7500) mixed
with 2 wt % EA surfactant ("REA20"). Concentrations of EA or RR
surfactant at 0.1 wt % or lower to 5% or greater. Other
formulations of oils and surfactants and other components added to
the aqueous phase are used to improved and optimized the
performance of the droplets performance. Those properties of the
oil-surfactant mixture include simple mixtures (i.e., one oil and
one surfactant, with varied surface concentration), co-surfactants,
oil mixtures and additives, such as zonyl and TFA.
[0116] Oil and surfactant mixture properties include surfactant
solubility, critical micelle concentration (CMC), surfactant
diffusivity, and interfacial tension, i.e., dynamic and
equilibrium. Emulsion properties are also accounted for, those
properties include size (absolute and size distribution),
stability, transport, and biocompatibility. Stability relates
directly to the coalesced droplets and their deformability/breaking
and shredding ability. More particularly, the stability of the
droplets in their generation, storage and shipping.
[0117] In general, production of surfactant and oils begins with
the synthesis of surfactants and starting materials, such as
PEG-diamine, EA and RR and also accounts for the purification
processes, characterization, quality control, mixing and
packaging.
[0118] 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.
[0119] In another embodiment, a quaternary ammonium salt at the
terminus of a hydrophilic oligomer is linked to a
perfluoropolyether tail as shown in the following formula:
[0120]
PFPE-C(O)NH--CH.sub.2CH.sub.2CH.sub.2--(OCH.sub.2CH.sub.2).sub.3O---
CH.sub.2CH.sub.2CH.sub.2--N(CH.sub.3).sub.3+I--Some specific
molecular features of the present invention include, but are not
limited to, PFPE is from Krytox 157 FSH (Mn-6500), amide bond
linking PFPE to hydrophile, propyl group immediately adjacent to
the amide, propyl group immediately adjacent to the trimethylamino
terminus. Specific structural formations can alter performance
relationships, for example, PFPE chain is sufficiently long for
molecule to be soluble in perfluorinated oils, amide linker
provides hydrolytic stability and hydrogen bonding site, and a
combination of PEG and quaternary ammonium cation provide high
anchoring strength to the aqueous phase as well as electrostatic
repulsion and steric hindrance to minimize reagent transport.
[0121] Variables in the molecular structure include, but are not
limited to, PFPE molecular weight and polydispersity, PFPE
structure, alternative perfluorinated tail chemistries, PEG
molecular weight and polydispersity, shorter hydrocarbon linkers
(ethyl or methyl versus propyl), longer hydrocarbon spacers (C4 or
higher), alternative counterions, such as monovalent anions,
monovalent, polyatomic anions and di- or tri-valent counterions (to
produce two or more tail fluorosurfactants). Further variables in
the molecule structure include alternative linker chemistries
(e.g., ether, ester, etc), alternative hydrophilic oligomers (e.g.,
polyalcohol, polyacrylamide, etc.), alternative quaternary ammonium
cations, and alternative ionic groups (e.g., anionic
terminus--carboxylate etc.; alternative cations).
[0122] The present invention is also directed to the coupling of
PEG-diamines with carboxylic acid-terminated perflouropolyether
(Krytox 157) to form surfactants. Specifically, the present
invention is directed to a fluorosurfactant molecule made by the
ionic coupling of amine-terminated polyethyleneglycol (PEG-amine)
with the carboxylic acid of DuPont Krytox perfluoropolyether
(PFPE). The resulting structure conveys good performance in the
stabilization of aqueous droplets in fluorinated oil in a
microfluidic system. Examples of specific surfactants are shown in
Examples 1 and 2. Preferred surfactants are also described in WO
2008/021123.
[0123] The present invention provides droplets with a
fluorosurfactant interface separating the aqueous droplet and its
contents from the surrounding immiscible fluorocarbon oil. In one
example, DNA amplification reactions occurring inside these
droplets generate material that does not interact with the channel
walls, and collection of the DNA-containing droplets for subsequent
manipulation and sequencing is straightforward. This technology
provides a solution for amplification of DNA from single cells,
allowing for both genotyping and whole genome amplification. In
addition, use within a microfluidic device or platform as described
herein achieves very high throughput, with droplets processed at
rates in excess of 5000 droplets per second, enabling greater than
1.times.10.sup.6 single-cell droplets to be formed and manipulated
per hour.
[0124] Other examples of materials related to this invention
include the formation of salts made by combination of various
primary, secondary, or tertiary amines with PFPE carboxylic acid.
The resulting amphiphilic structure could be useful as a
stand-alone surfactant or a cosurfactant. Similarly, fluorinated
materials with carboxylic acids other than Krytox PFPE could be
used to form ionic fluorosurfactants with various amine containing
compounds.
[0125] Alternative amine-containing compounds for use with the
present invention include, but are not limited to, PEG-monoamine
(molecular weights range from 200 to 1,000,000 or more),
PEG-diamine (molecular weights range from 200 to 1,000,000 or
more), Multifunctional PEG amines (e.g., branched or dendritic
structures), other hydrophilic polymers terminated with amines.
Sugars include, but are not limited to, Sugars, Peptides,
Biomolecules, Ethanolamine or Alkyl amines--primary, secondary, or
tertiary (e.g., triethylamine, trimethylamine, methylamine,
ethylamine, butylamine)
[0126] Alternative fluorinated groups for use with the present
invention include, but are not limited to, Krytox FSL and FSM
(alternative molecular weights), Demnum PFPE materials, Fluolink
PFPE materials or Fluorinated small molecules with carboxylic
acids.
[0127] The data described herein show that the fluorosurfactants
comprised of PEG amine salts provide better performance (compared
to other fluorosurfactants) for stabilization of aqueous droplets
in fluorinated oils in droplet-based microfluidics applications.
These novel surfactants are useful either in combination with other
surfactants or as a stand-alone surfactant system.
[0128] Driving Forces
[0129] 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 as described in Fulwyer, Science 156, 910
(1974); Li and Harrison, Analytical Chemistry 69, 1564 (1997);
Fiedler, et al. Analytical Chemistry 70, 1909-1915 (1998) and 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.
[0130] Positive displacement pressure driven flow is a preferred
way of controlling fluid flow and dielectrophoresis is a preferred
way of manipulating droplets within that flow. 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. Examples of
driving pressures and methods of modulating flow are as described
in WO 2006/040551; WO 2006/040554; WO 2004/002627; WO 2004/091763;
WO 2005/021151; WO 2006/096571; WO 2007/089541; WO 2007/081385 and
WO 2008/063227; U.S. Pat. No. 6,540,895 and U.S. Patent Application
Publication Nos. 20010029983 and 20050226742
[0131] Inlet Module
[0132] The microfluidic device of the present invention includes
one or more inlet modules. An "inlet module" is an area of a
microfluidic substrate device that receives molecules, cells, small
molecules or particles for additional 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 substrate. A substrate may contain more than one inlet module
if desired. Different sample inlet channels can communicate with
the main channel at different inlet modules. Alternately, different
sample inlet channels can communication with the main channel at
the same inlet module. The inlet module is in fluid communication
with the main channel. 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. 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 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).
[0133] Filters
[0134] An important element in making libraries utilizing the
microfluidic device of the present invention is to include features
in the channels of the device to remove particles that may effect
the microfluidic system. When emulsions are injected or re-injected
onto a microfluidic device, they carry contaminants that collect at
the nozzle and either clog the nozzle and/or induce uncontrolled
coalescence up to the complete shredding of the emulsion.
Debris/contaminants include small debris, such as dust or TCS,
fibers, goop (glue and/or surfactant) and large debris such as PDMS
skins/shavings. In one example, the present invention provides a
post trap for large debris, a pocket trap for small debris, a
serpentine trap for fibers and a step trap for large
droplets/debris. EAP filters work well to filter out the
contaminants.
[0135] The filter system filters out these contaminants and most
importantly traps the contaminants out of the main pathway and
allow the droplets to pass by so the contaminants cannot induce
uncontrolled coalescence. The present invention comprises two
distinct parts that specifically address two different scales. The
first filters contaminants that are larger than the droplet size.
The second filters contaminants that are smaller than the droplet
and nozzle sizes. The large contaminants are easily trapped but are
responsible for inducing uncontrolled coalescence, the small
contaminants tend to stick to the nozzle and most probably induce
wetting that results in the shredding of the emulsion.
[0136] To address the issue of large contaminants, a triangular
shape filter is used that contains an internal-collection channel
and smaller lateral channels connected to the internal-collection
channel with a specific angle. On each side of the triangle are
pockets to collect the contaminants that have been deflected by the
triangle and directed there by the flow of the droplets due to the
specific angle of the filter. In addition, the collection pockets
are connected to a channel of high hydrodynamic resistance so that
some of the flow will still go through and maintain the
contaminants in the collection pockets. The lateral collection
channels are located at a stepwise transition between a shallow
layer and a deep layer. In one example, he droplets are collected
in the Droplet Collection Channel through the lateral angled
channels. The contaminants are deflected toward the Contaminant
Collection Pocket because of the triangular shape and the droplet
flow. Because of the use of high resistance channel for the
Contaminants Collection Pockets, the droplets go through them only
marginally, but enough to force the trapped contaminants to stay
there.
[0137] To address the issue of the small contaminants, a series of
posts are used, each one being offset by a half-period to the
adjacent ones. This geometry intends to create a region of
null-recirculation flow at the tip of each post due to the symmetry
and contaminants are trapped in that region. In addition, the posts
have an indentation to both increase the effect of the flow pattern
described above and to trap the contaminants so that they are out
of the way of the droplets. The posts can be designed just with an
indentation or with a flow-through restriction of high hydrodynamic
resistance so that the contaminants will be directed and trapped
deep in the structure. The symmetrical design creates a region
where there is almost no flow, in this region creates the
conditions to trap the contaminants that are smaller than the
droplets. The droplets follow the main flow because of the high
hydrodynamic resistance conditions. The posts on one side of the
channel have a flow-through to ensure that the contaminants stay
trapped there; on the other side the posts have only an
indentation. Several series of these posts, offset by half of a
period are used to increase both the filter capacity and the odds
of trapping any given contaminant.
[0138] Droplet Interdigitation
[0139] 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, described in further
detail herein. 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 aqueous stream, an emulsion
stream, and an oil stream, or (iii) two emulsion streams and an oil
stream. The term "interdigitation" as used herein means pairing of
droplets from separate aqueous streams, or from two separate inlet
nozzles, for eventual coalescence.
[0140] Various nozzle designs enhance 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.
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). Examples of preferred nozzle designs are as described in
WO 2007/081385 and WO 2008/063227.
[0141] Reservoir/Well
[0142] A device of the invention can include 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. Reservoirs and wells used for
loading one or more samples onto the microfluidic device of the
present invention, include but are not limited to, syringes,
cartridges, vials, eppendorf tubes and cell culture materials
(e.g., 96 well plates). A reservoir may facilitate introduction of
molecules or cells into the device and into the sample inlet
channel of each analysis unit.
[0143] Coalescence Module
[0144] The microfluidic device of the present invention also
includes 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 fuse,
coalesce or combine their contents. The coalescence module can also
include an apparatus, for generating an electric force.
[0145] The electric force exerted on the fluidic droplet may be
large enough to cause the droplet to move within the liquid. In
some cases, the electric force exerted on the fluidic droplet may
be used to direct a desired motion of the droplet within the
liquid, for example, to or within a channel or a microfluidic
channel (e.g., as further described herein), etc. The electric
field can be generated from an electric field generator, i.e., a
device or system able to create an electric field that can be
applied to the fluid. The electric field generator may produce an
AC field (i.e., one that varies periodically with respect to time,
for example, sinusoidally, sawtooth, square, etc.), a DC field
(i.e., one that is constant with respect to time), a pulsed field,
etc. The electric field generator may be constructed and arranged
to create an electric field within a fluid contained within a
channel or a microfluidic channel. The electric field generator may
be integral to or separate from the fluidic system containing the
channel or microfluidic channel, according to some embodiments. As
used herein, "integral" means that portions of the components
integral to each other are joined in such a way that the components
cannot be in manually separated from each other without cutting or
breaking at least one of the components. Techniques for producing a
suitable electric field (which may be AC, DC, etc.) are known to
those of ordinary skill in the art. For example, in one embodiment,
an electric field is produced by applying voltage across a pair of
electrodes, which may be positioned on or embedded within the
fluidic system (for example, within a substrate defining the
channel or microfluidic channel), and/or positioned proximate the
fluid such that at least a portion of the electric field interacts
with the fluid. The electrodes can be fashioned from any suitable
electrode material or materials known to those of ordinary skill in
the art, including, but not limited to, silver, gold, copper,
carbon, platinum, copper, tungsten, tin, cadmium, nickel, indium
tin oxide ("ITO"), etc., as well as combinations thereof.
[0146] Preferred electrodes and patterned electrically conductive
layers are described in WO 2007/081385 and WO 2008/063227 and 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 forces to
manipulate and control the droplets and their contents.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] The electric field generator can be constructed and arranged
(e.g., positioned) to create an electric field applicable to the
fluid of at least about 0.01 V/micrometer, and, in some cases, at
least about 0.03 V/micrometer, at least about 0.05 V/micrometer, at
least about 0.08 V/micrometer, at least about 0.1 V/micrometer, at
least about 0.3 V/micrometer, at least about 0.5 V/micrometer, at
least about 0.7 V/micrometer, at least about 1 V/micrometer, at
least about 1.2 V/micrometer, at least about 1.4 V/micrometer, at
least about 1.6 V/micrometer, or at least about 2 V/micrometer. In
some embodiments, even higher electric field intensities may be
used, for example, at least about 2 V/micrometer, at least about 3
V/micrometer, at least about 5 V/micrometer, at least about 7
V/micrometer, or at least about 10 V/micrometer or more. As
described, an electric field may be applied to fluidic droplets to
cause the droplets to experience an electric force. The electric
force exerted on the fluidic droplets may be, in some cases, at
least about 10.sup.-16 N/micrometer.sup.3. In certain cases, the
electric force exerted on the fluidic droplets may be greater,
e.g., at least about 10.sup.-15 N/micrometer.sup.3, at least about
10.sup.-14 N/micrometer.sup.3, at least about 10.sup.-13
N/micrometer.sup.3, at least about 10.sup.-12 N/micrometer.sup.3,
at least about 10.sup.-11N/micrometer.sup.3, at least about
10.sup.-10 N/micrometer.sup.3, at least about 10.sup.-9
N/micrometer.sup.3, at least about 10.sup.-8 N/micrometer.sup.3, or
at least about 10.sup.-7 N/micrometer.sup.3 or more. The electric
force exerted on the fluidic droplets, relative to the surface area
of the fluid, may be at least about 10.sup.-15 N/micrometer.sup.2,
and in some cases, at least about 10.sup.-14 N/micrometer.sup.2, at
least about 10.sup.-13 N/micrometer.sup.2, at least about
10.sup.-12 N/micrometer.sup.2, at least about 10.sup.-11
N/micrometer.sup.2, at least about 10.sup.-10 N/micrometer.sup.2,
at least about 10.sup.-9 N/micrometer.sup.2, at least about
10.sup.-8 N/micrometer.sup.2, at least about 10.sup.-7
N/micrometer.sup.2, or at least about 10.sup.-6 N/micrometer.sup.2
or more. In yet other embodiments, the electric force exerted on
the fluidic droplets may be at least about 10.sup.-9 N, at least
about 10.sup.-8 N, at least about 10.sup.-7 N, at least about
10.sup.-6 N, at least about 10.sup.-5 N, or at least about 10.sup.4
N or more in some cases.
[0152] Channel Expansion Geometries
[0153] In preferred embodiments described herein, droplet
coalescence is presently carried out by having two droplet forming
nozzles emitting droplets into the same main channel. The size of
the nozzles allow for one nozzle to form a large drop that fills
the exhaust line while the other nozzle forms a drop that is
smaller than the first. The smaller droplet is formed at a rate
that is less than the larger droplet rate, which insures that at
most one small droplet is between big droplets. Normally, the small
droplet will catch up to the larger one over a relatively short
distance, but sometimes the recirculation zone behind the large
drop causes the small drop to separate from the large drop
cyclically. In addition, the small drop occasionally does not catch
up with the large one over the distance between the nozzles and the
coalescing electrodes. Thus, in some situations is a need for a
more robust coalescence scheme.
[0154] Geometric alterations in the coalescence module can create a
more robust, reliable coalescence or fusing of droplets over a
wider range of sizes and flows. The solution to improve the
performance is to place an expansion in the main channel between
the electrodes. Optionally, a small constriction (neckdown) just
before this expansion can be used to better align the droplets on
their way into the coalescence point. This optional neckdown can
help center the small droplet in the channel stream lines, reducing
the chance that it will flow around the larger droplet prior to
coalescing in the expansion. The electrode pair may be placed on
either one side of the channel or on both sides.
[0155] The expansion in the coalescing region allows for a dramatic
catching up of the small drop to the large drop, as shown through
micrographs taken on an operating device. The volume of the
expansion is big enough to slow the large droplet down so that the
small drop always catches up to the large drop, but doesn't allow
the next large drop to catch up and make contact with the pair to
be coalesced. The electrodes allow for coalescence to take place
when the drops are in contact with each other and passing through
the field gradient.
[0156] Detection Module
[0157] The microfluidic device of the present invention can also
include one or more detection modules. 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. However, other detection
techniques can also be employed
[0158] The terms "detecting" or "determining," as used herein,
generally refers to the analysis or measurement of a species, for
example, quantitatively or qualitatively, and/or the detection of
the presence or absence of the species. "Detecting or "determining"
may also refer to the analysis or measurement of an interaction
between two or more species, for example, quantitatively or
qualitatively, or by detecting the presence or absence of the
interaction. Examples of suitable techniques include, but are not
limited to, spectroscopy such as infrared, absorption,
fluorescence, UV/visible, FTIR ("Fourier Transform Infrared
Spectroscopy"), or Raman; gravimetric techniques; ellipsometry;
piezoelectric measurements; immunoassays; electrochemical
measurements; optical measurements such as optical density
measurements; circular dichromism; light scattering measurements
such as quasielectric light scattering; polarimetry; refractometry;
or turbidity measurements as described further herein.
[0159] 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. 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. Precise
boundaries for the detection module are not required, but are
preferred.
[0160] Sensors
[0161] One or more detections sensors and/or processors may be
positioned to be in sensing communication with the fluidic droplet.
"Sensing communication," as used herein, means that the sensor may
be positioned anywhere such that the fluidic droplet within the
fluidic system (e.g., within a channel), and/or a portion of the
fluidic system containing the fluidic droplet may be sensed and/or
determined in some fashion. For example, the sensor may be in
sensing communication with the fluidic droplet and/or the portion
of the fluidic system containing the fluidic droplet fluidly,
optically or visually, thermally, pneumatically, electronically, or
the like. The sensor can be positioned proximate the fluidic
system, for example, embedded within or integrally connected to a
wall of a channel, or positioned separately from the fluidic system
but with physical, electrical, and/or optical communication with
the fluidic system so as to be able to sense and/or determine the
fluidic droplet and/or a portion of the fluidic system containing
the fluidic droplet (e.g., a channel or a microchannel, a liquid
containing the fluidic droplet, etc.). For example, a sensor may be
free of any physical connection with a channel containing a
droplet, but may be positioned so as to detect electromagnetic
radiation arising from the droplet or the fluidic system, such as
infrared, ultraviolet, or visible light. The electromagnetic
radiation may be produced by the droplet, and/or may arise from
other portions of the fluidic system (or externally of the fluidic
system) and interact with the fluidic droplet and/or the portion of
the fluidic system containing the fluidic droplet in such as a
manner as to indicate one or more characteristics of the fluidic
droplet, for example, through absorption, reflection, diffraction,
refraction, fluorescence, phosphorescence, changes in polarity,
phase changes, changes with respect to time, etc. As an example, a
laser may be directed towards the fluidic droplet and/or the liquid
surrounding the fluidic droplet, and the fluorescence of the
fluidic droplet and/or the surrounding liquid may be determined.
"Sensing communication," as used herein may also be direct or
indirect. As an example, light from the fluidic droplet may be
directed to a sensor, or directed first through a fiber optic
system, a waveguide, etc., before being directed to a sensor.
[0162] Non-limiting examples of detection sensors useful in the
invention include optical or electromagnetically-based systems. For
example, the sensor may be a fluorescence sensor (e.g., stimulated
by a laser), a microscopy system (which may include a camera or
other recording device), or the like. As another example, the
sensor may be an electronic sensor, e.g., a sensor able to
determine an electric field or other electrical characteristic. For
example, the sensor may detect capacitance, inductance, etc., of a
fluidic droplet and/or the portion of the fluidic system containing
the fluidic droplet. In some cases, the sensor may be connected to
a processor, which in turn, cause an operation to be performed on
the fluidic droplet, for example, by sorting the droplet.
[0163] Characteristics
[0164] Characteristics determinable with respect to the droplet and
usable in the invention can be identified by those of ordinary
skill in the art. Non-limiting examples of such characteristics
include fluorescence, spectroscopy (e.g., optical, infrared,
ultraviolet, etc.), radioactivity, mass, volume, density,
temperature, viscosity, pH, concentration of a substance, such as a
biological substance (e.g., a protein, a nucleic acid, etc.), or
the like.
[0165] A corresponding signal is then 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 means of changing the flow
path can be accomplished through mechanical, electrical, optical,
or some other technique as described herein.
[0166] 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.
[0167] Fluorescence Polarization/Fluorescence Lifetime
[0168] As described herein, the biological/chemical entity to be
analyzed 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.
[0169] 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. CdSe--ZnS QDs also
have high quantum yields, are resistant to photodegradation, and
can be detected optically at concentrations comparable to organic
dyes.
[0170] 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. Three different q-dots in several
concentrations each can be placed in a microdroplet, and can then
be used with a microfluidic device to decode what is in the drop.
The Q-dot readout extension to the fluorescence station can be
incorporated into the design of the microfluidic device. A series
of dichroic beamsplitters, emission filters, and detectors can be
stacked onto the system, allowing measurement of the required five
emission channels (two fluorescence polarization signals and three
q-dot bands).
[0171] Fluorescence Polarization (FP) detection technology enables
homogeneous assays suitable for high throughput screening assays in
the Drug Discovery field. The most common label in the assays is
fluorescein. In FP-assay the fluorophore is excited with polarized
light. Only fluorophores parallel to the light absorb and are
excited. The excited state has a lifetime before the light emission
occurs. During this time the labeled fluorophore molecule rotates
and the polarization of the light emitted differs from the
excitation plane. To evaluate the polarization two measurements are
needed: the first using a polarized emission filter parallel to the
excitation filter (S-plane) and the second with a polarized
emission filter perpendicular to the excitation filter (P-plane).
The Fluorescence Polarization response is given as mP
(milli-Polarization level) and is obtained from the equation:
Polarization (mP)=1000*(S-G*P)/(S+G*P)
[0172] Where S and P are background subtracted fluorescence count
rates and G (grating) is an instrument and assay dependent
factor.
[0173] The rotational speed of a molecule is dependent on the size
of the molecule, temperature and viscosity of the solution.
Fluorescein has a fluorescence lifetime suitable for the rotation
speeds of molecules in bio-affinity assays like receptor-ligand
binding assays or immunoassays of haptens. The basic principle is
that the labeled compound is small and rotates rapidly (low
polarization). When the labeled compound binds to the larger
molecule, its rotation slows down considerably (polarization
changes from low to high polarization). Thus, FP provides a direct
readout of the extent of tracer binding to protein, nucleic acids,
and other biopolymers.
[0174] 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. 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.
[0175] 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.
[0176] Accordingly, the present invention provides methods to label
droplets and/or nanoreactors formed on a microfluidic device by
using only a single dye code to avoid cross-talk with other dyes
during FP. Additionally, the present invention provides methods to
create FP dye codes to label compounds contained within liquids
(including droplets and/or nanoreactors) where the compound is
designed to be differentiated by FP on a microfluidic device. In
this manner, dye codes having the same color, absorption, and
emission could be used to label compounds within liquids.
[0177] In one aspect, the present invention is directed to the use
of fluorescence polarization to label liquids. Droplets can be
labeled using several means. These labeling means include, but are
not limited to, the use of different dyes, quantum dots,
capacitance, opacity, light scattering, fluorescence intensity
(FI), fluorescence lifetime (FL), fluorescence polarization (FP),
circular dichroism (CD), fluorescence correlation and combinations
of all of these previous labeling means. The following disclosure
describes the use of FP and FI as a means to label droplets on a
microfluidic device. In addition, the use of FL as a means to
adjust the overall FP of a solution, and by varying the
concentration of the total FI, to create a 2-dimensional encoding
scheme is demonstrated.
[0178] In general, molecules that take up more volume will tumble
slower than a smaller molecule coupled to the same fluorophore. FP
is independent of the concentration of the dye; liquids can have
vastly different concentrations of FITC in them yet still have
identical FP measurements.
[0179] In a preferred embodiment, a FP dye is an organic dye that
does not interfere with the assay dye is used. Furthermore, since
the total intensity of the FP dye can be quantified, a second
dimension in which to label the droplet is provided. Thus, one can
exploit the differences in FP to create an encoding scheme of dye
within a liquid solution, including droplets. Examples of ways to
exploit the differences in FP are described in WO 2007/081385 and
WO 2008/063227. In a single dimension, FP can be used to create an
encoding scheme. However, the present invention can also use
Fluorescence Intensity (FI) of the overall solution to create even
more labels in a second dimension. Interestingly, the differences
of the fluorescence lifetime (FL) of two dyes with spectral overlap
in the detected emission wavelength to change the overall FP of the
combined solution can also be exploited.
[0180] Although the use of multiple compounds to which a dye
molecule is attached to span a range of FP can be utilized, it is
also possible to span the range using a high and low molecular
weight compound set. For example, a dye can be attached to a large
compound (for example streptavidin) and kept at a fixed
concentration, to which a smaller compound (for example, a free dye
molecule) would be titrated into the same solution. The FP of the
solution can be adjusted to be in discernable increments from the
value of the large molecule to somewhere slightly greater than the
FP of the smaller molecule. The [total] dye intensity can be varied
by varying the concentration of the mixture of the two dye-attached
compounds. By varying total dye concentration and the FP, two
dimensions can be used to generate the FP dye codes (FPcodes).
Accordingly, many FPcodes can be generated using only two
compounds.
[0181] This could also include use of large fluorescent proteins
such as GFP and the phycobiliproteins combined with a smaller
molecule.
[0182] Examples of dyes commonly used in biological dyes are listed
in Table 2 below.
TABLE-US-00002 TABLE 2 Excitation Emission Examples of Wavelength
Wavelength Compatible Dyes 450 500 Cyan 500 483 533 SYBR Green, FAM
523 568 HEX, VIC 558 610 RED 610 615 640 RED 640 650 670 CY5
[0183] In another aspect, the present invention is directed
labeling solids using properties other than dye emission and dye
concentration. In one embodiment the solid can include, for
example, a bead or location on a solid support or chip. As
demonstrated above for liquids, FI and FL can be two of many
dimensions of characteristics used as labels. By way of
non-limiting example, it is possible to use two dyes with different
FL to change the overall FP for a solid such as a bead or other
mobile solid support.
[0184] In another embodiment, a linker can be used to couple the
dye to the bead. The linker can be varied so as to allow the dye to
have differing degrees of freedom in which to rotate (i.e.,
tumble). Varying the linker in this manner can change the FP of the
attached dye, which in unique combinations can be used as a label.
In some embodiments, the beads can be swollen in organic solvent
and the dyes held in place by hydrophobic forces. In this case, the
FP, FI, FL methods described above for liquid labeling can also be
used as a means for labeling the beads. A quenching molecule can
also be used to change the characteristics of a dye. Such quenching
can be continuous or brought about through the interaction of a
molecule, such as a peptide or nucleic acid linker, with differing
means of bringing molecules together depending on the strength of
linker-internal interaction (e.g., a nucleotide stem loop structure
of varying lengths).
[0185] The reactions analyzed on the virtual, random and non-random
arrays (discussed briefly below) can be also increased beyond the
two (cy3 and cy5 intensities) commonly used for multiplexing. For
example, different FP, FI, etc can be used as a read-out.
[0186] Random array decoding: Beads of the prior art use one or
more pre-attached oligonucleotide-coupled beads that are held in
place in a fiber-optic faceplate (for example, those used by
Illiumina). The oligos on the beads are decoded using sequential
hybridization of a labeled complementary oligo. The assay of the
prior art uses a separate oligonucleotide complementary zipcode
(`Illumacode`) attached to each type of bead.
[0187] The invention described herein is superior to the methods of
the prior art in that the FP, FI, FL-labeled bead or mobile solid
support can be placed into a random array (e.g., a chip as
manufactured by Illumina) and the FP, FI, FL used to decode the
bead. The FP, FI, FL of the bead can be decoded before using the
chip and the different beads `mapped` as to their specific
locations. Alternatively, the bead can be decoded during attachment
of the assay read-out. Significantly, the methods described by the
present invention can be used to pre-determine the location of each
bead-type either before, or during analysis.
[0188] Virtual array decoding: Methods of the prior art use 2
lasers and 3 detectors to differentiate a set of 100 bead-types.
The beads-types are differentiated by the FI of two different dyes
present in 1 of 10 concentrations (per dye) contained within the
bead, and the assay detector is used to measure fluorescein
concentration on the bead. The dyes, which are added to
organic-solvent swollen beads, are not directly attached to the
beads, but remain held within the bead by hydrophobic forces.
[0189] Using the methods of the present invention as described
herein, a second detector to the machines of the prior art used to
measure FP can be added, thereby adding a third dimension and
extending the encoding scheme beyond the 100 available in the prior
art.
[0190] Non-random array decoding: In chips of the prior art (such
as those used by Affymetrix) oligonucleotides are synthesized
directly on the chip. Decoding is simply a matter of knowing the
location of the assay on the chip.
[0191] The methods as described herein can be advantageously used
in conjunction with such chips to increase the number of things
that can be simultaneously analyzed (i.e., multiplexed) on the
chip. By way of non-limiting example, Cy3, Cy5, FL and FP can be
used as analysis markers for hybridization reactions.
[0192] The present invention also provides methods for labeling
micro or nano-sized droplets using Radio Frequency Identification
(RFID). RFID tags can improve the identification of the contents
within the droplets. Preferably, the droplets are utilized within a
microfluidic device.
[0193] RFID is an automatic identification method, relying on
storing and remotely retrieving data using devices called RFID tags
or transponders. An RFID tag is an object that can be attached to
or incorporated into a product, animal, or person for the purpose
of identification using radio waves. Chip-based RFID tags contain
silicon chips and antennae. Passive tags require no internal power
source, whereas active tags require a power source. Hitachi has
"powder" 0.05 mm.times.0.05 mm RFID chips. The new chips are 64
times smaller than the previous record holder, the 0.4 mm.times.0.4
mm mu-chips, and nine times smaller than Hitachi's last year
prototype, and have room for a 128-bit ROM that can store a unique
38-digit ID number.
[0194] In one embodiment, a solution containing RFID tags are
emulsified into droplets and are used as a label for the
identification of the material within the droplet solution.
Applications include, but are not limited to; genetics, genomics,
proteomics, chemical synthesis, biofuels, and others.
[0195] In some embodiments, fluorescent polarization is used for
digital assays. In this method, positive (enzyme-containing)
droplets are identified and counted using changes in the
fluorescence polarization of fluorescent molecules in the droplets.
Fluorescence polarization (FP) detection technology can use a label
such as fluorescein. In an FP-assay the fluorophore is excited with
polarized light. Only fluorophores parallel to the light absorb and
are excited. The excited state has a lifetime before the light
emission occurs. During this time the labeled fluorophore molecule
rotates and the polarization of the light emitted differs from the
excitation plane. In some embodiments, to evaluate the polarization
two measurements are used: the first using a polarized emission
filter parallel to the excitation filter (S-plane) and the second
with a polarized emission filter perpendicular to the excitation
filter (P-plane). Fluorescence Lifetime (FL) changes can be
detected based on the chemical environment of the fluorophore such
that bound and unbound antibodies can be distinguished by FL
measurements well known to one skilled in the art.
[0196] The rotational speed of a molecule is dependent on the size
of the molecule, temperature and viscosity of the solution. The
principle here is that the labeled compound is small and rotates
rapidly (low polarization). When the labeled compound binds to the
larger molecule, its rotation slows down considerably (polarization
changes from low to high polarization). In general, molecules that
take up more volume will tumble slower than a smaller molecule
coupled to the same fluorophore. FP is independent of the
concentration of the dye; liquids can have vastly different
concentrations of FITC in them yet still have identical FP
measurements. Thus, FP provides a direct readout of the extent of
binding to protein, nucleic acids, and other biopolymers or
targets.
[0197] FP offers advantages in the context of multiplexing
(discussed in more detail elsewhere herein). 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.
[0198] The dyes chosen for FP include any suitable dye such as, for
example, fluorescein, Cy3, Cy5, etc. Suitable dyes include (name
followed by [excitation wavelength, emission wavelength]): Cyan 500
[450, 500]; SYBR Green, FAM [483, 533], HEX, VIC [523, 568]; RED
610 [558, 610]; RED 640 [615, 640]; and CY5 [650, 670].
[0199] In some embodiments, an FP dye is an organic dye that does
not interfere with other labels or dyes in an assay. Furthermore,
since the total intensity of the FP dye can be quantified, a second
dimension in which to label the partition is provided. Thus, one
can exploit the differences in FP to create an encoding scheme of
dye within a liquid solution, including droplets. Examples of ways
to exploit the differences in FP are described in WO 2007/081385
and WO 2008/063227. Fluorescence polarization is discussed in U.S.
Pub. 2010/0022414, the contents of which are hereby incorporated by
reference in their entirety.
[0200] In the example shown in FIG. 15A, the enzyme Src kinase
(Src) phosphorylates a Src substrate peptide and creates a binding
surface for a fluorescent reporter. A reporter construct containing
the Src homology 2 domain (SH2) and a fluorescein isothiocyanate
(FITC) serves as a fluorescent reporter (SH2-FITC). When SH2-FITC
is added, it will bind to the phosphorylated Src substrate peptide.
FIG. 15B shows the same sequence of steps but in which Src
substrate peptide is bound to a bead.
[0201] When the fluorescent reporter SH2-FITC is free in solution
it has a low fluorescence polarization, whereas when bound to a
phosphorylated peptide (e.g., the last step shown in each of FIGS.
15A and 15B) it has a measurably higher fluorescence polarization.
Detection of fluorescent polarization indicates a reaction-positive
fluid partition. Many other enzymes, binding motifs, and
fluorescent reporters can be used.
[0202] Lasers
[0203] 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 analysis or microchip such that the laser light
from the diodes shines on the detection module(s).
[0204] An integrated semiconductor laser and/or an integrated
photodiode detector can be included on the substrate 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.
[0205] 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).
[0206] 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 (FACS) 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.
[0207] Processors
[0208] As used herein, a "processor" or a "microprocessor" is any
component or device able to receive a signal from one or more
sensors, store the signal, and/or direct one or more responses
(e.g., as described above), for example, by using a mathematical
formula or an electronic or computational circuit. The signal may
be any suitable signal indicative of the environmental factor
determined by the sensor, for example a pneumatic signal, an
electronic signal, an optical signal, a mechanical signal, etc.
[0209] 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.
[0210] Beads
[0211] 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
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.
[0212] The present invention provides methods of screening
molecular libraries on beads through limited-dilution-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.
[0213] 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.
[0214] Sorting Module
[0215] The microfluidic 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. The main channel can communicate
with two or more branch channels at the sorting module or "branch
point", forming, for example, a T-shape or a Y-shape. Other shapes
and channel geometries may be used as desired. 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.
[0216] The device of the present invention can further include one
or more outlet modules. An "outlet module" is an area of the 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.
A characteristic of a fluidic droplet may be sensed and/or
determined in some fashion, for example, as described herein (e.g.,
fluorescence of the fluidic droplet may be determined), and, in
response, an electric field may be applied or removed from the
fluidic droplet to direct the fluidic droplet to a particular
region (e.g. a channel). A fluidic droplet is preferably sorted or
steered by inducing a dipole in the uncharged fluidic droplet
(which may be initially charged or uncharged), and sorting or
steering the droplet using an applied electric field. The electric
field may be an AC field, a DC field, etc. Methods of sorting or
steering droplets in an electric field are as described in WO
2006/040551; WO 2006/040554; WO 2004/002627; WO 2004/091763; WO
2005/021151; WO 2006/096571; WO 2007/089541; WO 2007/081385 and WO
2008/063227. Improvements in the efficiency, accuracy, and
reliability of the preferred dielectric droplet sorting technique
described above are possibly by utilizing additional channel and
electrode geometries, as described in WO 2007/081385 and WO
2008/063227.
[0217] Alternately, a fluidic droplet may be directed by creating
an electric charge (e.g., as previously described) on the droplet,
and steering the droplet using an applied electric field, which may
be an AC field, a DC field, etc. As an example, an electric field
maybe selectively applied and removed (or a different electric
field may be applied) as needed to direct the fluidic droplet to a
particular region. The electric field may be selectively applied
and removed as needed, in some embodiments, without substantially
altering the flow of the liquid containing the fluidic droplet. For
example, a liquid may flow on a substantially steady-state basis
(i.e., the average flowrate of the liquid containing the fluidic
droplet deviates by less than 20% or less than 15% of the
steady-state flow or the expected value of the flow of liquid with
respect to time, and in some cases, the average flowrate may
deviate less than 10% or less than 5%) or other predetermined basis
through a fluidic system of the invention (e.g., through a channel
or a microchannel), and fluidic droplets contained within the
liquid may be directed to various regions, e.g., using an electric
field, without substantially altering the flow of the liquid
through the fluidic system.
[0218] In some embodiments, the fluidic droplets may be sorted into
more than two channels. Alternately, a fluidic droplet may be
sorted and/or split into two or more separate droplets, for
example, depending on the particular application. Any of the
above-described techniques may be used to spilt and/or sort
droplets. As a non-limiting example, by applying (or removing) a
first electric field to a device (or a portion thereof), a fluidic
droplet may be directed to a first region or channel; by applying
(or removing) a second electric field to the device (or a portion
thereof), the droplet may be directed to a second region or
channel; by applying a third electric field to the device (or a
portion thereof), the droplet may be directed to a third region or
channel; etc., where the electric fields may differ in some way,
for example, in intensity, direction, frequency, duration, etc. In
a series of droplets, each droplet may be independently sorted
and/or split; for example, some droplets may be directed to one
location or another, while other droplets may be split into
multiple droplets directed to two or more locations.
[0219] In some cases, high sorting speeds may be achievable using
certain systems and methods of the invention. For instance, at
least about 1 droplet per second may be determined and/or sorted in
some cases, and in other cases, at least about 10 droplets per
second, at least about 20 droplets per second, at least about 30
droplets per second, at least about 100 droplets per second, at
least about 200 droplets per second, at least about 300 droplets
per second, at least about 500 droplets per second, at least about
750 droplets per second, at least about 1000 droplets per second,
at least about 1500 droplets per second, at least about 2000
droplets per second, at least about 3000 droplets per second, at
least about 5000 droplets per second, at least about 7500 droplets
per second, at least about 10,000 droplets per second, at least
about 15,000 droplets per second, at least about 20,000 droplets
per second, at least about 30,000 droplets per second, at least
about 50,000 droplets per second, at least about 75,000 droplets
per second, at least about 100,000 droplets per second, at least
about 150,000 droplets per second, at least about 200,000 droplets
per second, at least about 300,000 droplets per second, at least
about 500,000 droplets per second, at least about 750,000 droplets
per second, at least about 1,000,000 droplets per second may be
determined and/or sorted in such a fashion.
[0220] Sample Recovery
[0221] The present invention proposes methods for recovering
aqueous phase components from aqueous emulsions that have been
collected on a microfluidic device in a minimum number of steps and
in a gentle manner so as to minimize potential damage to cell
viability.
[0222] In one aspect, a stable aqueous sample droplet emulsion
containing aqueous phase components in a continuous phase carrier
fluid is allowed to cream to the top of the continuous phase
carrier oil. By way of nonlimiting example, the continuous phase
carrier fluid can include a perfluorocarbon oil that can have one
or more stabilizing surfactants. The aqueous emulsion rises to the
top or separates from the continuous phase carrier fluid by virtue
of the density of the continuous phase fluid being greater than
that of the aqueous phase emulsion. For example, the
perfluorocarbon oil used in one embodiment of the device is 1.8,
compared to the density of the aqueous emulsion, which is 1.0.
[0223] The creamed emulsion is then placed onto a second continuous
phase carrier fluid which contains a de-stabilizing surfactant,
such as a perfluorinated alcohol (e.g.,
1H,1H,2H,2H-Perfluoro-1-octanol). The second continuous phase
carrier fluid can also be a perfluorocarbon oil. Upon mixing, the
aqueous emulsion begins to coalesce, and coalescence is completed
by brief centrifugation at low speed (e.g., 1 minute at 2000 rpm in
a microcentrifuge). The coalesced aqueous phase can now be removed
(cells can be placed in an appropriate environment for further
analysis).
[0224] Additional destabilizing surfactants and/or oil combinations
can be identified or synthesized to be useful with this
invention.
[0225] Additional Modules
[0226] The microfluidic devices of the present invention can
further include one or more mixing modules, one or more delay
modules, one or more acoustic actuators and/or UV-release modules,
as described in WO 2007/081385 and WO 2008/063227.
[0227] Assays
[0228] The droplet generation rate, spacing and size of the water
droplets made on a microfluidic device are tuned to the desired
size, such as picoliter to nanoliter volumes. Additionally, droplet
libraries of the present invention can be introduced back onto a
medium for additional processing. Multicomponent droplets can
easily be generated by bringing together streams of materials at
the point where droplets are made (co-flow). Alternatively, one can
combine different droplets, each containing individual reactants.
This is achieved by selecting droplet sizes such that one droplet
is roughly wider than the channel width and the other droplet is
smaller so that the small droplets rapidly catch up to the larger
droplets. An electric field is then used to induce dipoles in the
droplet pairs, forcing them to combine into a single droplet and
permitting them to intermix the contents.
[0229] Optics for fluorescence detection capable of measuring
fluorophores within the aqueous droplets, while simultaneously
permitting visual monitoring via a high speed video microscope.
Specifically, three separate lasers provide excitation at 405 nm,
488 nm, and 561 nm wavelengths focused to a spot approximately 17
microns in diameter, illuminating each droplet as it enters the
detection zone. The system is configured to detect emitted light
using a series of photomultiplier tubes, and is able to detect less
than 10,000 FITC molecule equivalents at a 5 kHz droplet rate.
[0230] A critical component for isolating sub-populations or rare
cells from a heterogeneous cell mixture is a fluorescence-activated
microfluidic droplet sorter as described in greater detail herein.
Sorting in microfluidic devices can be done using a
dielectrophoretic force on neutral droplets. Providing an alternate
means that can be precisely controlled, can be switched at high
frequencies, and requires no moving parts. After the contents of
individual droplets are probed in the fluorescence detection zone,
selected droplets can be sorted into discreet streams for recovery
and further processing.
[0231] A key feature for improving genomic characterization of the
heterogeneous mixture of cell types present in a typical tissue or
biopsy would be the ability to fractionate the initial cell
population into sub-populations, permitting analysis of rare cells
and enabling molecular correlation studies. The microfluidic device
provides the ability to sort cell-containing droplets based on
fluorescent signals. A number of immediate uses for this capability
include: 1) sorting cell-containing droplets away from empty
droplets; 2) sorting sub-populations based on specific nucleic acid
hybridization; 3) sorting sub-populations based on cell surface
binding properties; 4) sorting sub-populations based on secreted
activities or reporter enzyme products. A number of these
approaches have already been tested in preliminary experiments,
using either bacterial or mammalian cells.
[0232] For example, Sort-on-Generation is a combination of modules
that generates single cell containing-droplets (along with
approximately 10 times more empty droplets, from Poisson
distribution as described herein and subsequently sorts the
cell-containing droplets away from the empty droplets, based on
fluorescent signals.
[0233] Also, it has been demonstrated the ability to
sort-on-generation using DNA-intercalating dyes. This approach is
enabled for any stained cell.
[0234] Determining the volume of an individual drop from a 2-D
image in a microfluidic channel can be accomplished relatively
easily with tools typically associated with microfluidics. The
basic equipment needed are; simple optics with a camera, a
fluorescent laser detector, a microfluidic device, and pumps.
[0235] A 10 pt calibration is done by plotting the average
projected area vs. the average volume of a drop. The average
projected area is determined by real-time image analysis of
droplets during emulsion generation in a specific region of the
chip. This region is clearly marked and called the calibration
region. Calibration is accomplished by simultaneously logging the
projected area of individual droplets for 60 s and calculating the
average, and using a laser is to count the total number of droplets
that pass through the channel at the calibration region. From this
count, one can determine the average frequency and the average
volume of a droplet. Where,
f = Drops t ##EQU00001## V _ = F Buffer uL hr + 10 6 pL uL f [
Drops s ] + 3600 s hr ##EQU00001.2##
[0236] Plotting this data for all points yields a calibration
curve.
[0237] During reinjection of an emulsion, using image analysis, one
can log the projected area of each individual droplet and estimate
the volume of each droplet by using the calibration curve. From
this data, one can calculate the average volume and size
distribution for a given population of droplets.
[0238] The microfluidic device of the present invention can be
utilized to conduct numerous chemical and biological assays,
including but not limited to, creating emulsion libraries, flow
cytometry, gene amplification, isothermal gene amplification, DNA
sequencing, SNP analysis, drug screening, RNAi analysis,
karyotyping, creating microbial strains with improved biomass
conversion, moving cells using optical tweezer/cell trapping,
transformation of cells by electroporation, .mu.TAS, and DNA
hybridization.
[0239] PCR in droplets
[0240] An emulsion library comprising at least a first aqueous
droplet and at least a second aqueous droplet within a fluorocarbon
oil comprising at least one fluorosurfactant, wherein the at least
first and the at least second droplets are uniform in size and
wherein at least first droplet comprises at least a first pair or
oligonucleotides and the at least second droplet comprises at least
a second pair or oligonucleotides, wherein the first and second
pair of oligonucleotides is different.
[0241] In an embodiment, the oligonucleotide is DNA; in another
embodiment, the oligonucleotide is RNA. In a further embodiment,
the oligonucleotide has at least 5 nucleotides, e.g., 5 to 100, 10
to 90, 12 to 80, 14 to 70, 15 to 60, 15 to 50, 15 to 40, 15 to 35,
15 to 30, 15 to 28, 15 to 25, 15 to 23, and 15 to 20 nucleotides.
In one embodiment, the two oligonucleotides in the pair of
oligonucleotides have the same number of nucleotides; in another
embodiment, the two oligonucleotides in the pair of
oligonucleotides have different number of nucleotides.
[0242] The present invention provides a method for amplifying a
genomic DNA, comprising (a) providing a first sample fluid wherein
said first sample fluid comprises an emulsion library comprising at
least a first aqueous droplet and at least a second aqueous droplet
within a fluorocarbon oil comprising at least one fluorosurfactant,
wherein the at least first and the at least second droplets are
uniform in size and wherein at least first droplet comprises at
least a first pair or oligonucleotides and the at least second
droplet comprises at least a second pair or oligonucleotides,
wherein the first and second pair of oligonucleotides is different;
(b) providing a second sample fluid wherein said second sample
fluid comprises a plurality of aqueous droplets comprising said
genomic DNA within an immiscible fluorocarbon oil comprising at
least one fluorosurfactant; (c) providing a microfluidic substrate
comprising at least two inlet channels adapted to carry at least
two dispersed phase sample fluids and at least one main channel
adapted to carry at least one continuous phase fluid; (d) flowing
the first sample fluid through a first inlet channel which is in
fluid communication with said main channel at a junction, wherein
said junction comprises a first fluidic nozzle designed for flow
focusing such that said first sample fluid forms a plurality of
droplets of a first uniform size in said continuous phase; (e)
flowing the second sample fluid through a second inlet channel
which is in fluid communication with said main channel at a
junction, wherein said junction comprises a second fluidic nozzle
designed for flow focusing such that said second sample fluid forms
a plurality of droplets of a second uniform size in said continuous
phase, wherein the size of the droplets of the second sample fluid
are smaller than the size of the droplets of the first sample
fluid; (f) providing a flow and droplet formation rate of the first
and second sample fluids wherein the droplets are interdigitized
such that a first sample fluid droplet is followed by and paired
with a second sample fluid droplet; (g) providing channel
dimensions such that the paired first sample fluid and the second
sample fluid droplet are brought into proximity; (h) coalescing the
paired first and second sample droplets as the paired droplets pass
through an electric field, and (i) amplifying said genomic DNA
comprised within the coalesced droplets.
[0243] A unique feature of the described droplet-based microfluidic
approach for working with nucleic acids is that it uses immiscible
oil-encapsulated aqueous droplets to shield the DNA from the inner
surfaces of the microfluidic chip, with a surfactant interface
separating the aqueous droplet and its contents from the
surrounding immiscible fluorocarbon oil. Therefore, DNA
amplification reactions occurring inside these droplets generate
material that does not interact with the channel walls, and
collection of the DNA-containing droplets for subsequent
manipulation and sequencing is straightforward. This technology
provides a solution for amplification of DNA from single cells,
allowing for both genotyping and whole genome amplification.
[0244] Evidence shows that specific loci can be amplified by PCR in
droplets generated on the microfluidic device, either by performing
PCR on-chip (with droplets moving through a serpentine channel
across several different temperatures under microfluidic control),
or by placing the collected droplets into a standard thermocycler.
Droplets generated containing DNA and reagents required for
PCR-based amplification (thermostable polymerase, dNTPs, Mg,
appropriate buffer) have been demonstrated to be extremely robust,
showing high stability for both on-chip and off-chip (standard
thermocycler) amplification. Each droplet remains intact and
separate during cycling, including during the denaturation steps at
98.degree. C. In one embodiment, a microfluidic device as described
herein, fuses droplets with individual primer pairs for PCR
amplification and preparation of many exons in parallel for high
throughput re-sequencing.
[0245] Antibodies and ELISA
[0246] The present invention provides a method for performing an
ELISA assay, comprising (a) providing a first sample fluid wherein
said first sample fluid comprises an emulsion library comprising a
plurality of aqueous droplets within an immiscible fluorocarbon oil
comprising at least one fluorosurfactant, wherein each droplet is
uniform in size and comprises at least a first antibody, and a
single element linked to at least a second antibody, wherein said
first and second antibodies are different; (b) providing a second
sample fluid wherein said second sample fluid comprises a plurality
of aqueous droplets within an immiscible fluorocarbon oil
comprising at least one fluorosurfactant, said droplets comprising
a test fluid; (c) providing a third sample fluid wherein said third
sample fluid comprises a plurality of aqueous droplets within an
immiscible fluorocarbon oil comprising at least one
fluorosurfactant, said droplets comprising at least one enzyme; (d)
providing a fourth sample fluid wherein said fourth sample fluid
comprises a plurality of aqueous droplets within an immiscible
fluorocarbon oil comprising at least one fluorosurfactant, said
droplets comprising at least one substrate; (e) providing a
microfluidic substrate comprising at least two inlet channels
adapted to carry at least two dispersed phase sample fluids and at
least one main channel adapted to carry at least one continuous
phase fluid; (f) flowing the first sample fluid through a first
inlet channel which is in fluid communication with said main
channel at a junction, wherein said junction comprises a first
fluidic nozzle designed for flow focusing such that said first
sample fluid forms a plurality of droplets of a first uniform size
in said continuous phase; (g) flowing the second sample fluid
through a second inlet channel which is in fluid communication with
said main channel at a junction, wherein said junction comprises a
second fluidic nozzle designed for flow focusing such that said
second sample fluid forms a plurality of droplets of a second
uniform size in said continuous phase, wherein the size of the
droplets of the second sample fluid are smaller than the size of
the droplets of the first sample fluid; (h) providing a flow and
droplet formation rate of the first and second sample fluids
wherein the droplets are interdigitized such that a first sample
fluid droplet is followed by and paired with a second sample fluid
droplet; (i) providing channel dimensions such that the paired
first sample fluid and the second sample fluid droplet are brought
into proximity; (j) coalescing the paired first and second sample
droplets as the paired droplets pass through an electric field,
forming at least a first coalesced droplet; (k) flowing the third
sample fluid through a third inlet channel which is in fluid
communication with said main channel at a junction, wherein said
junction comprises a third fluidic nozzle designed for flow
focusing such that said third sample fluid forms a plurality of
droplets of a third uniform size in said continuous phase, wherein
the size of the droplets of the third sample fluid are smaller than
the size of the droplets of at least first coalesced droplet; (1)
providing a flow and droplet formation rate of the third sample
fluid wherein the third sample fluid droplet and at least first
coalesced droplet are interdigitized such that the at least first
coalesced droplet is followed by and paired with the third sample
fluid droplet; (m) providing channel dimensions such that the
paired at least first coalesced droplet and the third sample fluid
droplet are brought into proximity; (n) coalescing the paired at
least first coalesced droplet and third sample droplets as the
paired droplets pass through an electric field, forming at least a
second coalesced droplet; (o) flowing the fourth sample fluid
through a fourth inlet channel which is in fluid communication with
said main channel at a junction, wherein said junction comprises a
fourth fluidic nozzle designed for flow focusing such that said
fourth sample fluid forms a plurality of droplets of a fourth
uniform size in said continuous phase, wherein the size of the
droplets of the fourth sample fluid are smaller than the size of
the droplets of at least second coalesced droplet; (p) providing a
flow and droplet formation rate of the fourth sample fluid wherein
the fourth sample fluid droplet and at least second coalesced
droplet are interdigitized such that the at least second coalesced
droplet is followed by and paired with the fourth sample fluid
droplet; (q) providing channel dimensions such that the paired at
least second coalesced droplet and the fourth sample fluid droplet
are brought into proximity; (r) coalescing the paired at least
second coalesced droplet and fourth sample droplets as the paired
droplets pass through an electric field, forming at least a third
coalesced droplet, and (s) detecting the conversion of said
substrate to a product by said enzyme within the at least a third
coalesced droplet.
[0247] The present invention also provides a method for performing
an ELISA assay, comprising (a) providing a first sample fluid
wherein said first sample fluid comprises an emulsion library
comprising a plurality of aqueous droplets within an immiscible
fluorocarbon oil comprising at least one fluorosurfactant, wherein
each droplet is uniform in size and comprises at least a first
element linked to at least a first antibody, and at least a second
element linked to at least a second antibody, wherein said first
and second antibodies are different; (b) providing a second sample
fluid wherein said second sample fluid comprises a plurality of
aqueous droplets within an immiscible fluorocarbon oil comprising
at least one fluorosurfactant, said droplets comprising a test
fluid (c) providing a third sample fluid wherein said third sample
fluid comprises a plurality of aqueous droplets within an
immiscible fluorocarbon oil comprising at least one
fluorosurfactant, said droplets comprising at least one substrate;
(d) providing a microfluidic substrate comprising at least two
inlet channels adapted to carry at least two dispersed phase sample
fluids and at least one main channel adapted to carry at least one
continuous phase fluid; (e) flowing the first sample fluid through
a first inlet channel which is in fluid communication with said
main channel at a junction, wherein said junction comprises a first
fluidic nozzle designed for flow focusing such that said first
sample fluid forms a plurality of droplets of a first uniform size
in said continuous phase; (f) flowing the second sample fluid
through a second inlet channel which is in fluid communication with
said main channel at a junction, wherein said junction comprises a
second fluidic nozzle designed for flow focusing such that said
second sample fluid forms a plurality of droplets of a second
uniform size in said continuous phase, wherein the size of the
droplets of the second sample fluid are smaller than the size of
the droplets of the first sample fluid; (g) providing a flow and
droplet formation rate of the first and second sample fluids
wherein the droplets are interdigitized such that a first sample
fluid droplet is followed by and paired with a second sample fluid
droplet; (h) providing channel dimensions such that the paired
first sample fluid and the second sample fluid droplet are brought
into proximity; (i) coalescing the paired first and second sample
droplets as the paired droplets pass through an electric field,
forming at least a first coalesced droplet, wherein if the two
antibodies bind an antigen in the test sample the at least first
and at least second elements interact to form a functional enzyme;
(j) flowing the third sample fluid through a third inlet channel
which is in fluid communication with said main channel at a
junction, wherein said junction comprises a third fluidic nozzle
designed for flow focusing such that said third sample fluid forms
a plurality of droplets of a third uniform size in said continuous
phase, wherein the size of the droplets of the third sample fluid
are smaller than the size of the droplets of at least first
coalesced droplet; (k) providing a flow and droplet formation rate
of the third sample fluid wherein the third sample fluid droplet
and at least first coalesced droplet are interdigitized such that
the at least first coalesced droplet is followed by and paired with
the third sample fluid droplet; (1) providing channel dimensions
such that the paired at least first coalesced droplet and the third
sample fluid droplet are brought into proximity; (m) coalescing the
paired at least first coalesced droplet and third sample droplets
as the paired droplets pass through an electric field, forming at
least a second coalesced droplet, and (n) detecting the conversion
of said substrate to a product by said enzyme within the at least a
second coalesced droplet.
[0248] Small sample volumes are needed in performing immunoassays.
Non-limiting examples include cases where the sample is precious or
limited, i.e., serum archives, tissue banks, and tumor biopsies.
Immunoassays would ideally be run in droplets where only 10 to 100
pL of sample were consumed for each assay. Specifically, the lack
of a robust convenient wash step has prevented the development of
ELISA assays in droplets. The present invention provides for
methods in which beads can be used to perform ELISA assays in
aqueous droplets within channels on a microfluidic device. The
advantage of utilizing microfluidic devices is it greatly reduces
the size of the sample volume needed. Moreover, a benefit of
droplet based microfluidic methods is the ability to run numerous
assays in parallel and in separate micro-compartments.
[0249] In the examples shown herein, there are several non-limiting
read-outs that can be applied to signal amplification in a
microfluidic device. The amplification methods include enzyme
amplification and rolling circle amplification of signal that uses
a nucleic-acid intermediate. In addition, a non-enzymatic means for
signal amplification can also be used.
[0250] Cell Libraries
[0251] The present invention provides a method for generating an
enzyme library, comprising (a) providing a first sample fluid
wherein said first sample fluid comprises an emulsion library
comprising a plurality of aqueous droplets within an immiscible
fluorocarbon oil comprising at least one fluorosurfactant, said
droplets comprising at least one cell transformed with at least one
nucleic acid molecule encoding for an enzyme, wherein said cells
replicate within said droplets thereby secreting produced enzymes
within the droplets; (b) providing a second sample fluid wherein
said second sample fluid comprises a plurality of aqueous droplets
within an immiscible fluorocarbon oil comprising at least one
fluorosurfactant, said droplets comprising at least one substrate;
(c) providing a microfluidic substrate comprising at least two
inlet channels adapted to carry at least two dispersed phase sample
fluids and at least one main channel adapted to carry at least one
continuous phase fluid; (d) flowing the first sample fluid through
a first inlet channel which is in fluid communication with said
main channel at a junction, wherein said junction comprises a first
fluidic nozzle designed for flow focusing such that said first
sample fluid forms a plurality of droplets of a first uniform size
in said continuous phase; (e) flowing the second sample fluid
through a second inlet channel which is in fluid communication with
said main channel at a junction, wherein said junction comprises a
second fluidic nozzle designed for flow focusing such that said
second sample fluid forms a plurality of droplets of a second
uniform size in said continuous phase, wherein the size of the
droplets of the second sample fluid are smaller than the size of
the droplets of the first sample fluid; (f) providing a flow and
droplet formation rate of the first and second sample fluids
wherein the droplets are interdigitized such that a first sample
fluid droplet is followed by and paired with a second sample fluid
droplet; (g) providing channel dimensions such that the paired
first sample fluid and the second sample fluid droplet are brought
into proximity; (h) coalescing the paired first and second sample
droplets as the paired droplets pass through an electric field, and
(i) detecting enzyme activity within the coalesced droplets,
wherein the conversion of substrate to product indicates the
presence of an enzyme library.
[0252] In a small library, the use of microfluidic system to
emulsify a library of 3-5 bacteria strains that encode a single
protease with a known range of activity against a designated
substrate in microdroplets, and sort via a fluorescence assay to
demonstrate the ability to identify and sort one of the cell
strains that expresses a protease that is more active against a
specified substrate than the other strains.
[0253] Further in a full library screen, the use of a microfluidic
system to emulsify a library of mutagenized bacteria cells in
microdroplets, identify and sort via a fluorescence assay a
subpopulation of cells to produce a 10.sup.4 fold enrichment of
cells expressing a designated enzyme variant, and recover viable
cells and enriched library.
[0254] The present invention provides a method for sorting a
plurality of cells, comprising (a) providing a first sample fluid
wherein said first sample fluid comprises an emulsion library
comprising a plurality of aqueous droplets within an immiscible
fluorocarbon oil comprising at least one fluorosurfactant, said
droplets comprising at least one cell labeled with an enzyme; (b)
providing a second sample fluid wherein said second sample fluid
comprises a plurality of aqueous droplets within an immiscible
fluorocarbon oil comprising at least one fluorosurfactant, said
droplets comprising at least one substrate; (c) providing a
microfluidic substrate comprising at least two inlet channels
adapted to carry at least two dispersed phase sample fluids and at
least one main channel adapted to carry at least one continuous
phase fluid; (d) flowing the first sample fluid through a first
inlet channel which is in fluid communication with said main
channel at a junction, wherein said junction comprises a first
fluidic nozzle designed for flow focusing such that said first
sample fluid forms a plurality of droplets of a first uniform size
in said continuous phase; (e) flowing the second sample fluid
through a second inlet channel which is in fluid communication with
said main channel at a junction, wherein said junction comprises a
second fluidic nozzle designed for flow focusing such that said
second sample fluid forms a plurality of droplets of a second
uniform size in said continuous phase, wherein the size of the
droplets of the second sample fluid are smaller than the size of
the droplets of the first sample fluid; (f) providing a flow and
droplet formation rate of the first and second sample fluids
wherein the droplets are interdigitized such that a first sample
fluid droplet is followed by and paired with a second sample fluid
droplet; (g) providing channel dimensions such that the paired
first sample fluid and the second sample fluid droplet are brought
into proximity; (h) coalescing the paired first and second sample
droplets as the paired droplets pass through an electric field; (i)
detecting enzyme activity within the coalesced droplets, and (j)
selecting cells where the enzyme has converted substrate to
product.
[0255] Whole Genome Amplification
[0256] Whole Genome Amplification (WGA) is a method that amplifies
genomic material from minute samples, even from a single cell,
enabling genome sequencing. A number of commercially available WGA
methodologies have been developed, including PCR-based methods like
degenerate oligonucleotide primed PCR (DOP-PCR) and primer
extension pre-amplification (PEP-PCR), and multiple displacement
amplification (MDA) which uses random hexamers and using high
fidelity 129 or Bst DNA polymerases to provide isothermal
amplification. Several analyses have shown that MDA products
generate the least amplification bias and produce a higher yield of
amplified DNA. This method has been used recently to amplify
genomic DNA for sequencing from single cells, with partial genome
sequencing demonstrated. MDA-based WGA has also been performed on
cell populations selected using flow-FISH.
[0257] Non-specific DNA synthesis due to contaminating DNA and
non-template amplification (NTA) are characteristic problems
associated with WGA. Recent evidence demonstrates that NTA and also
amplification bias are reduced when using very small reaction
volumes, with one group using 60 nanoliter microfluidic chambers
for single cell WGA reactions. Based on these findings, the use of
picoliter-volume droplets in a microfluidic system reduces NTA even
further. In addition, amplification from contaminating DNA
templates will be constrained to individual compartments
(droplets), minimizing the overwhelming effects of contamination in
bulk WGA reactions.
[0258] Next Generation Sequencing
[0259] Next generation sequencing instruments offer two distinct
advantages in the pursuit of microbiome characterization. First,
they do not require conventional clone-based approaches to DNA
sequencing, and thus ensure that the commonly experienced biasing
against specific sequences in the E. coli host system does not
impact the representation of genomes being sequenced. Second, they
offer a streamlined and robust workflow for preparing DNA for
sequencing that has far fewer steps than conventional workflows.
Hence, a library can be prepared for sequencing in about 2 days.
The Roche/454 FLX pyrosequencer was the first "next generation",
massively parallel sequencer to achieve commercial introduction (in
2004) and uses a sequencing reaction type known as "pyrosequencing"
to read out nucleotide sequences. In pyrosequencing, each
incorporation of a nucleotide by DNA polymerase results in the
release of pyrophosphate, which initiates a series of downstream
reactions that ultimately produce light by the firefly enzyme
luciferase. The light amount produced is proportional to the number
of nucleotides incorporated (up to the point of detector
saturation). In the Roche/454 instrument, the DNA fragments to be
sequenced first have specific A and B adapter oligos ligated to
their ends, and then are mixed with a population of agarose beads
whose surfaces carry oligonucleotides complementary to 454-specific
adapter sequences on the DNA fragments, such that each bead is
associated with a single DNA fragment. By isolating each of these
fragment:bead complexes into individual oil:water micelles that
also contain PCR reactants, thermal cycling ("emulsion PCR") of the
micelles produces approximately one million copies of each DNA
fragment on the surface of each bead. These amplified single
molecules are then sequenced en masse by first arraying them into a
PicoTiter Plate (PTP-a fused silica capillary structure), that
holds a single bead in each of several hundred thousand single
wells, providing a fixed location at which each sequencing reaction
can be monitored. Enzyme-containing beads that catalyze the
downstream pyrosequencing reaction steps then are added to the PTP,
and centrifuged to surround the agarose beads. On instrument, the
PTP acts as a flow cell, into which each pure nucleotide solution
is introduced in a stepwise fashion, with an imaging step after
each nucleotide incorporation step. Because the PTP is seated
opposite a CCD camera, the light emitted at each bead that is being
actively sequenced is recorded. In practice, the first four
nucleotides (TCGA) on the adapter fragment adjacent to the
sequencing primer added in library construction are first to be
sequenced, and this sequence corresponds to the sequential flow of
nucleotides into the flow cell. This strategy allows the 454 base
calling software to calibrate the light emitted by a single
nucleotide incorporation, which then enables the software to call
novel bases downstream according to the light emission levels.
However, the calibrated base calling cannot properly interpret long
stretches (>6) of the same nucleotides occurring in a stretch
("homopolymer" run), due to detector saturation, so these stretches
are prone to base insertion and deletion errors during base
calling. By contrast, since each incorporation step is nucleotide
specific, substitution errors are rarely encountered in Roche/454
sequence reads.
[0260] Enzyme Inhibitor Screening
[0261] The present invention provides a method for screening for an
enzyme inhibitor, comprising (a) providing a first sample fluid
wherein said first sample fluid comprises an emulsion library
comprising a plurality of aqueous droplets within an immiscible
fluorocarbon oil comprising at least one fluorosurfactant, said
droplets comprising at least one compound; (b) providing a second
sample fluid wherein said second sample fluid comprises a plurality
of aqueous droplets within an immiscible fluorocarbon oil
comprising at least one fluorosurfactant, said droplets comprising
at least one enzyme and substrate; (c) providing a microfluidic
substrate comprising at least two inlet channels adapted to carry
at least two dispersed phase sample fluids and at least one main
channel adapted to carry at least one continuous phase fluid; (d)
flowing the first sample fluid through a first inlet channel which
is in fluid communication with said main channel at a junction,
wherein said junction comprises a first fluidic nozzle designed for
flow focusing such that said first sample fluid forms a plurality
of droplets of a first uniform size in said continuous phase; (e)
flowing the second sample fluid through a second inlet channel
which is in fluid communication with said main channel at a
junction, wherein said junction comprises a second fluidic nozzle
designed for flow focusing such that said second sample fluid forms
a plurality of droplets of a second uniform size in said continuous
phase, wherein the size of the droplets of the second sample fluid
are smaller than the size of the droplets of the first sample
fluid; (f) providing a flow and droplet formation rate of the first
and second sample fluids wherein the droplets are interdigitized
such that a first sample fluid droplet is followed by and paired
with a second sample fluid droplet; (g) providing channel
dimensions such that the paired first sample fluid and the second
sample fluid droplet are brought into proximity; (h) coalescing the
paired first and second sample droplets as the paired droplets pass
through an electric field, and (i) detecting enzyme activity within
the coalesced droplets, wherein the failure of the enzyme to
convert the substrate to product indicates the compound is an
enzyme inhibitor.
[0262] The present invention provides compositions and methods for
generating, manipulating, and analyzing aqueous droplets of
precisely defined size and composition. These microfluidic
device-generated droplets can encapsulate a wide variety of
components, including those that are used in enzymatic assays.
Kinases are a therapeutically important class of enzymes, and this
collaboration examines the feasibility of performing analysis and
interrogation of kinases with potentially inhibitory compounds
using the described microfluidic platform and systems.
[0263] High-Throughput Droplet Live-Dead Assay Screening
[0264] The present invention provides a method for screening for a
live cell, comprising (a) providing a first sample fluid wherein
said first sample fluid comprises an emulsion library comprising a
plurality of aqueous droplets within an immiscible fluorocarbon oil
comprising at least one fluorosurfactant, said droplets comprising
at least one cell; (b) providing a second sample fluid wherein said
second sample fluid comprises a plurality of aqueous droplets
within an immiscible fluorocarbon oil comprising at least one
fluorosurfactant, said droplets comprising at least one
cell-membrane-permeable fluorescent dye and at least one
cell-membrane-impermeable fluorescent dye; (c) providing a
microfluidic substrate comprising at least two inlet channels
adapted to carry at least two dispersed phase sample fluids and at
least one main channel adapted to carry at least one continuous
phase fluid; (d) flowing the first sample fluid through a first
inlet channel which is in fluid communication with said main
channel at a junction, wherein said junction comprises a first
fluidic nozzle designed for flow focusing such that said first
sample fluid forms a plurality of droplets of a first uniform size
in said continuous phase; (e) flowing the second sample fluid
through a second inlet channel which is in fluid communication with
said main channel at a junction, wherein said junction comprises a
second fluidic nozzle designed for flow focusing such that said
second sample fluid forms a plurality of droplets of a second
uniform size in said continuous phase, wherein the size of the
droplets of the second sample fluid are smaller than the size of
the droplets of the first sample fluid; (f) providing a flow and
droplet formation rate of the first and second sample fluids
wherein the droplets are interdigitized such that a first sample
fluid droplet is followed by and paired with a second sample fluid
droplet; (g) providing channel dimensions such that the paired
first sample fluid and the second sample fluid droplet are brought
into proximity; (h) coalescing the paired first and second sample
droplets as the paired droplets pass through an electric field, and
(i) detecting fluorescence within the coalesced droplets, wherein
the detection of fluorescence of cell-membrane-permeable dye
indicates a droplet comprising a dead cell and the detection of
fluorescence of cell-membrane-impermeable dye indicates a droplet
comprising a live cell.
[0265] Single-cell analysis in the context of cell populations
avoids the loss of information on cellular systems that is inherent
with averaged analysis. In recent years, this type of analysis has
been aided by the development of sophisticated instrumentation.
Microfluidic technologies have the potential to enhance the
precision and throughput of these single-cell assays by integrating
and automating the cell handling, processing, and analysis steps.
However, major limitations in microfluidic systems hinder the
development of high-throughput screening platforms. One challenge
is to achieve sufficiently short mixing times. Mixing under the
laminar flow conditions typically found in microfluidic devices
occurs by diffusion, a relatively slow process for biological
material and biochemical reactants. Most importantly, as the scale
of these reactors shrinks, contamination effects due to surface
adsorption and diffusion limit both the smallest sample size and
the repeated use of channels for screening different conditions.
These limitations are major hurdles when this technology is to be
applied for screening libraries containing thousands of different
compounds each corresponding to different experimental
conditions.
[0266] The confinement of reagents in droplets in an immiscible
carrier fluid overcomes these limitations. The droplet technology
is an essential enabling technology for a high-throughput
microfluidic screening platform. Droplet isolation allows the cells
to be exposed to discrete concentrations of chemicals or factors.
Most importantly, the droplet format ensures that the sample
materials never touch the walls of the microfluidic channels and
thus eliminates the risk of contamination. The reagents can be
mixed within a droplet and sample dispersion is simultaneously
minimized. The advantages of this technique include the physical
and chemical isolation of droplets from one another and the ability
to digitally manipulate these droplets at very high-throughput.
Finally, the absence of any moving parts and in particular valves
brings the degree of robustness required for screening
applications.
[0267] Possible cell applications include screen for combinatorial
cell assays, cloning, FACS-like assays, and polymer encapsulation
for cell-based therapies. As a small number of cells are consumed
per sample, this technology is particularly suitable for working
with cells of limited availability, like primary cells. In
addition, for rare cell sorting, the dilution factor in the
collection droplets can be orders of magnitude smaller than for a
standard bench-scale flow cytometer. Finally, the use of
fluorocarbons that can dissolve large amount of oxygen as carrier
fluids is regarded as a key feature for long-term survival of
encapsulated cells.
[0268] Numerous modules have been developed for performing a
variety of key tasks on droplets. They include the generation of
monodisperse aqueous droplets and its use for cell encapsulation.
Droplets can be fused or coalesced, their content mixed, incubated
on-chip, and their incubation time tuned with an oil-extractor,
their fluorescent content can be interrogated, and finally they can
be sorted. The assembly of such modules into complete systems
provides a convenient and robust way to construct droplet
microfluidic devices that would fulfill the promises of the droplet
technology as a screening platform.
[0269] Example 9 illustrates some examples of live-dead assays. The
device has been designed to sequentially accomplish six different
functions: (i) separated cell and dye encapsulations, (ii) fusion
of droplets containing cells and droplets containing dyes, (iii)
mixing of cell with dyes in each fused droplet, (iv) oil-extraction
to modulate on-chip incubation of droplets, (v) droplet incubation
on-chip and (vi) interrogation of the fluorescent signal of each
droplet. Furthermore, encapsulated cells can be collected into a
syringe and re-inject the emulsion for on-chip scoring.
[0270] Kits
[0271] 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.
[0272] 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.
[0273] Enzyme Quantification
[0274] Described herein are methods for counting enzyme molecules
in fluid partitions such as, for example, microdroplets. A number
of readout modes and multiplexing formats are illustrated, and
examples of assays coupled to the digital readout are shown.
[0275] In some embodiments, methods of the invention include a
sandwich immunoassay, allowing for absolute counting of protein
molecules in a sample (digital droplet ELISA). Any upfront assay
that uses a chemical reaction at least one component of which
includes a detectable label (e.g., a reporter enzyme system) can be
used. Any suitable detectable label may be included (e.g., a
fluorescent product, or other optical or detectable non-optical
product) can be used with methods of the invention.
[0276] In certain embodiments, the invention provides methods for
the direct detection and quantification of enzymatically active
molecules potentially contained in samples (e.g.,
"bio-prospecting"). Reporter substrates specific for the target
enzyme or enzyme class are used to assay for enzyme-containing
samples (or coupled enzyme and substrates report to report on
another enzyme molecule). For example, a sample can be obtained
that is suspected to contain a target molecule of interest or a
target molecule member of a class of interest. The sample can be
distributed into a plurality of fluid partitions. Further, where a
specific activity or moiety is of interest or if enzymes having a
range or threshold of specific activity are of interest, a large
number of targets can be assayed using methods and systems of the
invention. The targets can be distributed among a plurality of
fluid partitions, and each partition can be provided with reagents
for a certain enzyme-catalyzed reaction. The occurrence of the
reaction in certain partitions can be detected. Optionally, using
sorting methods, enzyme-positive partitions can be isolated for
further analysis. Thus, a single (or very low amount of) target,
even an unknown target, can be identified and isolated according to
activity. Further discussion can be found in Miller, et al., PNAS
109(2):378-383 (2012); Kiss, et al., Anal. Chem. 80(23):8975-8981
(2008); and Brouzes, et al., Droplet microfluidic technology for
single-cell high-throughput screening, 10.1073/PNAS.0903542106
(Jul. 15, 2009), the contents of which are hereby incorporated by
reference in their entirety.
[0277] Methods of the invention allow for the detection of two or
more enzyme molecules or other molecules in a complex that can be
assayed using an enzymatic reporter or other activatable and
readable reporter (e.g. protein aggregation assay for mis-folded or
disease associated molecules). Methods include providing separately
detectable substrates for each enzyme species or where
complexes/aggregates are detected by different product
concentrations using the same enzyme type. A complex can be
detected as both product signals are detected in the same fluid
partition even when the fractional occupancy is low. For example,
at very low fractional occupancies, there is a vanishing
probability of the two enzymes being found in some number of the
same fluid partitions if not in a complex together (modeled
according to Poisson statistics). Thus, the detection of both
product signals reveals a protein-protein interaction between the
two enzymes.
[0278] A number of examples are shown utilizing `endpoint` type
digital counting, where the enzymatic reaction has reached a
plateau. More than one endpoint can also be used for detection of
multiple species. The invention further includes measurements at
earlier time points during the reaction (`kinetic` measurements vs.
`endpoint` measurements) such that different signal intensities
from single enzyme molecules reflect differences in enzymatic
specific activity, the presence of inhibitory or activating
molecules, or the presence of more than one enzyme molecule per
droplet. Kinetic mode measurements can be made following droplet
incubation at the appropriate temperature either off chip or on
chip. For on-chip measurements, a `timing module`, such as a delay
line, can be used to keep droplets on-chip for an appropriate
length of time, and one or multiple measurement points along the
length of the microfluidic channel can enable very precise kinetic
measurements. A delay line can include, for example, a channel in a
chip through which droplets flow. A delay line may include
locations for stopping droplets, or locations for moving droplets
out of the may stream of flow, or locations for droplets to
separate from the oil by buoyancy differences between the oil and
the droplets. A delay line may include a means of adding or
removing oil to speed up or slow down the rate of travel of
droplets through the delay line. A delay line may include
neck-downs or other features to homogenize the average velocity of
the droplets and minimize dispersion effects as droplets travel
through the channel. A droplet trap may be utilized to trap
droplets for a fixed period of time before releasing the droplets.
Such a trap may include a valve, or require the reversal of the
direction of flow through the trap region to release the trapped
droplets or it may require that the chip be flipped over to reverse
the direction that the droplets move in the gravitational field.
One skilled in the art will recognize a number of ways to control
the timing of the reaction. A portion of the channel may have a
much broader cross-sectional area that upstream or downstream
portions. Thus, for a certain volume per time flow rate, the
distance per time flow rate in the broad portion will be much
slower. Working with fluidic chips and known droplet behaviors, a
channel can be designed with a delay line that delays the flow for
a predetermined amount of time. This can allow reactions to
incubate or progress for the appropriate amount of time.
Temperature control of specific regions may be included in chip
designs and interfaces with the chips.
[0279] Analyte or reporter molecule may also include non-enzyme
species, provided the molecule or complex participates in
generation of a readable signal (e.g. an enzymatic activator or
inhibitor).
[0280] In general, the invention provides methods and systems for
measuring a molecular target. A plurality of fluid partitions are
formed. Fluid partitions can be any known in the art such as, for
example, wells on a plate or water-in-oil droplets. A detectable
reaction, such as an enzyme-catalyzed reaction, is performed in
some subset of the fluid partitions. For example, where a sample
suspected of containing the molecular target is separated into the
fluid partitions (including via an optional dilution or serial
dilution step), the subset of partitions that contain the target
will include a certain number of partitions. That number can be
associated with the amount of target in the sample.
[0281] A detectable reaction occurs in the subset of partitions
that contain the molecular target. In some embodiments, the
molecular target is an enzyme, and all of the partitions are
provided with a fluorescently labeled substrate. The
enzyme-catalyzed reaction can release the fluorescent label from
the substrate such that the fluor becomes un-quenched or can be
quantified by its location in the partition. In certain
embodiments, the target is a substrate, and all of the partitions
are provided with an enzyme and optionally an additional substrate
or a co-factor. One of the reaction ingredients contains a
molecular label that is released when the reaction occurs.
[0282] Because a productive reaction only takes place within the
subset of partitions that contain the target, determining the
number of partitions within which the reaction takes place (i.e.,
determining the number of the subset) allows one to determine the
amount of target in the original sample. Since each partition is
counted as reaction-positive or reaction-negative (e.g., in the
subset or not), this detection is said to be digital. This includes
cases where the positives can be further quantified as containing
quantized numbers of targets.
[0283] In certain embodiments, digital detection operates through a
reaction that includes a number of stages including, in various
embodiments, enzymes that are themselves substrates for other
enzyme-catalyzed reactions and substrates and/or enzymes or targets
that are dark (i.e., not reporting) when participating in reactions
and that are detectable when not. To illustrate, in certain
embodiments, a partition includes an enzyme and a substrate which
together will report the presence and number of target molecules.
The substrate is labeled such that it gives a dark state as long as
the enzyme is present. A target molecule that inhibits the enzyme
can be assayed for according to the steps described herein. Since
the target inhibits the reporting enzyme, then presence of the
target will cause the reporter to generate a readable signal.
[0284] In certain embodiments, a substrate is included in a
reaction mixture along with an enzyme that catalyzes a readable
reaction of the substrate, but the included enzyme is in an
inactive form and exposure of the included enzyme to the target
molecule will catalyze its conversion to an active form. In this
fashion, the presence or absence of target initiates an enzyme
cascade that results in a readable assay (the cascade can include
multiple steps) and quantified. In one embodiment, an apoenzyme is
used to detect the presence of a protease. The apoenzyme is
provided in a fluid partition with a substrate of the active form
of the enzyme. The apoenzyme will only be cleaved to form the
active form if the protease is present in the target sample.
[0285] The invention further provides methods and systems for
detecting or quantifying the presence of an enzyme inhibitor or
activator. For example, fluid partitions can be provided with an
enzyme and its substrate. A sample suspected to contain an
inhibitor or activator is separated into the partitions (with
optional dilution). Release of a reporter via an enzyme catalyzed
activity indicates the presence of an activator or absence of an
inhibitor. Further, enzyme kinetics as well as inhibition or
activation can be studied with methods described herein.
[0286] Methods of the invention can be used with any suitable
enzyme(s) or substrate(s). For example, beyond the variety of
examples given herein, the invention can further be used to detect
cleavage of a peptide by a protease. Where a sample is suspected to
contain a protease, a fluorescently labeled peptide substrate can
be provided in the fluid partitions.
[0287] As another example, methods of the invention can include use
of a polymerase enzyme and fluorescently labeled nucleotides to
detect activity of a ligase. Given the appropriate conditions, the
polymerase will only act on a product of a reaction catalyzed by
the ligase (e.g., a polynucleotide). When the polymerase catalyzes
a reaction it releases the fluorescent reporter as a readable
signal.
[0288] In one other illustrative example, the presence, type, and
number of restriction enzyme(s) can be quantified by providing a
construct that includes an oligonucleotide with a fluor and a
quencher close enough to each other that the quencher quenches the
fluor but is separated by a restriction site. The presence of the
restriction enzyme in the target sample will light up what is
otherwise a dark fluid partition. The presence, type, and number of
a set of analytes can be assayed using restriction enzymes that are
specific for different readable substrates, or can be configured
into a `one-of-many` type of assay where all the analytes have the
same restriction enzyme and substrate, or can be grouped into
different classes using enzyme/substrate classes.
[0289] In certain embodiments, the invention provides systems and
methods for detecting and quantifying classes of enzymes or
substrates. Any class of target can be the subject of an assay
including, for example, all enzymes exhibiting a certain activity
or all enzymes in a certain taxonomic group.
[0290] Another embodiment includes a downstream reporter that is
split (e.g., split green fluorescent protein or a split enzyme
reporter) into multiple parts that do not interact productively in
the absence of the upstream reporter (e.g. cleavage of the modified
downstream reporter by the upstream reporter enzyme-oligo-modified
reporter activated by the action of a restriction enzyme or a
peptide-modified reporter activated by protease reporter), or that
require being brought into close proximity for activity (e.g.
following cleavage of the inhibiting modification, two halves of a
split enzyme are brought into proximity for folding and
activation). Two subsections of the reporter can be separated by an
oligo containing a restriction site such that if the restriction
enzyme (or, remembering that an enzyme can in turn be a substrate
for another enzyme, an enzyme that activates the restriction
enzyme) is present, the oligo is cleaved and the two subsections
can come together to form the active reporter. Alternatively,
cleavage of linked moieties can release inhibition of an activity
that produces a readable signal.
[0291] One of skill in the art will recognize that any number of
the examples given herein can be combined to create multi-step
assays. Thus, for example, a restriction enzyme can cleave an oligo
that is separating two halves of a protease. The active protease
can cleave an apoenzyme to release an active phosphorylase that
phosphorylates and de-activates a downstream enzyme. If the
downstream enzyme is an inhibitor of a final enzyme, then
deactivation of a downstream enzyme can result in activity of the
final enzyme. One of skill in the art will recognize the wide
variety of combinations of the scenarios given herein that can be
used.
[0292] FIGS. 8A-8G show a workflow example for a digital droplet
reporter enzyme assay. In FIG. 8A, the enzyme molecules to be
counted are mixed with a fluorogenic substrate and loaded into
droplets via introduction into a microfluidic nozzle. The aqueous
mixture of enzyme and substrate flows down inlet channel 101 and
forms an emulsion when merged with oil from carrier fluid channels
103a and 103b. In FIG. 8A, arrows indicate the direction of flow.
The co-infusion of an immiscible oil segments the aqueous stream
into a number of uniformly sized droplet 109.
[0293] The droplet emulsion is collected into a suitable container
for off-chip incubation at an appropriate temperature for enzymatic
function.
[0294] FIG. 8B illustrates a schematic overview of a reaction
according to certain embodiments of the invention. In general, as
shown in FIG. 8B, an enzyme 113 catalyzes the conversion of a
fluorogenic substrate 111 to a fluorescent product 115.
[0295] In one exemplary embodiment, enzyme 113 was
streptavidin-conjugated beta galactosidase (.beta.-gal) (Calbiochem
product #569404 from Merck KGaA (Darmstadt, Germany)) and
fluorogenic substrate 111 was fluorescein
di-.beta.-D-galactopyranoside (FDG) sold under the trademark
MOLECULAR PROBES as product number F1179 by Life Technologies
(Carlsbad, Calif. ), with active enzyme able to cleave the
substrate to release, as fluorescent product 115, fluorescein
isothiocyanate (FITC) and two galactose.
[0296] As shown in FIG. 8C, after incubation of the droplet
emulsion at 37.degree. C. for a determined time, the droplet
temperature can be changed such that the enzyme is no longer
affecting the readable signal, and the droplets can be infused into
a second microfluidic nozzle 117, spaced into a train of individual
droplets using an immiscible oil, and run past laser spot 119
focused in microfluidic channel 121. FIG. 8D is an illustration
depicting the detection step when no enzyme is present (or after
enzyme is loaded but before incubation). The droplets in FIG. 8D
all exhibit uniformly low fluorescence intensity, indicating a lack
of conversion of substrate 111 to product 115.
[0297] As discussed with reference to FIGS. 8C-8E, detection can
include droplets flowing past a detector. However, any suitable
method of detecting an enzymatic reaction in a fluid partition can
be used including, for example, optical or non-optical detection
such as pH change or change in impedance or conductivity within a
fluid partition, or any other suitable detection method. In some
embodiments, non-optical detection includes nuclear magnetic
resonance (NMR) analysis of materials from fluid partitions.
Detection after release of the digitally generated reporter
moieties from the partitions may also be used (e.g. array,
electrode, magnet, sequencer, mass spectrometer, other
methods).
[0298] FIG. 8E is an illustration depicting the detection step when
a low concentration of enzyme is present, after incubation. Laser
spot 119 is used to detect a number n of positive droplets 125a,
125b, . . . , 125n. By counting the results of laser detection, the
number of partitions (here, droplets) in the subset of partitions
in which an enzyme-catalyzed reaction occurred is determined.
[0299] The resulting signal time traces from detection
photomultiplier tubes show examples for when either there were no
enzyme molecules loaded or after loading enzyme molecules but
before incubation (shown in FIG. 8F with an insert showing a zoomed
image of 10 individual droplet traces), and droplets generated with
a low concentration of enzyme after incubation (FIG. 8G). The
droplets that have no enzyme molecules (or have enzyme molecules
that were not incubated to allow for enzymatic activity) show
uniformly low fluorescent signal intensity, coming from the
unconverted fluorogenic substrate. For the case where a low
concentration of enzyme was used (see FIG. 8G) loading of enzyme
molecules into droplets occurs in a quantized manner, with the
signal time trace showing droplets that have no enzyme molecules
(with similar signal intensity to that seen at generation) and
droplets with enzyme molecules (showing quantized levels of signal
intensity that correspond to different numbers of enzyme molecules
per droplet).
[0300] The distribution of the number of enzyme molecules per
droplet (i.e. 0, 1, 2, 3, etc. molecules per droplet) is dependent
on the starting concentration of enzyme loaded into the droplets
and the whether the enzyme molecules are in un-dissociated
complexes. FIGS. 9A-9D and FIGS. 10A-10D illustrate this
phenomenon, showing the time traces (FIGS. 9A-9D) and histogram
distributions (FIGS. 10A-10D) for increasing concentrations of
.beta.-gal, as shown by fluorescent intensity of FITC (lowest
concentration shown in FIGS. 9A and 10A, highest concentration
shown in FIGS. 9D and 10D). These measurements were made on
droplets that had been incubated for about an hour. As the starting
enzyme concentration is increased, the time traces show the number
of `negative` (no enzyme) droplets decrease, the number of
`positive` droplets increase, and the number of enzyme molecules in
any positive droplet increases (seen as a higher signal
intensity).
[0301] The distribution of enzyme molecules into droplets can occur
according to a Poisson Distribution, or can occur in a non-Poisson
fashion. FIG. 10A shows a non-Poisson distribution. In some cases
the degree to which the distribution varies from a Poisson
distribution will be indicative of a degree of aggregation of the
component. In some cases the interpretation of the variation from a
Poisson distribution will be diagnostic. In FIG. 10A, the x-axis is
given in volts indicting reporter intensity as measured through a
photomultiplier. An appropriate scaling factor can be used (e.g.,
determined separately) to convert V to number of molecules per
droplet. As shown in FIG. 10A, the first peak at about 0.1 V can
indicate that a number of the droplets (i.e., Drop Count) that had
no .beta.-gal activity in them. The second peak about 0.23 V can
indicate a number of droplets that each had one active unit of
enzyme. The third peak about 0.42 V can indicate a number of
droplets that each contained two active units of enzyme. The fourth
peak, at about 0.58 V, can indicate a number of droplets that
contained three units of active enzyme. The fifth peak, at about
0.76 V, can indicate a number of droplets that contained four
active units of enzyme. FIGS. 10B-10C show greater concentration of
enzyme (i.e., less dilution). In certain embodiments, for an enzyme
that exhibits no aggregation or covalent or non-covalent complex
formation, a plot at the concentration illustrated in FIG. 10A
would show a Poisson distribution. FIG. 10A can indicate that a
substantial and statistically significant number of droplets
contain more than 1 enzyme unit than is predicted by Poisson. Thus,
FIG. 10A can show that .beta.-gal exhibits aggregation. When the
starting enzyme concentration is 1.6 pM (FIGS. 9D and 10D, there
are no negative droplets, and the histogram shows most droplet
signals centered around a single mean value, with a much smaller
number showing a quantized distribution like that seen at lower
concentrations (several small peaks close to the origin).
[0302] FIGS. 11A and 11B show an example of how this data can be
used to quantify the concentration of active enzyme molecules
loaded into droplets. FIG. 4A shows a readout histogram from an
enzyme concentration that was calculated to be 0.0128 pM. Using the
histogram from an enzyme concentration calculated to be 0.0128 pM,
based on multiplying the starting concentration and the dilution
factor (FIG. 11A), each peak's mean is determined and plotted as a
function of integer enzyme molecules to show linearity (FIG. 11B).
The number of droplets within each peak and the number of active
enzyme molecules within each peak are counted and tabulated. The
results are listed in Table 1.
TABLE-US-00003 TABLE 1 Distribution of #Droplets with #Molecules
Enzyme/droplet Droplets #enzymes 0 113458 0 1 15249 15249 2 3637
7274 3 1356 4068 4 536 2144 5 280 1400 6 139 834 7 73 511 >7 138
1405 Total: 137866 32885
[0303] By dividing the totals from Table 1 (number of active enzyme
molecules over total number of droplets counted) a number of
molecules per droplet can be calculated as shown in Equation 1.
(#molecules/droplet)=(32885/134866)=0.24 (1)
[0304] By multiplying by the appropriate scaling factors, the
measured concentration (MC) can be calculated using Equation 2:
M C ( p M ) = # molecules droplet .times. droplet volume ( pL )
.times. 1.0 pM 0 . 6 023 molecules / pL ( 2 ) ##EQU00002##
[0305] Using the value given by Equation 1 in Equation 2, gives the
result shown in Equation 3.
MC ( pM ) = 0 . 2 4 molecules droplet .times. droplet 30 pL .times.
1.0 pM 0 . 6 023 molecules / pL = 0 . 0 1275 pM ( 3 )
##EQU00003##
[0306] For this example, the measured concentration was 0.01275 pM,
with the expected concentration based on dilution factor being
0.0128 pM.
[0307] The dynamic range of the assay can span regimes where the
number of enzyme molecules is discretely quantized in all droplets
or where the majority of droplets (or all droplets) contain a mean
(with a distribution around the mean) number of enzyme molecules.
For the specific format described in the example (i.e. droplet
size, enzyme and substrate used) typically enzyme concentrations
greater than .about.pM can be analyzed using the mean distribution
(and also the small quantized tail seen near the origin of the
graph shown in FIG. 10D) and enzyme concentrations lower than
.about.pM can be analyzed using digital counting of the total
number of droplets, the number of enzyme-containing droplets, and
using the quantized signals from enzyme-containing droplets to
count the number of enzyme molecules per droplet. Thus, the dynamic
range of the assay is extremely wide, with the lower limit of
detection determined by the number of detectable molecules present
in the sample and the length of time required to run a sufficient
number of droplets through the detector (e.g. if the droplet system
runs at 10.sup.6 droplets per hour, the limit of detection is 1 in
10.sup.6 in an hour, and the limit of detection is 1 in 10.sup.7 in
10 hours), and the upper limit determined by the amount of
substrate converted to product (as enzyme concentrations get
higher, the substrate concentration will have to increase in order
for the product fluorescence to remain linearly (or correlatively)
related to enzyme mean concentration). Additional parameters that
can be adjusted include the time and temperature of incubation, as
well as the droplet volume used, and additional reaction
components. In certain embodiments, fluid partitions are droplets
and assays are performed in systems in which droplets are run past
a detector at 3,000 s. In some embodiments, droplets are run at
10,000/s or at about 100,000 per second. In some embodiments, a
lower limit of detection is 1 in 10.sup.9 and a flow rate is
10.sup.9 per hour.
[0308] FIG. 12 shows an illustration of the concept and workflow
for a digital droplet ELISA assay, one example of an upfront assay
that can be coupled to the digital reporter enzyme assay readout.
When protein concentrations are too low for standard detection
methods (typically low-sub-picomolar), this invention enables
protein quantification by counting individual protein molecules
with a fluorescent readout. Droplets containing a single molecule
(e.g. in an ELISA sandwich) will be fluorescent, and the number of
fluorescent droplets in a population of total droplets will yield a
digital count of molecules per volume (i.e. concentration) down to
a limit of detection dependent only on the number of droplets
examined.
[0309] FIG. 12 shows one example ELISA assay format and should not
be considered the only or preferred format (e.g. magnetic beads
could be added following antibody binding in solution). The
protein-containing sample (three proteins shown as diamonds with
the rare target protein to be counted shown as solid diamonds) is
combined with the binding reagents and incubated for a sufficient
time to bind into productive complexes.
[0310] In the "ELISA Sandwich Formation" step, each target protein
molecule is bound to two affinity reagents (each binding separate
epitopes of the same target molecule), generating an immunoaffinity
"sandwich" complex. In the example shown, one of the affinity
reagents (e.g. antibody) is immobilized onto a magnetic bead while
the other biotinylated antibody is free in solution. In certain
embodiments, the number of magnetic beads (with immobilized
antibody) is significantly greater than the number of target
proteins in solution, so that single target proteins are bound by
single beads. If the second antibody is used at the same time, its
concentration should be greater than the number of target
molecules, but less than the number of immobilized antibodies.
Alternatively, the second antibody can be added following the first
binding step (ensuring that all target molecules are bound to the
immobilized antibody first).
[0311] After the target proteins are bound into sandwich complexes,
the magnetic beads are retained by a magnetic field to allow
removal of unbound non-target proteins and free antibodies, and
washed to remove non-specific binders. Addition of the reporter
enzyme (e.g. streptavidin-beta galactosidase) results in binding to
the second biotinylated antibody and assembly of the final ELISA
sandwich, which is again washed to remove unbound reporter enzyme.
The final material (see, e.g., FIG. 13A) is re-suspended in a small
volume, along with a fluorogenic substrate, for processing in the
digital droplet readout.
[0312] FIGS. 13A-13D show a number of different readout `modes` for
running the digital droplet readout, following the ELISA sandwich
complex construction. In FIG. 13A, more than one magnetic bead is
in each generated droplet, but only a single ELISA sandwich is in
any single droplet (e.g. in this case sub-micron magnetic beads are
used).
[0313] FIG. 13B shows a mode where at most a single bead is in each
droplet, with at most one ELISA sandwich.
[0314] FIG. 13C shows a mode where the second antibody complexed to
the reporter enzyme has been eluted off of the magnetic bead, and
the droplets are loaded such that at most one antibody-reporter
complex is present in any droplet.
[0315] In FIG. 13D, the reporter enzyme itself is released off of
the magnetic bead, with droplets loaded such that at most one
enzyme molecule is present in any droplet.
[0316] Any suitable method can be used for releasing the enzyme
from the ELISA sandwich. Exemplary methods include: 1) competition
of a desthiobiotin-streptavidin interaction using biotin; 2)
reduction of a linker that contains a disulfide bond; 3) enzymatic
cleavage of a linker group. Other variations can be considered, and
Poisson and non-Poisson models can be used to enable high occupancy
loading while still providing quantitative counting.
[0317] With reference to FIGS. 14A and 14B, the invention provides
methods for multiplexing digital droplet reporter enzyme readout.
Several modes for multiplexing a digital assay are provided.
[0318] In certain embodiments, methods including generating
droplets that contain different fluorogenic substrates and enzymes
that produce different fluorescent products. For example, beta
galactosidase and FDG produce FITC, whereas horseradish peroxidase
and Amplex Red produce resorufin. The first method uses completely
separate enzyme and fluorogenic substrate pairs loaded into
droplets at the same time. For example, beta galactosidase and FDG
(FITC is the fluorescent product, with a peak emission wavelength
of 518 nm) can be used to count one set of target molecules, while
horseradish peroxidase and Amplex Red (Resorufin is the fluorescent
product, with a peak emission wavelength of 582 nm) can be used to
simultaneously report on a second set of target molecules, as the
detection wavelengths can be easily distinguished with standard
laser/filter setups.
[0319] Some combinations of different reporter enzymes and
different substrates producing the same fluorescent product can be
used. For example, beta galactosidase catalyzes reactions of FDG
while alkaline phosphatase catalyzes reactions of FDP, with each of
these combinations producing the same fluorescent product (FITC).
Nonetheless, the endpoint product concentration for each single
enzyme can be discriminated when multiplexing.
[0320] FIG. 14A illustrates discrimination by signal strength at
endpoint. While different enzymes and substrates are used, the
substrates generate the same fluorescent product (e.g. FITC).
Careful titration of the endpoint product concentrations can enable
separate counting of each target (e.g., traces with distinct
intensities in FIG. 14A).
[0321] FIG. 14B illustrates discrimination by running at different
time points. In these embodiments, a kinetic assay can be used
rather than an endpoint assay (e.g. alkaline phosphatase and
fluorescein diphosphate yield FITC as a product with much faster
kinetics than beta galactosidase and FDG). One assay (assay #1)
runs for a period of time and produces a detectable product. After
an amount of time, the detectable product hits a plateau in
intensity. Assay #1 is multiplexed with (i.e., run simultaneously
with) assay #2. Assay #2 proceeds more slowly than assay #1. By the
time that assay #2 begins any substantially uptick in activity, the
product of assay #1 has plateaued. Thus, the level of detectable
product from the plateau of assay #1 provides a baseline for the
level of product of assay #2. Such a pattern can be further
multiplexed to any suitable level of plexity.
[0322] In certain embodiments, all droplets within an assay have a
substantially identical size (e.g., even where optical labeling is
used). The same nozzle 105 can be used to generate droplets of
identical size (discussed in greater detail below). Further, since
all droplets are labeled separately for multiplexing, the droplets
can be incubated identically, due to the fact that they can be
handled in the same chamber or apparatus. Similarly, all droplets
can be read with the same optical mechanism (e.g., they all flow
through the same channel past the same detection point on-chip).
Thus, optical sample indexing allows for higher throughput and
better data comparisons.
[0323] While some descriptions herein illustrate digital enzyme
quantification in droplets, systems and methods of the invention
are applicable to any suitable fluid partition. Fluid volumes for
partitions can be provided by chambers made from closing valves,
SLIP-chips, wells, spontaneous breakup to form droplets on a
structured surface, droplets formed using electrowetting methods,
etc.
[0324] Allele-Specific Assay
[0325] FIG. 17 shows an illustration of the concept and workflow
for a digital droplet competitive allele specific enzyme
hybridization (CASE) assay, another example of an upfront assay
that can be coupled to the digital reporter assay readout. The CASE
hybridization assay uses allele-specific oligonucleotide probe
hybridization to select rare genomic targets for binding to the
reporter enzyme and subsequent digital counting.
[0326] Two probe types are used. Wild-type probe 130 is
complimentary to the abundant wild-type allele. Wild type probe 130
includes a minor groove binding motif 134 (either 5' or 3' to the
targeting oligonucleotide).
[0327] Mutant probe 131 is complimentary to the rare mutant allele.
Mutant probe 131 includes an immunoaffinity tag 135 (e.g., TAG) on
one end and a biotin on the other end. Other binding motifs can be
used, but in this example a DIG TAG (which can be bound by an
anti-DIG antibody) and biotin (which can be bound by streptavidin)
are used.
[0328] Wild-type probe 130 with minor groove binder 134
out-competes any non-specific binding of mutant probe 131 to the
wild-type sample DNA, thus limiting hybridization and duplex
formation such that only two duplex species form: wild-type DNA
hybridized to wild-type probe 130, and mutant DNA hybridized to
mutant probe 131. An excess of the two probes over sample DNA is
used, ensuring that each single strand of mutant sample DNA is in a
duplex with one mutant allele probe.
[0329] Following duplex formation, a single-strand nuclease is
added such as S1 nuclease. The nuclease digests any unbound mutant
probe 131 such that tag 135 is dissociated from the biotin.
[0330] Magnetic beads coupled to anti-TAG antibodies are next added
in excess, such that each bead will bind at most one complex of
probe 131 and tag 135. The beads are immobilized using magnet 137
and washed to remove non-specifically bound material (digested
biotinylated probe and the single strand nuclease).
[0331] Streptavidin-coupled reporter enzyme 139 is next added.
After washing, the only enzyme remaining immobilized on the magnet
is present in a one-to-one stoichiometry with the original rare
mutant allele present in the sample DNA. Finally, the reporter
enzyme is counted using the digital droplet reporter enzyme assay,
as above.
[0332] While shown in FIG. 17 in a certain embodiment, a CASE assay
can include any suitable probes for a particular assay. The
anti-TAG antibodies can be provided on any suitable solid
substrate. Reporter enzyme 139 can be any suitable enzyme, such as
any of those discussed herein.
[0333] Optical Labeling
[0334] In certain aspects, the invention provides methods and
devices for optical labeling of samples or assays. Methods of
optical labeling include adding a dye to a sample or assay before
droplet formation. Different samples or assays can be multiplexed
by adding one dye at different concentrations. FIG. 18A shows a
plot in which the x-axis corresponds to the droplet assay signal
and the y-axis corresponds to different concentrations of dye. As
can be seen from FIG. 18A, fluid partitions can include dye in six
different concentrations and still be clearly optically resolvable
from one another. More than six different concentrations can be
used such as, for example, seven, ten, fifty, or more.
[0335] Optical labeling can include further multiplexing by using
additional dyes. For example, FIG. 18B shows a plot in which the
x-axis shows 10 different concentrations of a dye that emits at 590
nm, while the y-axis shows 10 different concentrations of day that
emits at 650 nm. By including two dyes at 10 concentrations each,
100 samples can be separately labeled and run through a single
assay (99 samples are shown in FIG. 18B).
[0336] Multiplexing by these methods provides a high throughput
readout. In certain embodiments, a single dye laser set allows for
6-7 or more (e.g., 10, 25, more) index levels per run. An
additional laser then allows greater numbers (e.g., >30). By
multiplexing at these levels, positive and negative controls can be
run in an assay.
[0337] FIG. 18C shows the high levels of "plexity" for multiplexed
reactions using different concentrations of an optical dye and
different combinations of fluorescently labeled antibodies. As
shown in FIG. 18C, each circle represents a fluid partition. Each
inverted "Y" member represents an antibody. The labels "AB#1",
"AB#2", . . . , "AB#20" refer to twenty different antibodies, and
indicate that in any given fluid partition, all of the antibodies
are the same. The antibodies are shown with two different
fluorescent markers at their heads. A spiky marker indicates a
first color (e.g., green), while a globular marker indicates a
second color (e.g., red). "Optical Dye [1]" indicates a first dye
concentration; "Optical Dye [2]" indicates a second dye
concentration; "Optical Dye [3]" indicates a third dye
concentration; and "Optical Dye [0]" indicates no dye.
[0338] Noting that in any given fluid partition in FIG. 18C all of
the antibodies are the same, it can be seen that each fluid
partition is nonetheless distinctly labeled by a combination of
colored fluorescent markers and dye concentration. That is, no two
partitions in one column contain the same combination of colors of
fluorescent labels. Further, two different optical dyes can be
included in each fluid partition, each separately detectable and
each separately able to be provided at different concentrations.
Imaging an idealized axis extending normal to the surface of FIG.
18C, it can be appreciated that a second optical dye provided in 3
distinct concentrations will allow the assay to be multiplexed with
a "plexity" of 48--i.e., 48 different antibodies can be separately
and distinctly labeled.
[0339] Localized fluorescence
[0340] FIGS. 16A and 16B relate to methods of reaction detection in
fluid partitions that employ a localized fluorescence method of
detection. In these methods, positive (enzyme-containing)
partitions are identified and counted using changes in the
localized fluorescence of fluorescent molecules in the partitions.
In general, methods of the invention employ any detection method
that detects a localized concentration of a target in a fluid
partition. For example, in certain embodiments, enzyme activity
creates a binding surface for fluorescent molecules that can be
monitored by localized fluorescent readout.
[0341] In the example shown in FIG. 16A, the enzyme Src kinase
(Src) phosphorylates a Src substrate peptide that is immobilized on
a bead. Phosphorylation of the Src substrate peptide creates
binding motifs for the fluorescent reporter SH2-FITC. Thus,
SH2-FITC binds to the bead-bound Src substrate phospho-peptide as
shown in the last step of FIG. 16A.
[0342] FIG. 16B illustrates a reaction-negative fluid partition on
the left side and a reaction-positive fluid partition on the right.
Localization of the fluorescent molecules onto the bead surface can
be detected as an increased signal on the bead surface, a decreased
signal in a portion of the fluid partition (e.g., throughout the
partition volume), or both. Many other enzymes, binding motifs, and
fluorescent reporters can be used.
[0343] Any suitable method can be used to detect the pattern of
localized fluorescence within a fluid partition. For example, in
certain embodiments, fluid droplets are flowed through a narrow
channel that forces the droplets to exhibit an elongated shape (as
shown in FIG. 16B). As the droplets pass a laser detector,
reaction-negative droplets give a uniform low-level fluorescence
intensity along their length, while reaction-positive droplets show
a spike corresponding to when the bead-bound fluorescent reporter
passes the laser detector. In addition, when fluors become
localized onto particle(s) within the droplet (e.g. on a bead or a
cell surface) there is a coordinate depletion of the initial
fluorescence in the regions of the droplet that do not contain the
particle(s). Various signal processing algorithms can be used to
combine local signal increases and background signal decreases into
a more robust detection method.
[0344] FIGS. 19A and 19B show a workflow for a localized
fluorescence binding assay. Cell sample 201 provides cells that are
flowed into droplet generator 203. An optically labeled library 211
(e.g., according to multiplexing embodiments discussed elsewhere
herein) is flowed such that droplets from library 211 meet cell
droplets from sample 201 in droplet merger 207. Droplets leave
merger 207 having optical labels and reagents (e.g., fluorescent
antibody to a biomarker of interest) and incubated (not shown) and
then flow past detector 211. In certain embodiments, detector 211
includes a narrow channel portion 225 as discussed below in
reference to FIG. 20. Libraries are discussed in U.S. Pub.
2010/0022414, the contents of which are hereby incorporated by
reference in their entirety for all purposes.
[0345] FIG. 19B illustrates the principle of detection in detector
211. A fluid partition at the top of FIG. 19B includes a plurality
of fluorescently labeled antibody 215 and a cell presenting an
antigen of interest. After incubation, the fluid partition appears
as at the bottom of FIG. 19B, where it is shown containing only one
concentration of fluorescent marker 219, as all of the labeled
antibody has bound to the cell.
[0346] FIG. 13 shows detection of CD45 on the surface of U937
monocytes with systems and methods of the invention. An optically
labeled antibody library is flowed into the system from one input
to merge with cells from the other (tracing the cell path across
the figure from left to right). The streams merge and the droplets
combine, after which the droplets are incubated "off chip", as
indicated by the middle panel, in which the droplets can be seen
without a surrounding microfluidic channel. After incubation, the
droplets are flowed back onto a chip into a channel, after which
they are flowed through narrow channel portion 225. Flowing through
the narrow area causes the cells to elongate. A laser detector
reads across the channel in the middle of narrow portion 225.
[0347] As the cells pass the laser, the detector detects
fluorescence and creates a digital trace that can be stored and
analyzed on a computer. The digital trace can be viewed on a
display as a graph, in which the x-axis represents time (and
corresponds to the length of the droplets as they pass the
detector), and the y-axis corresponds to signal intensity (see
example traces in FIGS. 21A-21C).
[0348] Throughput of the assay is adjustable and can be performed
at, for example, about 1500 droplets per second (in the illustrated
example, corresponding to about 150 cells/second). Incubation time
(e.g., off chip, as shown in middle panel) can be adjusted. On
chip, incubation can be adjusted by use of a delay line (i.e.,
wider portion of channel where flow slows). The interior diameter
of narrow portion 225 can be adjusted to tune droplet elongation.
For example, interior diameter can be about 10 micrometers or 15
micrometers.
[0349] In certain embodiments, localized fluorescence is used to
screen for single-chain variable fragment (scFv) peptides in
droplets. For example, a library of bacterially produced scFv's can
be developed. Following a procedure as outlined in FIG. 19A,
transformed bacteria cells 201 are encapsulated and incubated at
37.degree. C. After incubation the droplets are combined with
droplets that contain beads, antigens and a detection antibody from
library 211.
[0350] The droplet based binding assays utilize localized
fluorescence detection of scFv binding as shown in FIG. 19B.
Specifically, the diffuse signal becomes bright and localized on
the capture bead.
[0351] Positive droplets are detected according to a localized
fluorescence method such as the one illustrated in FIG. 20. In
order to get a reading of the droplet and fluorescent level
therein, the droplet is elongated for detection in narrow channel
portion 225 as shown in FIG. 20.
[0352] The positive droplets can then be broken and the contents
recovered and sequenced. The process is able to screen for scFv's
in droplets at a rate of about 1.times.10.sup.6 per hour. Localized
fluorescence and scFv screening is discussed in U.S. Pub.
2010/0022414, the contents of which is hereby incorporated by
reference in its entirety.
[0353] FIGS. 21A-21C illustrate single droplet traces including
optical labels in a localized fluorescence assay. Note that the
trace in each of FIGS. 14A-14C may be shown in a separate color for
example on a single display. In each figure, the axes are the same
and the traces can be obtained in a single run detecting three
differently colored labels simultaneously. FIG. 21A shows a trace
indicating measurement of cell viability stain. Here, calcein AM
can be used, which gives a low intensity signal until it is cleaved
by esterases located only inside a cell. The two spikes in FIG. 21A
(at approximately 110 ms and approximately 300 ms) indicate the
second and fourth droplets, respectively, to pass the fluorescent
detector. These two spikes indicate that those droplets included a
viable cell.
[0354] FIG. 21B shows a signal strength of FITC signal for droplets
that include a labeled binder (e.g., anti-CD45-FITC). A spike
(e.g., at 30 ms) indicates binding (i.e., a localized increase in
fluorescence) while "bulges" centered on about 60 ms, 125 ms, 190
ms, 355 ms, 420 ms, and 480 ms (corresponding to the second, third,
fourth, sixth, seventh, and eight droplets to flow past the
detector, respectively) indicate dispersed, low-level fluorescence
and thus indicate no binding. In conjunction with to FIG. 21A,
which indicate the presence of a viable cell in the fourth droplet,
FIG. 21B indicates that the viable cell is binding anti-CD45.
[0355] It will appreciated in viewing FIG. 21B that the spike at
about 300 ms is surrounded by a low-level "bulge" spanning about
275 ms to about 320 ms. A positive assay can be detected by the low
level of the signal across this "bulge" as compared to the signal
level in the reaction-negative bulges (e.g., consistently above
about 0.1). Thus, localized fluorescence can be detected (and
positive or negative reaction fluid partitions identified) by
localized increases of fluorescence, partition-wide decreases in
fluorescence, or both. For example, a ratio could be calculated
between the localized increase and the partition-wide decrease, and
this ratio would be a sensitive indicator and/or measurement of
localization within the partition. Moreover, measurement of the
total volume of the partition may also be taken into consideration
to further scale or normalize the partition-wide decrease.
[0356] FIG. 21C illustrates optical labeling that was included with
the droplets shown in FIG. 14B (shown here as a blue trace). Here,
control droplets were labeled with a low concentration of dye,
while test droplets (including the anti CD45-FITC) were labeled
with a high concentration of the dye. Thus, the first three
signals, the fifth signal, and the sixth signal indicate that the
corresponding fluid partitions are negative controls. while the
fourth and seventh droplets were test partitions. An independent
(e.g., downstream or upstream) channel can be used to count cells,
count droplets, sort cells, or perform other steps.
[0357] FIGS. 22A-22C give single droplet traces with a scatter plot
and histogram. These figures illustrate the results of an assay for
IgG and CD45. Here, two traces are superimposed. One trace
indicates a level of fluorescence from FITC at locations within a
fluid partition (similar to that shown in FIG. 21B). The other
trace indicates a dye used in different concentrations to optically
label the antiCD45 test partition distinctly from the IgG test
partitions (i.e., the trace is similar to that in FIG. 21C). In
FIG. 22A, the appearance of a signal at about 125 ms with no
"shoulders" (i.e., lacking the trace that corresponds to FIG. 21B
as discussed above) indicates a fluid partition that includes no
FITC-labeled target. This indicates an unmerged cell droplet, i.e.,
a droplet that passed through merger 207 without receiving reaction
reagents.
[0358] The signal at about 725 ms indicates an antiCD45-FITC
positive droplet while the signal at about 1050 ms indicates an
IgG-FITC positive droplet (the tenth and fourteenth droplets to
pass the detector, respectively). Here, the tenth and fourteenth
droplets are distinguished based on the concentration of the
optical labeling dye (corresponding to the trace shown in FIG.
21C).
[0359] FIG. 22B gives a scatter-plot of results relating the traces
shown in FIG. 22A. Along the x-axis is plotted an intensity of the
antibody label and the y-axis corresponds to an intensity of a live
cell stain. Population 305 corresponds to a CD45 positive
population of fluid partitions and population 301 corresponds to an
IgG positive population of fluid partitions. As unmerged droplets
are also counted (e.g., second signal in FIG. 22A), the total
number of droplets that is prepared can be accounted for. FIG. 22C
shows a histogram corresponding to the plot shown in FIG. 22B. The
x-axis is the unitless Bin and the y-axis is frequency. Such
histograms are known in the art for display of flow cytometry
results.
[0360] FIGS. 23A-23C show ways of controlling or adjusting the
dynamic range of a localized fluorescence assay. As shown in FIG.
23A, an amount of fluorescently labeled antibody 215 can be
provided in a determined relationship to a known or expected number
of cell surface markers on cell 219. By controlling the
concentration of antibody added (e.g., through a series of
calibration runs), a desired signal of bound antibody can be
established that is greater than the dispersed background
signal.
[0361] FIG. 23B shows a method for lowering a strength of a
background signal (shown to scale with the diagram shown in FIG.
23A). By making the fluid partitions larger, fluorescent antibody
215 will be more dispersed when in the unbound mode. However, when
bound on cell 219, the bound signal will be substantially the same
as between FIGS. 23A and 23B. Thus, by controlling a volume of a
fluid partition, the signal gain can be modulated and the dynamic
range of the assay adjusted.
[0362] FIG. 23C shows another way of adjusting the dynamic range by
attenuating the background signal. Here, partitions included
fluorescent antibody 215 are merged with partitions (only one is
shown) that each contain at least one cell 219 (which may or may
not be expressing the cell surface marker, i.e., positive and
negative reaction partitions, where only positive is shown). A
buffer is added to the fluid partitions. For example, droplets
containing buffer are merged with assay droplets according to
methods described herein. The buffer effectively dilutes the
background signal. In certain embodiments, addition of the buffer
does not substantially change the localized fluorescence signal
(e.g., associated with reaction-positive instances of cell 219).
The approach illustrated in FIG. 23C allows for a binding or
incubation step to proceed at a substantially higher concentration
(e.g., corresponding to concentration illustrated by left panel of
FIG. 23A), while the detection step can employ a background
(non-localized) fluorescence at a different concentration.
[0363] Localized fluorescence is applicable in combination with
other assays and methods described herein including, for example,
bead-capture based assays that involve rupturing or opening a
partition to release the contents (as the signal may remain
localized on the captured bead when it is, for example, but back
into a fluid partition.
[0364] Digital Distribution Assays
[0365] In some aspects, the invention provides systems and methods
for detecting distributions of molecules. For example, methods of
the invention are useful to detect amounts of gene expression and
proteins. In particular, methods of the invention are useful to
detect protein aggregation or complexes. In one example, methods of
the invention are used to detect anomalous or unexpected
distributions of protein by conducting a chemical reaction that
produces a detectable report indicative of the presence and amount
of the protein (e.g., an ELISA or similar assay). The results of
these assays are compared to expected results or control samples in
order to determine whether there is an anomalous distribution or
amount of a particular protein.
[0366] In certain embodiments, the invention is used to determine
anomalous protein aggregation. In samples from the anomalous
subset, peptide aggregation or complexes can be detected by the
distribution of reactions in fluid partitions (e.g., digitally), as
compared to the distribution from the non-anomolous rest of the
population. Such a phenomenon may arise where a protein is known or
suspected to exhibit alternative splicing, binding, or folding
pathways or is in a stable complex. For example, one protein may be
known to be differently processed (e.g., cleaved by hydrolysis or
proteolysis; modified such as by phosphorylation; cross-linked;
etc.) such that a complex or aggregate is formed when individuals
have some disease propensity or state.
[0367] In Alzheimer's disease, for example, when amyloid precursor
protein (APP) undergoes proteolysis, the resulting fragments can
form aggregations such as fibrils. It is particularly hypothesized
that a protein called tau becomes hyper-phosphorylated, causing the
aggregation that results in neurofibrillary tangles and neuron
disintegration. Thus, Alzheimer's disease may generally be
associated with an aggregation of beta-amyloid protein.
Accumulation of aggregated amyloid fibrils may induce apoptosis and
may inhibit other critical enzyme functions.
[0368] This aggregation among proteins can result in there being an
anomalous (e.g., non-Poisson) distribution of those proteins when
assayed under dilution conditions which should produce Poisson-like
statistic distributions, and this non-standard distribution can
provide the basis for a digital detection assay.
[0369] Thus, digital assays are provided that relate to
distributions of proteins and other cellular components. Assays
according to methods of the invention involve determining an actual
distribution and comparing that to an expected distribution.
[0370] FIG. 26 shows histograms corresponding to three different
stages of protein aggregation. The histogram labeled "normal"
indicates an expected distribution of proteins that are not
aggregating. The histogram labeled "early" indicates a detected
distribution in which proteins have started exhibiting aggregation.
The histogram labeled "late" indicates a detected distribution of
proteins that exhibit extensive aggregation.
[0371] Assays for digital distribution assays include any assay
that can indicate a number of protein molecules per fluid
partition. For example, where a protein has one epitope that is
available while in either monomer or aggregated form, an assay can
include antibodies bound to that epitope(s) coupled to reporters in
each fluid partition. Each antibody can be linked (covalently or
non, e.g., through biotin) to an enzyme. Each fluid partition is
provided with fluorescently labeled substrate for the enzyme that
fluoresces when the enzyme catalyzes a reaction on the
substrate.
[0372] A sample is taken from a subject (e.g., a blood sample from
a patient). Any desired concentration, isolation, or preparation
steps are performed such that the target molecules become
associated with a reporter for digital detection in partitions. The
sample including the protein of interest and reporter is
partitioned into fluid partition (this illustrative example is
discussed in terms of a protein, but it will be appreciated that
methods of the invention can detect a distribution of any target
thus indicating aggregation or a complex).
[0373] Where the fluorescent reporter is the same for all
antibodies, the number of proteins in each fluid is indicated by
the fluorescent intensity of the signal. This can be run in
"kinetic" mode, or "end point" mode. In end-point mode, each
reaction is allowed to run to substantially completion, at which
point the amount of product has substantially plateaued. The amount
of product is then detected in each fluid partition and the number
of proteins in each partition is correlated to the strength of the
signal. The same correlation applies in kinetic mode. However,
measurements are made at one or more time points before the amount
of product plateaus. Where a whole series of time points is
collect, that time series also gives information about enzyme
kinetics.
[0374] In certain embodiments, where an assay is to be run in
kinetic mode, a time point for measurement is first established in
an independent calibration run. A series of droplets are made and
incubated. The droplets are provided with a dilution of a protein
that is known to aggregate and a reporter system (e.g., an
enzyme-linked antibody that binds to an epitope of the protein).
Fluorescence intensity is measured at multiple time points. For
example, where measurement is on-chip and incubation is-off chip,
all steps are performed at a temperature at which the enzyme
exhibits no activity but incubation, which involves a temperature
at which the enzyme exhibits activity. For example, .beta.-gal
exhibits no activity at 4.degree. C., and is active at 37.degree.
C. For any given N-mer of aggregated protein, the fluid partitions
that include one such N-mer will exhibit a characteristic sigmoidal
time curve after a series of measurements of fluorescence are
taken. To discriminate among some set of N-mers, a point along the
time trace graph at which the corresponding sigmoidal time curves
exhibit distinct heights is take for the measurement time.
[0375] In some embodiments, an expected distribution (i.e., from a
healthy individual) is known, and an assay need only discriminate
between a detected distribution and an expected distribution. Thus
where, for example, an expected distribution has a known ratio of
partitions that include 1 target to partitions that include more
than one target, an assay can include detecting a ratio of
partitions that include 1 target to partitions that include more
than one target that is statistically significantly different than
the known ratio. In some embodiments, a non-aggregating particle is
expected to exhibit a Poisson or near-Poisson distribution, and an
assay include detecting a number of droplets that contain greater
than one target molecule, wherein the detected number does not
agree with Poisson or near-Poisson distribution. In some
embodiments, a Poisson distribution at a certain dilution is
expected to yield a vanishingly small number of fluid partitions
that include two target molecules and zero fluid partitions that
include greater than two. Thus, an assay can detect a statistically
significant number of partitions that includes more than one
molecule to indicate the presence of protein aggregation and thus
indicate the presence of a physiological condition. For reference,
FIG. 10A shows a measurement result that may indicate protein
aggregation (i.e., not match the expected distribution or
Poisson).
[0376] In an alternative embodiment, each fluid partition is
provided with enzyme linked antibodies in which all of the
antibodies are the same, but a fraction (e.g., half) is linked to
one enzyme that operates on one substrate to generate one reporter,
and another portion of the antibodies are linked to another enzyme
that catalyzes a reaction that produces a different reporter. In
this example, assuming one reporter is blue and one is yellow, some
number of fluid partitions that have more than one protein will
produce both the blue and the yellow reporter. If the protein is
not aggregating, at a certain dilution, it can be expected that
blue and yellow will be found together in only some number of
droplets (e.g., zero, or 0.00001% of them). In this example,
detecting a blue with yellow in a greater number of droplets (e.g.,
.05%, 1%, etc.) indicates the protein is aggregating.
[0377] In distribution assays in which signal strength indicates a
number of proteins in the droplet, for a given dilution of sample
into droplets, assuming random distribution of a non-aggregating
protein, there will be a characteristic expected distribution. Even
for a protein that exhibits some aggregation in normal conditions,
there will be a characteristic expected distribution. In certain
embodiments, the expected distribution is predicted by Poisson or
is Poisson-like. A substantially large number of droplets will
contain zero proteins. A substantially majority of the droplets
that contain any protein will contain 1 protein. Some small (maybe
vanishingly small) number of protein-containing droplets will
contain 2 or more.
[0378] In the case where the proteins aggregate, such an expected
distribution will not obtain. For example, for a fully aggregated
protein (e.g., "late" stage) that aggregates into 4-mers, a
substantially large number of droplets will contain zero proteins
and a substantial majority of the droplets that contain any protein
will contain four proteins, as is illustrated in FIG. 26.
[0379] Furthermore, aggregation can be detected over time. That is,
early stages of aggregation will exhibit an non-expected
distribution. In some embodiments, protein folding into tertiary
structures and/or quaternary assembly is studied over the course
of, for example, minutes, hours, or days. In certain embodiments, a
distribution of aggregation in a sample indicates a stage of
progression of an aggregation-related condition that takes months,
years, or decades to progress. For example, late stage
distributions may only be expected after about 50 or 75 years.
However, an assay run at an earlier time period (e.g., 15 or 25
years) may indicate an "early" stage aggregation distribution, as
shown in FIG. 26.
[0380] In certain aspects, the invention provides a method for
detecting a physiological condition in a human that includes
forming fluid partitions that include components of a chemical
reaction, in which at least one of the components has a detectable
label that is acted on by the chemical reaction. The reaction
proceeds in the partitions, and a number of reaction-positive
partitions is identified and an amount of the component is
determined. A statistically expected distribution or amount of the
component is computed and compared to the actual distribution or
amount. The results of the comparison can indicate the presence of
an aggregation phenomenon.
[0381] In some embodiments, the invention provides a method for
testing a response to a treatment for a condition or for monitoring
a progression of a condition. A condition includes conditions that
characterized by aggregation such as, for example, Alzheimer's
disease. Determining a response to treatment is included in methods
for development of treatments such as, for example, drug
development. In some aspects, a candidate drug is tested (e.g.,
administered). A sample is taken and a distribution of molecules is
determined. The molecules can be a protein such as beta-amyloid.
Based on the determined distribution, an effectiveness of the drug
is evaluated and a recommendation can be made. In some aspects,
progress of a disease is monitored by methods that include taking a
sample and determining a distribution of molecules within the
sample according to methodologies described herein.
[0382] Digital distribution assays of the invention are highly
sensitive and can proceed quickly with very small samples. For
example, a 5 mL biological sample can be assayed in less than a day
(e.g., about an hour), and a stage of aggregation can be
determined.
[0383] In certain embodiments, the protein is beta-amyloid. A
sample of human blood can be taken for the assay. An expected
distribution can be calculated (e.g., statistically according to
Poisson), derived empirically (e.g., sampling numerous members of a
population for the majority pattern), or obtained from a reference
such as a digital file or chart. Accordingly, in some embodiments,
the invention offers a blood test for Alzheimer's disease. In
certain embodiments, an assay of the invention can be performed on
a patient at any life stage (e.g., childhood, teens, etc.). It will
be appreciated that any aggregation pattern may be subject to
detection by a digital distribution assay and aggregation generally
includes the reverse phenomenon of fragmentation. As such, targets
for a digital distribution assay may include peptides, nucleic
acids, carbohydrates, lipids, or other molecules. A distribution
assay can determine a stage of fragmentation as well as a stage of
polymerization (e.g., esterification, poly peptide formation,
carbohydrate cross-linking, synthetic polymerization, etc.).
[0384] Droplet Formation
[0385] Methods of the invention involve forming sample droplets.
The droplets are aqueous droplets that are surrounded by an
immiscible carrier fluid. Methods of forming such droplets are
shown for example in Link et al. (U.S. patent application numbers
2008/0014589, 2008/0003142, and 2010/0137163), Stone et al. (U.S.
Pat. No. 7,708,949 and U.S. patent application number
2010/0172803), Anderson et al. (U.S. Pat. No. 7,041,481 and which
reissued as RE41,780) and European publication number EP2047910 to
RainDance Technologies Inc. The content of each of which is
incorporated by reference herein in its entirety. Droplets can be
formed with various uniform sizes, a non-uniform size, or a range
of sizes.
[0386] FIG. 24 shows an exemplary embodiment of a device 100 for
droplet formation. Device 100 includes an inlet channel 101, and
outlet channel 102, and two carrier fluid channels 103 and 104.
Channels 101,102,103, and 104 meet at a junction 105. Inlet channel
101 flows sample fluid to the junction 105. Carrier fluid channels
103 and 104 flow a carrier fluid that is immiscible with the sample
fluid to the junction 105. Inlet channel 101 narrows at its distal
portion wherein it connects to junction 105 (See FIG. 25). Inlet
channel 101 is oriented to be perpendicular to carrier fluid
channels 103 and 104. Droplets are formed as sample fluid flows
from inlet channel 101 to junction 105, where the sample fluid
interacts with flowing carrier fluid provided to the junction 105
by carrier fluid channels 103 and 104. Outlet channel 102 receives
the droplets of sample fluid surrounded by carrier fluid.
[0387] The sample fluid 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 enzymes
can be used. The carrier fluid is immiscible with the sample fluid.
The carrier fluid can be a non-polar solvent, decane (e g.,
tetradecane or hexadecane), fluorocarbon oil, silicone oil or
another oil (for example, mineral oil).
[0388] In certain embodiments, the carrier fluid contains 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. In some applications, performance is improved by
adding a second surfactant. 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
a 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.
[0389] In certain embodiments, the droplets may be coated with a
surfactant. Preferred surfactants that may be added to the carrier
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).
[0390] In certain embodiments, the carrier fluid may be caused to
flow through the outlet channel so that the surfactant in the
carrier fluid coats the channel walls. 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
carrier fluid.
[0391] Unknown or known analytes or compounds over a very wide
dynamic range of concentrations can be merged with droplets
containing single or multiple enzyme molecules and reporters via
the use of a Taylor dispersion that forms upstream of the droplet
forming nozzle. Co-encapsulation of an optical dye that has
similarly been dispersed via a Taylor flow profile can be used to
track the analyte/compound concentration. (see, e.g., Miller, et
al., PNAS 109(2):378-383 (2012); U.S. Pat. 7,039,527; and U.S. Pub.
2011/0063943, the contents of which are hereby incorporated by
reference in their entirety).
[0392] In certain embodiments, an aliquot of a sample is aspirated
into a PEEK tubing, such that a slug of the sample is in the PEEK
tubing with a substantially uniform concentration. The tubing is
inserted into a port into a microfluidic channel, and the sample
enters the channel forming a concentration gradient in the aqueous
phase, the gradient generally following Taylor-Aris dispersion
mechanics. The sample fluid joins the carrier fluid first having a
vanishingly small concentration and then increasing up a gradient
asymptotically until a maximum (e.g., 10 micromolar) is reached,
after which it decreases similarly The fluid is flowed to a droplet
nozzle, where a series of droplets are made. The sample can be
pushed into the channel via a pump, a plunger, or be driven by
pressure (e.g., from a gas tank).
[0393] In certain embodiments, the dispersion is created for each
partition of a plurality (e.g., for numerous or all wells from a 96
well plate, in which every well has a target sample such as a small
molecule). Systems and methods of the invention provide a series of
microdroplets in which the contents have a controlled concentration
gradient through a simple injection procedure. Further, the sample
can be spiked with a dye having a known concentration, and the dye
concentration can be measured downstream (e.g., during or after any
other assay). The measured dye concentrations can be used to
determine the sample concentration.
[0394] By methods and systems provided here, a controlled gradient
of concentrations of one or more sample aliquots can be merged with
droplets that include a single target such as a single enzyme
molecule. Enzyme activity can be assayed and kinetics studied. For
example, individual enzyme molecules can be tested against a
concentration range of activators, inhibitors, etc.
[0395] Substrates or other reaction or reporter components may be
co-encapsulated into droplets with the enzyme without mixing before
droplet formation by `co-flow` of the separate components. When
co-flow is used, each separate component is flowed to the
microfluidic channel upstream of the droplet-forming nozzle, and
both components flow in a laminar fashion to the nozzle without
mixing. In co-flow methods, components are flowed in parallel
streams through a channel. Due to flow dynamics, the contents of
the streams do not mix until they hit the lambda injector or
droplet forming nozzle. Two separate streams can be co-flowed, or
three, four, or more. Where hardware is configured for N streams,
and N-1 streams containing reaction components are desired, a
"dummy" stream of water or saline can be included.
[0396] Another technique for forming droplets including enzymes and
substrates from different fluids or previously generated droplets
involves droplet merging. The merging of droplets can be
accomplished using, for example, one or more droplet merging
techniques described for example in Link et al. (U.S. patent
application numbers 2008/0014589, 2008/0003142, and 2010/0137163)
and European publication number EP2047910 to Raindance Technologies
Inc. In embodiments involving merging of droplets, two droplet
formation modules are used. A first droplet formation module
produces the droplets including enzymes. A second droplet formation
module produces droplets that contain substrate. The droplet
formation modules are arranged and controlled to produce an
interdigitation of droplets flowing through a channel. Such an
arrangement is described for example in Link et al. (U.S. patent
application numbers 2008/0014589, 2008/0003142, and 2010/0137163)
and European publication number EP2047910 to Raindance Technologies
Inc.
[0397] Droplets are then caused to merge, producing a droplet that
includes enzymes and substrates. Droplets may be merged for example
by: producing dielectrophoretic forces on the droplets using
electric field gradients and then controlling the forces to cause
the droplets to merge; producing droplets of different sizes that
thus travel at different velocities, which causes the droplets to
merge; and producing droplets having different viscosities that
thus travel at different velocities, which causes the droplets to
merge with each other. Each of those techniques is further
described in Link et al. (U.S. patent application numbers
2008/0014589, 2008/0003142, and 2010/0137163) and European
publication number EP2047910 to Raindance Technologies Inc. Further
description of producing and controlling dielectrophoretic forces
on droplets to cause the droplets to merge is described in Link et
al. (U.S. patent application number 2007/0003442) and European
Patent Number EP2004316 to Raindance Technologies Inc. Additional
methods may be used for controlled droplet merging, for example by
altering the flow profiles of paired droplets via properly
constrained microfluidic channel design. Merges can be performed in
a successive fashion, enabling step-wise addition of substrates,
reagents, or reaction step components.
[0398] Another approach to forming a droplet including enzymes and
substrates involves forming a droplet including enzymes, and
contacting the droplet with a fluid stream including substrate, in
which a portion of the fluid stream integrates with the droplet to
form a droplet including enzymes and substrates. In this approach,
only one phase needs to reach a merge area in a form of a droplet.
Further description of such method is shown in the co-owned and
co-pending U.S. patent applications to Yurkovetsky, (U.S. patent
application Ser. No. 61/441,985 and U.S. patent application Ser.
No. 13/371,222), the content of which is incorporated by reference
herein in its entirety.
[0399] A droplet is formed as described above. After formation of
the droplet is contacted with a flow of a second sample fluid
stream. Contact between the droplet and the fluid stream results in
a portion of the fluid stream integrating with the droplet to form
a droplet including nucleic acid from different samples.
[0400] The monodisperse droplets of the first sample fluid flow
through a first channel separated from each other by immiscible
carrier fluid and suspended in the immiscible carrier fluid. The
droplets are delivered to the merge area, i.e., junction of the
first channel with the second channel, by a pressure-driven flow
generated by a positive displacement pump. While droplet arrives at
the merge area, a bolus of a second sample fluid is protruding from
an opening of the second channel into the first channel.
Preferably, the channels are oriented perpendicular to each other.
However, any angle that results in an intersection of the channels
may be used.
[0401] The bolus of the second sample fluid stream continues to
increase in size due to pumping action of a positive displacement
pump connected to channel, which outputs a steady stream of the
second sample fluid into the merge area. The flowing droplet
containing the first sample fluid eventually contacts the bolus of
the second sample fluid that is protruding into the first channel.
Contact between the two sample fluids results in a portion of the
second sample fluid being segmented from the second sample fluid
stream and joining with the first sample fluid droplet to form a
mixed droplet. In certain embodiments, each incoming droplet of
first sample fluid is merged with the same amount of second sample
fluid.
[0402] In certain embodiments, an electric charge is applied to the
first and second sample fluids. Description of applying electric
charge to sample fluids is provided in Link et al. (U.S. patent
application number 2007/0003442) and European Patent Number
EP2004316 to Raindance Technologies Inc. (Lexington, Mass.), the
content of each of which is incorporated by reference herein in its
entirety. Electric charge may be created in the first and second
sample fluids within the carrier fluid using any suitable
technique, for example, by placing the first and second sample
fluids within an electric field (which may be AC, DC, etc.), and/or
causing a reaction to occur that causes the first and second sample
fluids to have an electric charge, for example, a chemical
reaction, an ionic reaction, a photocatalyzed reaction, etc.
[0403] The electric field, in some embodiments, is generated from
an electric field generator, i. e., a device or system able to
create an electric field that can be applied to the fluid. The
electric field generator may produce an AC field (i.e., one that
varies periodically with respect to time, for example,
sinusoidally, sawtooth, square, etc.), a DC field (i.e., one that
is constant with respect to time), a pulsed field, etc. The
electric field generator may be constructed and arranged to create
an electric field within a fluid contained within a channel or a
microfluidic channel. The electric field generator may be integral
to or separate from the fluidic system containing the channel or
microfluidic channel, according to some embodiments.
[0404] Techniques for producing a suitable electric field (which
may be AC, DC, etc.) are known to those of ordinary skill in the
art. For example, in one embodiment, an electric field is produced
by applying voltage across a pair of electrodes, which may be
positioned on or embedded within the fluidic system (for example,
within a substrate defining the channel or microfluidic channel),
and/or positioned proximate the fluid such that at least a portion
of the electric field interacts with the fluid. The electrodes can
be fashioned from any suitable electrode material or materials
known to those of ordinary skill in the art, including, but not
limited to, silver, gold, copper, carbon, platinum, copper,
tungsten, tin, cadmium, nickel, indium tin oxide ("ITO"), etc., as
well as combinations thereof. In some cases, transparent or
substantially transparent electrodes can be used.
[0405] The electric field facilitates rupture of the interface
separating the second sample fluid and the droplet. Rupturing the
interface facilitates merging of bolus of the second sample fluid
and the first sample fluid droplet. The forming mixed droplet
continues to increase in size until it a portion of the second
sample fluid breaks free or segments from the second sample fluid
stream prior to arrival and merging of the next droplet containing
the first sample fluid. The segmenting of the portion of the second
sample fluid from the second sample fluid stream occurs as soon as
the shear force exerted on the forming mixed droplet by the
immiscible carrier fluid overcomes the surface tension whose action
is to keep the segmenting portion of the second sample fluid
connected with the second sample fluid stream. The now fully formed
mixed droplet continues to flow through the first channel.
[0406] Droplet Sorting
[0407] Methods of the invention may further include sorting the
droplets. A sorting module may be a junction of a channel where the
flow of droplets can change direction to enter one or more other
channels, e.g., a branch channel, depending on a signal received in
connection with a droplet interrogation in the detection module.
Typically, a sorting module is monitored and/or under the control
of the detection module, and therefore a sorting module may
correspond to the detection module. The sorting region is in
communication with and is influenced by one or more sorting
apparatuses.
[0408] A sorting apparatus includes 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 that is in communication with a sorting region and a main
channel. The main channel can communicate with two or more branch
channels at the sorting module or branch point, forming, for
example, a T-shape or a Y-shape. Other shapes and channel
geometries may be used as desired. Typically, a branch channel
receives droplets 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.
[0409] A characteristic of a fluidic droplet may be sensed and/or
determined in some fashion, for example, as described herein (e.g.,
fluorescence of the fluidic droplet may be determined), and, in
response, an electric field may be applied or removed from the
fluidic droplet to direct the fluidic droplet to a particular
region (e.g. a channel). In certain embodiments, a fluidic droplet
is sorted or steered by inducing a dipole in the uncharged fluidic
droplet (which may be initially charged or uncharged), and sorting
or steering the droplet using an applied electric field. The
electric field may be an AC field, a DC field, etc. For example, a
channel containing fluidic droplets and carrier fluid, divides into
first and second channels at a branch point. Generally, the fluidic
droplet is uncharged. After the branch point, a first electrode is
positioned near the first channel, and a second electrode is
positioned near the second channel. A third electrode is positioned
near the branch point of the first and second channels. A dipole is
then induced in the fluidic droplet using a combination of the
electrodes. The combination of electrodes used determines which
channel will receive the flowing droplet. Thus, by applying the
proper electric field, the droplets can be directed to either the
first or second channel as desired. Further description of droplet
sorting is shown for example in Link et al. (U.S. patent
application numbers 2008/0014589, 2008/0003142, and 2010/0137163)
and European publication number EP2047910 to Raindance Technologies
Inc.
[0410] Droplet sorting relates to methods and systems described
herein by allowing one to detect the effect of an enzymatic
reaction in a fluid partition (or absence thereof) and to
selectively examine that specific partition further. For example,
where a specific class of molecule is being assayed for, an
enzyme-positive droplet can be sorted and separated from the rest.
That specific droplet can be "broken open" and its contents further
examined. For example, a cDNA library can be prepared from all RNA
(e.g., mRNA) in that droplet. Nucleic acids can then be
sequenced.
[0411] Release from Droplets
[0412] Methods of the invention may further involve releasing the
enzymes or products or other identifiable material from the
droplets for further analysis. Methods of releasing contents from
the droplets are shown for example in Link et al. (U.S. patent
application numbers 2008/0014589, 2008/0003142, and 2010/0137163)
and European publication number EP2047910 to Raindance Technologies
Inc.
[0413] In certain embodiments, sample droplets are allowed to cream
to the top of the carrier fluid. By way of non-limiting example,
the carrier fluid can include a perfluorocarbon oil that can have
one or more stabilizing surfactants. The droplet rises to the top
or separates from the carrier fluid by virtue of the density of the
carrier fluid being greater than that of the aqueous phase that
makes up the droplet. For example, the perfluorocarbon oil used in
one embodiment of the methods of the invention is 1.8, compared to
the density of the aqueous phase of the droplet, which is 1.0.
[0414] The creamed liquids are then placed onto a second carrier
fluid which contains a de-stabilizing surfactant, such as a
perfluorinated alcohol (e.g. 1H,1H,2H,2H-Perfluoro-l-octanol). The
second carrier fluid can also be a perfluorocarbon oil. Upon
mixing, the aqueous droplets begins to coalesce, and coalescence is
completed by brief centrifugation at low speed (e.g., 1 minute at
2000 rpm in a microcentrifuge). The coalesced aqueous phase can now
be removed and further analyzed.
Definitions
[0415] 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. 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. 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. The term "molecule" means any
distinct or distinguishable structural unit of matter comprising
one or more atoms, and includes for example polypeptides and
polynucleotides.
[0416] 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.
[0417] 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.
[0418] 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.
[0419] 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.
[0420] 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.
[0421] "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.
[0422] 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.
[0423] 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.
[0424] 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.
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.
[0425] A "small molecule" or "small molecule chemical compound" as
used herein, is meant to refer to a composition that has a
molecular weight of less than 500 Daltons. Small molecules are
distinguished from polynucleotides, polypeptides, carbohydrates and
lipids.
[0426] 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,
orparamagnetic 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.
[0427] 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.
[0428] 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.
[0429] 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, saccharrides,
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.
The invention is further described below, by way of the following
examples. The examples also illustrate useful methodology for
practicing the invention. These examples do not limit the claimed
invention.
EXAMPLES
Example 1
[0430] Example 1 shows methods of surfactant syntheses.
[0431] Below of the reaction scheme for creating the surfactants
utilized in stabilizing the droplet libraries provided by the
instant invention.
##STR00001##
[0432] Reagent Table is as follows:
TABLE-US-00004 Amount Other (density, Name MW Moles/Equiv. used
purity, safety, misc.) Krytox 6500 1.54 mmol (1 eq) 10.0 g FSH
Oxalyl 126.93 15.4 mmol (10 eq) 1.3 mL (1.95 g) d = 1.5 g/mL;
Chloride b.p. 62-65.degree. C. HFE 7100 250.06 50 mL b.p.
61.degree. C.
[0433] The procedure includes, adding 10.0 g (1.54 mmol; 1 eq) of
Krytox acid FSH in 50 mL HFE 7100 (not anhydrous) at right under Ar
was added 1.95 g (15.4 mmol; 10 eq) of Oxalyl Chloride dropwise.
Stirred 10 min, then the reaction was warmed to gentle reflux (note
boiling points of solvent and reagent). Some bubbling was noted,
even before reaction had reached reflux. Continued overnight.
[0434] The following day, the reaction was very slightly cloudy,
and contained a very small amount of a yellow solid. Cooled, HFE
and excess Oxalyl chloride evaporated. Residue dissolved in 40 mL
fresh HFE, then filtered to remove the solid. HFE evaporated again,
residue placed under hivac for 1 hr. Yield of a cloudy white oil
10.14 g. Used without further purification.
##STR00002##
[0435] The reagent table is as follows:
TABLE-US-00005 Amount Other (density, Name MW Moles/Equiv. used
purity, safety, misc.) JSJ 73-019 6518 1.55 mmol (2 eq) 10.14 g
used crude JSJ 73-006 568 0.77 mmol (1 eq) 0.441 g Triethylamine
101.19 2.33 mmol (3 eq) 0.325 mL d = 0.726; b.p. 88.degree. C.
Tetrahydrofuran 20 mL b.p. 66.degree. C. FC 3283 521 40 mL b.p.
123-33.degree. C.; dried over CaSO.sub.4
[0436] The procedure includes, drying the amine was by placing
under hivac rotovap at a bath temp of 60.degree. C. for 4 hrs. To a
solution of 0.441 g (0.77 mmol; 1 eq) of PEG 600 diamine J and
0.325 mL (2.33 mmol; 3 eq) of Et.sub.3N in 20 mL anhydrous THF at
rt under Ar was added a solution of 10.14 g (1.55 mmol; 2 eq) of
crude JSJ 73-019 in 40 mL FC 3283. A white precipitate
(Et.sub.3eHCl) was noted to form in the reaction and on the flask
walls. The milky-white two-phase suspension was stirred well
overnight.
[0437] The THF and most FC was evaporated. This left a solid
residue dispersed in the FC solvent (Et.sub.3N HCl). Oil residue
diluted with 50 mL FC 3283, then filtered through Celite. Celite
washed with 2.times.30 mL FC 3283. The solid left in the flask was
found to be water soluble, suggesting that it was Et.sub.3N HCl.
The filtrate was cloudy white. FC 3283 evaporated using hivac and
60.degree. C. bath. Kept as such for .about.1.5 hrs to evaporate
all solvent.
[0438] Sample submitted for 19F NMR. Peak for the CF was in the
correct position, indicating amide had formed
##STR00003##
[0439] The reagent table is as follows:
TABLE-US-00006 Amount Other (density, Name MW Moles/Equiv. used
purity, safety, misc.) PEG 600 600 0.125 (1 eq).sup. 75.0 g Avg. MW
600; (Fluka) CAS 25322-65-3; m.p. 17-22.degree. C. p-Toluene 190.65
0.283 (2.3 eq) 53.93 g Sulfonyl chloride Tetrahydrofuran 525 mL
b.p. 66.degree. C., not anhydrous Sodium 40.00 0.506 (4.05 eq)
20.25 g Hydroxide Water 156 mL
[0440] The procedure included adding to a solution of 20.25 g
(0.506 mol; 4.05 eq) of NaOH in 156 mL water cooled in an ice-bath
to .about.0.degree. C. (not under inert atmosphere) was added a
solution of 75.0 g (0.125 mol; 1 eq, 2 eq of hydroxyl) of PEG 600
in 300 mL of THF dropwise via addition funnel. Internal temp was
kept .about.5.degree. C. during the addition. After complete
addition, the slightly cloudy reaction was stirred while warming to
rt over 1 hr. Following this, the reaction was again cooled to
.about.0.degree. C. and a solution of 53.93 g (0.283 mol; 2.3 eq)
of Tosyl chloride in 225 mL THF was added dropwise via addition
funnel, again keeping the internal temp .about.10.degree. C. during
the addition. Reaction allowed to stir overnight while warming to
rt.
[0441] Layers allowed to separate in an addition funnel, THF
separated from aqueous layer and evaporated. Residue dissolved in
600 mL EtOAc and recombined w/aqueous from above. Shaken,
separated. Organic washed 3.times.125 mL water, then with brine,
dried over MgSO.sub.4. Stirred over the weekend.
[0442] Filtered, solvent evaporated to give a colorless oil, which
was dried under hivac rotovap 3 hrs at .about.60.degree. C. This
gave 91.95 g of a colorless oil (81% yield). This was a typical
yield for this reaction.
##STR00004##
[0443] The reagent table includes:
TABLE-US-00007 Other (density, Name MW Moles/Equiv. Amount used
purity, safety, misc.) JSJ 73-078 910 102 mmol (1 eq).sup. 92.75 g
used crude Potassium 185.22 224 mmol (2.2 eq) 41.5 g Phthalimide
Dimethylformamide 73.09 600 mL b.p. 153.degree. C.
[0444] The procedure includes adding to a solution of 92.75 g (102
mmol; 1 eq) of JSJ 73-078 in 600 mL anhydrous DMF at rt under Ar
was added 41.5 g (224 mmol; 2.2 eq) of Potassium phthalimide as a
solid in 2 portions. The heterogeneous solution was then warmed
slowly to 85-90.degree. C. (internal temp) and the rxn stirred
overnight. The phthalimide slowly went into solution and the color
became more yellow. A small amount of the phthalimide wasn't
consumed, remaining a solid dispersed in the reaction.
[0445] While still warm, the reaction was poured into a separate
flask and most of the DMF was evaporated (hivac at 80.degree. C.).
Any remaining solution was kept warm while the DMF was being
evaporated. The resulting sludgy solid was diluted EtOAc (.about.1
L in total) and filtered. The sludge was triturated in a smaller
portion of EtOAc (200 mL) and filtered again to ensure all product
was recovered. Solid was triturated w/another 200 mL EtOAc.
Filtrate was then concentrated to give a yellow oil. Continued
pumping on oil under hivac at 80.degree. C. for several hours to
remove residual DMF. This gave 57.93 g of orange-yellow oil. This
was typical yield for this reaction.
##STR00005##
[0446] The reagent table includes:
TABLE-US-00008 Other (density, Name MW Moles/Equiv. Amount used
purity, safety, misc.) JSJ 73-081 860 67 mmol (1 eq) 57.93 g used
crude Hydrazine, 32.05 471 mmol (7 eq) 15 g d = 1.021 g/mL,
anhydrous (15 mL) b.p. 113.degree. C. Tetrahydrofuran 300 mL b.p.
66.degree. C. Methanol 300 mL b.p. 65.degree. C.
[0447] The procedure includes adding to a solution of 57.93 g (67
mmol; 1 eq) of crude JSJ 73-081 in 300 mL anhydrous MeOH and 300 mL
anhydrous THF at rt under Ar was added 15 mL (471 mmol; 7 eq) of
Hydrazine and the reaction stirred (mechanical stirring used) at rt
for 1.5 hrs. After .about.45 min, white solid began to form from
the homogeneous solution. This was thought to be the byproduct of
the hydrazine-phthalimide reaction. The reaction was warmed to
40.degree. C. and stirred overnight under Ar. The stirring became
slightly more difficult as more solid formed, so the stirring was
increased slightly.
[0448] The reaction appeared the same, very thick with the solid.
Rxn cooled, THF and MeOH evaporated. The residue was diluted
w/EtOAc (300 mL) then filtered. Filtration was relatively easy,
solid washed well w/EtOAc (200 mL). Filtrate concentrated, upon
which additional solid was noted. Residue diluted back in EtOAc
(300 mL), giving a slightly cloudy solution (diamine may not be
completely soluble in this volume of EtOAc). Filtered, solvent
evaporated, giving a yellow oil.
[0449] This oil was placed under hivac rotovap at .about.70.degree.
C. for several hours to remove residual solvents, Hydrazine, and
also possibly any residual DMF brought from the SM in the previous
step.
Example 2
[0450] Example 2 shows PEG-Amine derived fluorosurfactant
syntheses
[0451] A PEG-amine derived fluorosurfactant can be made by the
following process: 10.0 g of Krytox 157 FSH (PFPE, 6500 g/mol,
0.00154 mole) was dissolved in 25.0-mL of FC-3283 (521 g/mol, 45.5
g, 0.0873 mole). 0.567 g PEG 600 Diamine (566.7 g/mol, 0.001 mole,
0.65 mol eq.) was dissolved in 10.0-mL of THF (72.11 g/mol, 8.9 g,
0.1234 mole). The resulting solutions were then combined and
emulsified. The resulting emulsion was spun on a BUCHI rota-yap. at
.about.75% for .about.20 hours. The crude reaction mixture was then
placed in centrifuge tubes with equal volumes of DI H2O, emulsified
and centrifuged at 15,000 rpm for 15-minutes. Once the emulsion was
broken, the oil layer was extracted, dried with anhydrous sodium
sulfate and filtered over a 0.45-um disposable nylon filter. The
filtered oil was then evaporated on a BUCHI rota-vap. model R-200
fitted with a B-490 water bath for .about.2-hours at 70.degree.
C.
[0452] The procedure is depicted in Scheme 1:
##STR00006##
[0453] FIG. 5 shows Ammonium Carboxylate Salt of Krytox 157 FSH 2
Wt % in FC 3283 without PEG amine salt (Panel A) and with PEG 600
Diammonium Carboxylate Salt of Krytox 157 FSH at 4.0% by volume
(Panel B) (Flow Rates: 2000 ul/hr (FC oil), 500 ul/hr (aq)). The
difference in the number of coalesced drops in the right image
indicates that the PEG amine salt is effective in stabilizing
emulsions. Emulsions made with the Ammonium Carboxylate Salt of
Krytox 157 FSH 2 Wt % in FC 3283 with PEG 600 Diammonium
Carboxylate Salt of Krytox 157 FSH co-surfactant added at 4.0% by
volume were shown to remain stable when reinjected into a
microfluidic channel.
Example 3
[0454] Primer Library Generation
[0455] The primer droplet library generation is a Type IV library
generation. FIG. 6 shows a schematic of the primer library
generation. Step 1 of the library formation is to design primers
for loci of interest. There are no constraints on primer design
associated with traditional multiplex PCR. Step 2 requires
synthesis of the primer pairs using standard oligo synthesis. After
the library elements are created, the primer pairs are reformatted
as droplets, where only one primer pair present in each droplet.
Each droplet contains multiple copies of the single primer pair
directed to a single target of interest. After the droplets for
each type of primer pair is created, the emulsions are pooled as
primer library. The droplet stability prevents cross-contamination
of primer pairs.
[0456] Primer Library for Genome Selection
[0457] A pooled primer library can be placed onto a microfluidic
device as provided by the instant invention. Each primer library
droplet follows an inlet channel that intersects with another inlet
channel, which has droplets containing gDNA, Taq, dNTPs and any
other materials needed to perform PCR. At the intersection the two
inlet channels merge into a single main channel where the two
different types of droplets, i.e., the primer library droplet and
the PCR component droplets travel singularly until they are
coalesced. The droplets are coalesced within the main channel in
which they are traveling, at a widened portion of the main channel.
In addition to the widening in the channel, electrodes are used to
coalesce the droplets containing the primer libraries and the
droplets containing the PCR components. The coalesced droplets then
continue along the same main channel and are collected onto well
containing plates. The droplets in the plates are subjected to
thermocycling to permit PCR amplification. The amplified DNA can
then be sequenced by any means known in the art. The relative
number of sequencing reads from each of 20 targeted exons can be
plotted using primer libraries against human genomic DNA.
[0458] Primer Library for Digital qPCR Method
[0459] Disposable PDMS/glass microfluidic devices were designed
with regions maintained at 95.degree. C., and regions maintained at
67.degree. C. and further including interrogation neckdowns.
Aqueous fluid was infused into the microfluidic device
perpendicularly to two channels flowing immiscible oil which
generated 65 pL (50 um) droplets. The aqueous fluid contained the
following reagents:
[0460] 50 mM Tris/HCl (pH 8.3)
[0461] 10 mM KCl
[0462] 5 mM (NH4)2504
[0463] 3.5 mM MgCl2
[0464] 0.2 mM dNTP
[0465] 0.5% Tetronics
[0466] 0.1 mg/ml BSA
[0467] 0.2 units per .mu.L of FastStart Taq DNA polymerase
[0468] 0.5 .mu.M each of forward and reverse primers
[0469] 0.25 .mu.M FAM-labeled probe quenched with a 3' BHQ1
[0470] 1 .mu.M Alexa Fluor 594
[0471] Serial dilutions of the pAdeasy-1 vector (Stratagene, La
Jolla, Calif.) were made from 60 to 0.0006 ng/uL or from 600 copies
per/droplet to 0.006 copies per/droplet
[0472] The concentrations of the diluted DNA were verified by qPCR
using a traditional real-time thermocycler. PCR primers and probe
were designed to detect a 245 bp region of the Adenovirus genome.
Droplets were generated at a rate of 500 per second. The channels
on the microfluidic device conveyed the droplets through of 2
thermal zones at a 95.degree. C. and 67.degree. C. for 34 passes
which were the equivalent of 34 cycles of two step PCR with the
following cycling parameters:
[0473] 1 cycle--3 min hot start
[0474] 34 cycles:
[0475] 95.degree. C. for 15 seconds
[0476] 68.degree. C. for 40 seconds
[0477] PCR droplets were interrogated at specific "neckdowns" which
were 100 micron long regions of the microfluidic device where the
channel width and depth decreased forcing droplets into a single
file. A two wavelength laser excitation and detection system was
used to interrogate the fluorescence at each of the neckdowns at
cycles 4, 8, 11, 14, 17, 20, 23, 26, 29, 32, and 34. A fluorescent
dye, Alexa Fluor 594, provided a constant signal in each droplet
that was used for droplet detection without inhibiting PCR
amplification efficiency or yield.
[0478] The distribution of fluorescence signal among droplets was
determined as the droplets pass through the excitation lasers (488
nm and 561 nm) at the last interrogation neckdown (cycle 34). A
bimodal distribution of FAM fluorescence was observed for droplets
with starting template concentrations of less than one molecule per
droplet, indicating the presence of two populations corresponding
to empty droplets and droplets that supported amplification. A time
trace of fluorescence signals from those droplets is readily
plotted as those droplets pass one-by-one through the excitation
lasers. For each of the Adenovirus dilutions examined, the
percentage of PCR positive droplets was plotted versus cycle
number.
[0479] Successful amplification was detected at Adenovirus
concentrations as low as 0.006 copies per drop (0.003 pg/.mu.l).
Following amplification, the droplets collected from the
microfluidic device were broken and analyzed by automated
electrophoresis to confirm a product of the appropriate size.
Consistent with the fluorescence data, gel analysis showed an
increase in total product as the amount of starting material was
increased. The observed titers were compared with the average
percentage of positive reactions predicted for each starting
template concentration by Poisson statistics and by MPN (most
probable number) (see Table below).
TABLE-US-00009 TABLE 3 Comparison of Observed Amplification
Distribution to Poisson Statistics and MPN. Template Concentration
PCR Positive Droplets Copies per Expected Copies per Droplet (MPN
Expected (Poisson MPN Droplet adjusted).sup.a Observed (Poisson)
adjusted).sup.a 0.006 0.0050 (.+-.0.000082) 2.08% 0.60% 0.49-0.51%
0.06 0.050 (.+-.0.00082) 11.7% 5.82% 4.76-4.95% 0.3 0.25
(.+-.0.0041) 20.3% 25.9% 21.6-22.4% 0.6 5.0 (.+-.0.0082) 32.6%
45.1% 38.6-39.8% 6 5.0 (.+-.0.082) 89.0% 99.8% 99.2-99.4% 60 50
(.+-.0.82) 95.9% 100% .sup. 100% 600 500 (.+-.8.2 98.2% 100% .sup.
100% .sup.aMPN calculation based on the 4 lowest dilutions was 0.83
.+-. 0.017. Adjusted values are within 95% confidence.
[0480] A percentage of droplets that supported amplification was
plotted versus starting copy number compared to that predicted by
Poisson. Very good agreement was seen between the percentage of
droplets that supported amplification and the predicted Poisson
distribution. Given the accuracy of the data for endpoint analysis
this droplet-based strategy appears to be ideal for quantitative
PCR applications that require single molecule detection.
Example 4
[0481] Generating Single Element Droplets
[0482] As described in detail herein, the formation of Type II
library droplets, which encapsulate a library element, e.g., cell
or bead, follow a Poisson Distribution. Using the following,
equation, the distributions were calculated based on theory using
the following equation:
P(N)=((CV).sup.Ne.sup.-CV)/N!
[0483] where P is the probability of N particles per drop, C is the
injection concentration, and V is the droplet volume.
[0484] Experimental results are based on beads that were injected
at a concentration of 40 million/ml into 23 .mu.m drops (6.37 pL);
CV=0.2548. The results are shown in the following table:
TABLE-US-00010 Theoretical Poisson Experimental P(N) Distribution
Distribution # of Drops Counted P(0) 77.5 79.8 1010 P(1) 19.7 17.3
219 P(2) 2.5 2.5 32 P(3 and more) 0.3 0.4 5 Total 100% 100%
1266
Example 5
[0485] Antibody-Bead Libraries--ELISA in Droplets
[0486] The primer droplet library generation is a Type II library
generation. FIG. 3 shows a schematic of the antibody pair library
generation. Reagent solutions 1 through n, where n is the number of
parallel ELISA assays to be performed, are prepared in separate
vials. Each solution contains two antibodies, one bound to beads
and one free in solution. It is often desirable, but not essential,
for the unbound antibody to be biotinylated or for it to be
conjugated to an enzyme. The beads are optically labeled using
dyes, quantum dots or other distinguishing characteristics that
make each of the n bead types uniquely identifiable, these
characteristics can be also geometrical shape or features or
fluorescence intensity or fluorescence polarization. For the
emulsion library preparation: in separate locations, beads of each
type are encapsulated in reagent droplets; the reagent contains an
unbound second antibody matched for a specific immunoassay. It is
often desirable, but not essential to have exactly one bead in a
droplet. Droplets having exactly one bead are collected by sorting
on droplet generation in a microfluidic device. The emulsion
library consists of a pooled collection of the n different droplet
types. The droplet stability prevents cross-contamination of
antibody pairs.
[0487] FIG. 3 shows the test fluid is the sample fluid used to
analyze one or more immunoassays.