U.S. patent application number 14/163442 was filed with the patent office on 2014-07-10 for bead manipulation techniques.
This patent application is currently assigned to ADVANCED LIQUID LOGIC, INC.. The applicant listed for this patent is ADVANCED LIQUID LOGIC, INC.. Invention is credited to VAMSEE K. PAMULA, RAMAKRISHNA SISTA, ARJUN SUDARSAN.
Application Number | 20140193807 14/163442 |
Document ID | / |
Family ID | 51061230 |
Filed Date | 2014-07-10 |
United States Patent
Application |
20140193807 |
Kind Code |
A1 |
PAMULA; VAMSEE K. ; et
al. |
July 10, 2014 |
BEAD MANIPULATION TECHNIQUES
Abstract
The invention provides a method of redistributing magnetically
responsive beads in a droplet. The method may include providing a
droplet including magnetically responsive beads. The droplet may be
provided within a region of a magnetic field having sufficient
strength to attract the magnetically responsive beads to an edge of
the droplet or towards an edge of the droplet, or otherwise
regionalize or aggregate beads within the droplet. The method may
also include conducting on a droplet operations surface one or more
droplet operations using the droplet without removing the
magnetically responsive beads from the region of the magnetic
field. The droplet operations may in some cases be
electrode-mediated. The droplet operations may redistribute and/or
circulate the magnetically responsive beads within the droplet. In
some cases, the droplet may include a sample droplet may include a
target analyte. The redistributing of the magnetically responsive
beads may cause target analyte to bind to the magnetically
responsive beads. In some cases, the droplet may include unbound
substances in a wash buffer. The redistributing of the magnetically
responsive beads causes unbound substances to be freed from
interstices of an aggregated set or subset of the magnetically
responsive beads.
Inventors: |
PAMULA; VAMSEE K.; (DURHAM,
NC) ; SUDARSAN; ARJUN; (CARY, NC) ; SISTA;
RAMAKRISHNA; (MORRISVILLE, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ADVANCED LIQUID LOGIC, INC. |
RESEARCH TRIANGLE PARK |
NC |
US |
|
|
Assignee: |
ADVANCED LIQUID LOGIC, INC.
RESEARCH TRIANGLE PARK
NC
|
Family ID: |
51061230 |
Appl. No.: |
14/163442 |
Filed: |
January 24, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12985409 |
Jan 6, 2011 |
8637317 |
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14163442 |
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11639531 |
Dec 15, 2006 |
8613889 |
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12985409 |
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PCT/US2009/050101 |
Jul 9, 2008 |
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12985409 |
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60745058 |
Apr 18, 2006 |
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60745039 |
Apr 18, 2006 |
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60745043 |
Apr 18, 2006 |
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60745059 |
Apr 18, 2006 |
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60745914 |
Apr 28, 2006 |
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61079346 |
Jul 9, 2008 |
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Current U.S.
Class: |
435/6.1 ;
436/501 |
Current CPC
Class: |
B01L 3/502761 20130101;
B01L 2200/0647 20130101; B01L 2400/0427 20130101; B03C 5/026
20130101; B03C 5/00 20130101; B01L 3/50273 20130101; B01L 2300/0887
20130101; G01N 33/54326 20130101; B01L 2400/0424 20130101; B03C
2201/26 20130101; B03C 5/005 20130101; B03C 1/288 20130101; B03C
7/026 20130101; B03C 1/01 20130101 |
Class at
Publication: |
435/6.1 ;
436/501 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/543 20060101 G01N033/543 |
Goverment Interests
GOVERNMENT INTEREST
[0003] This invention was made with government support under
CA114993 awarded by the National Institutes of Health. The United
States Government has certain rights in the invention.
[0004] The foregoing statement applies only to aspects of this
disclosure originating in U.S. Patent Application No. 61/103,302,
entitled "Bead Incubation and Washing on a Droplet Actuator," filed
on Oct. 7, 2008, and U.S. Patent Application No. 61/122,791, "Bead
Incubation and Washing on a Droplet Actuator," filed Dec. 16, 2008.
Claims
1. A method of redistributing magnetically responsive beads in a
droplet, the method comprising: (a) providing a droplet comprising
magnetically responsive beads within a region of a magnetic field
having sufficient strength to attract the magnetically responsive
beads to an edge of the droplet; and (b) using electrodes to
conduct on a droplet operations surface a droplet operation using
the droplet without removing the magnetically responsive beads from
the region of the magnetic field, thereby redistributing the
magnetically responsive beads within the droplet.
2. The method of claim 1 wherein the droplet comprises a sample
droplet comprising a target analyte.
3. The method of claim 1 wherein the redistributing of the
magnetically responsive beads causes target analyte to bind to the
magnetically responsive beads.
4. The method of claim 1 wherein the droplet comprises unbound
substances in a wash buffer.
5. The method of claim 4 wherein the redistributing of the
magnetically responsive beads causes unbound substances to be freed
from interstices of an aggregated set or subset of the magnetically
responsive beads.
6. The method of claim 1 wherein the droplet operation is selected
to agitate contents of the droplet.
7. The method of claim 1 wherein the droplet operation comprises
transporting the droplet.
8. The method of claim 1 wherein the droplet operation comprises
elongating the droplet.
9. The method of claim 8 wherein elongating the droplet comprises
flowing the droplet onto a region of the droplet operations surface
atop two or more activated droplet electrodes causing the droplet
to take on an elongated configuration.
10. The method of claim 1 wherein the droplet operation comprises
merging the droplet with another droplet.
11. The method of claim 1 wherein the droplet operation comprises
splitting the droplet to yield two or more daughter droplets.
12. The method of claim 11 wherein two or more of the daughter
droplets each comprise a substantial subset of the magnetically
responsive beads.
13. The method of claim 11 wherein the droplet operation comprises
merging two or more of the daughter droplets.
14. The method of claim 1 further comprising removing the droplet
or a sub-droplet thereof including at least a subset of the
magnetically responsive beads from the magnetic field.
15. The method of claim 1 wherein one or more droplet operations is
repeated in a series of two or more incubation cycles.
16. The method of claim 1 wherein the droplet operations surface is
in a droplet operations gap of a droplet actuator.
17. The method of claim 1 wherein the droplet operations surface is
coated by a liquid filler fluid.
18. The method of claim 1 wherein the droplet is surrounded by a
liquid filler fluid.
19. The method of claim 1 wherein the droplet operation is
electrode-mediated.
20. The method of claim 1 wherein the droplet operation is
electrowetting-mediated.
21. The method of claim 1 wherein the droplet operation is
dielectrophoresis-mediated.
22. A method of redistributing magnetically responsive beads in a
droplet, the method comprising: (a) providing a droplet comprising
magnetically responsive beads within a region of a magnetic field
having sufficient strength to attract the magnetically responsive
beads to an edge of the droplet; and (b) using electrodes to
conduct on a droplet operations surface a droplet operation using
the droplet without removing the magnetically responsive beads from
the magnetic field, thereby redistributing the magnetically
responsive beads within the droplet.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of and incorporates by
reference U.S. patent application Ser. No. 12/985,409, entitled
"Bead Manipulation Techniques" filed on Jan. 6, 2011, which is a
continuation-in-part of and incorporates by reference U.S. patent
application Ser. No. 11/639,531, entitled "Droplet-based washing"
filed on Dec. 15, 2006, the application of which claims priority to
and incorporates by reference related provisional U.S. Patent
Application Nos. 60/745,058, entitled "Filler Fluids for
Droplet-Based Microfluidics" filed on Apr. 18, 2006; 60/745,039,
entitled "Apparatus and Methods for Droplet-Based Blood Chemistry,"
filed on Apr. 18, 2006; 60/745,043, entitled "Apparatus and Methods
for Droplet-Based PCR," filed on Apr. 18, 2006; 60/745,059,
entitled "Apparatus and Methods for Droplet-Based Immunoassay,"
filed on Apr. 18, 2006; 60/745,914, entitled "Apparatus and Method
for Manipulating Droplets with a Predetermined Number of Cells"
filed on Apr. 28, 2006; 60/745,950, entitled "Apparatus and Methods
of Sample Preparation for a Droplet Microactuator," filed on Apr.
28, 2006; 60/746,797 entitled "Portable Analyzer Using
Droplet-Based Microfluidics," filed on May 9, 2006; 60/746,801,
entitled "Apparatus and Methods for Droplet-Based Immuno-PCR,"
filed on May 9, 2006; 60/806,412, entitled "Systems and Methods for
Droplet Microactuator Operations," filed on Jun. 30, 2006; and
60/807,104, entitled "Method and Apparatus for Droplet-Based
Nucleic Acid Amplification," filed on Jul. 12, 2006.
[0002] In addition to the patent applications cited above, U.S.
patent application Ser. No. 12/985,409, entitled "Bead Manipulation
Techniques" filed on Jan. 6, 2011, is a continuation of and
incorporates by reference International Patent Application No.
PCT/US2009/050101, entitled "Bead Manipulation Techniques"
International filing date of Jul. 9, 2009, the application of which
claims priority to and incorporates by reference related
provisional U.S. Patent Applications 61/079,346, entitled "Digital
Microfluidic Spacio- and Spectral-Multiplexing of Assays," filed on
Jul. 9, 2008; 61/080,731, entitled "Dielectrophoresis on a Droplet
Actuator," filed on Jul. 15, 2008; 61/084,637, entitled "Digital
Microfluidics Multi-well Droplet Actuator Device and Methods,"
filed on Jul. 30, 2008; 61/103,302, entitled "Bead Incubation and
Washing on a Droplet Actuator," filed on Oct. 7, 2008; 61/108,997,
entitled "Adjustable Magnets and Magnetic Fields on a Droplet
Actuator," filed on Oct. 28, 2008; 61/122,791, entitled "Bead
Incubation and Washing on a Droplet Actuator," filed on Dec. 16,
2008; and 61/149,808, entitled "Droplet-Based Platform for
Evaluating Enzymatic Activity," filed on Feb. 4, 2009.
FIELD OF THE INVENTION
[0005] The present invention generally relates to bead manipulation
techniques. In particular, the present invention is directed to a
method of redistributing magnetically responsive beads in a
droplet.
BACKGROUND
[0006] Droplet actuators are used to conduct a wide variety of
droplet operations. A droplet actuator typically includes one or
more substrates configured to form a surface or gap for conducting
droplet operations. The one or more substrates include electrodes
for conducting droplet operations. Liquids that are subjected to
droplet operations are typically surrounded by an immiscible filler
fluid. When the droplet actuator is configured to form a gap, the
gap between the substrates is typically filled or coated with the
filler fluid. Droplet operations are controlled by electrodes
associated with the one or more substrates. Droplets containing
particles, such as beads or cells, may be subjected to various
droplet operations on a droplet actuator. Droplets associated with
particles may require various methods that may include structures,
to be manipulated by the droplet actuator.
[0007] Beads, whether or not magnetically responsive have a
tendency to settle and form aggregates due to one or more forces
that may include gravity, friction, electric and magnetic forces.
Aggregation may also occur due to surface interactions between
beads or between substances bound to beads or interactions between
beads and droplet actuator substrates. Regardless of the causes,
aggregation has a direct impact on the performance of assays.
Immunoassays for example, has critical time consuming stages like
incubation and washing that may be influenced by the aggregation of
beads.
[0008] During incubation, where interaction of different antibodies
and antigens result in binding events, the available surface area
on the beads for binding is reduced due to aggregation, thereby
impeding reaction kinetics and consequently increasing time to
result and/or reducing assay sensitivity. Protocols used for
incubation, including but not limited to duration of incubation may
be influenced by the mixing efficiency within the droplets and also
the reaction and binding kinetics, all of which may be impacted by
bead aggregation. When it comes to washing, unwanted unbound
substances that are trapped in the interstices of bead aggregates
are difficult to separate, remove or wash away, thereby resulting
in reduced assay sensitivity. Time to results is impacted if more
number of washes are required.
[0009] Therefore, there is a need in droplet actuators for
resuspending and/or circulating beads within a droplet to break up
or loosen up aggregates when required to improve the overall assay
performance without having to compromise on sensitivity and the
overall time to result.
BRIEF DESCRIPTION OF THE INVENTION
[0010] The invention provides a method of redistributing
magnetically responsive beads in a droplet. The method may include
providing a droplet including magnetically responsive beads. The
droplet may be provided within a region of a magnetic field having
sufficient strength to attract the magnetically responsive beads to
an edge of the droplet or towards an edge of the droplet, or
otherwise regionalize or aggregate beads within the droplet. The
method may also include conducting on a droplet operations surface
one or more droplet operations using the droplet without removing
the magnetically responsive beads from the region of the magnetic
field. The droplet operations may in some cases be
electrode-mediated. The droplet operations may redistribute and/or
circulate the magnetically responsive beads within the droplet. In
some cases, the droplet may include a sample droplet may include a
target analyte. The redistributing of the magnetically responsive
beads may cause target analyte to bind to the magnetically
responsive beads. In some cases, the droplet may include unbound
substances in a wash buffer. The redistributing of the magnetically
responsive beads causes unbound substances to be freed from
interstices of an aggregated set or subset of the magnetically
responsive beads.
[0011] In certain embodiments, the droplet operation may be
selected to agitate contents of the droplet. The droplet operation
may include transporting the droplet. The droplet operation may
include elongating the droplet. In some cases, elongating the
droplet may include flowing the droplet onto a region of the
droplet operations surface atop two or more activated droplet
electrodes causing the droplet to take on an elongated
configuration. The droplet operation may include merging the
droplet with another droplet. The droplet operation may include
splitting the droplet to yield two or more daughter droplets. In
some cases, two or more of the daughter droplets each may include a
substantial subset of the magnetically responsive beads. In some
cases, the droplet operation may include merging two or more of the
daughter droplets. In some cases, further may include removing the
droplet or a sub-droplet thereof including at least a subset of the
magnetically responsive beads from the magnetic field. In certain
embodiments, one or more droplet operations may be repeated in a
series of two or more incubation cycles. The droplet operations
surface may be in a droplet operations gap of a droplet actuator.
The droplet operations surface may be coated by a liquid filler
fluid. The droplet may be surrounded by a liquid filler fluid.
[0012] The invention also provides a method of incubating
magnetically responsive beads in a droplet. The droplet including
magnetically responsive beads may include one or more substances
having affinity for one or more of the magnetically responsive
beads. The method may include redistributing the magnetically
responsive beads in the droplet in accordance with the method of
any of the methods described herein.
[0013] Further, the invention provides a method of washing
magnetically responsive beads in a droplet. The droplet including
magnetically responsive beads provided may also include one or more
unbound substances selected for removal. The method may include
merging the droplet including magnetically responsive beads with a
wash droplet to yield a combined droplet. The method may include
redistributing the magnetically responsive beads in the droplet in
accordance with the method of any of the methods described herein.
The method may include splitting the combined droplet to yield a
droplet including substantially all of the magnetically responsive
beads and a reduced concentration of the unbound substances
relative to the starting droplet, and a droplet substantially
lacking magnetically responsive beads. The method may be repeated
as necessary until a predetermined concentration or quantity of the
unbound substances being removed is achieved.
[0014] In another method of washing magnetically responsive beads
in a droplet, the method may include merging the droplet with
magnetically responsive beads with a wash droplet in the magnetic
field to yield a combined droplet and to redistribute the
magnetically responsive beads within the combined droplet, and
splitting the combined droplet to yield a droplet including
substantially all of the magnetically responsive beads and a
reduced concentration of the unbound substances relative to the
starting droplet, and a supernatant droplet substantially lacking
magnetically responsive beads.
[0015] In another method of washing magnetically responsive beads
in a droplet, the method may include conducting one or more droplet
operations using the droplet in the magnetic field to redistribute
the magnetically responsive beads in the droplet in accordance with
any of the other methods described herein, and merging the droplet
including the redistributed magnetically responsive beads with a
wash droplet to yield a combined droplet. Further, the method may
include splitting the combined droplet to yield a first daughter
droplet including substantially all of the magnetically responsive
beads and a reduced concentration of the unbound substances
relative to the starting droplet, and a second daughter droplet
substantially lacking magnetically responsive beads.
[0016] The invention provides a method of redistributing
magnetically responsive beads in a droplet, which method may
include providing a droplet including magnetically responsive beads
within a first region of a magnetic field having sufficient
strength to attract the magnetically responsive beads to an edge of
the droplet, and using electrodes to transport droplet to a second
region of a droplet operations surface in which the magnetic field
may be sufficiently reduced to permit the magnetically responsive
beads to circulate in the droplet during the conduct of one or more
droplet operations. The method may also include conducting the one
or more droplet operations to cause the magnetically responsive
beads to circulate in the droplet. In some cases, in the second
region of the droplet operations surface, the beads are
substantially free from the influence of the magnetic field. In
some embodiments, at least a subset of the beads in the starting
droplet are magnetically aggregated. The droplet may include a
sample droplet including a target analyte. Circulation of the
magnetically responsive beads may cause target analyte to bind to
the magnetically responsive beads. In some cases, the droplet may
include unbound substances in a wash buffer. In some cases, the
circulation of the magnetically responsive beads causes
disaggregation of an aggregated set or subset of the magnetically
responsive beads freeing of unbound substances from interstices of
the aggregated set or subset of the magnetically responsive beads.
The one or more droplet operations may be selected to agitate
contents of the droplet. The one or more droplet operations may
include transporting the droplet. The one or more droplet
operations may include elongating the droplet. In some cases,
elongating the droplet may include flowing the droplet onto a
region of the droplet operations surface atop two or more activated
droplet electrodes causing the droplet to take on an elongated
configuration. The droplet operation may include merging the
droplet with another droplet. The droplet operation may include
splitting the droplet to yield two or more daughter droplets. In
some cases, two or more of the daughter droplets each may include a
substantial subset of the magnetically responsive beads. The
droplet operation may include merging two or more of these daughter
droplets. One or more droplet operations may be repeated in a
series of two or more incubation cycles. The droplet operations
surface may be in a droplet operations gap of a droplet actuator.
The droplet operations surface may be coated by a liquid filler
fluid. The droplet may be surrounded by a liquid filler fluid.
[0017] In another method of incubating magnetically responsive
beads in a droplet may include merging the droplet including
magnetically responsive beads with a wash droplet to yield a
combined droplet, redistributing the magnetically responsive beads
in the combined droplet in accordance with the method of any of the
methods described herein, and reintroducing the magnetically
responsive beads into the first region of the magnetic field or
into a region of another magnetic field having sufficient strength
to attract the magnetically responsive beads to an edge of the
droplet. In yet another method of washing magnetically responsive
beads in a droplet, the method may include redistributing the
magnetically responsive beads in the droplet in accordance with the
method of any of the methods described herein and reintroducing the
magnetically responsive beads into the first region of the magnetic
field or into a region of another magnetic field having sufficient
strength to attract the magnetically responsive beads to an edge of
the droplet. These methods may also include splitting the combined
droplet to yield a droplet including substantially all of the
magnetically responsive beads and a reduced concentration of the
unbound substances relative to the starting droplet, and a droplet
substantially lacking magnetically responsive beads.
[0018] The invention also provides a method of incubating a
droplet, including providing a droplet including magnetically
responsive beads within a region of a magnetic field in which the
magnetically responsive beads are caused to become aggregated;
using electrodes to conduct on a droplet operations surface droplet
operations using the droplet wherein the droplet operations may
include: one or more droplet operations transporting the droplet
away from the magnetic field to a locus of the droplet operations
surface in which the magnetically responsive beads are resuspended
in the droplet; and one or more droplet operations effecting an
incubation cycle in the locus in which the magnetically responsive
beads are resuspended in the droplet.
[0019] Further, the invention provides a method of washing beads in
a droplet, including providing an elongated bead-containing droplet
may include one or more unbound substances; providing an elongated
wash droplet; restraining movement of beads within the elongated
bead-containing droplet; merging end-to-end the elongated
bead-containing droplet with the elongated bead containing droplet
to yield a combined droplet; and splitting the combined droplet to
form a droplet including substantially all of the beads and a
droplet substantially lacking in beads. In some cases, restraining
movement of beads within the elongated bead containing droplet may
include restraining the beads in an end region of the elongated
bead containing droplet. The method may also include conducting a
resuspension cycle using the bead-containing droplet prior to
conducting the merging step. Restraining movement of beads may be
accomplished by providing the elongated bead-containing droplet in
a magnetic field having a field strength which is sufficient to
restrain movement of the beads. In some cases, merging end-to-end
the elongated bead-containing droplet with the elongated bead
containing droplet causes circulation within the combined droplet
which redistributes the beads. In some cases, the restraining,
merging and splitting steps are completed in less than about 30
seconds, or less than about 15 seconds, or less than about 10
seconds, or less than about 5 seconds.
[0020] The invention provides another method of washing beads,
including providing the beads in a sample droplet may include a
target substance on a droplet operations substrate within a
magnetic field; transporting the sample droplet away from the
beads, causing the droplet to split, yielding a supernatant droplet
and leaving behind a daughter droplet including substantially all
of the magnetically responsive beads; and subjecting the daughter
droplet to a merge-and-split bead washing protocol. In some cases,
the supernatant droplet includes more than 50% of the unbound
substances being removed. In some cases, the supernatant droplet
includes more than 75% of the unbound substances being removed.
[0021] The steps of any of the washing processes described herein
may be repeated until the unbound substances selected for removal
from the droplet are reduced by a predetermined amount. In some
cases, the predetermined amount will be at least about 99%, or at
least about 99.9%, or at least about 99.99%, or at least about
99.999%. The predetermined reduction may in some cases be achieved
in 15 or fewer wash cycles, or 10 or fewer wash cycles, or 5 or
fewer wash cycles. Further, the predetermined reduction may be
achieved while retaining substantially all of the beads. In some
cases, at least about 99.9% of the beads are retained, or at least
about 99.99% of the beads are retained, or at least about 99.999%
of the beads are retained.
[0022] The invention also provides a method of removing beads from
a region of a magnetic field. The method may include providing a
droplet including the beads in a region of the magnetic field in
which the beads are aggregated by the magnetic field; elongating
the droplet; transporting the droplet away from the region of the
magnetic field in which the beads are aggregated out of the
magnetic field or into a region of the magnetic field which may be
sufficiently weak that the beads become disaggregated within the
droplet. The droplet may be provided on a droplet operations
surface of a droplet actuator. Elongating the droplet may include
activating one or more electrodes to cause the droplet to take on
an elongated conformation atop a droplet operations surface of a
droplet actuator. In some cases, the droplet operations surface may
be situated in a droplet operations gap of the droplet actuator. In
certain embodiments, the transporting may include
electrowetting-mediated droplet transporting. In certain
embodiments, the transporting may include transporting the droplet
away from the region of the magnetic field in a direction which
follows an approximately lengthwise axis of the droplet.
[0023] The invention also provides a method of multiplexing
detection in an assay. The method may include providing a set of
two or more detection-ready droplets. Each droplet may include two
or more sets of assay products. Each set of assay products may
include a unique optical marker, such as a color-based marker. The
method may include spectrally analyzing each of the two or more
droplets to quantify the assay products. In some cases, no single
droplet includes the same unique optical marker for two different
analytes. In certain embodiments, two different droplets may
include the same unique optical marker for two different analytes,
one of such analytes in each of the droplets. The spectrally
analyzing step may make use of a multi-channel spectral analyzer.
The multi-channel spectral analyzer may include an excitation light
source arranged to direct light in an excitation spectra into each
of the droplets. The multi-channel spectral analyzer may include a
electromagnetic radiation sensing device arranged to sense
electromagnetic radiation emitted from the droplets. In certain
embodiments, each droplet may include four or more sets of assay
products, or ten or more sets of assay products. In certain
embodiments, the method may include providing a set of five or more
of the detection-ready droplets, or set of 25 or more of the
detection-ready droplets, or a set of 50 or more of the
detection-ready droplets. In certain embodiments, the unique
optical marker may include a quantum dot marker. In some cases, the
quantum dot marker may include a core material coated with a high
bandgap material. In some cases, method may be executed on a
fluorescing background substrate, and the quantum dot markers
fluoresce at an excitation wavelength which differs from the
excitation wavelength of the fluorescing background substrate. In
some cases, assay products are bound to fluorescing beads, and the
quantum dot markers fluoresce at an excitation wavelength which
differs from the excitation wavelength of the fluorescing beads. In
some cases, the method may be executed on a fluorescing background
substrate, and the quantum dot markers fluoresce at an emission
wavelength which differs from the emission wavelength of the
fluorescing background substrate. In some cases, assay products are
bound to fluorescing beads, and the quantum dot markers fluoresce
at an emission wavelength which differs from the emission
wavelength of the fluorescing beads. In certain embodiments, the
assay products may include products of a droplet-based assay, such
as a droplet-based immunoassay. In certain embodiments, the assay
products may include products of a droplet-based assay executed on
a droplet actuator. In certain embodiments, the assay products may
include products of a droplet-based assay, and the detection-ready
droplet has a volume which may be less than about 1000 nL, or less
than about 500 nL. In certain embodiments, detection-ready droplets
are substantially surrounded by a liquid filler fluid. In some
cases, the liquid filler fluid may include an oil filler fluid. In
certain embodiments, detection-ready droplets are sandwiched
between two substrates. The method may also include analyzing light
from each droplet to identify and/or quantify assay products. In
some cases, analyzing light from each droplet may include
dispersing the light from each droplet along a dispersion axis. In
some cases, analyzing light from each droplet may include
separately binning light from each droplet to provide a spectrum
for each droplet. In some cases, analyzing light from each droplet
may include using filters to isolate signals from each droplet.
[0024] The invention provides a droplet actuator with a first
substrate including a droplet operations surface, electrodes
arranged for conducting one or more droplet operations on the
surface, and one or more dielectrophoresis electrode configurations
arranged for attracting and/or trapping one or more particles in a
droplet situated on the droplet operations surface. In some cases,
the droplet actuator may include a second substrate separated from
the droplet operations surface to form a droplet operations gap. In
some cases, the one or more dielectrophoresis electrode
configurations may include at least one dielectrophoresis electrode
configuration mounted on the second substrate. The
dielectrophoresis electrode configurations may include at least one
quadripole electrode configuration. In some cases, the quadripole
electrode configuration may include four opposing triangular
electrodes arranged to form a particle capture zone. In some cases,
the four opposing triangular electrodes are symmetrical. In some
cases, the four opposing triangular electrodes may include one or
more asymmetrical electrodes. In some cases, the quadripole
electrode configuration may include four wires terminating at a
particle capture zone. The dielectrophoresis electrode
configurations may include at least one configuration may include
two electrodes the two electrodes may include opposing fringed
regions separated by a gap. The dielectrophoresis electrode
configurations may include at least one configuration may include
multiple triangular electrodes arranged to form a particle trap
zone. The dielectrophoresis electrode configuration doubles as a
droplet operations electrode. The dielectrophoresis electrode
configuration may include a travelling wave configuration.
[0025] The invention also provides a method of dispensing a
droplet, including providing on a droplet operations surface a
first droplet may include a first concentration of particles
subject to dielectrophoretic forces, localizing the particles in a
region of the first droplet, and conducting an
electrowetting-driven droplet dispensing operation yielding a
second droplet may include a second concentration of the particles,
wherein the second concentration may be greater than the first
concentration, and a third droplet may include a third
concentration may include a third concentration of the particles,
wherein the third concentration may be less than the first
concentration.
DEFINITIONS
[0026] As used herein, the following terms have the meanings
indicated.
[0027] "Activate" with reference to one or more electrodes means
effecting a change in the electrical state of the one or more
electrodes which, in the presence of a droplet, results in a
droplet operation.
[0028] "Bead," with respect to beads on a droplet actuator, means
any bead or particle that is capable of interacting with a droplet
on or in proximity with a droplet actuator. Beads may be any of a
wide variety of shapes, such as spherical, generally spherical, egg
shaped, disc shaped, cubical and other three dimensional shapes.
The bead may, for example, be capable of being transported in a
droplet on a droplet actuator or otherwise configured with respect
to a droplet actuator in a manner which permits a droplet on the
droplet actuator to be brought into contact with the bead, on the
droplet actuator and/or off the droplet actuator. Beads may be
manufactured using a wide variety of materials, including for
example, resins, and polymers. The beads may be any suitable size,
including for example, microbeads, microparticles, nanobeads and
nanoparticles. In some cases, beads are magnetically responsive; in
other cases beads are not significantly magnetically responsive.
For magnetically responsive beads, the magnetically responsive
material may constitute substantially all of a bead or one
component only of a bead. The remainder of the bead may include,
among other things, polymeric material, coatings, and moieties
which permit attachment of an assay reagent. Examples of suitable
magnetically responsive beads include flow cytometry microbeads,
polystyrene microparticles and nanoparticles, functionalized
polystyrene microparticles and nanoparticles, coated polystyrene
microparticles and nanoparticles, silica microbeads, fluorescent
microspheres and nanospheres, functionalized fluorescent
microspheres and nanospheres, coated fluorescent microspheres and
nanospheres, dyed microparticles and nanoparticles, magnetic
microparticles and nanoparticles, superparamagnetic microparticles
and nanoparticles (e.g., DYNABEADS.RTM. particles, available from
Invitrogen Corp., Carlsbad, Calif.), fluorescent microparticles and
nanoparticles, coated magnetic microparticles and nanoparticles,
ferromagnetic microparticles and nanoparticles, coated
ferromagnetic microparticles and nanoparticles, and those described
in in U.S. Patent Publication No. 20050260686, entitled, "Multiplex
flow assays preferably with magnetic particles as solid phase,"
published on Nov. 24, 2005, the entire disclosure of which is
incorporated herein by reference for its teaching concerning
magnetically responsive materials and beads. Beads may be
pre-coupled with a biomolecule (ligand). The ligand may, for
example, be an antibody, protein or antigen, DNA/RNA probe or any
other molecule with an affinity for the desired target. Examples of
droplet actuator techniques for immobilizing magnetically
responsive beads and/or non-magnetically responsive beads and/or
conducting droplet operations protocols using beads are described
in U.S. patent application Ser. No. 11/639,566, entitled
"Droplet-Based Particle Sorting," filed on Dec. 15, 2006; U.S.
Patent Application No. 61/039,183, entitled "Multiplexing Bead
Detection in a Single Droplet," filed on Mar. 25, 2008; U.S. Patent
Application No. 61/047,789, entitled "Droplet Actuator Devices and
Droplet Operations Using Beads," filed on Apr. 25, 2008; U.S.
Patent Application No. 61/086,183, entitled "Droplet Actuator
Devices and Methods for Manipulating Beads," filed on Aug. 5, 2008;
International Patent Application No. PCT/US2008/053545, entitled
"Droplet Actuator Devices and Methods Employing Magnetic Beads,"
filed on Feb. 11, 2008; International Patent Application No.
PCT/US2008/058018, entitled "Bead-based Multiplexed Analytical
Methods and Instrumentation," filed on Mar. 24, 2008; International
Patent Application No. PCT/US2008/058047, "Bead Sorting on a
Droplet Actuator," filed on Mar. 23, 2008; and International Patent
Application No. PCT/US2006/047486, entitled "Droplet-based
Biochemistry," filed on Dec. 11, 2006; the entire disclosures of
which are incorporated herein by reference. The beads may include
one or more populations of biological cells adhered thereto. In
some cases, the biological cells are a substantially pure
population. In other cases, the biological cells include different
cell populations, e.g., cell populations which interact with one
another.
[0029] "Droplet" means a volume of liquid on a droplet actuator
that is at least partially bounded by filler fluid. For example, a
droplet may be completely surrounded by filler fluid or may be
bounded by filler fluid and one or more surfaces of the droplet
actuator. Droplets may, for example, be aqueous or non-aqueous or
may be mixtures or emulsions including aqueous and non-aqueous
components. Droplets may take a wide variety of shapes; nonlimiting
examples include generally disc shaped, slug shaped, truncated
sphere, ellipsoid, spherical, partially compressed sphere,
hemispherical, ovoid, cylindrical, and various shapes formed during
droplet operations, such as merging or splitting or formed as a
result of contact of such shapes with one or more surfaces of a
droplet actuator. For examples of droplet fluids that may be
subjected to droplet operations using the approach of the
invention, see International Patent Application No. PCT/US
06/47486, entitled, "Droplet-Based Biochemistry," filed on Dec. 11,
2006. In various embodiments, a droplet may include a biological
sample, such as whole blood, lymphatic liquid, serum, plasma,
sweat, tear, saliva, sputum, cerebrospinal liquid, amniotic liquid,
seminal liquid, vaginal excretion, serous liquid, synovial liquid,
pericardial liquid, peritoneal liquid, pleural liquid, transudates,
exudates, cystic liquid, bile, urine, gastric liquid, intestinal
liquid, fecal samples, liquids including single or multiple cells,
liquids including organelles, fluidized tissues, fluidized
organisms, liquids including multi-celled organisms, biological
swabs and biological washes. Moreover, a droplet may include a
reagent, such as water, deionized water, saline solutions, acidic
solutions, basic solutions, detergent solutions and/or buffers.
Other examples of droplet contents include reagents, such as a
reagent for a biochemical protocol, such as a nucleic acid
amplification protocol, an affinity-based assay protocol, an
enzymatic assay protocol, a sequencing protocol, and/or a protocol
for analyses of biological fluids.
[0030] "Droplet Actuator" means a device for manipulating droplets.
For examples of droplet actuators, see U.S. Pat. No. 6,911,132,
entitled "Apparatus for Manipulating Droplets by
Electrowetting-Based Techniques," issued on Jun. 28, 2005 to Pamula
et al.; U.S. patent application Ser. No. 11/343,284, entitled
"Apparatuses and Methods for Manipulating Droplets on a Printed
Circuit Board," filed on filed on Jan. 30, 2006; U.S. Pat. No.
6,773,566, entitled "Electrostatic Actuators for Microfluidics and
Methods for Using Same," issued on Aug. 10, 2004 and U.S. Pat. No.
6,565,727, entitled "Actuators for Microfluidics Without Moving
Parts," issued on Jan. 24, 2000, both to Shenderov et al.; Pollack
et al., International Patent Application No. PCT/US2006/047486,
entitled "Droplet-Based Biochemistry," filed on Dec. 11, 2006; and
Roux et al., U.S. Patent Pub. No. 20050179746, entitled "Device for
Controlling the Displacement of a Drop Between two or Several Solid
Substrates," published on Aug. 18, 2005; the disclosures of which
are incorporated herein by reference. Certain droplet actuators
will include a substrate, droplet operations electrodes associated
with the substrate, one or more dielectric and/or hydrophobic
layers atop the substrate and/or electrodes forming a droplet
operations surface, and optionally, a top substrate separated from
the droplet operations surface by a gap. One or more reference
electrodes may be provided on the top and/or bottom substrates
and/or in the gap. In various embodiments, the manipulation of
droplets by a droplet actuator may be electrode mediated, e.g.,
electrowetting mediated or dielectrophoresis mediated or Coulombic
force mediated. Examples of other methods of controlling liquid
flow that may be used in the droplet actuators of the invention
include devices that induce hydrodynamic fluidic pressure, such as
those that operate on the basis of mechanical principles (e.g.
external syringe pumps, pneumatic membrane pumps, vibrating
membrane pumps, vacuum devices, centrifugal forces,
piezoelectric/ultrasonic pumps and acoustic forces); electrical or
magnetic principles (e.g. electroosmotic flow, electrokinetic
pumps, ferrofluidic plugs, electrohydrodynamic pumps, attraction or
repulsion using magnetic forces and magnetohydrodynamic pumps);
thermodynamic principles (e.g. gas bubble
generation/phase-change-induced volume expansion); other kinds of
surface-wetting principles (e.g. electrowetting, and
optoelectrowetting, as well as chemically, thermally, structurally
and radioactively induced surface-tension gradients); gravity;
surface tension (e.g., capillary action); electrostatic forces
(e.g., electroosmotic flow); centrifugal flow (substrate disposed
on a compact disc and rotated); magnetic forces (e.g., oscillating
ions causes flow); magnetohydrodynamic forces; and vacuum or
pressure differential. In certain embodiments, combinations of two
or more of the foregoing techniques may be employed in droplet
actuators of the invention.
[0031] "Droplet operation" means any manipulation of a droplet on a
droplet actuator. A droplet operation may, for example, include:
loading a droplet into the droplet actuator; dispensing one or more
droplets from a source droplet; splitting, separating or dividing a
droplet into two or more droplets; transporting a droplet from one
location to another in any direction; merging or combining two or
more droplets into a single droplet; diluting a droplet; mixing a
droplet; agitating a droplet; deforming a droplet; retaining a
droplet in position; incubating a droplet; heating a droplet;
vaporizing a droplet; cooling a droplet; disposing of a droplet;
transporting a droplet out of a droplet actuator; other droplet
operations described herein; and/or any combination of the
foregoing. The terms "merge," "merging," "combine," "combining" and
the like are used to describe the creation of one droplet from two
or more droplets. It should be understood that when such a term is
used in reference to two or more droplets, any combination of
droplet operations that are sufficient to result in the combination
of the two or more droplets into one droplet may be used. For
example, "merging droplet A with droplet B," can be achieved by
transporting droplet A into contact with a stationary droplet B,
transporting droplet B into contact with a stationary droplet A, or
transporting droplets A and B into contact with each other. The
terms "splitting," "separating" and "dividing" are not intended to
imply any particular outcome with respect to volume of the
resulting droplets (i.e., the volume of the resulting droplets can
be the same or different) or number of resulting droplets (the
number of resulting droplets may be 2, 3, 4, 5 or more). The term
"mixing" refers to droplet operations which result in more
homogenous distribution of one or more components within a droplet.
Examples of "loading" droplet operations include microdialysis
loading, pressure assisted loading, robotic loading, passive
loading, and pipette loading. Droplet operations may be
electrode-mediated. In some cases, droplet operations are further
facilitated by the use of hydrophilic and/or hydrophobic regions on
surfaces and/or by physical obstacles.
[0032] "Filler fluid" means a liquid associated with a droplet
operations substrate of a droplet actuator, which liquid is
sufficiently immiscible with a droplet phase to render the droplet
phase subject to electrode-mediated droplet operations. The filler
fluid may, for example, be a low-viscosity oil, such as silicone
oil. Other examples of filler fluids are provided in International
Patent Application No. PCT/US2006/047486, entitled, "Droplet-Based
Biochemistry," filed on Dec. 11, 2006; International Patent
Application No. PCT/US2008/072604, entitled "Use of additives for
enhancing droplet actuation," filed on Aug. 8, 2008; and U.S.
Patent Publication No. 20080283414, entitled "Electrowetting
Devices," filed on May 17, 2007; the entire disclosures of which
are incorporated herein by reference. The filler fluid may fill the
entire gap of the droplet actuator or may coat one or more surfaces
of the droplet actuator. Filler fluid may be conductive or
non-conductive. Filler fluid may also be a wax-like material that
can be melted at elevated temperatures to fill the entire chip.
[0033] "Immobilize" with respect to magnetically responsive beads,
means that the beads are substantially restrained in position in a
droplet or in filler fluid on a droplet actuator. For example, in
one embodiment, immobilized beads are sufficiently restrained in
position to permit execution of a splitting operation on a droplet,
yielding one droplet with substantially all of the beads and one
droplet substantially lacking in the beads.
[0034] "Magnetically responsive" means responsive to a magnetic
field. "Magnetically responsive beads" include or are composed of
magnetically responsive materials, such as, for example,
DYNABEADS.RTM. MYONE.TM. beads. Examples of magnetically responsive
materials include paramagnetic materials, ferromagnetic materials,
ferrimagnetic materials, and metamagnetic materials. Examples of
suitable paramagnetic materials include iron, nickel, and cobalt,
as well as metal oxides, such as Fe.sub.3O.sub.4,
BaFe.sub.12O.sub.19, CoO, NiO, Mn.sub.2O.sub.3, Cr.sub.2O.sub.3,
and CoMnP. The magnetic field may be produced by any magnetic field
generating device which is suitable for causing the intended
effect. Examples of magnetic field generating devices include
permanent magnets and electromagnets. The product of the field
magnitude and the gradient generate the force on magnetically
responsive beads. In configuring systems of the invention, the
field magnitude or gradient may be altered as needed to achieve a
desired result. In some cases, a combination of electromagnet plus
rare earth magnet may be used to manipulate magnetically responsive
beads.
[0035] "Washing" with respect to washing a bead means reducing the
amount and/or concentration of one or more substances in contact
with the magnetically responsive bead or exposed to the
magnetically responsive bead from a droplet in contact with the
magnetically responsive bead. The reduction in the amount and/or
concentration of the substance may be partial, substantially
complete, or even complete. The substance may be any of a wide
variety of substances; examples include target substances for
further analysis, and unwanted substances, such as components of a
sample, contaminants, and/or excess reagent. In some embodiments, a
washing operation begins with a starting droplet in contact with a
magnetically responsive bead, where the droplet includes an initial
amount and initial concentration of a substance. The washing
operation may proceed using a variety of droplet operations. The
washing operation may yield a droplet including the magnetically
responsive bead, where the droplet has a total amount and/or
concentration of the substance which is less than the initial
amount and/or concentration of the substance. Examples of suitable
washing techniques are described in Pamula et al., U.S. Pat. No.
7,439,014, entitled "Droplet-Based Surface Modification and
Washing," granted on Oct. 21, 2008, the entire disclosure of which
is incorporated herein by reference. The unbound substances being
removed from the liquid surrounding the beads
[0036] The terms "top," "bottom," "over," "under," and "on" are
used throughout the description with reference to the relative
positions of components of the droplet actuator, such as relative
positions of top and bottom substrates of the droplet actuator. It
will be appreciated that the droplet actuator is functional
regardless of its orientation in space.
[0037] When a liquid in any form (e.g., a droplet or a continuous
body, whether moving or stationary) is described as being "on",
"at", or "over" an electrode, array, matrix or surface, such liquid
could be either in direct contact with the
electrode/array/matrix/surface, or could be in contact with one or
more layers or films that are interposed between the liquid and the
electrode/array/matrix/surface.
[0038] When a droplet is described as being "on" or "loaded on" a
droplet actuator, it should be understood that the droplet is
arranged on the droplet actuator in a manner which facilitates
using the droplet actuator to conduct one or more droplet
operations on the droplet, the droplet is arranged on the droplet
actuator in a manner which facilitates sensing of a property of or
a signal from the droplet, and/or the droplet has been subjected to
a droplet operation on the droplet actuator.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIGS. 1A-1E illustrate top views of an electrode/magnet
arrangement of a droplet actuator and a process of incubating
droplets including magnetically responsive beads.
[0040] FIGS. 2A-2E show the electrode/magnet arrangement of FIGS.
1A-1E and a different process of incubating droplets including
magnetically responsive beads.
[0041] FIGS. 3A-3C show the electrode/magnet arrangement of FIGS.
1A-1E and illustrate a process of incubating droplets by
transporting droplets back and forth.
[0042] FIGS. 4A-C show the results of work comparing incubation
time between on-magnet and off-magnet incubation protocols for an
immunoassay.
[0043] FIGS. 5A-5E illustrate top views of an electrode/magnet
arrangement of a droplet actuator (not shown) and a process of
washing magnetically responsive beads.
[0044] FIGS. 6A-6C show a comparison of washing protocols between
slug shaped and circular shaped wash droplets on immunoassay
performance measured in chemiluminescence.
[0045] FIGS. 7A-7C illustrate top views of the electrode/magnet
arrangement of FIGS. 5A-5E and show a process of resuspending
magnetically responsive beads during a wash protocol.
[0046] FIGS. 8A and 8B show plots comparing the results of washing
without resuspension cycles and with resuspension cycles,
respectively.
[0047] FIG. 9 illustrates a top view of an electrode/magnet
arrangement on a droplet actuator configured for efficient
washing.
[0048] FIGS. 10A-10C show a top view of an electrode/magnet
arrangement on a droplet actuator and illustrates a process of
separating beads from a droplet.
[0049] FIGS. 11A-11C show a top view of the electrode/magnet
arrangement shown in FIGS. 10A-10C and a process of transporting
beads within a droplet.
[0050] FIGS. 12A and 12B show a comparison of bench top and droplet
actuator immunoassay reagent ratios and a plot of reagent
concentration versus signal strength.
[0051] FIG. 13 shows a plot of the kinetics of a reaction between a
chemiluminescent substrate and ALP on magnetically responsive beads
for Troponin I (TnI).
[0052] FIG. 14 is a top view of a droplet actuator layout that may
be used for extracting DNA from a whole blood sample.
[0053] FIGS. 15A and 15B illustrate top views of an
electrode/magnet arrangement and show steps in an exemplary,
nonlimiting, immunoassay process.
[0054] FIG. 16 shows a plot of two 5-point standard curves for
cytokine IL-6.
[0055] FIG. 17 shows a plot of two 6-point standard curves for
cytokine TNF-.alpha..
[0056] FIG. 18 illustrates a perspective view of a microfluidics
assay multiplexing platform of the invention.
[0057] FIG. 19 illustrates the components of an example of a 4-plex
immunoassay that may be performed in a single droplet (not shown)
using quantum dots within the microfluidics assay multiplexing
platform of the invention.
[0058] FIGS. 20A and 20B illustrate the components of an example of
an immunoassay "sandwich" formation process that may be performed
in a single droplet (not shown) using quantum dots within the
microfluidics assay multiplexing platform of the invention.
[0059] FIG. 21 illustrates a perspective view of spectrometer
system of microfluidics assay multiplexing platform of the
invention.
[0060] FIG. 22 illustrates a concept for turning the information of
a 2D CCD array into multiple spectra.
[0061] FIG. 23 illustrates a perspective view of 12-channel
fiber-based readout head of FIG. 21, showing more details
thereof.
[0062] FIGS. 24A and 24B illustrate one configuration of a portion
of a droplet actuator of the invention.
[0063] FIGS. 25A-25E illustrate the configuration of FIGS. 24A and
24B in operation.
[0064] FIGS. 26A-26C illustrate an electrode path, including a
specialized electrode, which can be used as a droplet operations
electrode and as a DEP electrode.
[0065] FIG. 27A illustrates an octagon-shaped DEP electrode
configuration based on the use of 8 triangular shaped
electrodes.
[0066] FIG. 27B illustrates a hexagon-shaped DEP electrode
configuration based on the use of 8 triangular shaped
electrodes.
[0067] FIGS. 28A and 28B illustrate asymmetrical quadripole DEP
electrode arrangements, formed from differently sized trianglular
electrodes.
[0068] FIG. 29 illustrates an embodiment in which quadripole
electrodes are arranged in an electrode array.
[0069] FIG. 30 shows a dynamically tunable quadripole DEP electrode
arrangement in which each triangular electrode is further
subdivided into sections A, B, C and D.
[0070] FIGS. 31A-31C illustrate a configuration for applying a
travelling wave DEP within a droplet.
[0071] FIG. 32 shows a side view of the configuration illustrated
in FIGS. 31A-31C showing how the particles may congregate at an
edge of droplet.
[0072] FIG. 33 illustrates travelling wave DEP configurations in
which DEP electrodes are provided on a first substrate, and droplet
operations electrodes are provided on a second substrate.
[0073] FIG. 34 illustrates an alternative electrode
configuration.
[0074] FIGS. 35A-35C show an electrode path including DEP
electrodes.
[0075] FIGS. 36A-36E illustrate an embodiment which is similar to
the embodiment illustrated in FIGS. 35A-35C.
[0076] FIG. 37 illustrates an array of electrodes including DEP
electrodes.
[0077] FIGS. 38A and 38B illustrate several alternatives to
electrode described herein.
[0078] FIGS. 39A-39D illustrate a reservoir electrode having a DEP
electrode inset.
[0079] FIGS. 40A-40D illustrate a configuration useful for
dispensing a droplet including substantially all particles from a
particle-containing droplet on reservoir electrode onto a path or
array of electrodes.
[0080] FIG. 41 illustrates the use of DEP to separate particles
within a droplet for imaging.
DESCRIPTION
[0081] The invention provides devices and methods for resuspending
or circulating beads in a bead-containing droplet on a droplet
actuator. During an incubation or washing protocol, for example, a
bead-containing droplet may be subjected to one or more droplet
operations to resuspend or circulate beads within the droplet.
These droplet operations may, for example, be mediated by
electrowetting or other electric field mediated phenomena. Suitable
droplet operations may be selected to improve reaction kinetics,
such as by agitating, redistributing, and or circulating droplet
contents and/or controlling droplet temperature. Redistribution or
circulation of beads within a droplet may increase binding of a
target analyte to the beads and/or free up unbound substances from
within magnetically aggregated beads.
[0082] 8.1 Bead Incubation and Washing
[0083] Magnetically responsive beads have a tendency to settle and
form aggregates due to gravity and/or exposure to magnetic forces.
Non-magnetically responsive beads may also aggregate due to surface
interactions between beads or between substances bound to beads.
Aggregation reduces the available surface area for binding and
slows reaction kinetics, increasing time to result and/or reducing
assay sensitivity. Interstices in magnetically responsive bead
aggregates can also hold unbound substances. These trapped
substances may be difficult or impossible to separate from the
beads during washing processes, reducing sensitivity of assay
results. The invention provides techniques for circulating or
mixing beads within a droplet to overcome these issues. The
invention also provides incubation protocols that make use of these
recirculation techniques for improving binding of molecules to the
magnetically responsive beads. Moreover, the invention provides
washing protocols that make use of these recirculation techniques
for removing unbound molecules from the magnetically responsive
beads.
[0084] 8.1.1 Incubation Protocols
[0085] As observed above, beads in a droplet on a droplet actuator
are subject to bead aggregation issues. These bead-containing
droplets may be provided on a droplet operations surface of a
droplet actuator. The droplet operations surface may, in some
cases, be provided within a droplet operations gap of a droplet
actuator. The droplet may be partially or substantially completely
surrounded by a filler fluid. The droplet may be provided in a
reservoir associated with a droplet actuator. The reservoir may be
in fluid communication with a liquid path configured for
transporting liquid from the reservoir onto a droplet operations
surface of a droplet actuator. Here again, the droplet operations
surface may, in some cases, be provided within a droplet operations
gap of a droplet actuator.
[0086] The bead-containing droplet may be subjected to bead
resuspension protocols on the droplet actuator. During an
incubation or washing protocol, for example, the bead-containing
droplet may be subjected to one or more droplet operations to
resuspend or circulate beads within the droplet. These droplet
operations may, for example, be mediated by electrowetting or other
electric field mediated phenomena. Suitable droplet operations may
be selected to improve reaction kinetics, such as by agitating,
redistributing, and or circulating droplet contents and/or
controlling droplet temperature. Redistribution or circulation of
beads within a droplet may increase binding of a target analyte to
the beads and/or free up unbound substances from within
magnetically aggregated beads.
[0087] Droplet transport is an example of a droplet operation
selected to redistribute or circulate beads within a droplet.
During transport from electrode-to-electrode, contents of the
bead-containing are circulated and redistributed within the
droplet. Other examples of droplet operations suitable for
enhancing incubation or washing include splitting and merging
droplet operations. Any combination of droplet operations may be
used. Multiple droplet operations may be combined to provide a
complete incubation cycle (e.g., transport-split-merge,
transport-split-transport-merge-transport). Incubation cycles may
be repeated any number of times to achieve a desired result, such
as a desired degree of mixing of beads with contents of the
droplet.
[0088] The incubated droplet may include any suitable components
that require incubation. For example, the droplet may include
reagents and/or sample for conducting an immunoassay. A droplet
including beads having a binding affinity for an analyte may be
subjected to one or more incubation cycles to improve binding of
the analyte to the beads. Beads bound to an analyte may be
subjected to one or more incubation cycles in a droplet with
secondary antibody to improve binding of the secondary antibody to
the target. In another case, the magnetic beads already containing
the sample of interest can be incubated with an elution buffer to
elute the sample bound to the beads and transport it to further
processing. In that case, the beads would be transported to waste
reservoir after eluting off the sample. It should also be noted
that incubation cycles may be used to enhance the kinetics of
chemical reactions even in droplets where beads are not present. As
another example, a droplet including cells and reagents for
supplying one or more metabolic requirements of the cells may be
subjected to one or more incubation cycles to improve supply of the
metabolic reagent to the cells. In some cases, the cells may be
bound to beads. In another embodiment, the incubation can be
between a chemiluminescence or fluorescence producing reagent with
an enzyme on an immuno-complex bound to magnetic beads. Effective
resuspension of magnetic beads by incubating the enzyme labeled
magnetic beads would improve the sensitivity of the assay.
[0089] FIGS. 1A-1E illustrate top views of an electrode/magnet
arrangement 100 of a droplet actuator and a process of incubating
droplets including magnetically responsive beads. Arrangement 100
shows a path of electrodes 110. Droplet 118 is positioned in a
droplet operations gap (not shown) or on a droplet operations
surface where droplet 118 is subject to droplet operations mediated
by electrodes 110. Droplet 118 includes magnetically responsive
beads. Magnet 114 is provided in proximity to electrodes 110M.
Electrodes 110M are a subset of electrodes 110. Magnet 114 is
positioned relative to electrodes 110M such that when droplet 118
is atop one or more of electrodes 110M, magnetically responsive
beads 122 within droplet 118 are attracted by the magnetic field of
magnet 114. Alternatively, magnet 114 is positioned relative to
electrodes 110M such that when droplet 118 is subject to droplet
operations mediated by electrodes 110M, magnetically responsive
beads 122 within droplet 118 are attracted by the magnetic field of
magnet 114. The attraction of magnetically responsive beads 122 may
cause beads 122 to move within droplet 118 in the direction of
magnet 114. Magnetically responsive beads 122 may move towards an
edge of droplet 118 which is proximate magnet 114. The parameters
of the configuration may be adjusted such that beads 122 are
attracted towards an edge of droplet 118 without exiting droplet
118. In this and other examples described herein which make use of
magnetically responsive beads and magnets, the technique may be
optimized by adjusting properties such as interfacial tension of
droplet 118, properties and concentration of magnetically
responsive beads 122, and the pull force of exerted by magnet 114
on magnetically responsive beads 122. The incubation technique
shown in FIG. 1 illustrates the use of droplet operations to
redistribute magnetically responsive beads 122 within droplet 118.
One or more of the droplet operations may be conducted while the
magnetically responsive beads 122 are being influenced or attracted
by the magnetic field of magnet 114. Droplet 118 may be subjected
to droplet operations mediated by electrodes 110M while
magnetically responsive beads 122 within droplet 118 are being
attracted to magnet 114. For example, droplet 118 may be
transported along electrodes 110M by using electrodes 110M to
create an electrowetting effect on a droplet operations
surface.
[0090] In FIG. 1A, droplet 118 including beads 122 is positioned
adjacent to and overlapping droplet operations electrodes 110M.
Magnetically responsive beads 122 are attracted by the magnetic
field of magnet 114, causing a concentration of beads to form at
the edge of droplet 118 that is closest to magnet 114. In FIG. 1B,
droplet 118 is transported to and elongated along several
electrodes 110M using droplet operations mediated at least in part
by electrodes 110M. In this manner, droplet 118 is caused to
conform to an elongated geometry. The transformation of droplet 118
from a rounded configuration to an elongated configuration produces
a flow of liquid within droplet 118 that redistributes beads 122 in
droplet 118 allowing interaction of the beads with several parts of
the droplet more effectively. In FIG. 1C, elongated droplet 118 is
split using droplet operations to form daughter droplets 118A,
118B. Two daughter droplets 118A, 118B are illustrated here, but
any number of daughter droplets may be formed within the scope of
the invention. Splitting of droplet 118 redistributes beads 122
within the daughter droplets 118A, 118B. In FIG. 1D, daughter
droplets 118A,118B are merged using droplet operations mediated by
electrodes 110M to reform droplet 118. This merging is accomplished
while beads 122 are being attracted by the magnetic field of magnet
114. The transporting, elongation, splitting, and merging
operations of FIGS. 1B, 1C, and 1D are one example of an incubation
cycle. Multiple incubation cycles may be performed to provide for
resuspension and/or redistribution (i.e., mixing) of beads 122 of
droplet 118, e.g., during incubation and/or washing of droplet 118.
In FIG. 1E, droplet 118 is transported away from magnet 114 using
droplet operations to adjacent electrodes 110. Droplet 118 may, for
example, be transported a distance from magnet 114 sufficient to
reduce or substantially eliminate the attractive force of the
magnetic field of magnet 114 on magnetically responsive beads 122.
For example, the magnetic force may be sufficiently reduced to
permit beads 122 to be resuspended in droplet 118. Also, the
droplet can be transported at higher switching speeds allowing very
little time for the magnetic beads to get attracted. Higher droplet
switching speeds would enable better binding efficiency of the
analyte onto the magnetic beads thereby requiring lesser incubation
time. In this case the magnet is right underneath the droplet
containing magnetic beads, there is a higher chance of aggregation
of beads since the magnetic beads are always under the effect of
magnetic field gradient at every step of incubation. This effect
would be more pronounced in cases of longer incubation times or
multiple steps of incubation resulting in clumps of beads. However,
incubation of the beads right over the magnet would be useful when
the real estate available is very little and the sensitivity
requirements are not as stringent, wherein the typical dynamic
range of the analyte concentration is 1 ng/mL to 100 ng/mL and the
incubation times are in the range of 30 seconds to 300 seconds.
[0091] FIGS. 2A-2E show the electrode/magnet arrangement 100 of
FIG. 1 and a different process of incubating droplets including
magnetically responsive beads. FIG. 2 illustrates removal of
magnetically responsive beads 122 from the attraction of the
magnetic field of magnet 114, followed by execution of an
incubation cycle. At a sufficient distance from magnet 114, any
attractive force exerted by the magnetic field may be sufficiently
reduced to permit beads 122 to be resuspended and distributed
within droplet 118, as illustrated in FIGS. 2B-2E. A series of
droplet operations, such as split and merge droplet operations, may
be used to agitate and mix beads 122 within the droplet 118 after
droplet 118 has transported a sufficient distance from magnet 114
to permit resuspension of beads 122. In this case, since the
droplet is incubated away from the magnet surface, the maximum
magnetic field gradient is experienced only when the droplet is at
position 2A. However this can also be alleviated by transporting
the droplet at higher switching speeds allowing very little time
for the magnetic beads to settle and get influenced by the magnetic
field gradient.
[0092] In FIG. 2A, droplet 118 with beads 122 is positioned
adjacent to droplet operations electrodes 110M, such that beads 122
are attracted by the magnetic field of magnet 114. A concentration
of magnetically responsive beads 122 is formed at an edge of
droplet 118 that is closest to magnet 114. In FIG. 2B, droplet 118
is transported using electrode mediated droplet operations away
from magnet 114 and repositioned at a distance sufficient to permit
resuspension of beads 122 in droplet 118. FIGS. 2C, 2D, and 2E show
droplet elongation (i.e., formation of slug-shaped geometry),
droplet splitting, and droplet merging, respectively. The
incubation cycle cause spatial reorientation of the liquid of
droplet 118 and redistribution of beads 122 within droplet 118.
[0093] FIGS. 3A-3C show the electrode/magnet arrangement 100 of
FIG. 1 and illustrate a process of incubating droplets by
transporting droplets back and forth. The incubation steps take
place at a distance from the magnetic field of magnet 114 which is
sufficient to permit the magnetically responsive beads 122 remain
suspended within droplet 118 during incubation. A series of droplet
transport operations are used to resuspend the beads within the
droplet. Droplet 118 is transported using droplet operations along
a path of droplet operation electrodes 110. The transporting steps
agitate the liquid in droplet 118 and cause redistribution of
magnetically responsive beads 122 within droplet 118. This kind of
incubation sequence would allow for higher switching speeds than
the sequences described earlier. Since there is a split-merge
operation in the two incubation sequences (FIGS. 1 and 2), the
switching speed is limited since at higher speeds, the droplets
would not merge effectively. So, shuttling the droplet with no
split-merge operation at higher speeds would achieve the same
incubation efficiency.
[0094] FIGS. 4A-C show the results of work comparing incubation
time between on-magnet and off-magnet incubation protocols for an
immunoassay. Results are measured in chemiluminescence. The
sequence of droplet operations in the incubation protocol involved
shuttling the droplet along a linear path of electrodes with a
splitting and merging step inserted between transport cycles.
Immunoassays were performed on a 300 mL droplet that contained 5
ng/mL TnI as a model assay using two different incubation
protocols, among many other possibilities, with one performed
on-magnet as shown in FIG. 4B and the other off-magnet as shown in
FIG. 4C. The first incubation protocol was performed by shuttling a
merged droplet (sample droplet, capture antibody conjugated
magnetic beads droplet and ALP-labeled reporter antibody droplet)
across a set of seven electrodes (Steps i and ii in FIG. 4B)
followed by a split and merge sequence performed at the center of
the magnet (Steps iii, iv and v in FIG. 4B) so that the beads were
about equally distributed between the split droplets. Since the
droplets were transported at a switching frequency of 1 Hz, it
takes 18 seconds for the droplets to complete one incubation cycle.
Several such incubation cycles are repeated to obtain the required
incubation time as a multiple of 18 seconds. In the second
incubation protocol, the sequence of droplet operations and the
number of electrodes used for incubation is the same but the only
difference is that it is performed away from the magnet (the
nearest the droplet gets to the magnet is two electrode
widths--FIG. 4C(iii). Immunoassays were performed using both the
incubation protocols with varying incubation times and a plot of
incubation time versus signal was obtained for both the incubation
protocols, as shown in FIG. 4A1. Time-to-saturation in the
on-magnet incubation protocol was double that of the off-magnet
protocol. The difference in time-to-saturation may occur because of
the relatively greater recirculation of beads in the off-magnet
protocol. Off-magnet incubation circulates the magnetically
responsive beads in both the lateral (X-Y) and vertical (Z
direction) dimensions.
[0095] In certain point of care applications, where the size of the
droplet actuator and thereby the real estate on the droplet
actuator is restricted, incubation might need to be performed on
the magnet which will take about 10 minutes if 100% antigen has to
be captured leaving only 5 minutes for all other operations within
the time to result budget of 15 minutes. Therefore, in such a case,
incubation may be performed only for 5 minutes but still capturing
80% of the antigen. On the other hand, if real estate is not an
issue and if a few more electrodes off-magnet could be utilized for
incubation, then 100% of the antigen can be captured within 4
minutes. The same effect of off-magnet incubation could also be
obtained by mechanically moving the magnet away from the droplet
actuator.
[0096] FIG. 4A2 shows the result of work comparing the signal
obtained by using different switching speeds while incubating the
droplet using the sequence described in FIG. 3. Results were
measured in chemiluminescence. Immunoassays were performed on a 600
mL droplet that contained 10 pg/mL of Tumor necrosis factor-.alpha.
(TnF-.alpha.). The sequence of droplet operations in the incubation
protocol involved shuttling the droplet along a linear path of
electrodes with no split-merge operation as shown in FIG. 3. Effect
of switching speed on the signal obtained was studied by performing
the immunoassay using different switching speeds at the same
incubation time. Since the total incubation time was fixed, the
droplets had to be oscillated for a larger number of cycles at
higher switching speeds in order to maintain the same total
incubation time.
[0097] 8.1.2 Washing Protocols
[0098] The invention provides washing protocols for removing
unbound molecules from the magnetically responsive beads. The input
to a washing protocol is a bead-containing droplet including
unbound substances, and the output is typically a bead-containing
droplet in which the concentration and/or quantity of these unbound
substances is reduced relative to the concentration and/or quantity
present in the input droplet. Washing is thus a critical step in
the implementation of many assay protocols. In some embodiments,
washing is performed using a merge-and-split wash protocol. A
merge-and-split wash protocol generally involves merging a
bead-containing droplet with a wash droplet and then splitting off
a supernatant droplet which carries away at least a portion of the
unbound substances. In some cases, an initial droplet is subjected
to one or more splitting steps prior to the initial wash droplet
merge step. Droplet splitting steps are typically performed in the
presence of a magnet, so that the split yields one or more
bead-containing droplets in which the concentration and/or quantity
of unbound substances is reduced relative to the concentration
and/or quantity present in prior to the split and one or more
droplets without a substantial amount of beads wherein the
concentration and/or quantity of unbound substances is increased
relative to the concentration and/or quantity present prior to the
split. Bead retention is important, particularly when the process
involves multiple wash cycles, each cycle may potentially reduce
the number of retained beads. The washing steps may be repeated as
needed until the unbound substances are sufficiently depleted from
the liquid surrounding the beads. In some cases, the unbound
substances are substantially or completely depleted from the liquid
surrounding the beads.
[0099] FIGS. 5A-5E illustrate top views of an electrode/magnet
arrangement 500 of a droplet actuator (not shown) and a process of
washing magnetically responsive beads. Wash buffer droplet 516 and
magnetically responsive bead droplet 514 are elongated. A series of
merge and split operations are used to remove unbound material from
liquid surrounding the beads. The merge and split operations may
provide for substantially complete replacement of liquid in droplet
514 surrounding beads 522 in the bead droplet. Thus, substantially
all unbound supernatant in droplet 514 may be replaced with wash
buffer from droplets 516 during the washing operation.
[0100] Electrode/magnet arrangement 500 may include an arrangement
(e.g., a path or array) of droplet operations electrodes 510.
Droplets 514 and 516 are positioned in a droplet operations gap
(not shown) or on a droplet operations surface where droplets 514
and 516 are subject to droplet operations mediated by electrodes
510. Droplet 514 includes magnetically responsive beads 522. Magnet
512 is provided in proximity to electrodes 510M. Electrodes 510M
are a subset of electrodes 510. Magnet 512 is positioned relative
to electrodes 510M such that when droplet 514 is atop one or more
of electrodes 510M, magnetically responsive beads 522 within
droplet 514 are attracted by the magnetic field of magnet 512.
Alternatively, magnet 512 is positioned relative to electrodes 510M
such that when droplet 514 is subject to droplet operations
mediated by electrodes 510M, magnetically responsive beads 522
within droplet 514 are attracted by the magnetic field of magnet
512. The attraction of magnetically responsive beads 522 may cause
beads 522 to move within droplet 514 in the direction of magnet
512. Magnetically responsive beads 522 may move towards an edge of
droplet 514 which is proximate to magnet 512. The parameters of the
configuration may be adjusted such that beads 522 are attracted
towards an edge of droplet 514 without exiting droplet 514. In this
and other examples described herein which make use of magnetically
responsive beads and magnets, the technique may be optimized by
adjusting properties such as interfacial tension of droplets 514
and 516, properties and concentration of magnetically responsive
beads 522, and the pull force of exerted by magnet 512 on
magnetically responsive beads 522. The size, strength, orientation
relative to beads, and number of magnets may also be varied for the
purpose of optimization. The washing technique shown in FIG. 5
illustrates the use of droplet operations to redistribute
magnetically responsive beads 522 within droplet 514 during the
droplet washing operation. One or more of the droplet operations
may be conducted while the magnetically responsive beads 522 are
being influenced or attracted by the magnetic field of magnet 512.
Droplet 514 may be subjected to droplet operations mediated by
electrodes 510M while magnetically responsive beads 522 within
droplet 514 are being attracted to magnet 512. For example, droplet
514 may be transported along electrodes 510M by using electrodes
510M to create an electrowetting effect on a droplet operations
surface.
[0101] Droplet 516 may include a wash buffer. Droplet 514 may
include magnetically responsive beads 522. Bead droplet 514 and
wash buffer droplet 516 may, for example, be 2.times. droplets,
meaning that their footprint is approximately 2 times the area of
one droplet operations electrode 510. Bead droplet 514 and wash
buffer droplet 516 may be configured as slug-shaped droplets (i.e.,
elongated droplets) by performing droplet operations on the
2.times. droplets using two underlying active droplet operations
electrodes 510. Because the excess droplet volume is now spread
over a second active droplet operations electrode 510, the droplets
are elongated and conform to the shape of two electrodes.
[0102] FIG. 5A shows bead droplet 514 that has beads 522 therein
positioned such that beads 522 are attracted by the magnetic field
of magnet 512. A concentration of beads is formed at the edge of
bead droplet 514 that is closest to magnet 512. FIG. 5B shows bead
droplet 514 and buffer droplet 516 are merged to form merged
droplet 520 while beads 522 remain under the attractive influence
of magnet 512. Merging of bead droplet 514 and wash droplet 516
provides flow patterns within the merged droplet 520 that
redistribute beads 522. FIGS. 5C and 5D show elongation of droplet
520 in distally relative to magnet 512 and beads 522. Elongation
may be achieved by activating the contiguous droplet operations
electrodes 510. As droplet 520 is extended, beads 522 remain
concentrated on magnet 512. FIG. 5E shows droplet 520 split using
droplet operations to form supernatant droplet 532 and washed bead
droplet 534. Supernatant droplet 532 includes unbound particles and
reagents, such as unbound reporter antibody and sample
contaminants, from bead droplet 514. Supernatant droplet 532 is
typically discarded in a waste reservoir (not shown) or transported
into another process, e.g., into contact with a different bead set
for capturing a different target from the sample. FIGS. 5A through
5E show an example of a set of droplet operations that comprise a
wash cycle. Several wash cycles may be performed to provide for
sufficient removal of unbound material.
[0103] The wash cycle may yield a bead-containing droplet having a
decreased quantity or substantially decreased quantity of an
unwanted substance or substances relative to the starting
concentration of the unwanted substance or substances. The
resulting droplet may in some embodiments have a volume which is
approximately the same as the starting volume. In some embodiments,
the wash cycle may be repeated until a predetermined maximum
quantity of the one or more components is met or exceeded in the
resulting droplet. The predetermined amount may represent a
substantial reduction relative to the starting concentration. In
some cases, the resulting droplet may be substantially free of the
unwanted substance. For example, in some embodiments, the reduction
in amount of the unwanted substance exceeds 99, 99.9. 99.99,
99.999, 99.9999, 99.99999, 99.999999 percent on a molar basis.
[0104] Generally, each wash cycle results in retention of
sufficient beads for conducting the intended assay without unduly
detrimental effects on the results of the assay. In certain
embodiments, each execution of a wash cycle results in retention of
more than 99, 99.9. 99.99, 99.999, 99.9999, 99.99999, or 99.999999
percent of beads. In still other embodiments, the amount of
retained beads is calculated and the results are adjusted
accordingly.
[0105] In some cases, the wash cycle is repeated until the
reduction in amount of the unwanted substance exceeds 99, 99.9.
99.99, 99.999, 99.9999, 99.99999, 99.999999 percent on a molar
basis and more than 99, 99.9. 99.99, 99.999, 99.9999, 99.99999, or
99.999999 percent of beads is retained.
[0106] FIGS. 6A-6C show a comparison of washing protocols between
slug shaped and circular shaped wash droplets on immunoassay
performance measured in chemiluminescence. While incubation
benefits greatly from higher mixing efficiency, washing by serial
dilution benefits from no-mixing or low-mixing conditions. Ideally,
the wash droplet should maximize dilution of the liquid surrounding
the beads and minimize dilution of the supernatant that is carrying
away the unbound substances. Immunoassays were tested using only
two wash protocols to determine the optimum washing protocol
required to achieve a total time to result of <15 minutes.
Immunoassays were performed on a 300 mL droplet containing 0 ng/mL
TnI using either an elongated droplet or a circular droplet. 0
ng/mL TnI was chosen for this study because this sample would have
the greatest amount of unbound reporter antibodies that must be
removed during washing. Schematics of the washing protocols for the
elongated and circular cases are shown in FIGS. 6B and 6C,
respectively. In the first washing protocol, the wash buffer
droplet and the magnetic bead droplet are less rounded or circular
and more elongated, which was achieved by operating on the 2.times.
droplets using two electrodes each. By activating two electrodes,
the 2.times. droplet conforms to the shape of two electrodes as
illustrated in FIG. 6A. Even though the two protocols in FIGS.
6B(vi) and 6C(vi) appear similar, the effect of washing, as
depicted by the shading, is quite different. The second washing
protocol involves merging the wash droplet with the magnetic bead
droplet where the shape of the droplets is circular or almost
circular as shown in FIG. 6B. The circular shape of the droplet is
obtained by operating on a 2.times. (denotes two unit droplets)
droplet using only one electrode.
[0107] The magnetic bead droplets were washed with varying numbers
of wash cycles using the two wash protocols described above, and
the chemiluminescence was read with a PMT after adding the
chemiluminescence reagent. A plot of number of wash cycles versus
the chemiluminescent signal was obtained for both the washing
protocols, as illustrated in FIG. 6A. Wash cycles may typically
range from about 2 to about 30 seconds. The time for each wash
cycle generally depends on the distance a wash droplet has to
travel from the wash reservoir to the magnetic beads en route to
the waste reservoir, transport speed of the droplets, dispensing
and disposal rates of the droplets.
[0108] Since washing on droplet actuator involves several dilution
steps, the time to result can be seriously affected when several
wash cycles are required to achieve the desired wash efficiency.
FIG. 6A shows the chemiluminescence signal obtained from an
immunoassay after the droplets were subjected to different numbers
of wash cycles and different wash protocols. In both the cases
presented herein, incubation was performed using the off-magnet
incubation protocol for 3 minutes. Each wash cycle takes about 8-10
seconds in the slug-based protocol and 8-14 seconds in the circular
droplet protocol. It can be seen from FIG. 6A, that when washing
was performed using slugs of liquid (or elongated droplets as shown
in FIG. 6C) desired wash levels were achieved using fewer wash
cycles when compared to washing using circular shaped droplets. In
the former, mixing was minimized and the bulk of the unbound
material from the supernatant was replaced with fresh wash buffer,
whereas in the latter mixing was facilitated by operating the
2.times. droplets using only one electrode each. The shading used
in FIGS. 6B and 6C depict the situation achieved by operating the
2.times. droplet using two electrodes and one electrode
respectively. Also, it was observed visually that the dispersion of
magnetic beads in the lateral plane was higher in the elongated
droplet washing when the fresh wash buffer droplet merged with the
magnetic bead droplet. This would enable the breaking up of
aggregates and any unbound antibody trapped in the interstices to
diffuse into the supernatant and be washed away instantly or in
subsequent washes. Hence desired wash levels were achieved in
.about.10 washes using elongated droplet washing, as compared to
>18 washes in the rounded droplet washing. The washing behavior
has two distinct regimes, one regime where washing is very
pronounced and the second where the washing is more subtle. In the
slug based washing case, the washing is pronounced with each wash
cycle up to 9 cycles, after which the effect of washing is almost
subtle or negligible. In the rounded droplet protocol, the washing
effect is pronounced until the 15th wash, although the step wash
efficiency is less than that observed for the slug-based protocol.
Washing is only marginally effective for the circular droplet
protocol between the 15th and 18.sup.th cycles. This may occur
because the free unbound material may be washed away in the first
few cycles, after which washing only removes the unbound material
trapped between the beads. Removal of substances trapped between
the beads may be improved by including resuspension cycles in the
wash protocol.
[0109] FIGS. 7A-7C illustrate top views of the electrode/magnet
arrangement 500 of FIG. 5 and show a process of resuspending
magnetically responsive beads during a wash protocol. Droplet
resuspension cycles are used to resuspend the magnetically
responsive beads during or between wash cycles to free material
that would otherwise remain trapped in interstices of bead
aggregates. FIG. 7A shows bead droplet 514 that includes
magnetically responsive beads 522 being transported using droplet
operations away from magnet 512 in the direction of arrow A. FIGS.
7B and 7C show transporting of bead droplet 514 along a path of
droplet operation electrodes 510 in the direction of arrow A. Two
transport operations are shown in FIGS. 7B and 7C, but any number
of transport operations may be used to comprise a resuspension
cycle. Transporting of bead droplet 514 provides for sufficient
resuspension of beads 522 such that unbound material from the
interstices of bead aggregates may be effectively removed in
subsequent wash cycles. Other droplet operations, such as merging
and splitting, or rounding and elongating, may be included in the
resuspension cycle.
[0110] A complete wash protocol may include a series of wash
cycles, such as the slug based wash cycles of FIG. 5, interspersed
with a one or more resuspension cycles. Depending on the
sensitivity of the assay required and the time to result
requirement, any number of wash cycles may be interspersed with any
number of resuspension cycles. For example, a complete wash
protocol sequence may include, for example, four wash cycles, four
resuspension cycles, and four wash cycles.
[0111] FIGS. 8A and 8B show plots comparing the results of washing
without resuspension cycles and with resuspension cycles,
respectively. FIG. 8A illustrates that a washing protocol in the
absence of one or more resuspension cycles provides an initial drop
in signal after a number of wash cycles (A). As the number of wash
cycles increase (B), there is a further reduction in signal that
may be due to loss of unbound material from the interstices of bead
aggregates. FIG. 8B illustrates that a washing protocol including
resuspension cycles provides more efficient removal of unbound
material to a near zero level using fewer numbers of wash cycles
(A).
[0112] FIG. 9 illustrates a top view of an electrode/magnet
arrangement 900 on a droplet actuator configured for efficient
washing. Droplet actuator 900 includes an arrangement of droplet
operations electrodes 910. Electrodes 910 are arranged to provide
wash lanes 912 and waste lane 916. Wash lanes 912 are associated
with magnets 914 arranged to permit a droplet to be transported
into the field of the magnet for immobilizing or restraining
movement of magnetically responsive beads within the droplet. Wash
reservoirs 920 including reservoir electrodes 922 are provided for
dispensing wash droplets onto wash lanes 912. Wash reservoirs 920
may be associated with one or more openings 924 in a droplet
actuator substrate (not shown) for transporting wash liquid onto
reservoir electrode 922 for use in dispensing wash droplets. Waste
reservoir 918 including reservoir electrode 919 is provided for
disposing of waste droplets from waste lane 916. Waste reservoir
918 may be associated with one or more openings 925 in a droplet
actuator substrate (not shown) for transporting waste liquid out of
reservoir 918 to a locus which is exterior to the droplet
operations gap. Reservoirs 918 and 920 may be virtual reservoirs or
may be partially bounded by a physical barrier (not shown), such as
a gasket or spacer partially surrounding the reservoir electrode
and including an opening for dispensing of droplets along electrode
path 912 or disposal of droplets along path 916.
[0113] Droplet actuator 900 may be used to conduct a bead washing
protocol. Bead-containing droplets may be provided in wash lanes
912. Washing protocols, optionally including resuspension
protocols, may be conducted on lanes 912. Waste droplets may be
transported along lane 916, across lanes 912 into waste reservoir
918. Alternatively, each wash lane 912 may be associated with its
own waste reservoir. Supernatant (i.e., waste) droplets from wash
lanes 912 may be transported using droplet operations to wash lane
916. Supernatant droplets may then be transported in waste lane 916
to waste reservoir 918. Because waste lane 916 is common to wash
lanes 912, supernatant droplets must be transported serially (i.e.,
one after another).
[0114] In an alternative example, individual waste reservoirs 920
may be provided for each wash lane 912. Supernatant droplets may be
transported simultaneously to individual waste reservoirs.
Multiple, individual waste reservoirs provide for increased
efficiency (e.g., time to result) in a washing protocol. Multiple
waste reservoirs also provide for a reduction in the number of
droplet operations electrodes 910 that are required to transport a
supernatant droplet to a waste reservoir. Reducing the number of
operations electrodes 910 also reduces the potential for
cross-contamination between subsequent droplets used in a
protocol.
[0115] 8.1.3 Bead-Mediated Droplet Splitting
[0116] In some embodiments, the invention provides a means of
splitting a bead-containing droplet. In particular, it is sometimes
useful to split a bead-containing droplet in a manner which
concentrates the beads into a smaller droplet, thereby providing a
substantial reduction in unbound substances surrounding the
droplet. For example, in an assay a droplet comprising sample and
beads may be incubated together to permit a target substance from
the sample to bind to the beads. Following incubation, it may be
desirable to remove a large aliquot of sample from the beads prior
to initiating a merge-and-split wash protocol. The invention
provides techniques for conducting such separation.
[0117] FIGS. 10A-10C show a top view of an electrode/magnet
arrangement 1000 on a droplet actuator and illustrates a process of
separating beads from a droplet. Magnetically responsive beads are
split from a rounded or generally circular shaped droplet. In some
embodiments, droplet 1016 may have a generally rounded shape. In
some cases droplet 1016 has a length at its longest cross-section
is less than about 2 times the droplet's width measured along a
width axis which is arranged at a 90.degree. angle relative to the
lengthwise axis, e.g., as illustrated with respect to droplet 1016
in FIG. 10B. In another embodiment, the droplet's length at its
longest cross-section is less than about 1.5 times the droplet's
width.
[0118] Electrode/magnet arrangement 1000 includes an arrangement of
droplet operations electrodes 1010 configured for conducting
droplet operations. Droplet 1016 is provided in a droplet
operations gap (not shown) or on a droplet operations surface where
droplet 1016 is subject to droplet operations mediated by
electrodes 1010. Magnet 1014 is provided in proximity to electrodes
1010M. Electrodes 1010M are a subset of electrodes 1010. Magnet
1014 is positioned relative to electrodes 1010M such that when
droplet 1016 is atop one or more of electrodes 1010M, any
magnetically responsive beads 1022 within droplet 1018 are
attracted by the magnetic field of magnet 1014. Alternatively,
magnet 1014 is positioned relative to electrodes 1010M such that
when droplet 1016 is subject to droplet operations mediated by
electrodes 1010M, magnetically responsive beads 1022 within droplet
1016 are attracted by the magnetic field of magnet 1014. The
attraction of magnetically responsive beads 1022 may cause beads
1022 to move within droplet 1016 in the direction of magnet 1014.
Magnetically responsive beads 1022 may move towards an edge of
droplet 1016 which is proximate magnet 1014. The parameters of the
configuration may be adjusted such that beads 1022 are attracted
towards an edge of droplet 1016, and when droplet 1016 is
transported away from magnet 1014, a bead-containing droplet 1023
splits off of droplet 1016. In this and other examples described
herein which make use of magnetically responsive beads and magnets,
the technique may be optimized by adjusting properties such as
interfacial tension of droplet 1016, properties and concentration
of magnetically responsive beads 1022, and the pull force exerted
by magnet 1014 on magnetically responsive beads 1022. Droplet 1016
may be formed using a buffer having an interfacial tension which is
sufficiently low to permit magnetic beads 1022 to remain behind
atop magnet 1014 when bead-containing droplet 1023 is transported
away from magnet 114. The transporting away may be mediated by the
electrodes, e.g., by electrowetting-mediated or
dielectrophoresis-mediated droplet operations. In order to enhance
the "snapping off" of beads from a droplet that is being
transported away from magnetically restrained beads, higher
surfactant concentrations may be used. The magnetic bead
concentration and the pull force of the magnet may be relatively
high.
[0119] In general, the following parameters may be adjusted so that
transport of a magnetically responsive bead-containing droplet away
from the magnetic field will leave behind a highly concentrated
droplet including the magnetically responsive beads, which is
essentially snapped off as the bead-containing droplet moves away
from the magnetic field: size of the droplet relative to the
droplet operations electrode, interfacial tension of the droplet,
magnetic bead properties and concentration, pull force of the
magnet exerted on the magnetically responsive beads, and number,
size and orientation of magnets used. For example, the surfactant
may be Tween 20, and the concentration of Tween 20 may range from
about 0.02% to about 0.1%. Of course, the required concentration
will vary depending on the surfactant type. The desired interfacial
tension range may typically be in the range of about 1 dynes/cm to
about 4 dynes/cm. In general, the greater the size of the droplet
relative to the footprint of the electrode, the more favorable is
it for bead-mediated droplet splitting to occur. The magnetic bead
concentration range is typically from about 1 mg/mL to about 30
mg/mL. Pull force of the magnet may typically range from about 1
lbs to about 100 lbs.
[0120] FIG. 10A shows droplet 1016 with beads 1022 therein
positioned at a droplet operations electrode 1010M in proximity to
magnet 1014. Beads 1022 are attracted and aggregated by magnet
1014. Because a single droplet operations electrode 1010M is
active, droplet 1016 is generally circular in shape. FIG. 10B shows
droplet 1016 transported using droplet operations away from droplet
operations electrode 1010M to an adjacent droplet operations
electrode 1010. As droplet 1016 moves away from droplet operations
electrode 1010M, a concentration of beads 1022 is formed at an edge
of droplet 1016 that is closest to magnet 1014. As droplet 1016 is
transported away from magnet 1014, the geometry of droplet 1016 is
distorted as the concentration of beads 1022 is restrained while
the droplet moves away from magnet 1020. The bead-retaining force
of the interfacial tension of droplet 1016 is overcome by the
bead-attracting force of magnet 1014 on beads 1022, resulting in
the breaking away of a portion of the droplet including the beads.
FIG. 10C shows droplet 1016 transported using droplet operations
still further away from droplet operations electrode 1010M and to a
droplet operations electrode 1010. Droplet 1023 including beads
1022 breaks away (snaps off) from droplet 1016. A similar result
can be achieved using a barrier that permits a droplet including
magnetically responsive beads or substantially non-magnetically
responsive beads to be transported while restraining transport of
the beads with the main body of the droplet. The above described
technique may also be employed in wash protocols. For example,
after the merger of the wash droplet with the bead-containing
droplet, the bead-mediated droplet splitting can be employed to
result in a bead droplet 1022 with little or no unbound substances
and another droplet 1016 that contains most or all of the unbound
substances. The process can be required till sensitivity and time
to result requirements are met.
[0121] FIGS. 11A, 11B, and 11C show a top view of the
electrode/magnet arrangement 1000 shown in FIGS. 10A-10C and a
process of transporting beads within a droplet. Magnetically
responsive beads may be transported within the elongated droplet
away from a magnet. The steps shown in FIGS. 11A, 11B, and 11C are
substantially the same as those that are described in FIGS. 10A,
10B, and 10C except that, instead of processing a 1.times. droplet
using droplet operations mediated by single active electrodes,
droplet 1116 is a slug-shaped 3.times. droplet and is subjected to
droplet operations using three active electrodes for each droplet
operation, so that the droplet maintains an elongated form during
the droplet operations. Droplet 1116 may be subjected to droplet
operations in a manner which causes it to take on a generally
elongated shape. In some cases, droplet 1116 length at its longest
cross-section is greater than about 1.5 times the droplet's width
measured along a width axis which is arranged at a 90.degree. angle
relative to the lengthwise axis, e.g., as illustrated with respect
to droplet 1116 in FIG. 11A. In another embodiment, the droplet's
length at its longest cross-section is greater than about 2 times
the droplet's width. In yet another embodiment, the droplet's
length at its longest cross-section is greater than about 3 times
the droplet's width.
[0122] FIGS. 11A, 11B, and 11C show the process steps of
transporting beads 1022 within an elongated droplet 1116 away from
magnet 1014. FIG. 11A shows droplet 1116 with beads 1022 therein
positioned at droplet operations electrodes 1010 adjacent to magnet
1014. Beads 1022 are attracted and aggregated within an end region
of droplet 1116 by magnet 1014. Because three droplet operations
electrodes 1010 are active, droplet 1116 takes on an elongated
shape. FIG. 11B shows droplet 1116 transported using droplet
operations away from droplet operations electrode 1010M to an
adjacent droplet operations electrode 1010. As droplet 1116 moves
away from magnet 1014, beads 1022 remain within droplet 1116. In
general, the following parameters may be adjusted so that transport
of a magnetically responsive bead-containing droplet away from a
magnetic field will either leave behind or retain the magnetically
responsive beads: interfacial tension of the droplet, magnetic bead
properties and concentration, pull force of the magnet exerted on
the magnetically responsive beads, the number, size and orientation
of magnets. The method illustrated in FIG. 11 provides a means for
retaining beads in a droplet in which the parameters are such that
the beads would otherwise be lost from the droplet if the same
droplet were transported away from the magnet in a rounded droplet
configuration. Thus, the methods illustrated in FIGS. 10 and 11
provides techniques by which the droplet actuator may be used to
selectively leave the beads behind or retain the beads in the
droplet as the droplet is transported away from the magnet. In the
method illustrated in FIG. 11, the bead-retaining force of the
interfacial tension of droplet 1116 overcomes the bead-attracting
force of magnet 1014 on beads 1022, resulting in the retention of
the beads in the droplet. FIG. 11C shows droplet 1116 with beads
1022 resuspended therein.
[0123] 8.1.4 Component Ratios
[0124] FIGS. 12A and 12B show a comparison of bench top and droplet
actuator immunoassay reagent ratios and a plot of reagent
concentration versus signal strength. As shown in FIG. 12A, the
ratio of three components of an immunoassay, beads (i.e., capture
antibody conjugated to beads), sample (e.g., serum, plasma), and
secondary antibody (II.degree. Ab) are provided. For a bench top
immunoassay, a typical ratio is 1 part beads (60 .mu.L):1/2 part
sample (30 .mu.L):1 part II.degree. Ab (60 .mu.L). A reagent ratio
for a droplet actuator based immunoassay is typically 1/2 bead
droplet (150 mL):1 sample droplet (300 mL):2 II .degree. Ab
droplets (600 mL). The use of fewer beads (i.e., 1/2 bead droplet
or 1/2 concentration of beads) in a droplet actuator immunoassay
provides for increased efficiency of bead washing and a sufficient
reduction in non-specific binding of non-target analytes to the
capture beads. In addition, the concentration of secondary antibody
is the same in both bench top and droplet actuator immunoassays,
but the volume of secondary antibody solution is double in the
droplet actuator assay. FIG. 12B illustrates the improvement in
detection signal that is provided by the use of 2 droplets of
secondary antibody and 2 droplets of detection substrate in a
droplet actuator immunoassay.
[0125] 8.1.5 Incubation of Beads with Chemiluminescent
Substrate
[0126] Another parameter which may influence the time to result in
an immunoassay is the generation of a signal during the incubation
of a chemiluminescent substrate with the washed magnetically
responsive beads that include the antigen-antibody complex. FIG. 13
shows a plot of the kinetics of a reaction between a
chemiluminescent substrate and ALP on magnetically responsive beads
for Troponin I (TnI). Immunoassays were performed on TnI (100
ng/mL) using an on-magnet incubation protocol and a circular shaped
droplet washing protocol. As shown in FIG. 14, about 90% of the end
point signal was obtained in about 120 to about 130 seconds. For a
lower concentration of the analyte, maximum signal was achieved in
about <120 seconds. Based on this data, for the type of
substrate used, 2 minutes may be selected as an optimum incubation
time to generate maximum signal for the chemiluminescence reaction.
However, if the chemiluminescence reaction is observed to behave as
a flash signal instead of a glow reaction, the 2 minute incubation
may be reduced to about a few seconds. The peak intensity of flash
signal obtained is again a function of the mixing efficiency
between the magnetic beads and the trigger solution. Efficient
mixing can be obtained by oscillating the magnetic beads with the
substrate solution at high switching speeds.
[0127] Immunoassay kits were obtained from Beckman Coulter for
Troponin I (TnI) containing capture antibodies conjugated to
magnetic beads, reporter antibodies labeled with alkaline
phosphatase (ALP) and standards (0 ng/mL-100 ng/mL).
Chemiluminescence substrate for ALP (Lumigen APS-5) was obtained
from Lumigen Inc. (Southfield, Mich., USA). Wash buffer was 0.05 M
Tris-HCl, 0.1M NaCl, 0.02% Tween 20 and 0.1 mg/mL bovine serum
albumin, pH 9.5. Discarded whole blood samples (obtained from
anonymous healthy individuals) were procured from Duke University
Medical Center, Durham, USA. TnI standards were prepared by
dilution into whole blood at a ratio of 1 part TnI standard:4 parts
blood. The concentrations of the standards that were used to spike
the samples were 5, 25, and 100 ng/mL resulting in final TnI
concentrations of 1, 5 and 20 ng/mL in blood. A sample droplet was
mixed with a droplet containing magnetic beads with primary capture
antibodies and another droplet containing the secondary antibody
labeled with ALP (reporter antibody). All the droplets were
dispensed from their respective on-droplet actuator reservoirs and
transported to the reactor zone. During incubation, droplets were
shuttled, split and merged to improve binding efficiency. After the
formation of the capture antibody-antigen-reporter antibody
complex, the magnetic beads were immobilized with a magnet while
the unbound material was washed away. After the serial dilution
based wash steps, each droplet was transported into a detection
loop where a chemiluminescent reagent droplet was dispensed and
merged with the bead droplet to produce chemiluminescence from the
enzyme-substrate reaction. The chemiluminescent product droplet was
then transported to the detection spot and the end point glow of
chemiluminescence was detected using the PMT.
[0128] 8.1.6 Rapid Immunoassays
[0129] Using optimized protocols for incubation and washing, a full
immunoassay was performed on TnI (5 ng/mL). Magnetically responsive
beads were incubated with capture antibody, analyte and secondary
antibody labeled with ALP reporter using an off-magnet incubation
protocol. Ten slug-based washes were performed to remove the
unbound material from the supernatant (wash time approximately 2
minutes). The droplet with washed magnetically responsive beads
with the antigen-antibody complex was mixed with one droplet of a
chemiluminescent substrate and incubated for 2 minutes. The end
point chemiluminescence was detected using a photon counter. In
this example, the total time to result was approximately 10 minutes
per immunoassay.
[0130] 8.1.7 Extraction of Human Genomic DNA
[0131] FIG. 14 is a top view of a droplet actuator layout 1500 that
may be used for extracting DNA from a whole blood sample. Layout
1500 includes six on-actuator reservoirs, each with a capacity of
.about.2 .mu.L, which may be used for storing and dispensing
different reagents. A typical protocol for DNA extraction on a
droplet actuator may include the following steps. A droplet of
magnetically responsive beads, such as paramagnetic Dynabeads.RTM.
DNA Direct Universal from Dynal Biotech (1.05 .mu.m diameter),
suspended in a lysis buffer is dispensed from an on-droplet
actuator reservoir and transported using droplet operations to a
specific location on the droplet actuator. The beads, which are
magnetically responsive, are held by a permanent magnet placed
underneath the droplet actuator. Droplets of whole blood are
dispensed from a reservoir and mixed with droplets of lysis buffer
(including 10 M NaOH) dispensed from another on-droplet actuator
reservoir, into a mixing reservoir in the ratio of 1:6 and mixed
for about 10 seconds. Mixing can be performed by one of the several
means, for example, by dispensing a droplet and merging the droplet
back into the reservoir. Droplets of the cell lysate are
transported across the DNA capture beads in succession and the
supernatant is pinched off while holding the beads. Droplets of
wash buffer stored in separate on-droplet actuator reservoirs are
used to wash the beads to remove cell debris. Purified genomic DNA
captured on the beads is eluted and collected at the bead
collection reservoir. A modification of the protocol would be to
have the beads mixed with the cell lysate in the same reservoir and
then concentrate the beads into a droplet using a magnet positioned
closer to the reservoir and then transport the droplet with the
DNA-attached beads to a different location for washing and elution.
The collected DNA may be amplified either on the droplet actuator
as part of an integrated sample-to-answer droplet actuator or in a
commercial thermocycler for further DNA processing or diagnostic
applications.
[0132] 8.1.8 Immunoassay on a Droplet Actuator
[0133] FIGS. 15A and 15B illustrate top views of an
electrode/magnet arrangement 1500 and show steps in an exemplary,
nonlimiting, immunoassay process. In this non-limiting embodiment,
all steps involved in the immunoassay, including sample and reagent
aliquoting, incubation with antibodies, bead washing, and enzymatic
detection, are fully automated and under software control. The
protocol that is illustrated is only an example and the sequence of
addition of reagents may vary depending on the assay protocol.
[0134] Electrode/magnet arrangement 1500 includes an arrangement of
droplet operations electrodes 1510 configured for conducting
droplet operations. Droplet 1518 is provided in a droplet
operations gap (not shown) or on a droplet operations surface where
droplet 1518 is subject to droplet operations mediated by
electrodes 1510. Magnet 1514 is arranged in proximity to droplet
operations electrodes 1510M. Electrodes 1510M are a subset of
electrodes 1510. Magnet 1514 is positioned relative to electrodes
1510M such that when droplet 1518 is atop one or more of electrodes
1510M, any magnetically responsive beads 1522 within droplet 1518
are attracted by the magnetic field of magnet 1514. Alternatively,
magnet 1514 is positioned relative to electrodes 1510M such that
when droplet 1518 is subject to droplet operations mediated by
electrodes 1510M, magnetically responsive beads 1522 within droplet
1518 are attracted by the magnetic field of magnet 1514. The
attraction of magnetically responsive beads 1522 may cause beads
1522 to move within droplet 1518 in the direction of magnet 1514.
Magnetically responsive beads 1522 may move towards an edge of
droplet 1518 which is proximate magnet 1514. Various techniques
described herein for manipulating beads in droplets may also be
employed with electrode/magnet arrangement 1500. As illustrated,
droplet 1518 is a 3.times. droplet, meaning that its footprint is
approximately 3 times the area of one droplet operations electrode
1510. Droplet 1518 may be formed by merging a magnetic
bead-containing droplet with a sample droplet, e.g., by merging a
1.times. magnetic bead containing droplet with a 2.times. sample
droplet. Magnetically responsive beads 1522 are coated with a
primary antibody that has an affinity for a specific target
antigen. An example of a process of cytokine detection on a droplet
actuator may include one or more of the following steps:
[0135] Step A of FIG. 15A shows a droplet 1518 that has
magnetically responsive beads 1522 therein and is positioned at a
certain droplet operations electrode 1510. Droplet 1518 is formed
by merging a 1.times. magnetic bead containing droplet with a
2.times. sample droplet.
[0136] Steps B and C of FIG. 15A show an incubation process, in
which droplet 1518 is repeatedly transported back and forth via
droplet operations to adjacent electrodes 1510. Repeated
transporting of droplet 1518 is used during incubation of beads
1522 and sample in order to provide sufficient resuspension and
mixing of magnetically responsive beads 1522 for optimal antibody
and antigen binding. Typically, two or three droplet operations
electrodes 1510 may be used to transport a 3.times. droplet 1518,
which takes on an elongated or slug shaped geometry. In one
nonlimiting example, droplet 1518 may be incubated for 6 minutes
using 2 droplet operations electrodes 1510 and transporting droplet
1518 over a span of 8 electrodes at a switching speed of 5 Hertz
(Hz). The incubation cycle may include any combination of droplet
operations that provides for sufficient mixing of beads 1522 with
the contents of droplet 1518. It will be appreciated that timing
and steps of the incubation protocol may vary depending on the
sample content, the degree and/or specificity of affinity of the
beads for the target substance, and the purpose of the assay being
performed. Droplet 1518 is illustrated in the figure as a 2.times.
droplet; however, in other embodiments, the droplet may have a size
which ranges from about 1.times. to about 6.times., or even larger.
In some cases, the droplet may have a size which is approximately
1.times., 2.times., 3.times., 4.times., 5.times., 6.times., or
larger.
[0137] Step D of FIG. 15A shows droplet 1518 that has magnetically
responsive beads 1522 therein transported to droplet operations
electrode 1510M. A supernatant droplet 1524 is split off using
droplet operations. Because magnetically responsive beads 1522 are
attracted to magnet 1514, they are retained in droplet 1518 during
the splitting operation. Supernatant droplet 1524 is substantially
free of beads. In one example, supernatant droplet 1524 is a
1.times. droplet and droplet 1518 is now a 2.times. droplet.
Supernatant droplet 1524 may be discarded or transported downstream
for use in another process (e.g., merged with another set of beads
having affinity for a different target). Droplet 1518 may also be
subjected to a wash protocol, such as a merge-and-split wash
protocol, to remove additional unbound materials from the
beads.
[0138] Step E of FIG. 15A shows a reagent droplet 1528 that
includes secondary antibody being transported using droplet
operations to droplet operations electrode 1510M. Reagent droplet
1528 is merged with droplet 1518 (i.e., a 2.times. droplet) using
droplet operations to form, for example, a 3.times. reaction
droplet. In one example, reagent droplet 1528 is a 1.times. droplet
that includes biotinylated secondary antibody that has an affinity
to the target antigen. Merged droplet 1518 is subjected to one or
more on-magnet or off-magnet incubation cycles. In one embodiment,
merged droplet 1518 is incubated for about 4 minutes the incubation
cycle described in steps B and C. Following the incubation period,
droplet 1518 is transported using droplet operations to droplet
operations electrode 1510M, and a 1.times. supernatant droplet is
split off using droplet operations, as described in step D, in
order to yield a 2.times. droplet 1518. The supernatant droplet
(not shown) that includes unbound secondary antibody may be
discarded. In an alternative embodiment, droplet 1528 is parked on
the electrode path at a position which is outside the attractive
influence of the magnet, and droplet 1518 is transported away from
the magnet and merged with droplet 1528. The combined droplet may,
for example, be positioned in a manner which is similar to the
position of droplet 1518 in Step A.
[0139] Step F of FIG. 15B shows a bead washing step, in which a
wash droplet 1530 is transported from wash reservoir 1512 along
droplet operations electrodes 1510. Wash droplet 1530 merges with
droplet 1518. Beads 1522 may be restrained during a droplet
splitting operation in which one or more bead-free supernatant
droplets are removed from droplet 1518. The process may be
repeated, and in some cases beads may be resuspended during the
washing step, e.g., using the resuspension techniques discussed
herein. This is done to remove any unbound material trapped in
between the interstices of the magnetic beads. Here, as in other
steps and other processes described herein, supernatant droplets
may be discarded or transported elsewhere for use as input to
another process.
[0140] Step G of FIG. 15B shows one or more reagent droplets 1532
(e.g., 1532a, 1532b) transported to droplet operations electrode
1510M. In one example, reagent droplet 1532a that includes a
blocking agent (e.g., Elisa Synblock) and reagent droplet 1532b
that includes a streptavidin-enzyme conjugate (e.g.,
streptavidin-alkaline phosphatase (ALP) or streptavidin-horseradish
peroxidase) are transported to droplet operations electrode 1510M
and merged using droplet operations with droplet 1518. Merged
droplet 1518 is incubated for 4 minutes using droplet operations,
as described in steps B and C of FIG. 15A. Following the incubation
period, droplet 1518 is transported to droplet operations electrode
1510M and a supernatant droplet (i.e., a 1.times. droplet) is split
off using droplet operations, as described in step D of FIG. 15A,
in order to yield a 2.times. droplet 1518. The supernatant droplet
(not shown) that includes unbound streptavidin-enzyme conjugate may
be discarded. Droplet 1518 is subsequently washed, for example 15
times, as described in step F of FIG. 15B. Following bead washing,
a 1.times. supernatant droplet is split off droplet 1518, as
described in step D of FIG. 15A, in order to yield a 1.times.
droplet 1518. The supernatant droplet (not shown) is discarded.
Droplet 1518 that includes antibody-antigen sandwich is now ready
for detection. In an alternative embodiment, droplet 1528 is parked
on the electrode path at a position which is outside the attractive
influence of the magnet, and droplet 1518 is transported away from
the magnet and merged with droplet 1528. The combined droplet may,
for example, be positioned in a manner which is similar to the
position of droplet 1518 in Step A. Following this step, the merged
droplet may be subjected to an incubation protocol, followed by
immobilization of the beads at the magnet and splitting of the
droplet to yield a supernatant droplet.
[0141] Step H of FIG. 15B shows droplet 1534 (1.times. droplet)
that includes a detection substrate 1536 transported to droplet
operations electrode 1510M and merged using droplet operations with
droplet 1518. The detection substrate 1536 is converted by the
enzyme conjugate into a fluorescent signal (product formation time
about 15-20 seconds). The chemiluminescent signal is measured by a
detector (not shown) in order to determine the quantity of antigen
that is present. In some embodiments, wash buffer droplets may be
transported across the detection window following each
chemiluminescent droplet to clean up the detection window and the
detection loop prior to the next detection.
[0142] In a related embodiment, the invention may make use of an
enzyme or a series of enzymes to generate a signal amplification
cascade. The cascade improves the sensitivity of the detection
system. As an example, the signal cascade may terminate with
firefly luciferase converting luciferin to light in a "flash"
chemiluminescence reaction. In one example, .beta.-galactosidase
may be coupled to an antibody or streptavidin.
Luciferin-.beta.-galactoside, which is not a substrate for
luciferase, may be delivered to the immuno-complex, incubated and
hydrolyzed to free luciferin and galactose by the
.beta.-galactosidase. The luciferin is then delivered to the PMT
where it is mixed with excess ATP and firefly luciferase. All of
the luciferin is rapidly converted to light in a flash reaction.
Beta-galactosidase can form 700 pmole luciferin per ng enzyme per
minute which is equivalent to 7.sup.12 photons per second. In this
method the background is very low, and unlike the currently used
glow substrates, all of the assay signal may be captured in the
short time of the flash reaction. This method also reduces or
eliminates the currently observed contamination of long-lived glow
chemiluminescent products on-actuator because of the short life
time of the luciferin product. It just decays away spontaneously so
washing to remove glowing products is eliminated. This system is
not a signal regeneration loop like the one used in
pyrosequencing.
[0143] The steps in the flash assay may be achieved using droplet
operations. For example, a droplet protocol may include providing a
first droplet comprising .beta.-galactosidase-antibody or
.beta.-galactosidase-streptavidin. A second droplet including
luciferin-.beta.-galactoside, which is not a substrate for
luciferase, may be combined with the first droplet to yield a third
droplet. The third droplet may be incubated and hydrolyzed to free
luciferin and galactose by the beta-galactosidase. The third
droplet including freed luciferin may be transported using droplet
operations into the presence of a sensor, such as a PMT, where it
is combined using droplet operations with a droplet comprising
excess ATP and luciferase (e.g., firefly luciferase). The luciferin
is rapidly converted to light in a flash reaction.
[0144] The flash assay of the invention may be performed on a
droplet actuator, in oil. In some embodiments, a common detection
window is used for multiple assays. Where glow assays are used,
microdroplets from previous reactions may create background signal
that interferes with detection of subsequent droplets. The flash
assay of the invention provides a means whereby multiple droplets
may be processed for detection in a common detection window on a
droplet actuator in a filler fluid with little or no background
signal remaining between droplets. For example, little or no
background signal from a previous droplet may remain in oil or in
microdroplets in oil in proximity to the detection window. In some
cases, background signal interference from previous droplets is
substantially eliminated by using the flash procedure.
[0145] In flash assays, it may be useful to use wash droplets that
include the trigger solution to clean droplet transport lanes.
Electrode paths that have been used to transport the substrate may
be washed by transporting one or more wash droplets across some
portion or all of the same area. The wash droplets may include the
flash enzyme. For example, the wash droplet(s) may include
luciferase or luciferase and ATP.
[0146] As another example, acridinium ester (AE) may be used as a
chemiluminescent label in a flash assay of the invention. The AE
signal quickly rises to a high value, typically in less than about
10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 seconds upon addition of the
trigger solution. The signal decays to very low values, typically
in less than about 60, 30, 20, or 10 seconds. This decay may
eliminate contamination on the detection loop and the detection
spot. However, contamination may still be present on the wash lanes
and the incubation region by free secondary antibody bound with AE
which can potentially affect the subsequent assays performed on the
same lane. Transporting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
droplets of the AE trigger solution over the electrodes that are
contaminated with antibody bound with AE would produce
chemiluminescence which would decay quickly, substantially
eliminating AE contamination.
[0147] 8.1.9 IL-6 and TNF-.alpha. Example
[0148] A 1.times. droplet including beads with capture Ab was
combined using droplet operations with a 2.times. droplet sample to
yield a 3.times. reaction droplet. The 3.times. reaction droplet
was subjected to an off-magnet incubation protocol for 6 minutes
(shuttled the 3.times. droplet using 2 electrodes over a span of 8
electrodes with no split at a switching speed of 5 Hz). The
reaction droplet was transported to the magnet, and a 1.times.
supernatant droplet was split from the reaction droplet to yield a
2.times. bead-containing droplet. A 1.times. droplet including
biotinylated secondary Ab was added to the 2.times. droplet to
yield a 3.times. droplet, which was incubated for 4 minutes using
the same protocol at a switching speed of 5 Hz. The reaction
droplet was again transported to the magnet, and a 1.times.
supernatant droplet was split from the reaction droplet to yield a
2.times. bead-containing droplet. Beads were washed using a
merge-and-split wash protocol. Wash buffer droplets were 2.times.
slugs of IA wash buffer. This process was repeated 5 times to
remove most of the unbound secondary antibody and sample from the
supernatant. After washing, a 1.times. supernatant droplet was
split off of the bead droplet using droplet operations to yield a
1.times. reaction droplet. A 1.times. droplet including phosphate
free Synblock and a 1.times. droplet including Streptavidin-ALP
were added to the 1.times. bead droplet to yield a 3.times.
reaction droplet. Synblock acts as the blocking agent preventing
any non-specific adsorption of reagents onto the beads while the
streptavidin-ALP binds to the biotinylated secondary antibody. The
3.times. reaction droplet was incubated using the same incubation
protocol at 5 Hz for 4 minutes. The droplet was transported to the
magnet, and a 1.times.supernatant droplet was split off from the
3.times. reaction droplet to yield a 2.times. reaction droplet. The
2.times. reaction droplet was then subjected to merge-and-split
droplet washing protocol. The process was repeated 15 times to
ensure no unbound streptavidin-ALP floating in the supernatant
which would result in false positives. After washing is complete,
the 2.times. droplet was split at the magnet to yield a final
1.times. bead-containing droplet. The 1.times. droplet with the
magnetic beads containing the immuno-complex was merged with a
1.times. chemiluminescent substrate droplet and incubated for 120
seconds. Three wash droplets were transported over the detection
pathway and spot to remove any potential contaminants prior to
introducing the next droplet for detection.
[0149] FIG. 16 shows a plot of two 5-point standard curves for
cytokine IL-6. Two 5-point standard curves (0, 0.05, 0.5, 5, and 25
ng/mL of IL-6) were obtained in 2 runs for IL-6 performed on 2
separate droplet actuators.
[0150] FIG. 17 shows a plot of two 6-point standard curves for
cytokine TNF-.alpha.. In this example, two 6-point standard curves
(0, 0.01, 0.1, 1, 10, and 100 ng/mL of TNF-.alpha.) were obtained
in 2 runs for TNF-.alpha. performed on 2 separate droplet
actuators.
[0151] 8.2 Digital Microfluidic Spatio- and Spectral-Multiplexing
of Assays
[0152] The invention also provides a microfluidics assay
multiplexing platform that uses digital microfluidics and quantum
dots. The invention makes use, in some embodiments, of an
integrated multi-well droplet actuator in combination with a
spectrometer system. Immunoassays may be multiplexed using quantum
dots in droplets on a droplet actuator as optical reporters. For
example, the spectral multiplexing capability of quantum dots may
be combined with the spatial multiplexing of digital microfluidics,
in order to provide a unique, highly multiplexed platform for the
problem of cytokine profiling. The microfluidics assay multiplexing
platform of the invention may address the key technical barriers
that are associated with current state-of-the art technologies in
cytokine profiling, such as antibody cross-reactivity and sample
volume requirements.
[0153] 8.2.1 Assay Formats
[0154] FIG. 18 illustrates a perspective view of a microfluidics
assay multiplexing platform 1800 of the invention. Platform 1800
makes use of digital microfluidics to achieve spatial multiplexing
and quantum dots to achieve spectral multiplexing. Platform 1800
includes a multi-well droplet actuator 1810 in combination with a
spectrometer system 1814. The system is capable of processing
multiple droplets 1818 for performing various assays, such as
immunoassays. In one example, multi-well droplet actuator 1810 is a
12-well droplet actuator. In one example, spectrometer system 1814
is a 12-channel spectrometer system. An example of spectrometer
system 1814 is described in more detail with reference to FIGS. 21,
22, and 23.
[0155] In one example, platform 1800 of the invention may be used
to perform automated and multiplexed cytokine assays. For example
assays may be multiplexed using multiple reaction pathways and
multiple types of quantum dots on a single multi-well droplet
actuator 1810. Droplet actuator 1810 may, in one nonlimiting
example, include 8 reagent reservoirs and 12 sample reservoirs for
performing 8-plex immunoassays on 12 samples for a total of 96
immunoassays (i.e., 96-plex capability when all 12 samples are the
same or 8-plex assays on each of the 12 different samples).
Platform 1800 provides a spatio-spectral multiplexing platform by
which cytokine immunoassays may be performed by spatially dividing
a sample into, for example, 12 droplets and by performing, for
example, a further 4-plex immunoassays in each droplet using
quantum dots. When 4-plex spectral multiplexing is added, droplet
actuator 1810 can be used to perform up to 384-plex assays on a
single sample loaded into 12 sample reservoirs where a 4-plex
spectral multiplexing is performed on each of the 8-plex spatially
multiplexed samples. When 12 different samples are loaded into the
sample reservoirs, then 32-plex assays can be performed on each of
the 12 samples. Examples of immunoassays that may be performed by
use of the microfluidics assay multiplexing platform 1800 of the
invention are described in more detail with reference to FIGS. 19,
20A, and 20B. Additionally, an example of multi-well droplet
actuator 1810 that has 96-plex capability is illustrated in FIGS.
24A and 24B.
[0156] FIG. 19 illustrates the components of an example of a 4-plex
immunoassay 1900 that may be performed in a single droplet (not
shown) using quantum dots within the microfluidics assay
multiplexing platform of the invention. For example, FIG. 19 shows
multiple beads 1910 (1910a, 1910b, 1910c, 1910d), multiple types of
analytes 1914 (e.g., an analyte 1914a, 1914b, 1914c, and 1914d),
and multiple types of quantum dots 1918 (e.g., a quantum dot 1918a,
1918b, 1918c, and 1918d).
[0157] Beads 1910 may be any suitable size, including for example,
microbeads, microparticles, nanobeads and nanoparticles. In some
cases, beads 1910 may be magnetically responsive; in other cases
beads 1910 may not be significantly magnetically responsive.
Examples of suitable magnetically responsive beads are described in
U.S. Patent Publication No. 2005-0260686, entitled, "Multiplex flow
assays preferably with magnetic particles as solid phase." Beads
1910 have an affinity for a certain target substance, such as for a
certain type of cell, protein, DNA, and/or antigen. When the target
substance contacts beads 1910, the target substance may bind to
beads 1910. Analytes 1914a, 1914b, 1914c, and 1914d may be
different types of target substances to which beads 1910 have an
affinity. In one example, analytes 1914a, 1914b, 1914c, and 1914d
may be different protein cytokine analytes.
[0158] Quantum dots 1918, which are also known as semiconductor
nanocrystals, are generally composed of an inner semiconductor
core, usually cadmium sulfide (CdS) or cadmium telluride (CdTe),
that is surrounded by a high bandgap material, such as zinc sulfide
(ZnS). The emission spectrum of quantum dots 1918 is associated
with the size of quantum dots 1918, which usually ranges between
about 2 nanometers (nm) and about 20 nm. Quantum dots 1918 may be
commercially available from suppliers, such as, but not limited to,
Life Technologies (Carlsbad, Calif.) and Evident Technologies
(Troy, N.Y.). Quantum dots have unique optical properties including
narrow emission spectra, broad-range excitation, and high
photostability.
[0159] The use of quantum dots in digital microfluidics
immunoassays provides certain advantages. For example, sensitivity
may be increased by using quantum dots rather than using the
traditional organic dyes. The flexibility in choices of emission
spectra of quantum dots allows the choice of a type of quantum dot
that has a wavelength that differs from the autofluorescence
background of another key assay component, such as magnetically
responsive beads or a PCB substrate or the sample, thus improving
signal-to-background noise ratio. Referring to FIG. 19, quantum dot
1918a, 1918b, 1918c, and 1918d may be representative of quantum
dots that have different emission spectra, respectively. FIG. 19
shows beads 1910 that are conjugated to capture antibodies;
respective analytes 1914a, 1914b, 1914c, and 1914d; and four types
of quantum dots 1918 that are conjugated to their appropriate
detection antibody.
[0160] FIGS. 20A and 20B illustrate the components of an example of
an immunoassay "sandwich" formation process 2000 that may be
performed in a single droplet (not shown) using quantum dots within
the microfluidics assay multiplexing platform of the invention. For
example, FIG. 20A shows a certain bead 2010, certain analytes 2014,
and a certain quantum dot 2018. FIG. 20B shows analyte 2014 that is
bound to both the bead 2010 and to the quantum dot 2018. Analyte
2014 is therefore sandwiched between bead 2010 and quantum dot
2018, thereby forming the link between bead 2010 and a certain type
of quantum dot 2018.
[0161] The process that is shown in FIGS. 20A and 20B is an example
of an immunoassay sandwich format that incorporates quantum dots,
where the assay antibody sandwich occurs on a solid support (e.g.,
bead 2010) and the secondary optical labels (e.g., quantum dot
2018) may be imaged using a fluorescent microscope. Although
magnetic beads are shown in the figure for solid support, the
surface of the droplet actuator modified with capture elements,
such as nucleic acids, antibodies or antigens, can be used as a
fixed solid support instead of magnetic beads. In one example, the
binding and the number of immobilized quantum dots may be
correlated to the level of an analyte, such as prostate-specific
antigen (PSA), by fluorescent imaging of quantum dots on, for
example, a carbon substrate. Furthermore, a spatially multiplexed
cytokine approach using a protein microarray with a single type of
quantum dot may be used to detect certain cytokines (e.g.,
TNF-.alpha., IL-8, IL-6, MIP-1.beta., IL-13 and IL-1) in a single
solution down to a parts-per-million concentration level. Any
analytes detectable using a sandwich immunoassay or competitive
immunoassay format may be detected using the protocols of the
invention.
[0162] Separate pools of beads 2010 may be conjugated to the
corresponding capture antibody of the cytokine protein of interest.
By way of example, four separate conjugations occur for a 4-plex
assay. In addition, four detection antibodies may be separately
conjugated to four types of quantum dots (e.g., quantum dots 2018a,
2018b, 2018c, and 2018d). Referring again to FIG. 19, four pairs of
conjugates (e.g., four bead 2010/quantum dot 2018 pairs) with their
respective protein cytokine analytes 2014 is shown. These
conjugates represent separate droplet pools of conjugates (of
course, without analyte at this point) may be combined prior to
introduction on the multi-well droplet actuator 1810 of
microfluidics assay multiplexing platform 1800. When combined with
a sample that includes analytes 2014 (e.g., cytokine analytes), the
beads 2010, analytes 2014, and quantum dots 2018 form a sandwich.
After the conjugate and sample are mixed, multiple wash steps may
occur in order to remove unbound quantum dots 2018. After washing,
the sample may be resuspended in a droplet and transported to the
detection zone (e.g., the interface between multi-well droplet
actuator 1810 and spectrometer system 1814) of multi-well droplet
actuator 1810.
[0163] 8.2.2 Detection System
[0164] By using, for example, four types of quantum dots (e.g.,
quantum dots of different emission spectrum) and multiple separate
detection spots that are provided in microfluidics assay
multiplexing platform 1800, the multiplexing capability may be
expanded to n-plex. An important aspect of combining two
multiplex-schemes, is utilizing compatible antibody pairs. Certain
spectrometer tools and algorithms are provided for spectral
uncoupling of the assay signal from the respective types of quantum
dots.
[0165] FIG. 21 illustrates a perspective view of spectrometer
system 1814 of microfluidics assay multiplexing platform 1800 of
the invention. Spectrometer system 1814 may include multiple
optical fibers arranged to excite and collect quantum dot
fluorescence from multiple microfluidic droplets. In one
embodiment, spectrometer system 1814 may be a 12-channel
spectrometer system that includes a 12-channel fiber-based readout
head 2110. In this embodiment, 12 excitation fibers 2114 enter
fiber-based readout head 2110 and 12 collection fibers 2118 exit
fiber-based readout head 2110. Excitation fibers 2114 and
collection fibers 2118 are optical fibers. 12-channel fiber-based
readout head 2110 is designed to hold one end of each excitation
fiber 2114 and each collection fiber 2118 in alignment with a
respective liquid channel (not shown) of multi-well droplet
actuator 1810 and with a respective droplet 1818. An example of a
12-channel fiber-based readout head 2110 is described in more
detail in FIG. 23.
[0166] The opposite ends of the 12 excitation fibers 2114 are
optically coupled to a light source, such as an ultraviolet (UV)
source 2122. The opposite ends of the 12 collection fibers 2118 may
be arranged in a linear array, e.g., a spectrometer slit 2126. The
slit 2126 may be imaged through a diffraction grating 2130 onto a
two-dimensional (2D) charge-coupled device (CCD) array 2134. This
arrangement preserves the spatial information (y-axis) from each
droplet 1818 and disperses the spectral information onto the
x-axis.
[0167] While a spectrometer system, such as spectrometer system
1814, may be the preferred method of separating spectral
information from multiple quantum dots because of cost, size, and
performance advantages, microfluidics assay multiplexing platform
1800 is not limited to spectrometer system 1814 only.
Alternatively, an optical system for separating spectral
information may include dichroic beamsplitters in combination with
narrowband filters. For example, a filter-based design that may
measure 12 spots and 4 spectral channels may include 36 separate
dichroic beamsplitters (e.g., 3 per detection spot), 48 narrowband
filters, and 48 detectors. However, filter-based designs may be
considerably more costly and complex as compared with the
fiber-based spectrometer system, especially as the multiplexing
number increases, such as to 8 multiplexed quantum dots.
[0168] FIG. 22 illustrates a concept 2200 for turning the
information of a 2D CCD array 2134 into multiple spectra. A grid
area 2210 represents 2D CCD array 2134. Information from each spot
is dispersed along the x-axis, which is the dispersion axis.
Vertical pixels are binned in each vertical sub-section
corresponding to the different droplets (e.g., each of the 12
droplets 1818). These summed pixels create a spectrum for each
droplet.
[0169] FIG. 23 illustrates a perspective view of 12-channel
fiber-based readout head 2110 of FIG. 21, showing more details
thereof. 12-channel fiber-based readout head 2110 includes the 12
excitation fibers 2114 and 12 collection fibers 2118, as described
in FIG. 21. Additionally, 12-channel fiber-based readout head 2110
further includes support block 2310 in which are embedded one end
of the 12 excitation fibers 2114 and 12 collection fibers 2118. A
substrate, such as block 2310, may be formed, for example, of an
epoxy resin material. A substrate, such as block 2310, secures the
ends of excitation fibers 2114 and collection fibers 2118 in a
desired position relative to assay droplets on multi-well droplet
actuator 1810.
[0170] A coupling lens may be provided at the end of each
excitation fiber 2114 and collection fiber 2118. FIG. 23 shows
coupling lenses 2314 that are arranged to distribute light to,
and/or collect light from, droplets 1818. Excitation fibers 2114 in
combination with coupling lenses 2314 are used to focus the
excitation light from, for example, UV source 2122 onto droplets
1818. Collection fibers 2118 in combination with coupling lenses
2314 are used to collect the quantum dot fluorescence from each
droplet 1818.
[0171] The 12-channel fiber-based readout head 2110 makes use of
oblique incidence excitation and collection from each droplet.
Droplets 1818 represent microfluidic droplets each including
multiple types of quantum dots. FIG. 23 shows two fibers (e.g., one
excitation fiber 2114 and one collection fiber 2118) per droplet
1818. At the end of each excitation fiber 2114 and collection fiber
2118 are the coupling lenses 2314 that either focus the excitation
light to the quantum dots in the droplet 1818 or collect light from
the droplet 1818 to the collection fiber 2118. These excitation
fiber 2114 and collection fiber 2118 pairs are arranged linearly
along the length of the detection zone on multi-well droplet
actuator 1810 that includes droplets 1818; however, it will be
appreciated that the arrangement need not be linear at the
collection end. Any fiber array pattern (e.g., grid or other
preselected arrangement, such as an irregular arrangement) at the
sample end, and the fibers may coalesce into a linear format at the
spectrometer slit. In the specific embodiment illustrated here, the
droplets 1818 on multi-well droplet actuator 1810 may have a
spacing of, for example, about 4.5 millimeters (mm) between
assay-based droplets.
[0172] The operation of example spectrometer system 1814 is
generally as follows. In order to detect quantum dot emission from
12 separate spots without optical crosstalk, the design of
spectrometer system 1814 incorporates the 12 collection fibers 2118
from 12 droplets 1818 to a vertical position along the narrow slit
2126, as shown in FIG. 21. The collected light from the end of each
collection fiber 2118 at slit 2126 is imaged onto the 2D CDD array
2134, dispersed along the (x-axis) wavelength axis and confined to
a defined area along the y-axis. FIG. 22 shows the binning scheme
from 2D CDD array 2134. As shown in FIG. 22, light from each
droplet 1818 is confined to a vertical zone and dispersed along the
x-axis (dispersion axis). Each of these vertical zones is
separately binned to provide the spectra shown in FIG. 22.
Sub-vertical binning allows the separation of spatial and spectral
information over the 12 droplets 1818 that are being interrogated.
In the alternative embodiment of the dichroic system, separate
filters are provided for each quantum dot and each droplet
measurement channel.
[0173] 8.2.3 System Integration
[0174] Platform 1800 of the invention provides high immunoassay
multiplexability by dividing the multiplexability down into both
the spatial and spectral regime, by combining spatial multiplexing
in the digital microfluidic platform and spectral multiplexing in
quantum dots. Microfluidics assay multiplexing platform 1800
provides sandwich immunoassay capability with the ability to reach
detection limits that are clinically prognostic. While FIGS. 18
through 23 describe a combined multiplex approach of a 4-plex
spectral multiplex capability with a 12-plex spatial multiplex
capability, this is exemplary only. Microfluidics assay
multiplexing platform 1800 of the invention may be used to combine
n-plex spectral multiplex capability with a m-plex spatial
multiplex capability, where n and m denote two numbers for the
order of multiplexing and in a limiting case can be the same order.
Microfluidics assay multiplexing platform 1800 of the invention may
be, for example, a 96-plex protein assay system that provides
spectral multiplexing to an 8-plex spectral capability combined
with 12-plex spatial capability, thereby proving 96-plex capability
using only about 30 microliter (.mu.L) total sample. In existing
multiplexing technology, achieving multiplexability beyond 10-plex
in a single solution without assay cross-reactivity is an extremely
difficult exercise. However, by combining spatial and spectral
multiplexing in the microfluidics assay multiplexing platform 100
of the invention, this antibody-crosstalk barrier can be mitigated
and reduced to order of spectral multiplexing. For example, instead
of a 10-plex (where 10 antibody pairs resulting in 100 combinations
need to be tested for cross reactivity), with spectral
multiplexing, two 5-plex assays can be setup which broadens the
potential combinations of antibody pairs (where 5 antibody pairs
result only in 25 combinations).
[0175] 8.3 Dielectrophoresis
[0176] The invention provides a droplet actuator having unique
electrode structures for manipulating particles within a droplet on
the droplet actuator, as well as methods of performing such
manipulations. The invention makes use of dielectrophoresis (DEP).
Polarizable particles are concentrated at locations of highest or
lowest electrical field strength. The droplet actuator of the
invention includes electrodes configured to produce non-uniform
electrical fields, i.e., fields in which electrical field
intensities are spatially variable. In this manner, particles may
be concentrated, regionalized, isolated, or trapped within or
guided to a region of a droplet on a droplet actuator. The DEP
electrodes of the invention may be configured in association with
the top substrate (when present) and/or on the bottom substrate of
the droplet actuator. Typically, the DEP electrodes will be on a
surface of the top and/or bottom substrate and will be covered with
a dielectric coating. In certain embodiments, the electrodes used
for establishing a DEP effect may double as electrowetting
electrodes.
[0177] FIGS. 24A and 24B illustrate one configuration of a portion
of a droplet actuator of the invention. FIG. 24B shows a
cross-section of FIG. 24A along the line xy. The droplet actuator
includes a top substrate 2401 and a bottom substrate 2402 separated
by a gap 2403. DEP electrodes 2405A,B,C,D are associated with top
substrate 2401. A droplet operations electrode 2410, which may be
part of a path or array of droplet operations electrodes (not
shown), is associated with bottom substrate 2402. DEP electrodes
2405 and droplet operations electrode 2410 are each coated with a
dielectric 2420A, 2420B. A hydrophobic coating may also be provided
on the dielectric, rendering hydrophobic the surfaces of top
substrate 2401 and bottom substrate 2402 exposed to gap 2403.
[0178] DEP electrodes 2405A,B,C,D are wire electrodes having a
quadripolar DEP geometry. In the illustrated embodiment, they
terminate at DEP region 2425 which is centrally located relative to
droplet operations electrode 2410. Other arrangements are possible
within the scope of the invention. In DEP particle trapping
configuration, electrodes 2405A and D will have the same phase and
electrodes 2405B and C will have an opposite phase relative to the
phase of electrodes 2405A and D. However, other arrangements are
possible within the scope of the invention. For example, in one
embodiment, particles may be trapped and rotated by applying a
difference between each of adjacent electrodes 2405A,B,C,D, which
is less than 180.degree.. For example, it may be useful to apply a
90.degree. difference between each of electrodes 2405A,B,C,D, e.g.,
electrode 2405A is 0.degree., 2405B is 90.degree., 2405C is
180.degree., and 2405D is 270.degree..
[0179] Generally speaking, DEP region 2425 is configured such that
DEP fields can influence one or more particles within a droplet on
a droplet operations electrode. The size of DEP region 2425 may be
selected based on the number, size, and/or DEP properties of
particles to be influenced by the DEP fields. For example, where
particles are to be trapped, more particles can be trapped in a
larger DEP region 2425. Similarly, where it is desirable to trap a
single particle, the size of the DEP region 2425 may be selected
accordingly.
[0180] FIGS. 25A-25E illustrate the configuration of FIG. 24 in
operation. As shown in FIG. 25A, droplet 2505 comprising one or
more particles may be situated on a path of electrodes 2510
including electrode 2410. FIG. 25B illustrates that droplet 2505
may be transported along path 2510 onto electrode 2410 using
droplet operations. At electrode 2410, DEP electrodes may be
activated, thereby trapping one or more particles in DEP region
2425. FIG. 25C illustrates that droplet 2505 may be transported
using droplet operations along electrode path 2510 away from
electrode 2410, leaving behind daughter droplet 2515, including the
trapped one or more particles. A daughter droplet will be formed
where the DEP force is greater than the interfacial tension of the
droplet being transported away. Alternatively, a variety of droplet
operations may be used to remove one or more droplets including the
remaining (not trapped) particles. For example, this may be
accomplished using a wash protocol whereby a new droplet lacking
particles is combined with droplet 2505, and the combined droplet
is split to remove a droplet including untrapped particles. This
process can be repeated until only trapped particles remain in the
droplet on electrode 2410. In the process illustrated in FIGS. 25C,
25D and 25E, a new droplet 2520 (e.g., a buffer droplet, reagent
droplet, or sample droplet) is transported along electrode path
2510 onto electrode 2410 into contact with droplet 2515, yielding a
new combined droplet 2525. DEP electrodes 2405 may be deactivated
to release the one or more particles into droplet 2525. Droplet
2525 may be transported along electrode path 2510 or otherwise
subjected to additional droplet operations or analyses. Trapped
particles may have different DEP properties relative to droplets
that are not trapped. In this manner, particles having different
DEP properties may be separated.
[0181] FIGS. 26A-26C illustrate an electrode path 2600, including a
specialized electrode 2610, which can be used as a droplet
operations electrode and as a DEP electrode. Electrode 2610 is
configured to provide a quadripole trapping geometry having 4
electrodes 2610A, B, C and D. In the illustrated version, each of
the electrodes 2610A, B, C and D is a 45-45-90.degree. triangle,
with the 90.degree. angles facing each other to form a square
electrode or an electrode which is approximately square. It will be
appreciated that a variety of other electrode configurations are
possible to achieve the same effect. For example, in the
illustrated embodiment, particles are trapped at a location which
is central to the 4 electrodes, but they could be at any point on
or near the electrode, preferably within the footprint of the
droplet. Electrode configuration 2610 is positioned within a path
of square droplet operations electrodes 2605.
[0182] In normal droplet operations, the electrodes 2610A-D can be
operated in tandem as an ordinary droplet operations electrode.
However, in DEP mode of operation, the electrodes can be used to
trap particles. In this mode, electrodes 2610A and C will have a
first phase and electrodes 2610B and D will have a second phase
which is opposite (i.e., differs by 180.degree.) relative to the
first phase. FIGS. 26A, 26B and 26C illustrate that different gap
sizes can be used to establish trapping zones for differently sized
particles or for capturing different amounts of particles. Among
other things, by controlling the number of particles captured, it
is possible to aliquot particles from one droplet into multiple
sub-droplets, each sub-droplet having an approximately equal
quantity of the particles.
[0183] FIG. 27A illustrates an octagon-shaped DEP electrode
configuration 2700 based on the use of 8 triangular shaped
electrodes 2705. Opposite electrodes will generally have phases
which differ by about 180.degree.. Gaps between the triangular
electrodes can be selected to establish trapping zones for trapping
particles based on size or quantity. Electrode configuration 2700
is positioned within a path of square droplet operations electrodes
2710.
[0184] FIG. 27B illustrates a hexagon-shaped DEP electrode
configuration 2720 based on the use of 6 triangular shaped
electrodes 2725. Opposite electrodes in the will generally have
phases which differ by about 180.degree.. Gaps between the
triangular electrodes can be selected to establish trapping zones
for trapping particles based on size or quantity. Electrode
configuration 2720 is positioned within grid of hexagonal droplet
operations electrodes 2730.
[0185] FIGS. 28A and 28B illustrate asymmetrical quadrupole DEP
electrode arrangements 2800 and 2805, formed from differently sized
trianglular electrodes 2810.
[0186] FIG. 29 illustrates an embodiment in which quadrupole
electrodes 2610 are arranged in an electrode array 2900. In one
embodiment, each electrode 2610 has a different DEP voltage
configuration and can be used to trap a different type of particle.
In the specific non-limiting embodiment illustrated here, a droplet
including particles to be trapped may be transported to a DEP
electrode from any direction on the array, and a droplet including
the trapped one or more particles can be transported away from the
quadrupole electrodes in any direction. Further, it should be noted
that a droplet including particles to be trapped can be introduced
directly onto the DEP electrode via an opening in a substrate, such
as the top substrate (not shown). Similarly, a droplet including
the trapped one or more particles may be extracted via an opening
in a substrate, such as the top substrate (not shown). For example,
it may be useful in some circumstances to flow liquid including one
or more cells or particles to which cells are adhered into a
reservoir associated with the top substrate for culturing or for
collection and further processing. Further, the droplet operations
electrodes and DEP electrodes may be associated with the top and/or
bottom substrate.
[0187] FIG. 30 shows a dynamically tunable quadripole DEP electrode
arrangement 3000 in which each triangular electrode 3005, 3006,
3007, 3008 is further subdivided into sections A, B, and C. The
subdivision of the triangles is generally concentric relative to
the center of the square, i.e., breaks in each triangular electrode
3005, 3006, 3007, 3008 are generally parallel relative to the
triangle's hypotenuse. Further, they are generally evenly spaced
along a line extending from the right angle of each triangle to a
center point on the triangle's hypotenuse. Variations in geometry
are permissible, so long as the electrode arrangement achieves the
intended purpose.
[0188] In operation, each concentric group of sub-electrodes can be
activated independently to capture different numbers or sizes of
particles. In one embodiment, groups A, B and C may be activated to
capture the smallest quantity of particles; groups A and B may be
activated to capture a larger quantity of particles; and group A
may be activated to capture a still larger quantity of particles.
In another embodiment, group C may be activated to capture the
smallest number; group B may be activated to capture a larger
quantity; and group A may be activated to capture a still larger
quantity of particles. In one embodiment, each group of electrodes
(i.e., the A group, the B group, and the C group) is activated
together. In another embodiment, one or more members of any of the
groups may be operated independently. For example, in one
embodiment, the group C electrodes may be operated independently,
such that it is possible to activate groups A and B with any
combination of the group C electrodes. It is possible to have each
group of electrodes activated at different voltages and/or
frequencies (for example, group A can be activated at voltage V1
and frequency f1, B at voltage V2 and frequency f2, and C at
voltage V3 and frequency f3) so that different particles can be
segregated within a droplet at the gaps between the different
groups based on their polarizabilities. Among several applications
possible through this embodiment is the separation of dead and
viable cells within a droplet along a circular path of different
radii.
[0189] FIGS. 31A-31C illustrate a configuration for applying a
travelling wave DEP within a droplet. The travelling wave
configuration translates particles that are levitated by DEP along
the direction of a travelling wave. The phase of each adjacent DEP
electrode may be rotated by about 90.degree. relative to the
adjacent electrode to produce the travelling wave effect. A region
of a droplet actuator substrate includes DEP electrodes 3115
arranged alongside droplet operations electrode 3110. The
illustrated embodiment includes four pairs of DEP electrodes. Each
pair includes a first member on a first side of electrode 3110 and
a second member on an opposite side of electrode 3110. As
illustrated, the pairs are arranged sequentially in increasing
order of polarity (0.degree., 90.degree., 180, 270.degree.).
However, other arrangements are possible, depending on where in the
droplet it is desired for the particles of interest to be trapped.
Four pairs of electrodes are illustrated, but it will be
appreciated that more or less pairs are possible. The pairs are
illustrated as being arranged alongside a single electrode;
however, they may be arranged alongside a path of two or more
electrodes, which is particularly useful for applying a DEP force
in an elongated or slug-shaped droplet extended along a path of
electrodes (e.g., as illustrated in FIG. 36). A splitting
operation, such as the one shown in FIG. 36 may be used to divide
the slug into a droplet including the trapped particles and a
droplet substantially lacking the trapped particles. In this
manner, particles having specific DEP properties may be
regionalized within a first droplet and then split off into a
smaller daughter droplet, thereby concentrating and/or isolating
the beads.
[0190] Referring to FIGS. 31A-31C, a droplet 3120 including
particles 3125 may be transported using droplet operations along
electrode path 3130 onto electrode 3110. DEP electrodes 3115 may be
activated causing particles to congregate along an edge of droplet
3130. A new droplet 3121 including particles 3125 may be dispensed
using droplet operations from electrode 3110 onto electrode path
3135. In another embodiment, the order of polarity may be reversed
to localize particles at a location which is distal to dispensing
path 3135, and a droplet substantially lacking in particles may be
dispensed. In another embodiment, rather than being transported
into place, the droplet is loaded onto a reservoir electrode and
beads in the droplet are subjected to DEP to congregate beads in a
dispensing region of the droplet, such that a droplet with beads
may be dispensed. In another embodiment, rather than being
transported into place, the droplet is loaded onto a reservoir
electrode and beads in the droplet are subjected to DEP to
congregate beads away from a dispensing region of the droplet, such
that a droplet lacking beads may be dispensed.
[0191] In an alternative embodiment, DEP is used to focus the
particles between the oppositely facing electrodes and traveling
wave DEP is used to move through the droplet. In this embodiment,
the top DEP electrodes illustrated in FIG. 31 would be phase
shifted as they are illustrated: 0, 90, 180, 270.degree., and the
bottom electrodes would be 180, 270, 0, 90.degree., so that they
are also phase shifted by 90.degree., but they are opposite in
polarity to the opposing electrodes. In some embodiments, phases
may be changed during operation, such that beads are caused to
congregate in one region of a droplet, then travel to another
region of the droplet.
[0192] FIG. 32 shows a side view of the configuration illustrated
in FIG. 31 showing how the particles 3125 may congregate at an edge
of droplet 3120. Droplet 3121 including particles 3125 can be split
off using smaller unit droplet 3121. A mechanism similar to this
can be used to concentrate the beads into the dispensed droplet in
the DNA extraction application, explained in section 7.1.7, instead
of magnetic beads.
[0193] FIG. 33 illustrates travelling wave DEP configurations 3300,
3301 in which DEP electrodes 3305 are provided on a first
substrate, and droplet operations electrodes 3310 are provided on a
second substrate. In some embodiments, DEP electrodes 3305 are
associated with the substrate which is across the droplet
operations gap from the droplet operations electrode 3310.
Moreover, the DEP electrodes 3305 may overlap the droplet
operations electrode 3310, and the overlapping DEP electrodes 3305
may be positioned on the same or opposite sides of the droplet
operations gap in a manner similar to electrodes 2405 and 2110 as
shown in FIG. 24B. Configuration 3300 shows DEP electrodes 3305
arranged opposite a single droplet operations electrode 3310, such
as a reservoir electrode. This configuration may, for example, be
operated in a manner similar to electrodes 3110 and 3115
illustrated in FIG. 31. FIG. 33 shows DEP electrodes 3305 arranged
opposite a path of droplet operations electrodes 3310. In
operation, the electrodes 3310 may be activated, causing a
bead-containing droplet to take on an elongated configuration atop
the electrodes, e.g., as illustrated in FIG. 36. Each DEP electrode
may be phase shifted relative to its neighboring DEP electrodes,
thereby creating a traveling wave DEP effect, which transports the
beads to an end region of the droplet. Electrode 3310B may then be
deactivated, causing the droplet to split and yielding two daughter
droplets. DEP electrodes may be arranged alongside two, three or
more droplet operations electrodes. A slug-shaped droplet may be
provided on the droplet operations electrodes. The DEP electrodes
may be used to localize particles in one end of the slug. An
intermediate electrode may be deactivated to split the slug into
two droplets, one including substantially all of the particles and
one substantially lacking the particles. Typically, one of the
daughter droplets will include a higher concentration of the beads.
In some cases, substantially all of the beads will make their way
into one daughter droplet, while the other daughter droplets will
be substantially free of the beads. In other cases, the beads may
be distributed among two or more of the daughter droplets. Among
other things, this technique is useful for concentrating beads for
analysis or for conducting a merge-and-split bead washing
protocol.
[0194] FIG. 34 illustrates an alternative electrode configuration.
The configuration includes two electrodes 3410A,B with fringed
edges 3405 separated by a gap 3420. The two electrodes have
different polarities. The electrodes A and B have fringed edges
3405 designed to generate a DEP field. The electrodes A and B may
be operated together as a single droplet operations electrode or
separately as a set of DEP electrodes. In operation, particles will
line up in a DEP region 3415, generally along the gap 3420 between
the two electrodes 3410A and B.
[0195] FIGS. 35A-35C show an electrode path 3505 including DEP
electrodes 3410A & B. FIGS. 35A and 35B illustrate how a
particle-containing droplet 3520 can be transported using droplet
operations to electrode 3410, and the particles can be trapped
within droplet 3520 by electrodes 3410. FIG. 35C shows how liquid
can be exchanged around immobilized droplets, e.g., for washing or
introducing sample and/or reagents to the particles or for removing
supernatant from the particles for further analysis. Further, the
particles in the new droplets can be transported away by removing
the DEP field and using adjacent electrodes from electrode path
3505 to transport droplet 3520. In this manner, particles can be
localized in a droplet and concentrated by removing liquid from the
droplet without substantial loss of particles. The technique for
conducting this operation may, for example, be a merge-and-split
operation.
[0196] FIGS. 36A-36E illustrate an embodiment which is similar to
the embodiment illustrated in FIG. 35. DEP electrode 3410 is used
to trap particles during a droplet-splitting operation mediated by
electrodes 3610, which are configured in an electrode path. The
electrode path also includes a DEP electrode 3410. FIG. 36A
illustrates an electrode path including a particle-containing
droplet 3615. FIG. 36B shows the droplet elongated across three
activated electrodes 3610, with DEP electrode 3410 activated to
attract and trap particles 3616 within droplet 3615. FIGS. 36C and
D show deactivation of an intermediate electrode to split the
droplet, leaving a first droplet including substantially all of the
particles and a second droplet substantially lacking particles.
Either or both of these droplets may be subjected to further
droplet operations, e.g., as part of an assay protocol. FIG. 36E
shows the first droplet 3615 being transported away from DEP
electrode 3410 with the particles 3616. In a washing protocol, the
particle-containing droplet may be combined with a wash droplet and
split as described above multiple times until the wash is
sufficiently complete. In an alternative embodiment, the path of
electrodes may include droplet operations electrodes 3610, and DEP
electrode 3410 may be positioned on the substrate across the
droplet operations gap from the droplet operations electrode, e.g.,
as described with respect to electrodes 2405 and 2410 in FIG. 24.
In yet another embodiment, DEP electrodes 3410 may be positioned in
the path of droplet operations electrodes 3610 as illustrated, and
one or more droplet operations electrodes may be positioned on the
substrate across the droplet operations gap from DEP electrodes
3410 to hold the droplet in place during DEP operation.
[0197] FIG. 37 illustrates an array of electrodes 3715 including
DEP electrodes 3410, as described above. In one embodiment, each
electrode 3410 has a different DEP configuration and can be used to
differentially trap particles having different DEP characteristics.
In the specific non-limiting embodiment illustrated here, a droplet
including particles to be trapped may be transported to a DEP
electrode from any direction on the array, and a droplet including
the trapped one or more particles can be transported away from the
quadrupole electrodes in any direction. Further, it should be noted
that a droplet including particles to be trapped can be introduced
directly onto the DEP electrode via an opening in a substrate, such
as the top substrate (not shown). Similarly, a droplet including
the trapped one or more particles may be extracted via an opening
in a substrate, such as the top substrate (not shown). For example,
it may be useful in some circumstances to flow liquid including one
or more cells or particles to which cells are adhered into a
reservoir associated with the top substrate for culturing or for
collection and further processing. Further, the droplet operations
electrodes and DEP electrodes may be associated with the top and/or
bottom substrate.
[0198] FIGS. 38A and 38B illustrate several alternatives to
electrode 3410 described above. In column A, the fringes are
generally centrally located within the electrode configuration. In
column B, the fringes are asymmetrically located within the
electrode configuration. Various fringe types are also
illustrated.
[0199] FIGS. 39A-39D illustrate a reservoir electrode 3910 having a
DEP electrode inset 3911. DEP electrode inset 3911 includes a
fringed region which corresponds to a fringe region on reservoir
electrode 3910. The configuration is useful for dispensing a
supernatant droplet 3930 onto a path or array of electrodes 3915
from a particle-containing droplet 3920. In FIG. 39A, all
electrodes in the arrangement are off. In FIG. 39B, reservoir
electrode 3910 and electrodes 3915 are on, and DEP electrode inset
3911 is activated to effect a DEP field in the region of the
fringed edges 3913, trapping the particles 3930 in the DEP field.
In FIG. 39C, an intermediate one of the electrodes 3915 is
deactivated to cause formation of droplet 3930, shown in FIG. 39D.
In this manner, a supernatant droplet 3930 substantially lacking in
the particles is dispensed from a reservoir, while droplet 3920 in
the reservoir retains substantially all of the particles.
[0200] FIGS. 40A-40D illustrate a configuration useful for
dispensing a droplet 4030 including substantially all particles
4035 from a particle-containing droplet 4020 on reservoir electrode
4010 onto a path or array of electrodes 4015. Alternatively,
droplet 4030 includes a concentration of particles from
particle-containing droplet 4020, wherein the concentration of
particles in droplet 4030 is greater than the concentration in
parent droplet 4020. Electrodes 4015 include a DEP electrode 4025
at a location which is distal relative to reservoir electrode 4010.
Further, the fringe region 4026 of the DEP electrode is distal
within electrode 4025 relative to reservoir electrode 4010.
[0201] In FIG. 40A, all electrodes in the arrangement are off. In
FIG. 40B, reservoir electrode 4010 and electrodes 4015 are on, and
DEP electrode 4025 is activated to effect a DEP field in the fringe
region 4026, attracting and trapping the particles in the DEP
field. In FIG. 40C, an intermediate one of the electrodes 4015 is
deactivated to cause formation of droplet 4030, shown in FIG. 40D.
In this manner, a droplet including substantially all particles is
dispensed from reservoir electrode 4010, while droplet 4020 retains
substantially none of the particles. It will be appreciated that by
appropriate timing and selection of DEP field properties (e.g.,
size of the fringe region), a droplet including less than
substantially all of the particles may dispensed, leaving some
particles in droplet 4020. In this manner, droplets including
predetermined quantities of particles may be dispensed.
[0202] FIG. 41 illustrates the use of DEP to separate particles
within a droplet 4105 for imaging. In this embodiment DEP
electrodes 4110A,B are arranged on top and bottom substrates,
respectively, of the droplet actuator. The DEP force generated by
DEP electrodes 4110A,B causes particles with different properties
to separate vertically in planes 4115. A confocal microscope 4120
can be used to detect signal from particles in specific planes of
the droplet. For example, several assays for different analytes can
be performed on the droplet actuator, and particles with
fluorescing compounds from the assays can be separated in planes
4115 within droplet 4105. Ideally the planes 4115 are generally
horizontal with the surface 4125 of the bottom substrate 4130.
Confocal microscope 4120 can be used to detect fluorescence of each
particle set by eliminating fluorescence emanating from planes
other than the plane or planes in which the target particle set is
located. An analogous approach can also be used for quantifying
particles of each type. Particles in each plane can be imaged and
counted. Additionally, levitating particles within a droplet can be
used in a setting in which it is desirable to eliminate background
fluorescence. Particles can be levitated using the DEP arrangement
shown in FIG. 41, and confocal microscope 4120 can focus on signal
from the particles while eliminating background signal.
[0203] It should also be noted that DEP arrangements such as those
described herein can be used to agitate beads within a droplet. For
example, beads may settle in a droplet after time or may be
attracted to weak magnetic forces from magnets located elsewhere on
a droplet actuator. Beads can be resuspended within a reservoir by
alternating between negative and positive DEP to redistribute beads
within a droplet. A similar effect can be achieved using DEP
electrodes arranged on the top and bottom plates, e.g., as shown in
FIG. 41.
[0204] 8.4 Systems
[0205] As will be appreciated by one of skill in the art, the
invention may be embodied as a method, system, or computer program
product. Accordingly, various aspects of the invention may take the
form of hardware embodiments, software embodiments (including
firmware, resident software, micro-code, etc.), or embodiments
combining software and hardware aspects that may all generally be
referred to herein as a "circuit," "module" or "system."
Furthermore, the methods of the invention may take the form of a
computer program product on a computer-usable storage medium having
computer-usable program code embodied in the medium.
[0206] Any suitable computer useable medium may be utilized for
software aspects of the invention. The computer-usable or
computer-readable medium may be, for example but not limited to, an
electronic, magnetic, optical, electromagnetic, infrared, or
semiconductor system, apparatus, device, or propagation medium.
More specific examples (a non-exhaustive list) of the
computer-readable medium would include some or all of the
following: an electrical connection having one or more wires, a
portable computer diskette, a hard disk, a random access memory
(RAM), a read-only memory (ROM), an erasable programmable read-only
memory (EPROM or Flash memory), an optical fiber, a portable
compact disc read-only memory (CD-ROM), an optical storage device,
a transmission medium such as those supporting the Internet or an
intranet, or a magnetic storage device. Note that the
computer-usable or computer-readable medium could even be paper or
another suitable medium upon which the program is printed, as the
program can be electronically captured, via, for instance, optical
scanning of the paper or other medium, compiled, interpreted, or
otherwise processed in a suitable manner, if necessary, and stored
in a computer memory. In the context of this document, a
computer-usable or computer-readable medium may be any medium that
can include, store, communicate, propagate, or transport the
program for use by or in connection with the instruction execution
system, apparatus, or device.
[0207] Computer program code for carrying out operations of the
invention may be written in an object oriented programming language
such as Java, Smalltalk, C++ or the like. However, the computer
program code for carrying out operations of the invention may also
be written in conventional procedural programming languages, such
as the "C" programming language or similar programming languages.
The program code may execute entirely on the user's computer,
partly on the user's computer, as a stand-alone software package,
partly on the user's computer and partly on a remote computer or
entirely on the remote computer or server. In the latter scenario,
the remote computer may be connected to the user's computer through
a local area network (LAN) or a wide area network (WAN), or the
connection may be made to an external computer (for example,
through the Internet using an Internet Service Provider).
[0208] Certain aspects of invention are described with reference to
various methods and method steps. It will be understood that each
method step can be implemented by computer program instructions.
These computer program instructions may be provided to a processor
of a general purpose computer, special purpose computer, or other
programmable data processing apparatus to produce a machine, such
that the instructions, which execute via the processor of the
computer or other programmable data processing apparatus, create
means for implementing the functions/acts specified in the
methods.
[0209] The computer program instructions may also be stored in a
computer-readable memory that can direct a computer or other
programmable data processing apparatus to function in a particular
manner, such that the instructions stored in the computer-readable
memory produce an article of manufacture including instruction
means which implement various aspects of the method steps.
[0210] The computer program instructions may also be loaded onto a
computer or other programmable data processing apparatus to cause a
series of operational steps to be performed on the computer or
other programmable apparatus to produce a computer implemented
process such that the instructions which execute on the computer or
other programmable apparatus provide steps for implementing various
functions/acts specified in the methods of the invention.
CONCLUDING REMARKS
[0211] The foregoing detailed description of embodiments refers to
the accompanying drawings, which illustrate specific embodiments of
the invention. Other embodiments having different structures and
operations do not depart from the scope of the invention. The term
"the invention" or the like is used with reference to certain
specific examples of the many alternative aspects or embodiments of
the applicants' invention set forth in this specification, and
neither its use nor its absence is intended to limit the scope of
the applicants' invention or the scope of the claims. This
specification is divided into sections for the convenience of the
reader only. Headings should not be construed as limiting of the
scope of the invention. The definitions are intended as a part of
the description of the invention. It will be understood that
various details of the invention may be changed without departing
from the scope of the invention. Furthermore, the foregoing
description is for the purpose of illustration only, and not for
the purpose of limitation.
* * * * *