U.S. patent application number 13/686495 was filed with the patent office on 2013-06-13 for droplet actuator loading and target concentration.
This patent application is currently assigned to ADVANCED LIQUID LOGIC INC. The applicant listed for this patent is Vamsee K. Pamula, Michael G. Pollack, Vijay Srinivasan. Invention is credited to Vamsee K. Pamula, Michael G. Pollack, Vijay Srinivasan.
Application Number | 20130146461 13/686495 |
Document ID | / |
Family ID | 39639519 |
Filed Date | 2013-06-13 |
United States Patent
Application |
20130146461 |
Kind Code |
A1 |
Pamula; Vamsee K. ; et
al. |
June 13, 2013 |
Droplet Actuator Loading and Target Concentration
Abstract
A droplet actuator and method of providing a droplet comprising
an target substance on the droplet actuator, and including discrete
flow and continuous flow functionality. Discrete flow function is
controlled by electrodes arranged for conducting droplet operations
on a substrate surface. The continuous flow function includes a
fluid path arranged for flowing a fluid therethrough. The discrete
flow and continuous flow functions may be by a barrier, including a
second fluid path through the barrier. The continuous flow function
may include a capture surface comprising a component having an
affinity for the target substance. Methods of the invention may
include flowing a fluid comprising the target substance through the
fluid path; capturing the target substance on the capture surface;
and forming a droplet in the discrete flow region via the second
fluid path and the electrodes comprising the captured target
substance.
Inventors: |
Pamula; Vamsee K.; (Durham,
NC) ; Pollack; Michael G.; (Durham, NC) ;
Srinivasan; Vijay; (Durham, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pamula; Vamsee K.
Pollack; Michael G.
Srinivasan; Vijay |
Durham
Durham
Durham |
NC
NC
NC |
US
US
US |
|
|
Assignee: |
ADVANCED LIQUID LOGIC INC
Research Triangle Park
NC
|
Family ID: |
39639519 |
Appl. No.: |
13/686495 |
Filed: |
November 27, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12531835 |
Oct 15, 2009 |
8317990 |
|
|
PCT/US2008/057963 |
Mar 24, 2008 |
|
|
|
13686495 |
|
|
|
|
60896643 |
Mar 23, 2007 |
|
|
|
60980529 |
Oct 17, 2007 |
|
|
|
61017880 |
Dec 31, 2007 |
|
|
|
Current U.S.
Class: |
204/518 |
Current CPC
Class: |
B01L 3/502792 20130101;
B01L 2200/0673 20130101; B01L 2400/0487 20130101; G01N 33/5438
20130101; B01L 2300/0816 20130101; B01F 13/0071 20130101; B01L
2300/0861 20130101; B01L 2300/0867 20130101; B01L 2400/0427
20130101; B01L 2200/0605 20130101; B01L 2300/089 20130101; B01L
2300/0636 20130101; B01L 2200/0647 20130101; B01L 3/502761
20130101; C25B 15/00 20130101; B01L 2200/0668 20130101 |
Class at
Publication: |
204/518 |
International
Class: |
C25B 15/00 20060101
C25B015/00 |
Goverment Interests
1. GOVERNMENT INTEREST
[0001] This invention was made with government support under
DK066956-02 and GM072155-02 awarded by the National Institutes of
Health of the United States. The United States Government has
certain rights in the invention.
2. RELATED PATENT APPLICATIONS
[0002] This application claims priority to and incorporates by
reference U.S. patent application Ser. No. 12/531,835, filed Oct.
15, 2009, entitled "Droplet Actuator Loading and Target
Concentration," now U.S. Pat. No. 8,317,990, issued Nov. 27, 2012
which is a national phase application of PCT/US2008/057963, filed
on Mar. 24, 2008, entitled "Droplet Acutator Loading and Target
Concentration," which claims priority and incorporates by reference
to U.S. Patent Application No. 60/896,643, filed on Mar. 23, 2007,
entitled "Pre-concentration of an analyte"; U.S. Patent Application
No. 60/980,529, filed on Oct. 17, 2007, entitled "Pre-concentration
of an analyte"; and U.S. Patent Application No. 61/017,880, filed
on Dec. 3, 2007, entitled "Droplet actuator loading and target
concentration"; the entire disclosures of which are incorporated
herein by reference.
Claims
1-51. (canceled)
52. A method of providing a droplet on a droplet actuator, the
method comprising: (a) providing a droplet actuator comprising a
substrate comprising: (i) a discrete flow section comprising
electrodes arranged for conducting droplet operations on a
substrate surface; (ii) a continuous flow section comprising a
fluid path arranged for flowing a fluid therethrough, and separated
from the discrete flow section by a barrier; (iii) an opening in
the barrier arranged relative to the electrodes such that a droplet
from the fluid path passing through the opening comes into
proximity to one or more of the electrodes; (b) flowing a filler
fluid comprising the droplet through the fluid path; (c) causing
the droplet to pass through the opening into proximity with one or
more of the electrodes to permit one or more electrode mediated
droplet operations to be performed on the droplet.
53. The method of claim 52 further comprising recirculating the
filler fluid through the fluid path via a recirculation path.
54. The method of claim 53 wherein the fluid path and/or the
recirculation path comprises a filler fluid reservoir.
55. The method of claim 53 wherein the recirculation is facilitated
using a pump to cause filler fluid to flow through the
recirculation path.
56. The method of claim 52 wherein the droplet comprises a sample
droplet.
57. The method of claim 52 wherein the droplet comprises a reagent
droplet.
58. The method of claim 52 wherein step 52(c) is mediated by one or
more of the electrodes.
59. The method of claim 52 wherein step 52(c) is mediated by a
pressure forcing the droplet through the opening.
60. The method of claim 52 wherein the filler fluid is a low
viscosity oil.
61. The method of claim 52 wherein the filler fluid is silicone
oil.
Description
3. BACKGROUND
[0003] Droplet actuators are used to conduct a wide variety of
droplet operations. A droplet actuator typically includes a
substrate associated with droplet operation electrodes, which are
arranged in a manner which permits certain droplet operations to be
performed on a surface of the substrate. In some cases, the droplet
actuator also includes a top plate separated from the surface of
the substrate in a manner which creates a gap in which the droplet
operations may be performed. Typically the droplets are at least
partially surrounded by filler fluid that is immiscible with the
droplets. The surface of the substrate and other surfaces exposed
to the filler fluid and/or the droplet are typically made from or
coated with a material which is hydrophobic relative to the
droplets being manipulated.
[0004] Referring to the applicants unpublished patent applications,
which are cited herein, certain kinds of droplets may be subjected
to various droplet operations on a droplet actuator to conduct
certain kinds of droplet-based protocols. Where the droplet-based
protocols involve detection of an analyte that is present in an
initial sample in scarce amounts, there is a need for approaches to
concentrating the analyte for analysis in the very small droplets
that are used on the droplet actuator.
4. BRIEF DESCRIPTION OF THE INVENTION
[0005] In addition to the methods described herein, the invention
provides new droplet actuators. For example, in one embodiment, the
invention provides a droplet actuator with a substrate divided into
a discrete flow section and a continuous flow section. In another
embodiment, the invention provides a droplet actuator with a
discrete flow substrate and a continuous flow substrate fluidly
coupled by a fluid flow path. The discrete flow section may include
electrodes arranged for conducting droplet operations on a
substrate surface. The continuous flow section may include a fluid
path arranged for flowing a fluid therethrough. The discrete flow
and continuous flow sections may be separated by a barrier. The
barrier may include an opening arranged relative to the electrodes
such that fluid in the fluid path is in proximity to one or more of
the electrodes. The continuous flow section may include a capture
surface comprising a component having an affinity for the target
substance.
[0006] The invention also provides a method of providing a droplet
comprising an target substance on a droplet actuator. In operation,
a method of the invention may proceed as follows: flowing a fluid
comprising the target substance through the fluid path; capturing
the target substance on the capture surface; forming a droplet
comprising the captured target substance in the discrete flow
section. The method may also include transporting the target
substance through the opening and into the discrete flow section. A
droplet may be formed in the discrete flow section by conducting
droplet operations using the electrodes to draw fluid comprising
the sample of interest through the opening and forming a droplet
therefrom.
[0007] In some cases, the droplet actuator further includes sample
collection beads immobilized in the fluid path such that a target
substance in a fluid flowing through the fluid path is collected on
the beads. Forming a droplet in the discrete flow section may
involve transporting a droplet comprising the beads through the
opening and into the droplet actuation region.
[0008] The droplet including the captured target substance in the
discrete flow section is in certain embodiments at least partially
surrounded by a filler fluid. The droplet including the captured
target substance in the discrete flow section is in certain other
embodiments substantially completely surrounded by a filler fluid.
The droplet including the captured target substance in the discrete
flow section may include one or more beads and/or one or more
biological cells.
[0009] In some embodiments, the continuous flow section includes a
second set of electrodes. These electrodes may, for example, be
arranged for conducting droplet operations. Such an arrangement
facilitates forming one or more sample droplets on the second set
of electrodes, and transporting said one or more sample droplets
into the discrete flow section. In another embodiment, such an
arrangement facilitates forming one or more sample droplets on the
second set of electrodes, and using the second set of electrodes to
transport said one or more sample droplets into proximity with
electrodes in the discrete flow section.
[0010] In some cases, the continuous flow section includes two or
more capture surfaces. For example, the two or more capture
surfaces may each comprise a different population of beads, and
each population may have affinity for a different target
substance.
[0011] In some embodiments employing beads, the beads are
maintained in place by one or more physical barriers and/or one or
more magnetic fields (e.g., a magnetic field produced by an
electromagnet and/or by a permanent magnet).
[0012] Capturing the target substance on the capture surface may in
some cases may involve combining one or more beads having affinity
for the target substance with a fluid comprising the target
substance prior to introduction of the fluid into the fluid path.
The target substance may, for example, include an analyte of
interest and/or a product of a chemical reaction. The target
substance may in other cases include a bead and/or a biological
cell. The target substance may include a bead comprising a analyte
of interest and/or a product of a chemical reaction.
[0013] In some cases, the discrete flow section is fluidly coupled
to the continuous flow section by a fluid path including a
reservoir and a reservoir electrode associated with a surface
thereof. Such embodiments may facilitate a method involving flowing
a fluid comprising the substance of interest from the fluid path
into the reservoir; and dispensing droplets from the reservoir onto
one or more electrodes in the discrete flow section.
[0014] Flowing a fluid comprising the target substance through the
fluid path may in some cases involve forcing fluid through the
fluid path using a pressure source. The pressure source may, for
example, be a pump or a syringe coupled to the fluid path.
[0015] Forming a droplet comprising the captured target substance
in the discrete flow section may, in one embodiment, involve
activating one of more electrodes in the discrete flow section
adjacent to the opening to cause fluid to flow from the fluid path
into the discrete flow section and form the droplet. In another
embodiment, forming a droplet comprising the captured target
substance in the discrete flow section may involve forcing fluid
from the fluid path through the opening into the discrete flow
section into proximity with one or more electrodes, and conducting
droplet operations using the one of more electrodes to form the
droplet. The force applied may cause a pressure in the fluid path
which forces fluid through the opening into the discrete flow
section into proximity with the one or more electrodes.
[0016] In one embodiment, the method of the invention conducts a
chemical or biochemical reaction or interaction. Such a method may
comprise providing a droplet actuator of the invention; flowing a
fluid comprising the target substance through the fluid path;
capturing the target substance on the capture surface; and
conducting one or more droplet operations using the electrodes to
transport a reagent into contact with the target substance and
thereby conduct a chemical or biochemical reaction or interaction.
In certain embodiments, the target substance comprises a cell and
the biochemical interaction comprises interaction of the reagent
with the cell.
[0017] In another aspect of the invention the method involves
flowing a filler fluid comprising the droplet through the fluid
path; and causing the droplet to pass through the opening into
proximity with one or more of the electrodes to permit one or more
electrode mediated droplet operations to be performed on the
droplet.
[0018] Various embodiments may involve recirculating the filler
fluid through the fluid path via a recirculation path. The
recirculation path may in some embodiments comprises a filler fluid
reservoir. Recirculation may be facilitated using a pump to cause
filler fluid to flow through the recirculation path.
[0019] Droplet formation may be mediated by one or more of the
electrodes described above, and may in some cases be assisted by a
pressure forcing the droplet through the opening in the
barrier.
[0020] Various other embodiments of the invention will be apparent
from the description of the invention set forth in the ensuing
sections.
5. DEFINITIONS
[0021] As used herein, the following terms have the meanings
indicated.
[0022] "Activate" with reference to one or more electrodes means
effecting a change in the electrical state of the one or more
electrodes which results in a droplet operation.
[0023] "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; 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 are described in U.S. Patent Publication No. 2005-0260686,
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.
[0024] "Droplet" means a volume of liquid on a droplet actuator
which 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 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.
[0025] "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 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 size of the resulting
droplets (i.e., the size 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.
[0026] "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.
[0027] "Magnetically responsive" means responsive to a magnetic
field. 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.
[0028] The terms "top" and "bottom" are used throughout the
description with reference to the top and bottom substrates of the
droplet actuator for convenience only, since the droplet actuator
is functional regardless of its position in space.
[0029] When a given component such as a layer, region or substrate
is referred to herein as being disposed or formed "on" another
component, that given component can be directly on the other
component or, alternatively, intervening components (for example,
one or more coatings, layers, interlayers, electrodes or contacts)
can also be present. It will be further understood that the terms
"disposed on" and "formed on" are used interchangeably to describe
how a given component is positioned or situated in relation to
another component. Hence, the terms "disposed on" and "formed on"
are not intended to introduce any limitations relating to
particular methods of material transport, deposition, or
fabrication.
[0030] 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.
6. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] 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 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.
[0032] FIG. 1 illustrates a droplet actuator having target
substance concentrating capabilities.
[0033] FIG. 2 illustrates a droplet actuator, which is like droplet
actuator of FIG. 1, except that multiple target substance
collection surfaces are included in the fluid path, and further,
the droplet actuator includes multiple openings.
[0034] FIG. 3 illustrates a droplet actuator, which is like droplet
actuator of FIG. 1, except that a physical barrier is used to
capture beads.
[0035] FIG. 4 illustrates a droplet actuator, which is like droplet
actuator of FIG. 3, except that a dispensing reservoir is
included.
[0036] FIG. 5 illustrates droplet actuator, which is like droplet
actuator of FIG. 3, except that a series of bead populations is
provided, each trapped in place by physical barriers.
[0037] FIG. 6 illustrates droplet actuator, which is like droplet
actuator of FIG. 1, except that the fluid path includes electrodes
arranged for conducting droplet operations.
[0038] FIG. 7 illustrates droplet actuator, which is like the
droplet actuator shown in FIG. 4, except that laminar flow of two
liquids is employed to separate/concentrate target substance.
7. DESCRIPTION
[0039] The invention provides a droplet actuator configured for
processing input fluids to concentrate one or more target
substances (e.g., analytes, products and/or beads containing
analytes or products) and provide droplets including the
concentrated target substances. For example, the target substances
may be analytes or substances associated with one or more analytes
(e.g., antibodies bound to analytes), and the droplets may be used
in droplet-based protocols for analyzing the analytes. The
invention also provides methods of making and using such droplet
actuators. In general, the methods relate to preparing small volume
sample droplets from large volume samples. In certain embodiments,
the concentration of one or more target substances in the resulting
small volume sample droplets is greater than the concentration of
one or more target substances in the starting sample fluid.
[0040] The invention also relates to methods of loading reagents or
any kinds of droplets onto a droplet actuator. For example, a
filler fluid may be flowed through a continuous flow section of a
droplet actuator (as described herein), droplets may be introduced
into the continuous flow section and when the droplets are flowed
into proximity with a discrete flow section, the droplets may be
transported onto the discrete flow section and subjected to droplet
operations. In this arrangement, the filler fluid may circulate
back to a filler fluid reservoir.
[0041] The small volume fluid droplets containing the target
substance may be subjected to one or more droplet operations on the
droplet actuator. The droplet operations may be part of a
droplet-based assay protocol to analyze the droplets for the target
substance. In order to analyze larger volumes of starting fluid,
multiple unit sized droplets may be analyzed in parallel and/or in
sequence. Such an approach is suitable, for example, when the
target substance is present at a very low concentration. Prior to
introduction of a fluid into the droplet actuator of the invention,
various concentration methods may be employed to pre-concentrate
the target substance. Examples include centrifugation and/or
filtration.
7.1 Fluid Processing Designs
[0042] FIG. 1 illustrates a droplet actuator 100 having target
substance concentrating capabilities. Droplet actuator 100 is
divided into a continuous flow section and a discrete flow
section.
[0043] The continuous flow section is represented by fluid path 101
through which fluid containing a target substance may be flowed.
Fluid path 101 is defined by barriers 103 and 107. Barrier 103
separates the continuous flow section from the discrete flow
section of droplet actuator 100. One or more openings in barrier
103, shown in the example as opening 119, may provide a fluid
passage from the continuous flow section to the discrete flow
section of the device.
[0044] The discrete flow section is represented by the array 113 of
electrodes, which may be any arrangement of electrodes suitably
configured to conduct droplet operations on droplets formed from
fluid flowing through fluid path 101. In operation, a fluid may be
flowed through inlet 109, through fluid path 101, and out through
outlet 111, optionally via valve 117. One or more electrodes,
represented in the example as electrode 105, may be provided in
proximity to opening 119 to facilitate droplet operations using
fluid from the continuous flow section. For example, such
electrodes may be used to facilitate flow of fluid from the
continuous flow section into the discrete flow section of droplet
actuator 100 and/or formation of droplets from flow of droplets
from such fluid. In one embodiment, fluid is circulated through
fluid path 101.
[0045] As an example, a fluid containing an analyte of interest may
be flowed through fluid path 101. As the fluid passes opening 119,
electrode 105 may be activated, causing fluid from fluid path 101
to flow onto electrode 105. Additional electrodes from the array
113 of electrodes may be activated/deactivated in various patterns
to form a droplet from the fluid on electrode 105 and conduct
various droplet operations using the droplet. Flow of fluid onto
electrode 105 may also be facilitated by restricting outflow of
fluid path 101, relative to inflow, e.g., by constricting or
closing valve 117 while pressure source 115 continues to force
fluid into fluid path 101.
[0046] As another example, the fluid flowed through path 101 may be
a filler fluid including one or more droplets, such as reagent
droplets. The transport of reagent droplets from the continuous
flow channel 101 onto the discrete flow section may, for example,
be mediated by electrode 105 and adjacent electrodes. Impedance
sensing may be used to determine when a droplet flowing through
path 101 is in sufficient proximity to electrode 105 to be
subjected to droplet operations mediated by electrode 105. In
another embodiment, droplets in filler fluid in fluid path 101 may
be forced into the discrete flow section by closing valve 117
causing flow of fluid through path 101 to divert through opening
119 into the discrete flow portion. Similarly, a suction may be
applied to filler fluid in the discrete flow portion to force a
droplet from path 101 through opening 105 and into the discrete
flow section.
[0047] Filler fluid exiting fluid path 101, e.g., via outlet 111,
may be flowed into a waste reservoir or recirculated to inlet 109
via a fluid path (not shown), optionally including filler fluid
reservoir. Outside the droplet actuator, the droplet may be
introduced into a filler fluid line that flows into path 101 using
a variety of approaches. For example, the droplet may be injected
into the filler fluid line. Similarly, the droplet may be deposited
in a filler fluid reservoir having an outflow at a lower portion.
The droplet may be permitted to sink (due to relative density of
the filler fluid) to the bottom of the reservoir where it will flow
into the filler fluid line and into fluid path 101.
[0048] Various approaches may be used to concentrate the target
substance in fluid path 101 prior to introduction of fluid from
fluid path 101 into the discrete flow section of droplet actuator
100. Such approaches serve to provide a droplet on the droplet
actuator in which the target substance is more concentrated than in
a starting fluid. The means of concentrating the target substance
may, for example, involve capture of the target substance by a
binder. The binder may be attached to a surface. The surface may
be, for example, a surface of a bead, such as a magnetically
responsive bead or a non-magnetically responsive bead. The surface
may also alternatively be a fixed surface, such as an inner surface
of the droplet actuator.
[0049] In one embodiment, the fluid containing the target substance
is flowed through fluid path 101, where it is exposed to the
capture surface. The capture surface may be a surface that is
arranged at a fixed point within fluid path 101 and/or is otherwise
relatively stably positioned in fluid path 101. In another
embodiment, the surface (e.g., beads) may be combined with the
fluid before it is flowed into fluid path 101. The surface may be
captured as the fluid flows through fluid path 101 in order to
concentrate the target substance.
[0050] As an example, where the target substance in the fluid is an
analyte, beads having antibodies with specificity for the analyte
may be combined with the fluid where the antibodies will capture
the analyte. The fluid containing the beads with bound analyte
thereon, may be flowed through fluid path 101 where the beads are
captured, and fluid containing some or all of the beads is flowed
onto the discrete flow section of droplet actuator 100 and formed
into a droplet. The capture may for example, be accomplished using
a magnetic field for magnetically responsive beads and/or physical
barriers which may prevent beads from continuing to flow through
fluid path 101. Once formed, bead-containing droplet may then be
subjected to further droplet operations as part of a protocol for
analyzing the analyte.
[0051] In one embodiment, opening 119 may be configured so that in
the ordinary course of flowing fluid through fluid path 101, fluid
does not enter the discrete flow section of droplet actuator 100
without some further assistance. For example, opening 119 may be
configured so that it's width is smaller than the height of fluid
path 101. In this manner, fluid may flow through fluid path 101
without readily passing through opening 119. Fluid can be prompted
to flow through opening 119 by, for example, activating electrode
105 and/or restricting outflow from fluid path 101 while continuing
to provide pressure from pressure source 115.
[0052] Fluid may be flowed through fluid path 101 using a variety
of positive and/or negative pressure sources. For example, pressure
source 115 may be gravity, capillarity or a syringe pushing or
pulling fluid through fluid path 101. Various valves, such as valve
117, may be provided to control fluid flow through fluid path 101.
For example, fluid outflow may be controlled by a valve 117 at
outlet 111. A valve may be arranged to control application of a
positive and/or negative pressure source for flowing fluid through
fluid path 101. One or more valves may also be used to control the
rate of fluid flow through fluid path 101.
[0053] The digital portion 113 of droplet actuator 100 may include
electrode configurations for conducting any of a variety of droplet
operations.
[0054] Barriers 103 and 107 may, for example, be formed from gasket
material. Inferior portions of droplet actuator 100 may, in some
embodiment, be made from or coated with a hydrophobic material. In
another embodiment, surfaces of fluid path 101 may be made from
and/or coated with a hydrophilic material. In one embodiment, the
discrete flow section of droplet actuator 100 is covered or filled
with a filler fluid that is immiscible with fluid in fluid path
101. Similarly, the filler fluid may be selected to be immiscible
with droplets that are to be subjected to droplet operations
mediated by electrode array 113, which may be any arrangement of
electrodes suitable for conducting one or more droplet operations.
A filler fluid/sample fluid interface (e.g., an oil/water
interface) may be present at opening 119.
[0055] In the example illustrated in FIG. 1, fluid flows from inlet
109, through the fluid path 101, and out through outlet 111. Fluid
not transported into the discrete flow section may be recirculated
through the fluid path 101. Various other embodiments are
contemplated in which the fluid flow is different. For example,
there may be multiple openings 119 in barrier 103 which lead to
fluid path 101. Similarly, there may be multiple openings in
barrier 107 for introducing and/or removing fluid from fluid path
101. Further, fluid may be introduced and/or removed from fluid
path 101 through barrier 103, barrier 107, the base substrate of
droplet actuator 100 and/or the optional top plate of droplet
actuator 100. Similarly, fluid path 101 may be a capillary or tube
which passes through the discrete flow section of droplet actuator
100 and having openings therein for flowing fluid from inside the
capillary or two into proximity with one or more electrodes of
droplet actuator 100.
[0056] Fluid path 101 is illustrated as being adjacent to
electrodes 119 substantially on a common plane; however, it will be
appreciated that fluid path 101 may be oriented in any direction
relative to electrodes 119, e.g., above, alongside, and/or below
electrodes 119. Further, multiple fluid paths 101 may be used for
each electrode 119 and/or one or more of the electrodes 119 may be
associated with a different fluid path 101.
[0057] FIG. 2 illustrates a droplet actuator 200, which is like
droplet actuator 100 of FIG. 1, except that droplet actuator 200
includes multiple target substance collection surfaces 201, 203,
205 in fluid path 101, and further, droplet actuator 200 includes
multiple openings 119.
[0058] In operation, a fluid containing a target substance may be
floated into inlet 109, through fluid path 101, and out through
outlet 111. Target substances in the input fluid may be captured at
capture points 201, 203, and 205, etc. As described above, the
target substance may be captured using a binder which is specific
to the target substance. The surfaces may include beads (not
shown).
[0059] In one embodiment, fluid path 101 also includes droplet
operation electrodes arranged such that a droplet can be
transported from the discrete flow section into fluid path 101. In
this embodiment, a target substance may be captured on a surface of
fluid path 101, e.g., at a capture point 201, 203, 205. A droplet
comprising a fluid for releasing the target substance may be
transported using electrode-mediated droplet operations from the
discrete flow section onto the electrodes in fluid path 101 into
contact with a capture point 201, 203, 205, where the droplet may
release and absorb the target substance. The droplet, now including
the target substance, may be transported back to the discrete flow
section for further steps, e.g., in an assay to analyze the target
substance.
[0060] In another related embodiment, following capture on capture
point 201, 203, 205, a droplet containing assay reagents may be
transported using electrode-mediated droplet operations from the
discrete flow section onto the electrodes in fluid path 101 into
contact with a capture point 201, 203, 205, where the reaction may
occur and the product be detected using a detector in proximity
with the droplet actuator. In alternative embodiment, the detection
zone may be located in the discrete flow section, and a droplet
from the assay region may be transported into the discrete flow
section for detection.
[0061] In another related embodiment, following capture on capture
point 201, 203, 205, a fluid for releasing the target substance may
be flowed into contact with the capture point to release the
captured target substance into the fluid. The fluid may be then
flowed into the discrete flow section of the droplet actuator,
where it may be formed into one or more droplets for conducting
droplet operations.
[0062] Different capture sites may use the same or different
capture means. For example, capture site 201 may utilize an
antibody which binds to an antigen in the input fluid; capture site
203 may utilize an antigen which binds to an antibody in the input
fluid; capture site 205 may utilize a nucleic acid which binds to
nucleic acids in the input fluid. Accordingly, the different target
substances are concentrated and separated.
[0063] Further, the fluid flowed into contact with the capture
points 201, 203, 205 may be droplets contained in a filler fluid
which is flowed through fluid path 101.
[0064] FIG. 3 illustrates a droplet actuator 300, which is like
droplet actuator 100 of FIG. 1, except that droplet actuator 300
illustrates the use of a physical barrier to capture beads. In this
embodiment, beads having affinity for a target substance may be
combined with a fluid having the target substance. The target
substance will bind to the beads. The beads may be concentrated,
e.g., using a filter. In any case, a fluid comprising the beads is
flowed through fluid path 101 where the beads are captured by
barrier 301, while the fluid continues to flow through channel 101
and, in the embodiment illustrated, out outlet 111. Once sufficient
beads have been captured, electrode 105 may be activated to pull a
droplet into the discrete flow section of droplet actuator 300.
Other means for forcing fluid into the discrete flow section of the
droplet actuator, as described elsewhere, may also be used.
[0065] In some embodiment, the beads may include a mixture of bead
populations, each bead population having one or more beads having
specificity for a specific target substance. U.S. Patent
Application No. 60/896,393, filed on Mar. 22, 2007, entitled
"Sample preparation by beads sorting" and Related U.S. patent
application Ser. No. [Docket No. 040PRV2], filed contemporaneously
with this patent application, also entitled "Sample preparation by
bead sorting," the entire disclosures of which are incorporated
herein by reference, described droplet based approaches to sorting
beads. Further, beads may be sorted on the droplet actuator using
any approach which permits a difference in beads to be sensed in a
droplet on a droplet actuator. Examples include different colored
beads, beads with different levels of radioactivity, beads that
absorb light differently, and many other approaches.
[0066] FIG. 4 illustrates a droplet actuator 400, which is like
droplet actuator 300 of FIG. 3, except that droplet actuator 400
illustrates the use of a dispensing reservoir 401. Dispensing
reservoir 401 is located on the discrete flow section of droplet
actuator 400. Opening 119 provides a fluid path from fluid path 101
into reservoir 401 and into proximity with reservoir electrode 402.
Reservoir electrode 402 may optimally be bounded in part by a
barrier 403. In operation, once sufficient beads are captured at
capture point 205, fluid containing the beads may be flowed into
reservoir 401, into contact with reservoir electrode 402. Fluid
containing beads may then be dispensed from reservoir 401 as
discrete, unit-sized droplets for further droplet operations.
[0067] FIG. 5 illustrates droplet actuator 500, which is like
droplet actuator 300 of FIG. 3, except that droplet actuator 500
illustrates the use of a series of bead populations, each trapped
in place by physical barriers. In operation, fluid flowed through
fluid path 101 sequentially comes into contact with each of the
bead populations. The populations may contain the same or different
target substance binders. Once sufficient fluid has been contacted
with each of the bead populations, droplets comprising the beads
can be formed and manipulated using various droplet operations on
the discrete flow section of droplet actuator 500.
[0068] In a related embodiment, droplet manipulation electrodes are
provided in fluid path 101, and the electrodes are used to
manipulate discrete droplets into contact with each of the bead
populations, and into the discrete flow section of droplet actuator
500. In another related embodiment, filler fluid comprising
droplets therein is flowed through fluid path 101, and each droplet
is transported into the discrete flow section of the droplet
actuator once it has contacted its bead population. Thus, for
example, the first droplet may contact the first set of beads, and
be transported into the discrete flow section of the droplet
actuator. The second droplet may contact the second set of beads,
and be transported into the droplet actuator, and so on. This
approach has the advantage that each droplet contacts only one set
of beads, preventing interference that may be caused by nonspecific
binding.
[0069] FIG. 6 illustrates droplet actuator 600, which is like
droplet actuator 100 of FIG. 1, except that droplet actuator 600
illustrates an embodiment in which fluid path 101 includes
electrodes arranged for conducting droplet operations. One or more
of the electrodes may be associated with a target substance capture
site, e.g., capture sites A, B, C, D, associated with electrodes
603, 605, 607, 609. Fluid flowing through fluid path 101 interacts
with the sites, permitting target substance to be captured. As
described in other examples, capture may be accomplished by binding
factors on the beads or other surfaces associated with the capture
sites.
[0070] In one embodiment, fluid is flowed through fluid path 101
permitting target substance to be captured at capture sites A, B,
C, D. Once capture sites A, B, C, D have been sufficiently exposed
to input fluid, electrodes 603, 605, 607, 609, A, B, C, D are
activated, and the remaining fluid is flushed from fluid path 101,
e.g., by flowing oil through the fluid path. As a result, droplets
remain on the activated electrodes 603, 605, 607, 609. These
droplets may then be subjected to droplet operations using the
electrodes in fluid path 101 and/or transported into the discrete
flow section of the droplet actuator for further
processing/analysis.
[0071] The capture sites 603, 605, 607, and 609 may be a labeled
set of beads, or any other labeled surface attachment. An electrode
is activated at the capture sites. As the oil flows through fluid
path 101, the pressure is set to oust any sample liquid that is not
at the capture sites. The liquid at the capture sites remains due
to the activated electrode. Oil flows around the liquid at the
capture sites, creating droplets in fluid path 101. In one
embodiment, the capture sites include beads, and droplets including
the beads are subjected to further droplet operations.
[0072] FIG. 7 illustrates droplet actuator 700, which is like
droplet actuator 400 of FIG. 4, except that droplet actuator 700
illustrates an embodiment in which laminar flow of two liquids is
employed to separate/concentrate target substance. The fluids may
be flowed together through fluid path 101 of droplet actuator 700,
with one fluid being flowed along an inner portion of fluid path
101, which is generally proximate relative to the discrete flow
section of droplet actuator 700, and the other fluid being flowed
along an outer portion of fluid path 101, which is generally distal
relative to the discrete flowed section of droplet actuator 700. In
one embodiment, one fluid is an aqueous solution, and the second
fluid is a non-aqueous, such as acetone. Target substance may be
extracted from the aqueous solution into the nonaqueous solution or
vice versa. By controlling the flow of the solutions, fluid
containing target substance may accumulate in reservoir 707. The
flow of the solutions may be controlled by an H-filter. Droplets
may then be dispensed from the reservoir into the discrete flow
section of droplet actuator 700 for further
processing/analysis.
[0073] This embodiment makes use of laminar flow of the liquids,
which can result in a gradient in the concentration of materials.
The concentration of the material may be a function of its position
in fluid path 101. For example, one type of material concentrated
on one side of the reservoir and one type of material may be
concentrated on the other side of the reservoir. Sampling may be
controlled effectively obtain droplets enriched for one type of
material or another.
[0074] In an embodiment, the input fluid may be blood, and the
second solution may be buffer. As the solutions flow through fluid
path 101, blood components may adhere to surfaces along fluid path
101. Smaller molecules from the blood may diffuse into the buffer,
and larger cells such as white blood cells may remain in the blood.
Similarly, the flow may be controlled to permit transfer of the
white blood cells to the reservoir.
[0075] In any of the various embodiments described herein, fluid
may in some cases be recirculated through fluid path 101, e.g., to
improve capture of analytes.
[0076] Further, the fluid flowed through fluid path 101 may be a
filler fluid containing droplets. Some or all of the droplets may
be diverted into the discrete flow section of the droplet actuator
for processing using the various approaches described herein.
7.2 Droplet Actuator
[0077] For examples of droplet actuator architectures suitable for
use with the present invention, 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. Nos.
6,773,566, entitled "Electrostatic Actuators for Microfluidics and
Methods for Using Same," issued on Aug. 10, 2004 and 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/US 06/47486, entitled
"Droplet-Based Biochemistry," filed on Dec. 11, 2006, the
disclosures of which are incorporated herein by reference. Examples
of droplet actuator techniques for immobilizing magnetic beads
and/or non-magnetic beads are described in the foregoing
international patent applications and in Sista, et al., U.S. Patent
Application Nos. 60/900,653, filed on Feb. 9, 2007, entitled
"Immobilization of magnetically-responsive beads during droplet
operations"; Sista et al., U.S. Patent Application No. 60/969,736,
filed on Sep. 4, 2007, entitled "Droplet Actuator Assay
Improvements"; and Allen et al., U.S. Patent Application No.
60/957,717, filed on Aug. 24, 2007, entitled "Bead washing using
physical barriers," the entire disclosures of which is incorporated
herein by reference.
7.3 Fluids
[0078] For examples of fluids usefully processed according to the
approach of the invention, see the patents listed in section 7.2,
especially International Patent Application No. PCT/US 06/47486,
entitled "Droplet-Based Biochemistry," filed on Dec. 11, 2006. In
some embodiments, the input fluid includes or consists of a
biological sample, such as whole blood, lymphatic fluid, serum,
plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic
fluid, seminal fluid, vaginal excretion, serous fluid, synovial
fluid, pericardial fluid, peritoneal fluid, pleural fluid,
transudates, exudates, cystic fluid, bile, urine, gastric fluid,
intestinal fluid, fecal samples, fluidized tissues, fluidized
organisms, biological swabs and biological washes.
7.4 Filler Fluids
[0079] The gap will typically be filled with a filler fluid. 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/US 06/47486, entitled
"Droplet-Based Biochemistry," filed on Dec. 11, 2006.
[0080] 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.
[0081] It will be understood that various details of the present
invention may be changed without departing from the scope of the
present invention. Various aspects of each embodiment described
here may be interchanged with various aspects of other embodiments.
Furthermore, the foregoing description is for the purpose of
illustration only, and not for the purpose of limitation.
* * * * *