U.S. patent application number 17/519400 was filed with the patent office on 2022-04-28 for devices and methods for sample analysis.
The applicant listed for this patent is Abbott Laboratories. Invention is credited to Graham Davis, Sergey Gershtein, Mark A. Hayden, Jeffrey B. Huff.
Application Number | 20220126296 17/519400 |
Document ID | / |
Family ID | 1000006068782 |
Filed Date | 2022-04-28 |
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United States Patent
Application |
20220126296 |
Kind Code |
A1 |
Huff; Jeffrey B. ; et
al. |
April 28, 2022 |
DEVICES AND METHODS FOR SAMPLE ANALYSIS
Abstract
Integrated devices that include a sample preparation component
integrated with a detection component are disclosed. The sample
preparation component may be a digital microfluidics module or a
surface acoustic wave module which modules are used for combing a
sample droplet with a reagent droplet and for performing additional
sample preparation step leading to a droplet that contains
beads/particles/labels that indicate presence or absence of an
analyte of interest in the sample. The beads/particles/labels may
be detected by moving the droplet to the detection component of the
device, which detection component includes an array of wells.
Additional analyte detection devices configured to operate an
analyte detection chip to prepare a test sample and to detect an
analyte related signal from the prepared test sample in the analyte
detection chip are disclosed. The analyte detection chip may
include a digital microfluidics (DMF) region and an analyte
detection region which may overlap or may be spatially separated.
The analyte detection device may be configured for detection of
analyte by an optical or electrochemical means operably connected
with an analyte detection chip inserted into the device.
Inventors: |
Huff; Jeffrey B.;
(Lincolnshire, IL) ; Hayden; Mark A.; (Vernon
Hills, IL) ; Davis; Graham; (Princeton, NJ) ;
Gershtein; Sergey; (Skillman, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Abbott Laboratories |
Abbott Park |
IL |
US |
|
|
Family ID: |
1000006068782 |
Appl. No.: |
17/519400 |
Filed: |
November 4, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16850711 |
Apr 16, 2020 |
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17519400 |
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15726280 |
Oct 5, 2017 |
11198129 |
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16850711 |
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62404716 |
Oct 5, 2016 |
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62425006 |
Nov 21, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 27/44721 20130101;
G01N 27/3271 20130101; B01L 2300/0816 20130101; B01L 2300/0636
20130101; B01L 2300/0645 20130101; B01L 2300/0887 20130101; B01L
2400/0424 20130101; G01N 33/54366 20130101; B01L 2200/0668
20130101; B01L 3/502761 20130101; B01L 2300/0654 20130101; B01L
2300/0893 20130101; B01L 3/502715 20130101; G01N 33/5438 20130101;
B01L 3/502784 20130101; B01L 2400/0415 20130101; B01L 2200/0673
20130101; B01L 2400/0427 20130101; B01L 2200/10 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; G01N 33/543 20060101 G01N033/543; G01N 27/447 20060101
G01N027/447 |
Claims
1.-60. (canceled)
61. A method for electrochemical detection of an analyte in a
sample, the method comprising: (a) introducing the sample into a
cartridge, the cartridge comprising: a first substrate; a second
substrate; a gap separating the first substrate from the second
substrate; a plurality of electrodes to generate electrical
actuation forces on a liquid droplet; and an electrochemical
species sensing region comprising a working electrode and a
reference electrode; (b) actuating the plurality of electrodes to
provide a first liquid droplet comprising the analyte; (c)
actuating the plurality of electrodes to provide a second liquid
droplet comprising an enzyme selective for the analyte; (d)
actuating the plurality of electrodes to merge the first and second
droplets to create a mixture; (e) actuating the plurality of
electrodes to move all or a portion of the mixture to the
electrochemical sensing region; (f) detecting, via the working and
reference electrodes, an electrical signal of an electrochemical
species generated by action of the enzyme on the analyte.
62. The method of claim 61, wherein the second liquid droplet
comprises a redox mediator.
63. The method of claim 61, further comprising determining a
concentration of the analyte based on the electrical signal.
64. The method of claim 61, wherein the electrochemical sensing
region is located in a capillary region.
65. A method for electrochemical detection of an analyte in a
sample, the method comprising: (a) introducing the sample into a
cartridge, the cartridge comprising: a first substrate; a second
substrate; a gap separating the first substrate from the second
substrate, a plurality of electrodes to generate electrical
actuation forces on a liquid droplet; and an electrochemical
species sensing region comprising a working electrode and a
reference electrode; (b) actuating the plurality of electrodes to
provide a first liquid droplet comprising the analyte; (c)
actuating the plurality of electrodes to provide a second liquid
droplet comprising a solid substrate comprising a first binding
member that specifically binds to the analyte; (d) actuating the
plurality of electrodes to merge the first and second droplets to
create a mixture; (e) actuating the plurality of electrodes to
merge all or a portion of the mixture with a third liquid droplet
comprising a second binding member that specifically binds to the
analyte; (f) holding the solid substrate in place while actuating
the plurality of electrodes to remove any unbound analyte and/or
second binding member; (g) actuating the plurality of electrodes to
contact the solid substrate with a substrate molecule for the
enzyme conjugated to the second binding member; and (h) detecting,
via the working and reference electrodes, an electrical signal of
an electrochemical species generated by action of the enzyme on the
substrate molecule.
66. The method of claim 65, wherein the method comprises moving a
liquid droplet comprising the solid second substrate from step (f)
to the electrochemical sensing region prior to steps (g) and
(h).
67. The method of claim 65, wherein the method comprises moving a
liquid droplet comprising the solid second substrate and enzyme
substrate from step (g) to the electrochemical sensing region.
68. The method of claim 65, further comprising determining a
concentration of the analyte based on the electrical signal.
69. The method of claim 65, further comprising conducting an
immunoassay on a sample, using a single cartridge or a different
cartridge.
70. The method of claim 69, comprising spatially segregating single
molecules and optically detecting the segregated single molecules
to detect presence of an analyte in the sample.
71.-86. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 62/404,716, filed on Oct. 5, 2016, and U.S.
Provisional Application Ser. No. 62/425,006, filed on Nov. 21,
2016, the disclosures of which applications are herein incorporated
by reference.
INTRODUCTION
[0002] Analyte analysis is usually performed by carrying out a
sample preparation step that is either performed manually or using
complicated robotics. After sample preparation, the assaying of an
analyte in the prepared sample further involves use of expensive
and complicated systems for transporting the prepared sample to a
machine that then performs analysis of an analyte in the prepared
sample.
[0003] Integrated devices that can be used to prepare a sample and
assay the prepared sample are highly desirable in the field of
analyte analysis. Such integrated devices would offer a low cost
option and would considerably increase the ease of performing
analyte analysis, especially in clinical applications, such as
point-of-care applications.
[0004] As such, there is an interest in integrated devices for
performing analyte analysis.
SUMMARY
[0005] An integrated microfluidic and analyte detection device is
disclosed. Also provided herein are exemplary methods for using an
integrated microfluidic and analyte detection device and associated
systems. Analyte detection devices configured to operate an analyte
detection chip to prepare a test sample and to detect an analyte
related signal from the prepared test sample in the analyte
detection chip are disclosed. The analyte detection cartridge may
include a digital microfluidics (DMF) region and an analyte
detection region which may overlap or may be spatially separated.
The analyte detection device may be configured for detection of
analyte by an optical or electrochemical means operably connected
with an analyte detection chip inserted into the device.
[0006] Disclosed is a digital microfluidic and analyte detection
device, including a first substrate and a second substrate, wherein
the second substrate is separated from the first substrate by a
gap, the first substrate including a plurality of electrodes to
generate electrical actuation forces on a liquid droplet; and an
array of wells dimensioned to hold a portion of the liquid droplet,
wherein at least a portion of the array of wells is positioned
between one or more of the plurality of electrodes and the gap.
[0007] In some embodiments, the plurality of electrodes is
positioned on a surface of the first substrate. In certain
embodiments, the device further includes a first layer disposed on
the surface of the first substrate and covering the plurality of
electrodes. In some embodiments, the first substrate includes a
first portion at which the liquid droplet is introduced and a
second portion toward which a liquid droplet is moved. In certain
embodiments, the plurality of electrodes and the first layer extend
from the first portion to the second portion of the first
substrate. In certain embodiments, the array of wells is positioned
in the second portion of the first substrate. In certain
embodiments, the second substrate includes a first portion and a
second portion, wherein the first portion is in facing arrangement
with the first portion of the first substrate and the second
portion is in facing arrangement with the array of wells. In
certain embodiments, the second portion of the second substrate is
substantially transparent to facilitate optical interrogation of
the array of wells.
[0008] In some embodiments, the device further includes a second
layer disposed on a surface of the first layer. In certain
embodiments, the second layer extends over the first and second
portions of the first substrate. In certain embodiments, the first
layer is a dielectric layer and the second layer is a hydrophobic
layer. In certain embodiments, the array of wells is positioned in
the second layer. In certain embodiments, the array of wells is
positioned in the first layer. In certain embodiments, the array of
wells has a hydrophilic surface.
[0009] In some embodiments, the array of wells include a sidewall
that is oriented to facilitate receiving and retaining of beads or
particles present in droplets moved over the well array. In certain
embodiments, the array of wells include a first sidewall opposite
to a second side wall, wherein the first sidewall is oriented at an
obtuse angle with reference to a bottom of the wells, and wherein
the second sidewall is oriented at an acute angle with reference to
the bottom of the wells, wherein movement of droplets is in a
direction parallel to the bottom of the wells and from the first
sidewall to the second sidewall. In certain embodiments, the array
of wells have a frustoconical shape with a narrower part of the
frustoconical shape providing an opening of the array of wells. In
certain embodiments, the array of wells include a first sidewall
opposite to a second side wall, wherein a top portion of the first
sidewall is oriented at an obtuse angle with reference to a bottom
of the wells and a bottom portion of the sidewall is oriented
perpendicular to the bottom of the wells, and wherein the second
sidewall is oriented perpendicular with reference to the bottom of
the wells, wherein the movement of droplets is in a direction
parallel to the bottom of the wells and from the first sidewall to
the second sidewall, wherein the top portion of the first side wall
is at an opening of the wells.
[0010] Also disclosed is a digital microfluidic and analyte
detection device, including a first substrate and a second
substrate defining the device, wherein the second substrate is
separated from the first substrate by a gap, wherein the device
includes a first portion and a second portion; and the first
portion includes a plurality of electrodes to actuate combining of
a first liquid droplet containing an analyte of interest from a
biological sample and a second liquid droplet containing at least
one bead; and the second portion includes an array of wells
dimensioned to hold a portion of the liquid droplet.
[0011] In some embodiments, the plurality of electrodes are only
positioned in the first portion of the device. In certain
embodiments, the plurality of electrodes is positioned on a surface
of the first substrate. In some embodiments, the device further
includes a first layer disposed on the surface of the first
substrate and covering the plurality of electrodes. In certain
embodiments, the first substrate includes a first portion at which
the liquid droplet is introduced and a second portion toward which
a liquid droplet is moved. In certain embodiments, the plurality of
electrodes and the first layer extend from the first portion to the
second portion of the first substrate. In certain embodiments, the
array of wells is positioned in the second portion of the first
substrate.
[0012] In certain embodiments, the second substrate includes a
first portion and a second portion, wherein the first portion is in
facing arrangement with the first portion of the first substrate
and the second portion is in facing arrangement with the array of
wells.
[0013] In certain embodiments, the second portion of the second
substrate is substantially transparent to facilitate optical
interrogation of the array of wells. In certain embodiments, the
plurality of electrodes are configured to move a droplet placed in
the gap towards the second portion of the device, the device
includes a capillary portion fluidically connecting the first
portion to the second portion, wherein the capillary includes a
hydrophilic material to facilitate movement of the droplet from the
first portion to the second portion via the capillary portion in
absence of an electric force.
[0014] In some embodiments, the device further includes a second
layer is disposed on an upper surface of the first layer. In
certain embodiments, the second layer extends over the first
substrate. In certain embodiments, the first layer is a dielectric
layer and the second layer is a hydrophobic layer.
[0015] In some embodiments, the plurality of wells is positioned in
the second layer. In certain embodiments, the array of wells is
positioned in the first layer. In certain embodiments, the array of
wells has a hydrophilic surface. In certain embodiments, the wells
include a sidewall that is oriented to facilitate receiving and
retaining of nanobeads or nanoparticles present in droplets moved
over the well array. In certain embodiments, the wells include a
first sidewall opposite to a second side wall, wherein the first
sidewall is oriented at an obtuse angle with reference to a bottom
of the wells, and wherein the second sidewall is oriented at an
acute angle with reference to the bottom of the wells, wherein the
movement of droplets is in a direction parallel to the bottom of
the wells and from the first sidewall to the second sidewall. In
certain embodiments, the wells have a frustoconical shape with the
narrower part of the frustoconical shape providing the opening of
the wells. In certain embodiments, the wells include a first
sidewall opposite to a second side wall, wherein a top portion of
the first sidewall is oriented at an obtuse angle with reference to
a bottom of the wells and a bottom portion of the sidewall is
oriented perpendicular to the bottom of the wells, and wherein the
second sidewall is oriented perpendicular to the bottom of the
wells, wherein the movement of droplets is in a direction parallel
to the bottom of the wells and from the first sidewall to the
second sidewall, wherein the top portion of the first side wall is
at an opening of the wells.
[0016] Also disclosed herein is a surface acoustic wave
microfluidic and analyte detection device, including a first
substrate and a second substrate, wherein the second substrate is
separated from the first substrate by a gap, wherein the device
includes a first portion and a second portion, the first portion
including a superstrate coupled to a surface acoustic wave
generating component; and the second portion including a plurality
of wells positioned on the first substrate or the second
substrate.
[0017] In some embodiments, the superstrate includes phononic
structures on an upper surface of the superstrate. In certain
embodiments, the superstrate overlays a piezoelectric crystal
layer. In certain embodiments, the second substrate is
substantially transparent.
[0018] Also disclosed herein is a surface acoustic wave
microfluidic and analyte detection device, including a first
substrate and a second substrate, wherein the second substrate is
separated from the first substrate by a gap, the first substrate
including a plurality of wells, and the second substrate including
phononic structure, wherein the plurality of wells and the phononic
structures are located across to each other.
[0019] In some embodiments, the second substrate is a superstrate.
In certain embodiments, the superstrate is disposed on the second
substrate and the phononic structure are located on the
superstrate. In certain embodiments, the first substrate, second
substrate and superstrate are substantially transparent.
[0020] Also disclosed are methods of detecting or measuring an
analyte of interest in a liquid droplet. In certain embodiments,
the method involves the steps of providing a first liquid droplet
containing an analyte of interest, providing a second liquid
droplet containing at least one solid support which contains a
specific binding member that binds to the analyte of interest,
using energy to exert a force to manipulate the first liquid
droplet with the second liquid droplet to create a mixture, moving
all or at least a portion of the mixture to an array of wells,
wherein one or more wells of the array is of sufficient size to
accommodate the at least one solid support, adding a detectable
label to the mixture either before or after moving a portion of the
mixture to array of wells, and detecting the analyte of interest in
the wells.
[0021] In certain embodiments, the at least one solid support
include at least one binding member that specifically binds to the
analyte of interest. In certain embodiments, the method involves
adding a detectable label to the mixture before moving at least a
portion of the mixture to the array of wells. In certain
embodiments, the method involves adding a detectable label to the
mixture after moving at least a portion of the mixture to the array
of wells. In certain embodiments, the detectable label include at
least one binding member that specifically binds to the analyte of
interest. In certain embodiments, the detectable label includes a
chromagen, a fluorescent compound, an enzyme, a chemiluminescent
compound or a radioactive compound. In certain embodiments, the
binding member is a receptor or an antibody.
[0022] In certain embodiments, the energy used is an electric
actuation force or acoustic force. In certain embodiments, the
electric actuation force is droplet actuation, electrophoresis,
electrowetting, dielectrophoresis, electrostatic actuation,
electric field mediated, electrode mediated, capillary force,
chromatography, centrifugation, or aspiration. In certain
embodiments, the acoustic force is surface acoustic wave.
[0023] In certain embodiments, generating an electric actuation
force includes generating an alternating current. In certain
embodiments, the alternating current has a root mean squared (rms)
voltage of 10 V or more. In certain other embodiments, the
alternating current has a frequency in a radio frequency range.
[0024] In certain embodiments, the first liquid droplet is a
polarizable liquid, the second liquid droplet is a polarizable
liquid, the mixture is a polarizable liquid or both the first
liquid droplet and second liquid droplet are each polarizable
liquids.
[0025] In certain embodiments, the method further includes
positioning the at least a portion of the mixture over the array of
wells using an electric actuation force. In certain other
embodiments, the method further includes positioning the at least a
portion of the mixture over the array of wells using a capillary
element configured to facilitate movement of the mixture to the
array of wells.
[0026] In certain embodiments, the supports are magnetic solid
supports. In certain other embodiments, when magnetic solid
supports are used, an electric actuation force and a magnetic field
are applied from opposite directions relative to the at least a
portion of the mixture. In certain embodiments, the method further
includes mixing the mixture by moving the mixture back and forth,
moving the mixture in a circular pattern, splitting the mixture
into two or more submixtures and merging the submixtures. In
certain embodiments, the mixture is an aqueous liquid. In certain
other embodiments, the mixture is an immiscible liquid. In certain
other embodiments the liquid droplet is a hydrophobic liquid
droplet. In certain embodiments, the array of wells has a
hydrophilic surface. In certain other embodiments, the array of
wells has a hydrophobic surface. In certain embodiments, the
substrate includes a hydrophilic surface. In certain other
embodiments, the substrate includes a hydrophobic surface. In
certain embodiments, the method further includes generating an
electric actuation force with a series of electrodes to move the
mixture to the array of wells to seal the loaded wells.
[0027] In certain embodiments, one or more wells of the array are
loaded with at least one solid support. In certain other
embodiments, the loading includes applying a magnetic field to
facilitate movement of at least one solid support into the one or
more wells of the array. In certain other embodiments, the method
further includes removing any solid supports that are not loaded
into a well of the array after the loading. In certain other
embodiments, the removing includes generating an electric actuation
force with the series of electrodes to move a polarizable fluid
droplet to the array of wells to move the at least a portion of the
mixture to a distance from the array of wells. In certain other
embodiments, the removing includes generating an electric actuation
force with the series of electrodes to move an aqueous washing
droplet across the array of wells.
[0028] In certain embodiments, the method is performed using a
microfluidics device, digital microfluidics device (DMF), a surface
acoustic wave based microfluidic device (SAW), an integrated DMF
and analyte detection device, an integrated SAW and analyte
detection device, or robotics based assay processing unit.
[0029] In other embodiments, the method includes the steps of
providing a first liquid droplet containing an analyte of interest,
providing a second liquid droplet containing a detectable label
which contains a specific binding member that binds to the analyte
of interest, using energy to exert a force to manipulate the first
liquid droplet and the second liquid droplet to create a mixture,
moving all or at least a portion of the mixture to an array of
wells, and detecting the analyte of interest in the wells.
[0030] In certain embodiments, the detectable label includes a
chromagen, a fluorescent compound, an enzyme, a chemiluminescent
compound or a radioactive compound. In certain embodiments, the
binding member is a receptor or an antibody.
[0031] In certain embodiments, the energy used is an electric
actuation force or acoustic force. In certain embodiments, the
electric actuation force is droplet actuation, electrophoresis,
electrowetting, dielectrophoresis, electrostatic actuation,
electric field mediated, electrode mediated, capillary force,
chromatography, centrifugation, or aspiration. In certain
embodiments, the acoustic force is surface acoustic wave.
[0032] In certain embodiments, generating an electric actuation
force includes generating an alternating current. In certain
embodiments, the alternating current has a root mean squared (rms)
voltage of 10 V or more. In certain other embodiments, the
alternating current has a frequency in a radio frequency range.
[0033] In certain embodiments, the first liquid droplet is a
polarizable liquid, the second liquid droplet is a polarizable
liquid, the mixture is a polarizable liquid or both the first
liquid droplet and second liquid droplet are each polarizable
liquids.
[0034] In certain embodiments, the method further includes
positioning the at least a portion of the mixture over the array of
wells using an electric actuation force. In certain other
embodiments, the method further includes positioning the at least a
portion of the mixture over the array of wells using a capillary
element configured to facilitate movement of the mixture to the
array of wells.
[0035] In certain embodiments, the method further includes mixing
the mixture by moving the mixture back and forth, moving the
mixture in a circular pattern, splitting the mixture into two or
more submixtures and merging the submixtures. In certain
embodiments, the mixture is an aqueous liquid. In certain other
embodiments, the mixture is an immiscible liquid. In certain other
embodiments the liquid droplet is a hydrophobic liquid droplet. In
certain embodiments, the array of wells has a hydrophilic surface.
In certain other embodiments, the array of wells has a hydrophobic
surface. In certain embodiments, the substrate includes a
hydrophilic surface. In certain other embodiments, the substrate
includes a hydrophobic surface. In certain embodiments, the method
further includes generating an electric actuation force with a
series of electrodes to move the mixture to the array of wells to
seal the loaded wells.
[0036] In certain embodiments, one or more wells of the array are
loaded with at least one detectable label. In certain other
embodiments, the removing includes generating an electric actuation
force with the series of electrodes to move a polarizable fluid
droplet to the array of wells to move the at least a portion of the
mixture to a distance from the array of wells. In certain other
embodiments, the removing includes generating an electric actuation
force with the series of electrodes to move an aqueous washing
droplet across the array of wells.
[0037] In certain embodiments, the method is performed using a
microfluidics device, digital microfluidics device (DMF), a surface
acoustic wave based microfluidic device (SAW), an integrated DMF
and analyte detection device, an integrated SAW and analyte
detection device, or robotics based assay processing unit.
[0038] In other embodiments, the method includes the steps of
measuring an analyte of interest in a liquid droplet, the method
includes providing a first liquid droplet containing an analyte of
interest, providing a second liquid droplet containing at least one
solid support which contains a specific binding member that binds
to the analyte of interest, using energy to exert a force to
manipulate the first liquid droplet with the second liquid to
create a mixture, moving all or at least a portion of the mixture
to an array of wells, wherein one or more wells of the array is of
sufficient size to accommodate the at least one solid support,
adding a detectable label to the mixture either before or after
moving a portion of the mixture to array of wells, and measuring
the detectable label in the wells.
[0039] In certain embodiments, the at least one solid support
includes at least one binding member that specifically binds to the
analyte of interest. In certain embodiments, the method involves
adding a detectable label to the mixture before moving at least a
portion of the mixture to the array of wells. In certain
embodiments, the method involves adding a detectable label to the
mixture after moving at least a portion of the mixture to the array
of wells. In certain embodiments, the detectable label includes at
least one binding member that specifically binds to the analyte of
interest. In certain embodiments, the detectable label includes a
chromagen, a fluorescent compound, an enzyme, a chemiluminescent
compound or a radioactive compound. In certain embodiments, the
binding member is a receptor or an antibody.
[0040] In certain embodiments, the energy used is an electric
actuation force or acoustic force. In certain embodiments, the
electric actuation force is droplet actuation, electrophoresis,
electrowetting, dielectrophoresis, electrostatic actuation,
electric field mediated, electrode mediated, capillary force,
chromatography, centrifugation, or aspiration. In certain
embodiments, the acoustic force is surface acoustic wave.
[0041] In certain embodiments, generating an electric actuation
force includes generating an alternating current. In certain
embodiments, the alternating current has a root mean squared (rms)
voltage of 10 V or more. In certain other embodiments, the
alternating current has a frequency in a radio frequency range.
[0042] In certain embodiments, the first liquid droplet is a
polarizable liquid, the second liquid droplet is a polarizable
liquid, the mixture is a polarizable liquid or both the first
liquid droplet and second liquid droplet are each polarizable
liquids.
[0043] In certain embodiments, the method further includes
positioning the at least a portion of the mixture over the array of
wells using an electric actuation force. In certain other
embodiments, the method further includes positioning the at least a
portion of the mixture over the array of wells using a capillary
element configured to facilitate movement of the mixture to the
array of wells.
[0044] In certain embodiments, the supports are magnetic solid
supports. In certain other embodiments, when magnetic solid
supports are used, an electric actuation force and a magnetic field
are applied from opposite directions relative to the at least a
portion of the mixture.
[0045] In certain embodiments, the method further includes mixing
the mixture by moving the mixture back and forth, moving the
mixture in a circular pattern, splitting the mixture into two or
more submixtures and merging the submixtures.
[0046] In certain embodiments, the mixture is an aqueous liquid. In
certain other embodiments, the mixture is an immiscible liquid. In
certain other embodiments the liquid droplet is a hydrophobic
liquid droplet. In certain embodiments, the array of wells has a
hydrophilic surface. In certain other embodiments, the array of
wells has a hydrophobic surface. In certain embodiments, the
substrate includes a hydrophilic surface. In certain other
embodiments, the substrate includes a hydrophobic surface. In
certain embodiments, the method further includes generating an
electric actuation force with a series of electrodes to move the
mixture to the array of wells to seal the loaded wells.
[0047] In certain embodiments, one or more wells of the array are
loaded with at least one solid support. In certain other
embodiments, the loading includes applying a magnetic field to
facilitate movement of at least one solid support into the one or
more wells of the array. In certain other embodiments, the method
further includes removing any solid supports that are not loaded
into a well of the array after the loading. In certain other
embodiments, the removing includes generating an electric actuation
force with the series of electrodes to move a polarizable fluid
droplet to the array of wells to move the at least a portion of the
mixture to a distance from the array of wells. In certain other
embodiments, the removing includes generating an electric actuation
force with the series of electrodes to move an aqueous washing
droplet across the array of wells.
[0048] In certain embodiments, the method is performed using a
microfluidics device, digital microfluidics device (DMF), a surface
acoustic wave based microfluidic device (SAW), an integrated DMF
and analyte detection device, an integrated SAW and analyte
detection device, or robotics based assay processing unit.
[0049] In certain embodiments, the measuring involves determining
the total number of solid supports in the wells of an array. In
certain embodiments, the measuring involves determining the number
of solid supports in the wells of the array that contain the
detectable label. In certain embodiments, the measuring involves
subtracting the number of solid supports that contain a detectable
label from the total number of solid supports in the wells of the
array to determine the number of solid supports in the wells of the
array that do not contain any detectable label. In certain
embodiments, the measuring involves determining the ratio of solid
supports that contain a detectable label to the number of solid
supports that do not contain any detectable label.
[0050] Also disclosed herein is a method of loading wells with
particles, including generating an electric field with a plurality
of electrodes to move a liquid droplet containing microparticles to
an array of wells, wherein one or more wells of the array of wells
is of sufficient size to have loaded therein a particle; loading
one or more wells with a particle; and generating an electric field
with the plurality of electrodes to move a polarizable fluid
droplet to the array of wells to seal the array of wells.
[0051] In some embodiments, the method further includes positioning
the liquid droplet over the array of wells using the electric
field. In some embodiments, the method further includes positioning
the liquid droplet over the array of wells using a capillary
element configured to facilitate movement of the liquid droplet to
the array of wells. In some embodiments, the particle is a magnetic
bead. In some embodiments, the loading includes applying a magnetic
field to facilitate movement of the one or more magnetic beads into
the one or more wells of the array. In some embodiments, the array
of wells has a hydrophilic surface. In some embodiments, the array
of wells has a hydrophobic surface. In some embodiments, the
generating an electric field includes generating an alternating
current. In certain embodiments, the alternating current has a root
mean squared (rms) voltage of 10 V or more. In certain embodiments,
the alternating current has a frequency in a radio frequency
range.
[0052] Also disclosed herein is a method of forming a digital
microfluidic and analyte detection device, including unwinding a
first roll including a first substrate to position a first portion
of the first substrate at a first position; forming a plurality of
electrodes on the first portion of the first substrate at the first
position; and forming an array of wells on a second portion of the
first substrate at a second position.
[0053] In some embodiments, the method further includes unwinding
the first roll to position the second portion adjacent the first
portion of the first substrate at the second position prior to
forming the array of wells. In some embodiments, the method further
includes unwinding a second roll including a second substrate to
position a third portion of the third substrate at a third
position; and bonding the second substrate with the first substrate
at the third position in a manner sufficient to position the second
substrate spaced apart from the first substrate.
[0054] Also disclosed herein is a method of forming an integrated
digital microfluidic and analyte detection device, including
unwinding a first roll including a first substrate to position a
first portion of the first substrate at a first position; forming a
plurality of electrodes on the first portion of the first substrate
at the first position; unwinding a second roll including a second
substrate to position a second portion of the second substrate at a
second position; forming an array of wells on the second portion at
the second position; and bonding the second substrate with the
first substrate in a manner sufficient to position the second
substrate spaced apart from the first substrate; and position the
second portion above the first portion, or above a third portion
adjacent the first portion of the first substrate, wherein the
array of wells faces the first substrate.
[0055] In some embodiments, the forming the array of wells includes
using thermal or ultraviolet nanoimprint lithography, nanoimprint
roller, laser ablation, or by bonding a prefabricated substrate
including an array of wells onto the first portion of the first
substrate. In some embodiments, the method further includes
subjecting the first substrate to intense heat, pressure, or
ultraviolet light to form phononic structures on or within the
first substrate using a mold.
[0056] In some embodiments, the method further includes applying a
hydrophobic and/or a dielectric material on electrodes of the
series using a printer device. In some embodiments, the hydrophobic
and/or dielectric material includes a curing material. In some
embodiments, the method further includes applying heat or
ultraviolet light to cure the applied hydrophobic and/or dielectric
material. In some embodiments, the method further includes dicing
the first and second substrates to generate a bonded substrates
includes the first and second portions.
[0057] Also disclosed herein is a method of detecting an analyte of
interest in a liquid droplet, including, providing a first liquid
droplet including an analyte of interest; providing a second liquid
droplet including a specific binding member and a labeled analyte,
wherein the binding member is immobilized on at least one solid
support, the specific binding member specifically binds to the
analyte of interest, and the labeled analyte is an analyte of
interest labeled with a detectable label; using energy to exert a
force to manipulate the first liquid droplet with the second liquid
droplet to create a mixture; and moving all or at least a portion
of the mixture to an array of wells, wherein one or more wells of
the array is of sufficient size to accommodate the at least one
solid support.
[0058] Also disclosed herein is a method of detecting an analyte of
interest in a liquid droplet, including providing a first liquid
droplet including an analyte of interest; providing a second liquid
droplet including an immobilized analyte and at least one specific
binding member, wherein the immobilized analyte is an analyte of
interest immobilized on at least one solid support, the at least
one specific binding member specifically binds to the analyte of
interest, and the at least one specific binding member is labeled
with a detectable label; using energy to exert a force to
manipulate the first liquid droplet with the second liquid droplet
to create a mixture; moving all or at least a portion of the
mixture to an array of wells, wherein one or more wells of the
array is of sufficient size to accommodate the at least one solid
support; and detecting the analyte of interest in the wells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] FIG. 1A illustrates a side view of an integrated digital
microfluidic and analyte detection device according to one
embodiment.
[0060] FIG. 1B illustrates a side view of the integrated digital
microfluidic and analyte detection device according to another
embodiment.
[0061] FIG. 2A illustrates a side view of an integrated digital
microfluidic and analyte detection device according to an
embodiment.
[0062] FIG. 2B illustrates a side view of the integrated digital
microfluidic and analyte detection device according to another
embodiment.
[0063] FIG. 3A illustrates a side view of the device of FIG. 2A
with a liquid droplet being moved in the device.
[0064] FIG. 3B illustrate a side view of the device of FIG. 2B with
of droplet being moved in the device.
[0065] FIG. 4A illustrates a side view of the device of FIG. 2A
with a droplet containing particles/beads being moved onto an array
of wells.
[0066] FIG. 4B illustrates a side view of the device of FIG. 2B
with a droplet containing particles/beads being moved onto an array
of wells with a droplet of an immiscible fluid.
[0067] FIG. 5 illustrates an aqueous droplet being moved over the
array of wells using a hydrophilic capillary region of the
device.
[0068] FIG. 6 illustrates an aqueous droplet being moved over the
array of wells.
[0069] FIGS. 7A and 7B illustrate various exemplary orientations of
the sidewalls of the wells.
[0070] FIG. 8 illustrates an example of fabricating a second (e.g.,
bottom) substrate of the digital microfluidic and analyte detection
device.
[0071] FIG. 9 illustrates an example of fabricating a first (e.g.,
top) substrate of the digital microfluidic and analyte detection
device.
[0072] FIG. 10 illustrates an example of assembling the top and
bottom substrates to manufacture a plurality of digital
microfluidic and analyte detection devices.
[0073] FIGS. 11A and 11B show a view from the top of a bottom
substrate of exemplary digital microfluidic and analyte detection
devices of the present disclosure.
[0074] FIGS. 12A-12D illustrate examples of fabricating the array
of wells into the integrated digital microfluidic and analyte
detection device.
[0075] FIG. 13A illustrates a side view of one embodiment of the
surface acoustic component of the integrated microfluidic and
analyte device and array of wells.
[0076] FIG. 13B illustrates a side view of another embodiment of
the surface acoustic component of the integrated microfluidic and
analyte device and array of wells.
[0077] FIGS. 14A and 14B illustrate an example of fabricating the
sample preparation component and well array component.
[0078] FIG. 15 depicts an exemplary method of the present
disclosure.
[0079] FIG. 16 illustrates an exemplary method for removing beads
not located in the wells of the depicted device.
[0080] FIG. 17 illustrates another exemplary method for removing
beads not located in the wells of the depicted device.
[0081] FIG. 18 depicts a schematic of a fabrication process of a
low-cost DMF chip.
[0082] FIG. 19 depicts a single flexible chip fabricated according
to the schematic in FIG. 18.
[0083] FIG. 20 depicts actuation of droplets in a DMF chip,
according to embodiments of the present disclosure.
[0084] FIGS. 21, A-E depicts performance of an immunoassay in a DMF
chip, according to embodiments of the present disclosure.
[0085] FIGS. 22A and 22B are schematic diagrams showing a design
and fabrication method of DMF top electrode chips and well array,
according to embodiments of the present disclosure.
[0086] FIG. 23 shows a schematic diagram of a well design,
according to embodiments of the present disclosure.
[0087] FIGS. 24A and 24B are schematic diagram showing well spacing
formats, according to embodiments of the present disclosure.
[0088] FIG. 25 are a collection of magnified optical images of the
array of wells, according to embodiments of the present
disclosure.
[0089] FIG. 26 is a schematic diagram showing assembly of an
integrated DMF-well device from a DMF top electrode chip and a well
array, according to embodiments of the present disclosure.
[0090] FIGS. 27A-27G are a collection of schematic diagrams showing
an immunoassay performed on a integrated DMF-well device, according
to embodiments of the present disclosure.
[0091] FIG. 28 is a schematic diagram of an enzyme-linked
immunosorbent assay (ELISA)-based sandwich immunoassay, coupled
with digital fluorescence detection in a well array, according to
embodiments of the present disclosure.
[0092] FIG. 29 is a schematic showing components for DMF-directed
top loading of microparticles into a well array, according to
embodiments of the present disclosure.
[0093] FIG. 30, A-D are a collection of schematic diagrams showing
steps of a thyroid stimulating hormone (TSH) immunoassay using an
integrated DMF-well device, according to embodiments of the present
disclosure.
[0094] FIG. 31, A-F provides a schematic of an analyte detection
chip according to one embodiment.
[0095] FIG. 32, A-C provides a schematic of an analyte detection
chip according to another embodiment.
[0096] FIG. 33 provides a schematic of an analyte detection chip
according to one embodiment.
[0097] FIGS. 34A and 34B illustrates side views of an exemplary
analyte detection chip.
[0098] FIG. 35 illustrates a schematic of a top view of an analyte
detection chip according to another embodiment.
[0099] FIG. 36 illustrates a schematic of an alternate exemplary
analyte detection chip.
[0100] FIG. 37 provides a schematic of an exemplary hematology
chip.
[0101] FIGS. 38 and 39 illustrates alternate embodiments of DMF
chip with multiple detection regions.
[0102] FIGS. 40, A and B illustrate a schematic of exemplary
analyte detection devices. C is a schematic of a cartridge
compatible with the analyte detection devices in A and B. FIGS. 40D
and 40E illustrate cartridge adapters that allow insertion of
different types of cartridges into the same slot.
[0103] FIGS. 41, A and B depict embodiments of a cartridge (FIG.
41A) and an analyte detection device (FIG. 41B) that is compatible
with the cartridge.
[0104] FIGS. 42A-42E illustrate cartridges comprising DMF
electrodes and optical detection chamber.
[0105] FIGS. 43A and 43B illustrate exemplary analyte detection
systems with a plurality of instruments for conducting a plurality
of assays.
DETAILED DESCRIPTION OF THE INVENTION
[0106] An integrated microfluidic and analyte detection device is
disclosed. Also provided herein are exemplary methods for using an
integrated microfluidic and analyte detection device and associated
systems.
[0107] Before the present invention is described in greater detail,
it is to be understood that this invention is not limited to a
particular embodiment described, as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting, since the scope of the present invention
will be limited only by the appended claims.
[0108] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an" and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, refer to "an electrode" includes plurality of such
electrodes and reference to "the well" includes reference to one or
more wells and equivalents thereof known to those skilled in the
art, and so forth.
[0109] All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited. The present
disclosure is controlling to the extent there is a contradiction
between the present disclosure and a publication incorporated by
reference.
DETAILED DESCRIPTION
[0110] Embodiments of the present disclosure relate to methods,
systems, and devices for analysis of analyte(s) in a sample. In
certain embodiments, the sample may be a biological sample.
Definitions
[0111] Before the embodiments of the present disclosure are
described, it is to be understood that this invention is not
limited to particular embodiments described, as such may, of
course, vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting.
[0112] "Comprise(s)," "include(s)," "having," "has," "can,"
"contain(s)," and variants thereof, as used herein, are intended to
be open-ended transitional phrases, terms, or words that do not
preclude the possibility of additional acts or structures. The
singular forms "a," "and" and "the" include plural references
unless the context clearly dictates otherwise. The present
disclosure also contemplates other embodiments "comprising,"
"consisting of" and "consisting essentially of," the embodiments or
elements presented herein, whether explicitly set forth or not.
[0113] For the recitation of numeric ranges herein, each
intervening number there between with the same degree of precision
is explicitly contemplated. For example, for the range of 6-9, the
numbers 7 and 8 are contemplated in addition to 6 and 9, and for
the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,
6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
[0114] "Affinity" and "binding affinity" as used interchangeably
herein refer to the tendency or strength of binding of the binding
member to the analyte. For example, the binding affinity may be
represented by the equilibrium dissociation constant (K.sub.D), the
dissociation rate (k.sub.d), or the association rate (k.sub.a).
[0115] "Analog" as used herein refers to a molecule that has a
similar structure to a molecule of interest (e.g., nucleoside
analog, nucleotide analog, sugar phosphate analog, analyte analog,
etc.). An analyte analog is a molecule that is structurally similar
to an analyte but for which the binding member has a different
affinity.
[0116] The term "aptamer" as used herein refers to an
oligonucleotide or peptide molecule that can bind to pre-selected
targets including small molecules, proteins, and peptides among
others with high affinity and specificity. Aptamers may assume a
variety of shapes due to their propensity to form helices and
single-stranded loops. An oligonucleotide or nucleic acid aptamer
can be a single-stranded DNA or RNA (ssDNA or ssRNA) molecule. A
peptide aptamer can include a short variable peptide domain,
attached at both ends to a protein scaffold.
[0117] "Bead" and "particle" are used herein interchangeably and
refer to a substantially spherical solid support.
[0118] "Component," "components," or "at least one component,"
refer generally to a capture antibody, a detection reagent or
conjugate, a calibrator, a control, a sensitivity panel, a
container, a buffer, a diluent, a salt, an enzyme, a co-factor for
an enzyme, a detection reagent, a pretreatment reagent/solution, a
substrate (e.g., as a solution), a stop solution, and the like that
can be included in a kit for assay of a test sample, such as a
patient urine, serum, whole blood, tissue aspirate, or plasma
sample, in accordance with the methods described herein and other
methods known in the art. Some components can be in solution or
lyophilized for reconstitution for use in an assay.
[0119] "Digital microfluidics (DMF)," "digital microfluidic module
(DMF module)," or "digital microfluidic device (DMF device)" as
used interchangeably herein refer to a module or device that
utilizes digital or droplet-based microfluidic techniques to
provide for manipulation of discrete and small volumes of liquids
in the form of droplets. Digital microfluidics uses the principles
of emulsion science to create fluid-fluid dispersion into channels
(principally water-in-oil emulsion). It allows the production of
monodisperse drops/bubbles or with a very low polydispersity.
Digital microfluidics is based upon the micromanipulation of
discontinuous fluid droplets within a reconfigurable network.
Complex instructions can be programmed by combining the basic
operations of droplet formation, translocation, splitting, and
merging.
[0120] Digital microfluidics operates on discrete volumes of fluids
that can be manipulated by binary electrical signals. By using
discrete unit-volume droplets, a microfluidic operation may be
defined as a set of repeated basic operations, i.e., moving one
unit of fluid over one unit of distance. Droplets may be formed
using surface tension properties of the liquid. Actuation of a
droplet is based on the presence of electrostatic forces generated
by electrodes placed beneath the bottom surface on which the
droplet is located. Different types of electrostatic forces can be
used to control the shape and motion of the droplets. One technique
that can be used to create the foregoing electrostatic forces is
based on dielectrophoresis which relies on the difference of
electrical permittivities between the droplet and surrounding
medium and may utilize high-frequency AC electric fields. Another
technique that can be used to create the foregoing electrostatic
forces is based on electrowetting which relies on the dependence of
surface tension between a liquid droplet present on a surface and
the surface on the electric field applied to the surface.
[0121] "Drag-tag" refers to a mobility modifier. The drag-tag may
be genetically engineered, highly repetitive polypeptides ("protein
polymers") that are designed to be large, water-soluble, and
completely monodisperse. Positively charged arginines may be
deliberately introduced at regular intervals into the amino acid
sequence to increase the hydrodynamic drag without increasing
drag-tag length. Drag-tags are described in U.S. Patent Publication
No. 20120141997, which is incorporated herein by reference.
[0122] "Enzymatic cleavable sequence" as used herein refers to any
nucleic acid sequence that can be cleaved by an enzyme. For
example, the enzyme may be a protease or an endonuclease, such as a
restriction endonuclease (also called restriction enzymes).
Restriction endonucleases are capable of recognizing and cleaving a
DNA molecule at a specific DNA cleavage site between predefined
nucleotides. Some endonucleases, such as for example Fokl, comprise
a cleavage domain that cleaves the DNA unspecifically at a certain
position regardless of the nucleotides present at this position. In
some embodiments, the specific DNA cleavage site and the DNA
recognition site of the restriction endonuclease are identical.
[0123] "Globular protein" refers to a water soluble protein that
has a roughly spherical shape. Examples of globular proteins
include but are not limited to ovalbumin, beta-globulin, C-reactive
protein, fibrin, hemoglobin, IgG, IgM, and thrombin.
[0124] "Label" or "detectable label" as used interchangeably herein
refers to a moiety attached to a specific binding member or analyte
to render the reaction between the specific binding member and the
analyte detectable, and the specific binding member or analyte so
labeled is referred to as "detectably labeled." A label can produce
a signal that is detectable by visual or instrumental means.
Various labels include: (i) a tag attached to a specific binding
member or analyte by a cleavable linker; or (ii) signal-producing
substance, such as chromagens, fluorescent compounds, enzymes,
chemiluminescent compounds, radioactive compounds, and the like.
Representative examples of labels include moieties that produce
light, e.g., acridinium compounds, and moieties that produce
fluorescence, e.g., fluorescein. Other labels are described herein.
In this regard, the moiety, itself, may not be detectable but may
become detectable upon reaction with yet another moiety. Use of the
term "detectably labeled" is intended to encompass such
labeling.
[0125] "Microparticle(s)(s)" and "microbead(s)" are used
interchangeably herein and refer to a microbead or microparticle
that is allowed to occupy or settle in an array of wells, such as,
for example, in an array of wells in a detection module. The
microparticle and microbead may contain at least one specific
binding member that binds to an analyte of interest and at least
one detectable label. Alternatively, the microparticle and
microbead may containing a first specific binding member that binds
to the analyte and a second specific binding member that also binds
to the analyte and contains at least one detectable label.
[0126] "Nucleobase" or "base" means those naturally occurring and
synthetic heterocyclic moieties commonly known in the art of
nucleic acid or polynucleotide technology or peptide nucleic acid
technology for generating polymers. Non-limiting examples of
suitable nucleobases include: adenine, cytosine, guanine, thymine,
uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil,
5-methylcytosine, pseudoisocytosine, 2-thiouracil and
2-thiothymine, 2-aminopurine, N9-(2-amino-6-chloropurine),
N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine),
N9-(7-deaza-8-aza-guanine) and N8-(7-deaza-8-aza-adenine).
Nucleobases can be linked to other moieties to form nucleosides,
nucleotides, and nucleoside/tide analogs.
[0127] "Nucleoside" refers to a compound consisting of a purine,
deazapurine, or pyrimidine nucleobase, e.g., adenine, guanine,
cytosine, uracil, thymine, 7-deazaadenine, 7-deazaguanosine, that
is linked to the anomeric carbon of a pentose sugar at the 1'
position, such as a ribose, 2'-deoxyribose, or a
2',3'-di-deoxyribose.
[0128] "Nucleotide` as used herein refers to a phosphate ester of a
nucleoside, e.g., a mono-, a di-, or a triphosphate ester, wherein
the most common site of esterification is the hydroxyl group
attached to the C-5 position of the pentose.
[0129] "Nucleobase polymer" or "nucleobase oligomer" refers to two
or more nucleobases that are connected by linkages to form an
oligomer. Nucleobase polymers or oligomers include, but are not
limited to, poly- and oligonucleotides (e.g., DNA and RNA polymers
and oligomers), poly-and oligo-nucleotide analogs and poly- and
oligonucleotide mimics, such as polyamide or peptide nucleic acids.
Nucleobase polymers or oligomers can vary in size from a few
nucleobases to several hundred nucleobases or to several thousand
nucleobases. The nucleobase polymers or oligomers may include from
about 2 to 100 nucleobases or from about 8000 to 10000 nucleobases.
For example, the nucleobase polymers or oligomers may have at least
about 2 nucleobases, at least about 5 nucleobases, at least about
10 nucleobases, at least about 20 nucleobases, at least about 30
nucleobases, at least about 40 nucleobases, at least about 50
nucleobases, at least about 60 nucleobases, at least about 70
nucleobases, at least about 80 nucleobases, at least about 90
nucleobases, at least about 100 nucleobases, at least about 200
nucleobases, at least about 300 nucleobases, at least about 400
nucleobases, at least about 500 nucleobases, at least about 600
nucleobases, at least about 700 nucleobases, at least about 800
nucleobases, at least about 900 nucleobases, at least about 1000
nucleobases, at least about 2000 nucleobases, at least about 3000
nucleobases, at least about 4000 nucleobases, at least about 5000
nucleobases, at least about 6000 nucleobases, at least about 7000
nucleobases, at least about 8000 nucleobases, at least about 9000
nucleobases, or at least about 10000 nucleobases.
[0130] "Polymer brush" refers to a layer of polymers attached with
one end to a surface. The polymers are close together and form a
layer or coating that forms its own environment. The brushes may be
either in a solvent state, when the dangling chains are submerged
into a solvent, or in a melt state, when the dangling chains
completely fill up the space available. Additionally, there is a
separate class of polyelectrolyte brushes, when the polymer chains
themselves carry an electrostatic charge. The brushes may be
characterized by the high density of grafted chains. The limited
space then leads to a strong extension of the chains, and unusual
properties of the system. Brushes may be used to stabilize
colloids, reduce friction between surfaces, and to provide
lubrication in artificial joints
[0131] "Polynucleotides" or "oligonucleotides" refer to nucleobase
polymers or oligomers in which the nucleobases are connected by
sugar phosphate linkages (sugar-phosphate backbone). Exemplary
poly- and oligonucleotides include polymers of
2'-deoxyribonucleotides (DNA) and polymers of ribonucleotides
(RNA). A polynucleotide may be composed entirely of
ribonucleotides, entirely of 2'-deoxyribonucleotides or
combinations thereof. The term nucleic acid encompasses the terms
polynucleotide and oligonucleotides and includes single stranded
and double stranded polymers of nucleotide monomers.
[0132] "Polynucleotide analog" or "oligonucleotide analog" refers
to nucleobase polymers or oligomers in which the nucleobases are
connected by a sugar phosphate backbone comprising one or more
sugar phosphate analogs. Typical sugar phosphate analogs include,
but are not limited to, sugar alkylphosphonates, sugar
phosphoramidites, sugar alkyl- or substituted
alkylphosphotriesters, sugar phosphorothioates, sugar
phosphorodithioates, sugar phosphates and sugar phosphate analogs
in which the sugar is other than 2'-deoxyribose or ribose,
nucleobase polymers having positively charged sugar-guanidyl
interlinkages such as those described in U.S. Pat. Nos. 6,013,785
and 5,696,253.
[0133] "Receptor" as used herein refers to a protein-molecule that
recognizes and responds to endogenous-chemical signals. When such
endogenous-chemical signals bind to a receptor, they cause some
form of cellular/tissue-response. Examples of receptors include,
but not limited to, neural receptors, hormonal receptors, nutrient
receptors, and cell surface receptors.
[0134] As used herein, "spacer" refers to a chemical moiety that
extends the cleavable group from the specific binding member, or
which provides linkage between the binding member and the support,
or which extends the label/tag from the photocleavable moiety. In
some embodiments, one or more spacers may be included at the
N-terminus or C-terminus of a polypeptide or nucleotide-based tag
or label in order to distance optimally the sequences from the
specific binding member. Spacers may include but are not limited to
6-aminocaproic acid, 6-aminohexanoic acid; 1,3-diamino propane;
1,3-diamino ethane; polyethylene glycol (PEG) polymer groups and
short amino acid sequences, such as polyglycine sequences, of 1 to
5 amino acids.
[0135] "Specific binding partner" or "specific binding member" as
used interchangeably herein refer to one of two different molecules
that specifically recognizes the other molecule compared to
substantially less recognition of other molecules. The one of two
different molecules has an area on the surface or in a cavity,
which specifically binds to and is thereby defined as complementary
with a particular spatial and polar organization of the other
molecule. The molecules may be members of a specific binding pair.
For example, a specific binding member may include, but not limited
to, a protein, such as a receptor, an enzyme, an antibody and an
aptamer, a peptide a nucleotide, oligonucleotide, a polynucleotide
and combinations thereof.
[0136] As used herein, "tag" or "tag molecule" both refer to the
molecule (e.g., cleaved from the second binding member dissociated
from the target analyte) that is used to provide an indication of
the level of analyte in a sample. These terms refer to a single tag
molecule or a plurality of the same tag molecule. Likewise "tags",
unless specified otherwise, refers to one or one or more tags.
[0137] "Tracer" as used herein refers to an analyte or analyte
fragment conjugated to a tag or label, wherein the analyte
conjugated to the tag or label can effectively compete with the
analyte for sites on an antibody specific for the analyte. For
example, the tracer may be an analyte or analog of the analyte,
such as cyclosporine or its analog ISA247, vitamin D and its
analogs, sex hormones and their analogs, etc.
[0138] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. In case of conflict, the present
document, including definitions, will control. Preferred methods
and materials are described below, although methods and materials
similar or equivalent to those described herein can be used in
practice or testing of the present invention. All publications,
patent applications, patents and other references mentioned herein
are incorporated by reference in their entirety to disclose and
describe the methods and/or materials in connection with which the
publications are cited. The materials, methods, and examples
disclosed herein are illustrative only and not intended to be
limiting.
Methods for Analyte Analysis
[0139] Provided herein are methods for analyte analysis. The method
may involve single molecule counting. In certain embodiments, a
method for analyte analysis may involve assessing an analyte
present in a sample. In certain embodiments, the assessing may be
used for determining presence of and/or concentration of an analyte
in a sample. In certain embodiments, the method may also be used
for determining presence of and/or concentration of a plurality of
different analytes present in a sample.
[0140] Provided herein are methods for detecting an analyte of
interest in liquid droplet (wherein the analyte of interest is from
a test or biological sample). The method includes providing a first
liquid droplet containing an analyte of interest, providing a
second liquid droplet containing at least one solid support (such
as, for example, a magnetic solid support (such as a bead)) which
contains a specific binding member that binds to the analyte of
interest, using energy to exert a force to manipulate the first
liquid droplet (which contains the analyte of interest) with the
second liquid (containing the at least one solid support) to create
a mixture, moving all or at least a portion of the mixture to an
array of wells (where one or more wells of the array are of
sufficient size to accommodate the at least one solid support),
adding at least one detectable label to the mixture before, after
or both before or after moving a portion of the mixture to the
array of wells and detecting the analyte of interest in the wells.
In certain embodiments, "using energy to exert a force to
manipulate the first liquid droplet with the second liquid droplet"
refers to the use of non-mechanical forces (namely, for example,
energy created without the use of pumps and/or valves) to provide
or exert a force that manipulates (such as merges or combines) at
least the first and second liquid droplets (and optionally,
additional droplets) into a mixture. Example of non-mechanical
forces that can be used in the methods described herein include
electric actuation force (such as droplet actuation,
electrophoresis, electrowetting, dielectrophoresis, electrostatic
actuation, electric field mediated, electrode mediated, capillary
force, chromatography, centrifugation or aspiration) and/or
acoustic force (such as surface acoustic wave (or "SAW"). In
certain embodiments, the the electric actuation force generated is
an alternating current. For example, the alternating current can
have a root mean squared (rms) voltage of 10 V, 15 V, 20 V, 25 V,
30 V, 35V or more. For example, such alternating current can have a
rms voltage of 10 V or more, 15 V or more, 20 V or more, 25 V or
more, 30 V or more or 35 V or more. Alternatively, the alternating
current can have a frequency in a radio frequency range.
[0141] In certain embodiments, if magnetic solid supports are used,
an electric actuation force and a magnetic field can be applied and
applied from opposition directions, relative to the at least a
portion of the mixture. In certain other embodiments, the mixture
is mixed by moving it: back and forth, in a circular pattern or by
splitting it into two or more submixtures and then merging the
submixtures. In certain other embodiments, an electric actuation
force can be generated using a series or plurality of electrodes
(namely, at least two or more, at least three or more, at least
four or more, at least five or more, at least six or more, at least
seven or more, at least eight or more, at least nine or more, at
least ten or more, at least eleven or more, at least twelve or
more, at least thirteen or more, at least fourteen or more, at
least fifteen or more, etc.) to move the mixture to the array of
wells in order to seal the wells (which are loaded with at least
one solid support).
[0142] In certain embodiments, the moving of all or at least a
portion of the mixture to an array of wells results in the loading
(filling and/or placement) of the at least one solid support into
the array of wells. In certain embodiments, a magnetic field is
used to facilitate movement of the mixture and thus, at least one
solid support, into one or more wells of the array. In certain
embodiments, after the at least one solid supports are loaded into
the wells, any solid supports that are not loaded into a well can
be removed using routine techniques known in the art. For example,
such removing can involve generating an electric actuation force
(such as that described previously herein) with a series or
plurality of electrodes to move a fluid droplet (such as a
polarizable fluid droplet) to the array of wells to move at least a
portion of the mixture to a distance (the length of which is not
critical) from the array of wells. In certain embodiments, an
aqueous washing liquid can be used to remove the solid supports not
bound to any analyte of interest. In such embodiments, the removal
involves generating an electric actuation force with a series or
plurality of electrodes to move an aqueous wash (or washing)
droplet (a third droplet) across the array of wells. The amount and
type of aqueous liquid used for said washing is not critical.
[0143] In certain embodiments, the mixture in the method is an
aqueous liquid. In other embodiments, the mixture is an immiscible
liquid. In other embodiments, the liquid droplet is a hydrophobic
liquid droplet. In other embodiments, the liquid droplet is a
hydrophilic liquid droplet. In certain embodiments, the array of
wells used in the method have a hydrophobic surface. In other
embodiments, the array of wells has a hydrophilic surface.
[0144] In certain embodiments, the first liquid droplet used in the
method is a polarizable liquid. In certain embodiments, the second
liquid droplet used in the method is a polarizable liquid. In
certain embodiments, the first and second liquid droplets used in
the method are polarizable liquids. In certain embodiments, the
mixture is a polarizable liquid. In certain embodiments one or more
of the first droplet, second droplet and mixture is a polarizable
liquid.
[0145] In certain embodiments, the at least one solid support
comprises at least one binding member that specifically binds to
the analyte of interest. In certain embodiments, the detectable
label is added to the mixture before moving at least a portion of
the mixture to the array of wells. In certain other embodiments,
the detectable label is added to the mixture after the moving of at
least a portion of the analyte of interest. In certain embodiments,
the detectable label comprises at least one binding member that
specifically binds to the analyte of interest. In certain
embodiments, the detectable label comprises a chromagen, a
florescent compound, an enzyme, a chemiluminescent compound or a
radioactive compound. In certain embodiments, the binding member is
a receptor, aptamer or antibody. In certain embodiments, the method
further comprises positioning the at least a portion of the mixture
over the array of wells using a capillary element configured to
facilitate movement of the mixture to the array of wells.
[0146] In certain embodiments, the method described herein is
performed using a microfluidics device. In certain embodiments, the
method described herein is performed using a digital microfluidics
device (DMF). In certain embodiments, method described herein is
performed using a surface acoustic wave based microfluidics device
(SAW). In certain embodiments, method described herein is performed
using an integrated DMF and analyte detection device. In certain
embodiments, method described herein is performed using an
integrated surface acoustic wave based microfluidic device and
analyte detection device. In certain embodiments, method described
herein is performed using a Robotics based assay processing
unit.
[0147] Provided herein are methods for detecting an analyte of
interest in liquid droplet (wherein the analyte of interest is from
a test or biological sample). The method includes providing a first
liquid droplet containing an analyte of interest, providing a
second liquid droplet containing at least one detectable label
which contains a specific binding member that binds to the analyte
of interest, using energy to exert a force to manipulate the first
liquid droplet (which contains the analyte of interest) with the
second liquid (containing the at least one solid support) to create
a mixture (namely, an analyte/detectable label-specific binding
member complex), moving all or at least a portion of the mixture to
an array of wells (where one or more wells of the array are of
sufficient size to accommodate the at least one solid support) and
detecting the analyte of interest in the wells. In certain
embodiments, "using energy to exert a force to manipulate the first
liquid droplet with the second liquid droplet" refers to the use of
non-mechanical forces (namely, for example, energy created without
the use of pumps and/or valves) to provide or exert a force that
manipulates (such as merges or combines) at least the first and
second liquid droplets (and optionally, additional droplets) into a
mixture. Example of non-mechanical forces that can be used in the
methods described herein include electric actuation force (such as
droplet actuation, electrophoresis, electrowetting,
dielectrophoresis, electrostatic actuation, electric field
mediated, electrode mediated, capillary force, chromatography,
centrifugation or aspiration) and/or acoustic force (such as
surface acoustic wave (or "SAW"). In certain embodiments, the the
electric actuation force generated is an alternating current. For
example, the alternating current can have a root mean squred (rms)
voltage of 10 V, 15 V, 20 V, 25 V, 30 V, 35V or more. For example,
such alternating current can have a rms voltage of 10 V or more, 15
V or more, 20 V or more, 25 V or more, 30 V or more or 35 V or
more. Alternatively, the alternating current can have a frequency
in a radio frequency range.
[0148] In certain embodiments, the mixture is mixed by moving it:
back and forth, in a circular pattern or by splitting it into two
or more submixtures and then merging the submixtures. In certain
other embodiments, an electric actuation force can be generated
using a series or plurality of electrodes (namely, at least two or
more, at least three or more, at least four or more, at least five
or more, at least six or more, at least seven or more, at least
eight or more, at least nine or more, at least ten or more, at
least eleven or more, at least twelve or more, at least thirteen or
more, at least fourteen or more, at least fifteen or more, etc.) to
move the mixture to the array of wells in order to seal the wells
(which are loaded with at least one solid support).
[0149] In certain embodiments, the moving of all or at least a
portion of the mixture to an array of wells results in the loading
(filling and/or placement) of the an analyte/detectable
label-specific binding member complex into the array of wells. In
certain embodiments, a magnetic field is used to facilitate
movement of the mixture and thus, at least one an
analyte/detectable label-specific binding member complex into one
or more wells of the array. For example, such removing can involve
generating an electric actuation force (such as that described
previously herein) with a series or plurality of electrodes to move
a fluid droplet (such as a polarizable fluid droplet) to the array
of wells to move at least a portion of the mixture to a distance
(the length of which is not critical) from the array of wells. In
certain embodiments, an aqueous washing liquid can be used to
remove any detectable label-specific binding members not bound to
any analyte. In such embodiments, the removal involves generating
an electric actuation force with a series or plurality of
electrodes to move an aqueous wash (or washing) droplet (a third
droplet) across the array of wells. The amount and type of aqueous
liquid used for said washing is not critical.
[0150] In certain embodiments, the mixture in the method is an
aqueous liquid. In other embodiments, the mixture is an immiscible
liquid. In other embodiments, the liquid droplet is a hydrophobic
liquid droplet. In other embodiments, the liquid droplet is a
hydrophilic liquid droplet. In certain embodiments, the array of
wells used in the method have a hydrophobic surface. In other
embodiments, the array of wells has a hydrophilic surface.
[0151] In certain embodiments, the first liquid droplet used in the
method is a polarizable liquid. In certain embodiments, the second
liquid droplet used in the method is a polarizable liquid. In
certain embodiments, the first and second liquid droplets used in
the method are polarizable liquids. In certain embodiments, the
mixture is a polarizable liquid. In certain embodiments one or more
of the first droplet, second droplet and mixture is a polarizable
liquid.
[0152] In certain embodiments, the detectable label is bound to at
least one solid support. In certain embodiments, the detectable
label comprises a chromagen, a florescent compound, an enzyme, a
chemiluminescent compound or a radioactive compound. In certain
embodiments, the binding member is a receptor, aptamer or antibody.
In certain embodiments, the method further comprises positioning
the at least a portion of the mixture over the array of wells using
a capillary element configured to facilitate movement of the
mixture to the array of wells.
[0153] In certain embodiments, the method described herein is
performed using a microfluidics device. In certain embodiments, the
method described herein is performed using a digital microfluidics
device (DMF). In certain embodiments, method described herein is
performed using a surface acoustic wave based microfluidics device
(SAW). In certain embodiments, method described herein is performed
using an integrated DMF and analyte detection device. In certain
embodiments, method described herein is performed using an
integrated surface acoustic wave based microfluidic device and
analyte detection device. In certain embodiments, method described
herein is performed using a Robotics based assay processing
unit.
[0154] Provided herein are methods for measuring an analyte of
interest in liquid droplet (wherein the analyte of interest is from
a test or biological sample). The method includes providing a first
liquid droplet containing an analyte of interest, providing a
second liquid droplet containing at least one solid support (such
as, for example, a magnetic solid support (such as a bead)) which
contains a specific binding member that binds to the analyte of
interest, using energy to exert a force to manipulate the first
liquid droplet (which contains the analyte of interest) with the
second liquid (containing the at least one solid support) to create
a mixture, moving all or at least a portion of the mixture to an
array of wells (where one or more wells of the array are of
sufficient size to accommodate the at least one solid support),
adding at least one detectable label to the mixture before, after
or both before or after moving a portion of the mixture to the
array of wells and measuring the analyte of interest in the wells.
In certain embodiments, "using energy to exert a force to
manipulate the first liquid droplet with the second liquid droplet"
refers to the use of non-mechanical forces (namely, for example,
energy created without the use of pumps and/or valves) to provide
or exert a force that manipulates (such as merges or combines) at
least the first and second liquid droplets (and optionally,
additional droplets) into a mixture. Example of non-mechanical
forces that can be used in the methods described herein include
electric actuation force (such as droplet actuation,
electrophoresis, electrowetting, dielectrophoresis, electrostatic
actuation, electric field mediated, electrode mediated, capillary
force, chromatography, centrifugation or aspiration) and/or
acoustic force (such as surface acoustic wave (or "SAW"). In
certain embodiments, the the electric actuation force generated is
an alternating current. For example, the alternating current can
have a root mean squred (rms) voltage of 10 V, 15 V, 20 V, 25 V, 30
V, 35V or more. For example, such alternating current can have a
rms voltage of 10 V or more, 15 V or more, 20 V or more, 25 V or
more, 30 V or more or 35 V or more. Alternatively, the alternating
current can have a frequency in a radio frequency range.
[0155] In certain embodiments, if magnetic solid supports are used,
an electric actuation force and a magnetic field can be applied and
applied from opposition directions, relative to the at least a
portion of the mixture. In certain other embodiments, the mixture
is mixed by moving it: back and forth, in a circular pattern or by
splitting it into two or more submixtures and then merging the
submixtures. In certain other embodiments, an electric actuation
force can be generated using a series or plurality of electrodes
(namely, at least two or more, at least three or more, at least
four or more, at least five or more, at least six or more, at least
seven or more, at least eight or more, at least nine or more, at
least ten or more, at least eleven or more, at least twelve or
more, at least thirteen or more, at least fourteen or more, at
least fifteen or more, etc.) to move the mixture to the array of
wells in order to seal the wells (which are loaded with at least
one solid support).
[0156] In certain embodiments, the moving of all or at least a
portion of the mixture to an array of wells results in the loading
(filling and/or placement) of the at least one solid support into
the array of wells. In certain embodiments, a magnetic field is
used to facilitate movement of the mixture and thus, at least one
solid support, into one or more wells of the array. In certain
embodiments, after the at least one solid supports are loaded into
the wells, any solid supports that are not loaded into a well can
be removed using routine techniques known in the art. For example,
such removing can involve generating an electric actuation force
(such as that described previously herein) with a series or
plurality of electrodes to move a fluid droplet (such as a
polarizable fluid droplet) to the array of wells to move at least a
portion of the mixture to a distance (the length of which is not
critical) from the array of wells. In certain embodiments, an
aqueous washing liquid can be used to remove the solid supports not
bound to any analyte of interest. In such embodiments, the removal
involves generating an electric actuation force with a series or
plurality of electrodes to move an aqueous wash (or washing)
droplet (a third droplet) across the array of wells. The amount and
type of aqueous liquid used for said washing is not critical.
[0157] In certain embodiments, the mixture in the method is an
aqueous liquid. In other embodiments, the mixture is an immiscible
liquid. In other embodiments, the liquid droplet is a hydrophobic
liquid droplet. In other embodiments, the liquid droplet is a
hydrophilic liquid droplet. In certain embodiments, the array of
wells used in the method have a hydrophobic surface. In other
embodiments, the array of wells has a hydrophilic surface.
[0158] In certain embodiments, the first liquid droplet used in the
method is a polarizable liquid. In certain embodiments, the second
liquid droplet used in the method is a polarizable liquid. In
certain embodiments, the first and second liquid droplets used in
the method are polarizable liquids. In certain embodiments, the
mixture is a polarizable liquid. In certain embodiments one or more
of the first droplet, second droplet and mixture is a polarizable
liquid.
[0159] In certain embodiments, the at least one solid support
comprises at least one binding member that specifically binds to
the analyte of interest. In certain embodiments, the detectable
label is added to the mixture before moving at least a portion of
the mixture to the array of wells. In certain other embodiments,
the detectable label is added to the mixture after the moving of at
least a portion of the analyte of interest to the array of wells.
In certain embodiments, the detectable label comprises at least one
binding member that specifically binds to the analyte of interest.
In certain embodiments, the detectable label comprises a chromagen,
a florescent compound, an enzyme, a chemiluminescent compound or a
radioactive compound. In certain embodiments, the binding member is
a receptor, aptamer or antibody. In certain embodiments, the method
further comprises positioning the at least a portion of the mixture
over the array of wells using a capillary element configured to
facilitate movement of the mixture to the array of wells.
[0160] In certain embodiments, the method described herein is
performed using a microfluidics device. In certain embodiments, the
method described herein is performed using a digital microfluidics
device (DMF). In certain embodiments, method described herein is
performed using a surface acoustic wave based microfluidics device
(SAW). In certain embodiments, method described herein is performed
using an integrated DMF and analyte detection device. In certain
embodiments, method described herein is performed using an
integrated surface acoustic wave based microfluidic device and
analyte detection device. In certain embodiments, method described
herein is performed using a Robotics based assay processing
unit.
[0161] In certain embodiments, the measuring first involves
determining the total number of solid supports in the well of the
array ("total solid support number"). Next, the number of solid
supports in the wells of the array that contain the detectable
label are determined, such as, for example, determining the
intensity of the signal produced by the detectable label
("positives"). The positives are subtracted from the total solid
support number to provide the number of solid supports in the array
of wells that do not contain a detectable label or are not detected
("negatives"). Then, the ratio of positives to negatives in the
array of wells can be determined and then compared to a calibration
curve. Alternatively, digital quantitation using the Poission
equation P(x; .mu.) as shown below:
P(x;.mu.)=(e.sup.-.mu.)(.mu..sup.x)/x!
[0162] where:
[0163] e: A is a constant equal to approximately 2.71828,
[0164] .mu.: ix ghd mean number of successes that occur in a
specified region, and
[0165] x: is the tactual number of successes that occur in a
specified region.
[0166] The sample may be any test sample containing or suspected of
containing an analyte of interest. As used herein, "analyte",
"target analyte", "analyte of interest" are used interchangeably
and refer to the analyte being measured in the methods and devices
disclosed herein. Analytes of interest are further described
below.
[0167] "Contacting" and grammatical equivalents thereof as used
herein refer to any type of combining action which brings a binding
member into sufficiently close proximity with the analyte of
interest in the sample such that a binding interaction will occur
if the analyte of interest specific for the binding member is
present in the sample. Contacting may be achieved in a variety of
different ways, including combining the sample with a binding
member, exposing a target analyte to a binding member by
introducing the binding member in close proximity to the analyte,
and the like.
[0168] In certain cases, the first binding member may be
immobilized on a solid support. As used herein, the term
"immobilized" refers to a stable association of the first binding
member with a surface of a solid support. By "stable association"
is meant a physical association between two entities in which the
mean half-life of association is one day or more, e.g., under
physiological conditions. In certain aspects, the physical
association between the two entities has a mean half-life of two
days or more, one week or more, one month or more, including six
months or more, e.g., 1 year or more, in PBS at 4.degree. C.
According to certain embodiments, the stable association arises
from a covalent bond between the two entities, a non-covalent bond
between the two entities (e.g., an ionic or metallic bond), or
other forms of chemical attraction, such as hydrogen bonding, Van
der Waals forces, and the like.
[0169] The solid support having a surface on which the binding
reagent is immobilized may be any convenient surface in planar or
non-planar conformation, such as a surface of a microfluidic chip,
an interior surface of a chamber, an exterior surface of a bead (as
defined herein), or an interior and/or exterior surface of a porous
bead. For example, the first binding member may be attached
covalently or non-covalently to a bead, e.g., latex, agarose,
sepharose, streptavidin, tosylactivated, epoxy, polystyrene, amino
bead, amine bead, carboxyl bead, or the like. In certain
embodiments, the bead may be a particle, e.g., a microparticle. In
some embodiments, the microparticle may be between about 0.1 nm and
about 10 microns, between about 50 nm and about 5 microns, between
about 100 nm and about 1 micron, between about 0.1 nm and about 700
nm, between about 500 nm and about 10 microns, between about 500 nm
and about 5 microns, between about 500 nm and about 3 microns,
between about 100 nm and 700 nm, or between about 500 nm and 700
nm. For example, the microparticle may be about 4-6 microns, about
2-3 microns, or about 0.5-1.5 microns. Particles less than about
500 nm are sometimes considered nanoparticles. Thus, the
microparticle optionally may be a nanoparticle between about 0.1 nm
and about 500 nm, between about 10 nm and about 500 nm, between
about 50 nm and about 500 nm, between about 100 nm and about 500
nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about
300 nm, about 350 nm, about 400 nm, about 450 nm, or about 500
nm.
[0170] In certain embodiments, the bead may be a magnetic bead or a
magnetic particle. In certain embodiments, the bead may be a
magnetic nanobead, nanoparticle, microbead or microparticle.
Magnetic beads/particles may be ferromagnetic, ferrimagnetic,
paramagnetic, superparamagnetic or ferrofluidic. Exemplary
ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO.sub.2,
MnAs, MnBi, EuO, NiO/Fe. Examples of ferrimagnetic materials
include NiFe.sub.2O.sub.4, CoFe.sub.2O.sub.4, Fe.sub.3O.sub.4 (or
FeO.Fe.sub.2O.sub.3). Beads can have a solid core portion that is
magnetic and is surrounded by one or more non-magnetic layers.
Alternately, the magnetic portion can be a layer around a
non-magnetic core. The solid support on which the first binding
member is immobilized may be stored in dry form or in a liquid. The
magnetic beads may be subjected to a magnetic field prior to or
after contacting with the sample with a magnetic bead on which the
first binding member is immobilized.
[0171] After the contacting step, the sample and the first binding
member may be incubated for a sufficient period of time to allow
for the binding interaction between the binding member and analyte
to occur. In addition, the incubating may be in a binding buffer
that facilitates the specific binding interaction. The binding
affinity and/or specificity of the first binding member and/or the
second binding member may be manipulated or altered in the assay by
varying the binding buffer. In some embodiments, the binding
affinity and/or specificity may be increased by varying the binding
buffer. In some embodiments, the binding affinity and/or
specificity may be decreased by varying the binding buffer.
[0172] The binding affinity and/or specificity of the first binding
member and/or the second binding member may be measured using the
disclosed methods and device described below. In some embodiments,
the one aliquot of sample is assayed using one set of conditions
and compared to another aliquot of sample assayed using a different
set of conditions, thereby determining the effect of the conditions
on the binding affinity and/or specificity. For instance, changing
or altering the condition can be one or more of removing the target
analyte from the sample, adding a molecule that competes with the
target analyte or the ligand for binding, and changing the pH, salt
concentration, or temperature. Additionally or alternatively, a
duration of time can be the variable and changing the condition may
include waiting for a duration of time before again performing the
detection methods.
[0173] The binding buffer may include molecules standard for
antigen-antibody binding buffers such as, albumin (e.g., BSA),
non-ionic detergents (Tween-20, Triton X-100), and/or protease
inhibitors (e.g., PMSF). In certain cases, the binding buffer may
be added to the microfluidic chip, chamber, etc., prior to or after
adding the sample. In certain cases, the first binding member may
be present in a binding buffer prior to contacting with the sample.
The length of time for binding interaction between the binding
member and analyte to occur may be determined empirically and may
depend on the binding affinity and binding avidity between the
binding member and the analyte. In certain embodiments, the
contacting or incubating may be for a period of 5 sec to 1 hour,
such as, 10 sec-30 minutes, or 1 minute-15 minutes, or 5 minutes-10
minutes, e.g., 10 sec, 15 sec, 30 sec, 1 minute, 5 minutes, 10
minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour or 2 hours.
Other conditions for the binding interaction, such as, temperature,
salt concentration, may also be determined empirically or may be
based on manufacturer's instructions. For example, the contacting
may be carried out at room temperature (21.degree. C.-28.degree.
C., e.g., 23.degree. C.-25.degree. C.), 37.degree. C., or 4.degree.
C. In certain embodiments, an optional mixing of the sample with
the first binding member may be carried out during the contacting
step.
[0174] Following complex formation between the immobilized first
binding member and the analyte, any unbound analyte may be removed
from the vicinity of the first binding member along with the sample
while the complex of the first binding member and the analyte may
be retained due to its association with the solid support.
Optionally, the solid support may be contacted with a wash buffer
to remove any molecules non-specifically bound to the solid
support.
[0175] After the first contacting step, and the optional removal of
sample and/or optional wash steps, the complex of the first binding
member and the analyte may be contacted with a second binding
member, thereby leading to the formation of a sandwich complex in
which the analyte is bound by the two binding members. An optional
mixing of the second member with the first binding member-analyte
complex may be carried out during the second contacting step. In
some embodiments, immobilization of the analyte molecules with
respect to a surface may aid in removal of any excess second
binding members from the solution without concern of dislodging the
analyte molecule from the surface. In some embodiments, the second
binding member may include a detectable label comprising one or
more signal-producing substances, such as chromagens, fluorescent
compounds, chemiluminescent compounds, enzymes, radioactive
compounds, and the like.
[0176] As noted above, the second contacting step may be carried
out in conditions sufficient for binding interaction between the
analyte and the second binding member. Following the second
contacting step, any unbound second binding member may be removed,
followed by an optional wash step. Any unbound second binding
member may be separated from the complex of the first binding
member-analyte-second binding member by a suitable means such as,
droplet actuation, electrophoresis, electrowetting,
dielectrophoresis, electrostatic actuation, electric field
mediated, electrode mediated, capillary force, chromatography,
centrifugation, aspiration or SAW. Upon removal of any unbound
second binding member from the vicinity of the complex of the first
binding member-analyte-second binding member, the detectable label
attached to the second binding member present in the complex of the
first binding member-analyte-second binding member may be separated
by a suitable means or may be detected using techniques known in
the art. In some embodiments, the detectable label comprises a
detectable label comprising one or more signal-producing
substances, such as chromagens, fluorescent compounds, enzymes,
chemiluminescent compounds, radioactive compounds, and the like.
Alternatively, in some embodiments, if the detectable label
comprises a tag, the tag can be cleaved or disassociated from the
complex which remains after removal of unbound reagents. For
example, the tag may be attached to the second binding member via a
cleavable linker ("cleavable linker" as described herein. The
complex of the first binding member-analyte-second binding member
may be exposed to a cleavage agent that mediates cleavage of the
cleavable linker.
[0177] As noted herein, the tag may include a nucleic acid. In
certain embodiments, the quantification of the analyte does not
include determining the identity of the tag by determining identity
of at least a portion of the nucleic acid sequence present in the
tag. For example, the counting step may not include determining a
sequence of the tag. In other embodiments, the tag may not be
sequenced, however, identity of the tag may be determined to the
extent that one tag may be distinguished from another tag based on
a differentiable signal associated with the tag due its size,
conformation, charge, amount of charge and the like. Identification
of tag may be useful in methods involving simultaneous analysis of
a plurality of different analytes in a sample, for example, two,
three, four, or more different analytes in a sample.
[0178] In certain embodiments, the simultaneous analysis of
multiple analytes in a single sample may be performed by using a
plurality of different first and second binding members where a
pair of first and second binding members is specific to a single
analyte in the sample. In these embodiments, the detectable label
associated with the second binding member of a first pair of first
and second binding members specific to a single analyte may be
distinguishable from the detectable label associated with the
second binding member of a second pair of first and second binding
members specific to a different analyte. As noted above, a first
detectable label may be distinguishable from second detectable
label based on difference in signal-producing substances, etc.
[0179] In some embodiments, the concentration of an analyte in the
fluid sample that may be substantially accurately determined is
less than about 5000 fM (femtomolar), less than about 3000 fM, less
than about 2000 fM, less than about 1000 fM, less than about 500
fM, less than about 300 fM, less than about 200 fM, less than about
100 fM, less than about 50 fM, less than about 25 fM, less than
about 10 fM, less than about 5 fM, less than about 2 fM, less than
about 1 fM, less than about 500 aM (attomolar), less than about 100
aM, less than about 10 aM, less than about 5 aM, less than about 1
aM, less than about 0.1 aM, less than about 500 zM (zeptomolar),
less than about 100 zM, less than about 10 zM, less than about 5
zM, less than about 1 zM, less than about 0.1 zM, or less.
[0180] In some cases, the limit of detection (e.g., the lowest
concentration of an analyte which may be determined in solution) is
about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM,
about 2 fM, about 1 fM, about 500 aM (attomolar), about 100 aM,
about 50 aM, about 10 aM, about 5 aM, about 1 aM, about 0.1 aM,
about 500 zM (zeptomolar), about 100 zM, about 50 zM, about 10 zM,
about 5 zM, about 1 zM, about 0.1 zM, or less. In some embodiments,
the concentration of analyte in the fluid sample that may be
substantially accurately determined is between about 5000 fM and
about 0.1 fM, between about 3000 fM and about 0.1 fM, between about
1000 fM and about 0.1 fM, between about 1000 fM and about 0.1 zM,
between about 100 fM and about 1 zM, between about 100 aM and about
0.1 zM, or less.
[0181] The upper limit of detection (e.g., the upper concentration
of an analyte which may be determined in solution) is at least
about 100 fM, at least about 1000 fM, at least about 10 pM
(picomolar), at least about 100 pM, at least about 100 pM, at least
about 10 nM (nanomolar), at least about 100 nM, at least about 1000
nM, at least about 10 .mu.M, at least about 100 .mu.M, at least
about 1000 .mu.M, at least about 10 mM, at least about 100 mM, at
least about 1000 mM, or greater.
[0182] In some cases, the presence and/or concentration of the
analyte in a sample may be detected rapidly, usually in less than
about 1 hour, e.g., 45 minutes, 30 minutes, 15 minutes, 10 minutes,
5 minutes, 1 minute, or 30 seconds.
[0183] In certain embodiments, at least some steps of the methods
described herein may be carried out on a digital integrated
microfluidics and analyte detection device, such as the device
described herein. In certain embodiments, the methods of the
present disclosure are carried out using a digital integrated
microfluidics device in conjunction with an analyte detection
device. For example, the digital microfluidics device and the
analyte detection device may be separate devices and a droplet
containing the detectable label may be generated in the
microfluidics device and transported to the analyte detection
device.
[0184] In certain embodiments, the methods of the present
disclosure are carried out using a device in which a digital
microfluidics module is integrated with an analyte detection
device, such as the device described below. In certain embodiments,
the digital integrated microfluidics module and the analyte
detection device may be reversibly integrated. For example, the two
modules may be combined physically to form the integrated device
and which device could then be separated into the individual
modules. In certain embodiments, the methods of the present
disclosure are carried out using a disposable cartridge that
includes a microfluidics module with a built-in analyte detection
device. Exemplary embodiments of the devices used for performing
the methods provided herein are described further in the next
section.
[0185] Exemplary embodiments of the present method include merging
a sample droplet containing an analyte of interest with a droplet
containing a first binding member that binds to the analyte of
interest and that may be immobilized on a solid support (such as
magnetic particles or beads). The single merged droplet can be
incubated for a period of time sufficient to allow binding of the
first binding member to the analyte of interest. Optionally, the
single droplet may be agitated to facilitate mixing of the sample
with the first binding member. Mixing may be achieved by moving the
single droplet back and forth, moving the single droplet around
over a plurality of electrodes, splitting a droplet and then
merging the droplets, or using SAWs, and the like. Next, the single
droplet may be subjected to a magnetic force to retain the beads at
a location in the device while the droplet may be moved away and
replaced with a droplet containing a second binding member, which
second binding member can optionally contain a detectable label. An
optional wash step may be performed, prior to adding the second
binding member, by moving a droplet of wash buffer to the location
at which the beads are retained using the magnetic force. After a
period of time sufficient for the second binding member to bind the
analyte bound to the first binding member, the droplet containing
the second binding member may be moved away while the beads are
retained at the first location. The beads may be washed using a
droplet of wash buffer. Following the wash step, the magnetic force
may be removed and the droplet containing labeled beads (containing
the first specific binding member/analyte/second specific binding
member--an optional detectable label) are moved to a detection
module such as that described herein. The labeled beads are allowed
to settle into an array of wells in the detection module. The beads
may settle via gravitational force or by applying electric or
magnetic force. Following a wash step to remove any beads not
located inside the wells, the wells may be sealed using a
hydrophobic liquid. In the above embodiments, optionally, after the
combining, a droplet may be manipulated (e.g., moved back and
forth, moved in a circular direction, oscillated, split/merged,
exposed to SAW, etc.) to facilitate mixing of the sample with the
assay reagents, such as, the first binding member, second binding
member, etc. In embodiments where the detectable label is an
enzyme, a substrate can be added either before or after moving the
complex is moved to the array of wells.
[0186] The moving of the droplets in the integrated microfluidic
and analyte detection device may be carried out using electrical
force (e.g., electrowetting, dielectrophoresis, electrode-mediated,
opto-electrowetting, electric-field mediated, and electrostatic
actuation) pressure, surface acoustic waves and the like. The force
used for moving the droplets may be determined based on the
specifics of the device, which are described in the following
sections, and for the particular device described herein.
[0187] Multiplexing
[0188] The methods may include one or more (or alternately two or
more) specific binding members to detect one or more (or
alternately two or more) target analytes in the sample in a
multiplexing assay. Each of the one or more (or alternately two or
more) specific binding members binds to a different target analyte
and each specific binding member is labeled with a different
detectable label. For example, a first specific binding member
binds to a first target analyte, a second specific binding member
binds to a second target analyte, a third specific binding member
binds to a third target analyte, etc. and the first specific
binding member is labeled with a detectable label, the second
specific binding member is labeled with a second detectable label,
the third specific binding member is labeled with a third
detectable label, etc. For example the first, second and third
detectable labels can each have a different color. Alternatively,
different types of labels can be used, such as, for example, the
first label is an enzymatic label, the second label is a chromagen
and the third label is a chemiluminescent compound.
[0189] Exemplary Target Analytes
[0190] As will be appreciated by those in the art, any analyte that
can be specifically bound by a first and second binding member, may
be detected and, optionally, quantified using methods and devices
of the present disclosure.
[0191] In some embodiments, the analyte may be a biomolecule.
Non-limiting examples of biomolecules include macromolecules such
as, proteins, lipids, and carbohydrates. In certain instances, the
analyte may be hormones, antibodies, growth factors, cytokines,
enzymes, receptors (e.g., neural, hormonal, nutrient, and cell
surface receptors) or their ligands, cancer markers (e.g., PSA,
TNF-alpha), markers of myocardial infarction (e.g., troponin,
creatine kinase, and the like), toxins, drugs (e.g., drugs of
addiction), metabolic agents (e.g., including vitamins), and the
like. Non-limiting embodiments of protein analytes include
peptides, polypeptides, protein fragments, protein complexes,
fusion proteins, recombinant proteins, phosphoproteins,
glycoproteins, lipoproteins, or the like.
[0192] In certain embodiments, the analyte may be a
post-translationally modified protein (e.g., phosphorylated,
methylated, glycosylated protein) and the first or the second
binding member may be an antibody specific to a post-translational
modification. A modified protein may be bound to a first binding
member immobilized on a solid support where the first binding
member binds to the modified protein but not the unmodified
protein. In other embodiments, the first binding member may bind to
both the unmodified and the modified protein, and the second
binding member may be specific to the post-translationally modified
protein.
[0193] In some embodiments, the analyte may be a cell, such as,
circulating tumor cell, pathogenic bacteria, viruses (including
retroviruses, herpesviruses, adenoviruses, lentiviruses,
Filoviruses (ebola), hepatitis viruses (e.g., A, B, C, D, and E);
HPV etc.); spores, etc.
[0194] A non-limiting list of analytes that may be analyzed by the
methods presented herein include A.beta.42 amyloid beta-protein,
fetuin-A, tau, secretogranin II, prion protein, Alpha-synuclein,
tau protein, neurofilament light chain, parkin, PTEN induced
putative kinase 1, DJ-1, leucine-rich repeat kinase 2, mutated
ATP13A2, Apo H, ceruloplasmin, Peroxisome proliferator-activated
receptor gamma coactivator-1 alpha (PGC-1.alpha.), transthyretin,
Vitamin D-binding Protein, proapoptotic kinase R (PKR) and its
phosphorylated PKR (pPKR), CXCL13, IL-12p40, CXCL13, IL-8, Dkk-3
(semen), p14 endocan fragment, Serum, ACE2, autoantibody to CD25,
hTERT, CAI25 (MUC 16), VEGF, sIL-2, Osteopontin, Human epididymis
protein 4 (HE4), Alpha-Fetoprotein, Albumin, albuminuria,
microalbuminuria, neutrophil gelatinase-associated lipocalin
(NGAL), interleukin 18 (IL-18), Kidney Injury Molecule-1 (KIM-1),
Liver Fatty Acid Binding Protein (L-FABP), LMP1, BARF1, IL-8,
carcinoembryonic antigen (CEA), BRAF, CCNI, EGRF, FGF19, FRS2,
GREB1, and LZTS1, alpha-amylase, carcinoembryonic antigen, CA 125,
IL8, thioredoxin, beta-2 microglobulin levels--monitor activity of
the virus, tumor necrosis factor-alpha receptors--monitor activity
of the virus, CA15-3, follicle-stimulating hormone (FSH),
leutinizing hormone (LH), T-cell lymphoma invasion and metastasis 1
(TIAM1), N-cadherin, EC39, amphiregulin, dUTPase, secretory
gelsolin (pGSN), PSA (prostate specific antigen), thymosin
.beta.15, insulin, plasma C-peptide, glycosylated hemoglobin
(HBA1c), C-Reactive Protein (CRP), Interleukin-6 (IL-6), ARHGDIB
(Rho GDP-dissociation inhibitor 2), CFL1 (Cofilin-1), PFN1
(profilin-1), GSTP1 (Glutathione S-transferase P), S100A11 (Protein
S100-A11), PRDX6 (Peroxiredoxin-6), HSPE1 (10 kDa heat shock
protein, mitochondrial), LYZ (Lysozyme C precursor), GPI
(Glucose-6-phosphate isomerase), HIST2H2AA (Histone H2A type 2-A),
GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), HSPG2 (Basement
membrane-specific heparan sulfate proteoglycan core protein
precursor), LGALS3BP (Galectin-3-binding protein precursor), CTSD
(Cathepsin D precursor), APOE (Apolipoprotein E precursor), IQGAP1
(Ras GTPase-activating-like protein IQGAP1), CP (Ceruloplasmin
precursor), and IGLC2 (IGLC1 protein), PCDGF/GP88, EGFR, HER2,
MUC4, IGF-IR, p27(kip1), Akt, HER3, HER4, PTEN, PIK3CA, SHIP, Grb2,
Gab2, PDK-1 (3-phosphoinositide dependent protein kinase-1), TSC1,
TSC2, mTOR, MIG-6 (ERBB receptor feedback inhibitor 1), S6K, src,
KRAS, MEK mitogen-activated protein kinase 1, cMYC, TOPO II
topoisomerase (DNA) II alpha 170 kDa, FRAP1, NRG1, ESR1, ESR2, PGR,
CDKN1B, MAP2K1, NEDD4-1, FOXO3A, PPP1R1B, PXN, ELA2, CTNNB1, AR,
EPHB2, KLF6, ANXA7, NKX3-1, PITX2, MKI67, PHLPP, adiponectin
(ADIPOQ), fibrinogen alpha chain (FGA), leptin (LEP), advanced
glycosylation end product-specific receptor (AGER aka RAGE),
alpha-2-HS-glycoprotein (AHSG), angiogenin (ANG), CD14 molecule
(CD14), ferritin (FTH1), insulin-like growth factor binding protein
1 (IGFBP1), interleukin 2 receptor, alpha (IL2RA), vascular cell
adhesion molecule 1 (VCAM1) and Von Willebrand factor (VWF),
myeloperoxidase (MPO), TNF.alpha., perinuclear anti-neutrophil
cytoplasmic antibody (p-ANCA), lactoferrin, calprotectin, Wilm's
Tumor-1 protein, Aquaporin-1, MLL3, AMBP, VDAC1, E. coli
enterotoxins (heat-labile exotoxin, heat-stable enterotoxin),
influenza HA antigen, tetanus toxin, diphtheria toxin, botulinum
toxins, Shiga toxin, Shiga-like toxin I, Shiga-like toxin II,
Clostridium difficile toxins A and B, etc.
[0195] Samples
[0196] As used herein, "sample", "test sample", "biological sample"
refer to fluid sample containing or suspected of containing an
analyte of interest. The sample may be derived from any suitable
source. In some cases, the sample may comprise a liquid, fluent
particulate solid, or fluid suspension of solid particles. In some
cases, the sample may be processed prior to the analysis described
herein. For example, the sample may be separated or purified from
its source prior to analysis; however, in certain embodiments, an
unprocessed sample containing the analyte may be assayed directly.
The source of the analyte molecule may be synthetic (e.g., produced
in a laboratory), the environment (e.g., air, soil, fluid samples
e.g, water supplies, etc.), an animal, e.g., a mammal, a plant, or
any combination thereof. In a particular example, the source of an
analyte is a human bodily substance (e.g., bodily fluid, blood,
serum, plasma, urine, saliva, sweat, sputum, semen, mucus, lacrimal
fluid, lymph fluid, amniotic fluid, interstitial fluid, lung
lavage, cerebrospinal fluid, feces, tissue, organ, or the like).
Tissues may include, but are not limited to skeletal muscle tissue,
liver tissue, lung tissue, kidney tissue, myocardial tissue, brain
tissue, bone marrow, cervix tissue, skin, etc. The sample may be a
liquid sample or a liquid extract of a solid sample. In certain
cases, the source of the sample may be an organ or tissue, such as
a biopsy sample, which may be solubilized by tissue
disintegration/cell lysis.
[0197] A wide range of volumes of the fluid sample may be analyzed.
In a few exemplary embodiments, the sample volume may be about 0.5
nL, about 1 nL, about 3 nL, about 0.01 .mu.L, about 0.1 .mu.L,
about 1 .mu.L, about 5 .mu.L, about 10 .mu.L, about 50 .mu.L, about
100 .mu.L, about 1 mL, about 5 mL, about 10 mL, or the like. In
some cases, the volume of the fluid sample is between about 0.01
.mu.L and about 10 mL, between about 0.01 .mu.L and about 1 mL,
between about 0.01 .mu.L and about 100 .mu.L, between about 0.1
.mu.L and about 10 .mu.L, between about 1 .mu.L and about 100
.mu.L, between about 10 .mu.L and about 100 .mu.L, or between about
10 .mu.L and about 75 .mu.L.
[0198] In some cases, the fluid sample may be diluted prior to use
in an assay. For example, in embodiments where the source of an
analyte molecule is a human body fluid (e.g., blood, serum), the
fluid may be diluted with an appropriate solvent (e.g., a buffer
such as PBS buffer). A fluid sample may be diluted about 1-fold,
about 2-fold, about 3-fold, about 4-fold, about 5-fold, about
6-fold, about 10-fold, about 100-fold, or greater, prior to
use.
[0199] In some cases, the sample may undergo pre-analytical
processing. Pre-analytical processing may offer additional
functionality such as nonspecific protein removal and/or effective
yet cheaply implementable mixing functionality. General methods of
pre-analytical processing may include the use of electrokinetic
trapping, AC electrokinetics, surface acoustic waves,
isotachophoresis, dielectrophoresis, electrophoresis, or other
pre-concentration techniques known in the art. In some cases, the
fluid sample may be concentrated prior to use in an assay. For
example, in embodiments where the source of an analyte molecule is
a human body fluid (e.g., blood, serum), the fluid may be
concentrated by precipitation, evaporation, filtration,
centrifugation, or a combination thereof. A fluid sample may be
concentrated about 1-fold, about 2-fold, about 3-fold, about
4-fold, about 5-fold, about 6-fold, about 10-fold, about 100-fold,
or greater, prior to use.
[0200] In certain embodiments, the analyte is not amplified (i.e.,
the copy number of the analyte is not increased) prior to the
measurement of the analyte. For example, in cases where the analyte
is DNA or RNA, the analyte is not replicated to increase copy
numbers of the analyte. In certain cases, the analyte is a protein
or a small molecule.
[0201] Specific Binding Members
[0202] As will be appreciated by those in the art, the binding
members will be determined by the analyte to be analyzed. Binding
members for a wide variety of target molecules are known or can be
readily found or developed using known techniques. For example,
when the target analyte is a protein, the binding members may
include proteins, particularly antibodies or fragments thereof
(e.g., antigen-binding fragments (Fabs), Fab' fragments,
F(ab').sub.2 fragments, recombinant antibodies, chimeric
antibodies, single-chain Fvs ("scFv"), single chain antibodies,
single domain antibodies, such as variable heavy chain domains
("VHH"; also known as "VHH fragments") derived from animals in the
Camelidae family (VHH and methods of making them are described in
Gottlin et al., Journal of Biomolecular Screening, 14:77-85
(2009)), recombinant VHH single-domain antibodies, and V.sub.NAR
fragments, disulfide-linked Fvs ("sdFv"), and anti-idiotypic
("anti-Id") antibodies, and functionally active epitope-binding
fragments of any of the above, full-length polyclonal or monoclonal
antibodies, antibody-like fragments, etc.), other proteins, such as
receptor proteins, Protein A, Protein C, or the like. In case where
the analyte is a small molecule, such as, steroids, bilins,
retinoids, and lipids, the first and/or the second binding member
may be a scaffold protein (e.g., lipocalins) or a receptor. In some
cases, binding member for protein analytes may be a peptide. For
example, when the target analyte is an enzyme, suitable binding
members may include enzyme substrates and/or enzyme inhibitors
which may be a peptide, a small molecule and the like. In some
cases, when the target analyte is a phosphorylated species, the
binding members may comprise a phosphate-binding agent. For
example, the phosphate-binding agent may comprise metal-ion
affinity media such as those describe in U.S. Pat. No. 7,070,921
and U.S. Patent Application No. 20060121544.
[0203] In certain cases, at least one of the binding members may be
an aptamer, such as those described in U.S. Pat. Nos. 5,270,163,
5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337.
Nucleic acid aptamers (e.g., single-stranded DNA molecules or
single-stranded RNA molecules) may be developed for capturing
virtually any target molecule. Aptamers bind target molecules in a
highly specific, conformation-dependent manner, typically with very
high affinity, although aptamers with lower binding affinity can be
selected. Aptamers may distinguish between target analyte molecules
based on very small structural differences such as the presence or
absence of a methyl or hydroxyl group and certain aptamers can
distinguish between D- and L-enantiomers and diastereomers.
Aptamers may bind small molecular targets, including drugs, metal
ions, and organic dyes, peptides, biotin, and proteins. Aptamers
can retain functional activity after biotinylation, fluorescein
labeling, and when attached to glass surfaces and microspheres.
[0204] Nucleic acid aptamers are oligonucleotides that may be
single stranded oligodeoxynucleotides, oligoribonucleotides, or
modified oligodeoxynucleotide or oligoribonucleotides. The term
"modified" encompasses nucleotides with a covalently modified base
and/or sugar. For example, modified nucleotides include nucleotides
having sugars which are covalently attached to low molecular weight
organic groups other than a hydroxyl group at the 3' position and
other than a phosphate group at the 5' position. Thus modified
nucleotides may also include 2' substituted sugars such as
2'-O-methyl-; 2-O-alkyl; 2-O-allyl; 2'-S-alkyl; 2'-S-allyl;
2'-fluoro-; 2'-halo or 2-azido-ribose, carbocyclic sugar analogues
a-anomeric sugars; epimeric sugars such as arabinose, xyloses or
lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.
[0205] Peptide aptamers may be designed to interfere with protein
interactions. Peptide aptamers may be based on a protein scaffold
onto which a variable peptide loop is attached, thereby
constraining the conformation of the aptamer. In some cases, the
scaffold portion of the peptide aptamer is derived from Bacterial
Thioredoxin A (TrxA).
[0206] When the target molecule is a carbohydrate, potentially
suitable capture components (as defined herein) include, for
example, antibodies, lectins, and selectins. As will be appreciated
by those of ordinary skill in the art, any molecule that can
specifically associate with a target molecule of interest may
potentially be used as a binding member.
[0207] For certain embodiments, suitable target analyte/binding
member complexes can include, but are not limited to,
antibodies/antigens, antigens/antibodies, receptors/ligands,
ligands/receptors, proteins/nucleic acid, enzymes/substrates and/or
inhibitors, carbohydrates (including glycoproteins and
glycolipids)/lectins and/or selectins, proteins/proteins,
proteins/small molecules, etc.
[0208] In a particular embodiment, the first binding member may be
attached to a solid support via a linkage, which may comprise any
moiety, functionalization, or modification of the support and/or
binding member that facilitates the attachment of the binding
member to the support. The linkage between the binding member and
the support may include one or more chemical or physical (e.g.,
non-specific attachment via van der Waals forces, hydrogen bonding,
electrostatic interactions, hydrophobic/hydrophilic interactions;
etc.) bonds and/or chemical spacers providing such bond(s).
[0209] In certain embodiments, a solid support may also comprise a
protective, blocking, or passivating layer that can eliminate or
minimize non-specific attachment of non-capture components (e.g.,
analyte molecules, binding members) to the binding surface during
the assay which may lead to false positive signals during detection
or to loss of signal. Examples of materials that may be utilized in
certain embodiments to form passivating layers include, but are not
limited to: polymers, such as poly(ethylene glycol), that repel the
non-specific binding of proteins; naturally occurring proteins with
this property, such as serum albumin and casein; surfactants, e.g.,
zwitterionic surfactants, such as sulfobetaines; naturally
occurring long-chain lipids; polymer brushes, and nucleic acids,
such as salmon sperm DNA.
[0210] Certain embodiments utilize binding members that are
proteins or polypeptides. As is known in the art, any number of
techniques may be used to attach a polypeptide to a wide variety of
solid supports. A wide variety of techniques are known to add
reactive moieties to proteins, for example, the method outlined in
U.S. Pat. No. 5,620,850. Further, methods for attachment of
proteins to surfaces are known, for example, see Heller, Acc. Chem.
Res. 23:128 (1990).
[0211] As explained herein, binding between the binding members and
the analyte, is specific, e.g., as when the binding member and the
analyte are complementary parts of a binding pair. In certain
embodiments, the binding member binds specifically to the analyte.
By "specifically bind" or "binding specificity" it is meant that
the binding member binds the analyte molecule with specificity
sufficient to differentiate between the analyte molecule and other
components or contaminants of the test sample. For example, the
binding member, according to one embodiment, may be an antibody
that binds specifically to an epitope on an analyte. The antibody,
according to one embodiment, can be any antibody capable of binding
specifically to an analyte of interest. For example, appropriate
antibodies include, but are not limited to, monoclonal antibodies,
bispecific antibodies, minibodies, domain antibodies (dAbs) (e.g.,
such as described in Holt et al. (2014) Trends in Biotechnology
21:484-490), and including single domain antibodies sdAbs that are
naturally occurring, e.g., as in cartilaginous fishes and camelid,
or which are synthetic, e.g., nanobodies, VHH, or other domain
structure), synthetic antibodies (sometimes referred to as antibody
mimetics), chimeric antibodies, humanized antibodies, antibody
fusions (sometimes referred to as "antibody conjugates"), and
fragments of each, respectively. As another example, the analyte
molecule may be an antibody and the first binding member may be an
antigen and the second binding member may be a secondary antibody
that specifically binds to the target antibody or the first binding
member may be a secondary antibody that specifically binds to the
target antibody and the second binding member may be an
antigen.
[0212] In some embodiments, the binding member may be chemically
programmed antibodies (cpAbs) (described in Rader (2014) Trends in
Biotechnology 32:186-197), bispecific cpAbs, antibody-recruiting
molecules (ARMs) (described in McEnaney et al. (2012) ACS Chem.
Biol. 7:1139-1151), branched capture agents, such as a triligand
capture agent (described in Millward et al. (2011) J. Am. Chem.
Soc. 133:18280-18288), engineered binding proteins derived from
non-antibody scaffolds, such as monobodies (derived from the tenth
fibronectin type III domain of human fibronectin), affibodies
(derived from the immunoglobulin binding protein A), DARPins (based
on Ankyrin repeat modules), anticalins (derived from the lipocalins
bilin-binding protein and human lipocalin 2), and cysteine knot
peptides (knottins) (described in Gilbreth and Koide, (2012)
Current Opinion in Structural Biology 22:1-8; Banta et al. (2013)
Annu. Rev. Biomed. Eng. 15:93-113), WW domains (described in Patel
et al. (2013) Protein Engineering, Design & Selection
26(4):307-314), repurposed receptor ligands, affitins (described in
Behar et al. (2013) 26:267-275), and/or Adhirons (described in
Tiede et al. (2014) Protein Engineering, Design & Selection
27:145-155).
[0213] According to one embodiment in which an analyte is a
biological cell (e.g., mammalian, avian, reptilian, other
vertebrate, insect, yeast, bacterial, cell, etc.), the binding
members may be ligands having specific affinity for a cell surface
antigen (e.g., a cell surface receptor). In one embodiment, the
binding member may be an adhesion molecule receptor or portion
thereof, which has binding specificity for a cell adhesion molecule
expressed on the surface of a target cell type. In use, the
adhesion molecule receptor binds with an adhesion molecule on the
extracellular surface of the target cell, thereby immobilizing or
capturing the cell, the bound cell may then be detected by using a
second binding member that may be the same as the first binding
member or may bind to a different molecule expressed on the surface
of the cell.
[0214] In some embodiments, the binding affinity between analyte
molecules and binding members should be sufficient to remain bound
under the conditions of the assay, including wash steps to remove
molecules or particles that are non-specifically bound. In some
cases, for example in the detection of certain biomolecules, the
binding constant of the analyte molecule to its complementary
binding member may be between at least about 10.sup.4 and about
10.sup.6 M.sup.-1, at least about 10.sup.5 and about 10.sup.9
M.sup.-1, at least about 10.sup.7 and about 10.sup.9 M.sup.-1,
greater than about 10.sup.9 M.sup.-1, or greater.
[0215] Detectable Labels: Tags and Signal-Producing Substances
[0216] The methods described herein may include a specific binding
member bound to a detectable label, such as a tag to analyze an
analyte. The incorporated tag or labels do not substantially
interfere with the conduct of the reaction scheme. For example, the
incorporated tag or label does not interfere with the binding
constant of or the interaction between the analyte and its
complementary binding member. The size and number of incorporated
tags or labels may be related to the speed of capture and read
rate. The speed of capture and read rate may be increased by
increasing the size and/or number of incorporated tags or labels.
The incorporated tag or labels do not alter the binding member
kinetics, for example, antibody kinetics, or the reaction scheme.
Exemplary tags include polymers such as, an anionic polymer or a
cationic polymer (e.g., a polypeptide with a net positive charge,
such as, polyhistidine or polylysine), where the polymer is about
5-1000 residues in length; a protein (e.g., a globular protein)
which does not cross react with the binding member and/or interfere
with the assay, a dendrimer, e.g., a DNA dendrimer; and a charged
particle, e.g., a bead. A polymer tag may include a nucleic acid,
such as, a deoxyribonucleic acid or a ribonucleic acid. A polymer
tag may include a nucleobase polymer. In certain cases, the tag may
be DNA or a RNA aptamer, where the aptamer does not bind to the
analyte. A polymer tag or a particle (e.g., a bead) may be
sufficiently large to generate a reproducible signal. Aptamers may
be 20-220 bases in length, e.g., 20-60 bases long. The size of the
particle (e.g., a bead or a dendrimer) may range from about 1 nm to
about 950 nm in diameter for example, 10 nm-900 nm, 20 nm-800 nm,
30 nm-700 nm, 50 nm-600 nm, 80 nm-500 nm, 100 nm-500 nm, 200 nm-500
nm, 300 nm-500 nm, or 400 nm-500 nm in diameter, e.g., 10 nm, 20
nm, 30 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm,
700 nm, 800 nm, or 900 nm. In certain cases, the bead/particle may
be made of a material that has a net negative or positive charge or
can be treated to have a net negative or positive charge. Exemplary
beads/particles include those made from organic or inorganic
polymers. Organic polymers include polymers such as, polystyrene,
carbon, polyacrylamide, etc. Inorganic polymers include silicon or
metal beads/particles. In certain cases, the beads/particles may
not be magnetic.
[0217] In certain cases, the tag may be a single stranded DNA or
RNA. The single stranded DNA or RNA may be hybridized to a probe
molecule prior to. In certain cases, the method may include
analysis of multiple analytes in a single sample. The second
binding members that bind to the different analytes in a sample may
include different single stranded DNA or RNA attached thereto as
tags and the different single stranded DNA or RNA may be hybridized
to different probes that further distinguish the different single
stranded DNA or RNA from each other. In other embodiments, the tags
attached to the different second binding members may have different
hairpin structures (e.g., length of the hairpin structure) that are
distinguishable. In yet another embodiment, the tags attached to
the different second binding members may have different lengths
that are distinguishable--for example, the tags may be double
stranded DNA of different lengths (e.g., 25 bp, 50 bp, 75 bp, 100
bp, 150 bp, 200 bp, or more). In certain cases, the tags attached
to the different second binding members may have different lengths
of polyethylene glycol (PEG) or may be DNA or RNA modified
differentially with PEG.
[0218] It is noted that reference to a tag or a tag molecule
encompasses a single tag or a single tag molecule as well as
multiple tags (that all may be identical). The tag may be any size
or shape. In some embodiments, the tag may be a nanoparticle or a
nanobead about 10 and 950 nm in diameter, e.g., 20-900 nm, 30-800
nm, 40-700 nm, 50-600 nm, 60-500 nm, 70-400 nm, 80-300 nm, 90-200
nm, 100-150 nm, 200-600 nm, 400-500 nm, 2-10 nm, 2-4 nm, or 3-4 nm
in diameter. The tag may be substantially spherical, for example a
spherical bead or nanobead, or hemispherical. The tag may be a
protein about 0.5 kDa to about 50 kDa in size, e.g., about 0.5 kDa
to about 400 kDa, about 0.8 kDa to about 400 kDa, about 1.0 kDa to
about 400 kDa, about 1.5 kDa to about 400 kDa, about 2.0 kDa to
about 400 kDa, about 5 kDa to about 400 kDa, about 10 kDa to about
400 kDa, about 50 kDa to about 400 kDa, about 100 kDa to about 400
kDa, about 150 kDa to about 400 kDa, about 200 kDa to about 400
kDa, about 250 kDa to about 400 kDa, about 300 kDa to about 400
kDa, about 0.5 kDa to about 300 kDa, about 0.8 kDa to about 300
kDa, about 1.0 kDa to about 300 kDa, about 1.5 kDa to about 300
kDa, about 2.0 kDa to about 300 kDa, about 5 kDa to about 300 kDa,
about 10 kDa to about 300 kDa, about 50 kDa to about 300 kDa, about
100 kDa to about 300 kDa, about 150 kDa to about 300 kDa, about 200
kDa to about 300 kDa, about 250 kDa to about 300 kDa, about 0.5 kDa
to about 250 kDa, about 0.8 kDa to about 250 kDa, about 1.0 kDa to
about 250 kDa, about 1.5 kDa to about 250 kDa, about 2.0 kDa to
about 250 kDa in size, about 5 kDa to about 250 kDa, about 10 kDa
to about 250 kDa, about 50 kDa to about 250 kDa, about 100 kDa to
about 250 kDa, about 150 kDa to about 250 kDa, about 200 kDa to
about 250 kDa, about 0.5 kDa to about 200 kDa, about 0.8 kDa to
about 200 kDa, about 1.0 kDa to about 200 kDa, about 1.5 kDa to
about 200 kDa, about 2.0 kDa to about 200 kDa in size, about 5 kDa
to about 200 kDa, about 10 kDa to about 200 kDa, about 50 kDa to
about 200 kDa, about 100 kDa to about 200 kDa, about 150 kDa to
about 200 kDa, about 0.5 kDa to about 100 kDa, about 0.8 kDa to
about 100 kDa, about 1.0 kDa to about 100 kDa, about 1.5 kDa to
about 100 kDa, about 2.0 kDa to about 100 kDa, about 5 kDa to about
100 kDa, about 10 kDa to about 100 kDa, about 50 kDa to about 100
kDa, about 0.5 kDa to about 50 kDa, about 0.8 kDa to about 50 kDa,
about 1.0 kDa to about 50 kDa, about 1.5 kDa to about 50 kDa, about
2.0 kDa to about 50 kDa, about 5 kDa to about 50 kDa, about 10 kDa
to about 50 kDa. about 10 kDa to about 90 kDa, about 10 kDa to
about 80 kDa, about 10 kDa to about 70 kDa, about 10 kDa to about
60 kDa, about 20 kDa to about 90 kDa, about 20 kDa to about 80 kDa,
about 20 kDa to about 70 kDa, about 20 kDa to about 60 kDa, about
40 kDa to about 90 kDa, about 40 kDa to about 80 kDa, about 40 kDa
to about 70 kDa, or about 40 kDa to about 60 kDa.
[0219] In certain embodiments, the tag may be a nanoparticle or
nanobead. As noted herein, the nanoparticle may be reversibly
(e.g., cleavably) attached to the second binding member. In certain
aspects, the nanoparticle may be a nanobead of a defined diameter.
In certain cases, the methods, systems, and devices of the present
disclosure may be used to simultaneously analyze a plurality of
different analytes in a sample. For such analysis a plurality of
second binding members that each specifically bind to a cognate
analyte may be used. Each of the different second binding member
may be attached to a different sized nanobead that may be used to
identify the second binding member. For example, the different
nanobead tags may have different diameters, such as, 1 nm, 2 nm, 4
nm, 6 nm, 8 nm, 10 nm, 12 nm, 14 nm, or larger, such as up to 20
nm, 30 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm,
700 nm, 800 nm, 900 nm, 950 nm, or 990 nm.
[0220] Exemplary nanoparticles that may be used as tags in the
present methods include gold nanoparticles or polystyrene
nanoparticles ranging in diameter from 5 nm-950 nm.
[0221] In certain cases, the tag may be a polymer, such as, a
nucleic acid. The presence of the tag may be determined by
detecting a signal characteristic of the tag, such as a signal
related to the size or length of the polymer tag. The size or
length of the polymer tag can be determined by measuring its
residence time in the pore or channel, e.g., by measuring duration
of transient blockade of current.
[0222] Elements which can be part of, all of, associated with, or
attached to the tag or label include: a nanoparticle; gold
particle; silver particle; silver, copper, zinc, or other metal
coating or deposit; polymer; drag-tag (as defined herein); magnetic
particle; buoyant particle; metal particle; charged moiety;
dielectrophoresis tag, silicon dioxide, with and without impurities
(e.g., quartz, glass, etc.); poly(methylmethacrylate) (PMMA);
polyimide; silicon nitride; gold; silver; quantum dot (including
CdS quantum dot); carbon dot; a fluorophore; a quencher; polymer;
polystyrene; Janus particle; scattering particle; fluorescent
particle; phosphorescent particle; sphere; cube; insulator;
conductor; bar-coded or labeled particle; porous particle; solid
particle; nanoshell; nanorod; microsphere; analyte such as a virus,
cell, parasite and organism; nucleic acid; protein; molecular
recognition element; spacer; PEG; dendrimer; charge modifier;
magnetic material; enzyme; DNA including aptamer sequence;
amplifiable DNA; repeated sequence of DNA; fusion or conjugate of
detectable elements with molecular recognition elements (e.g.,
engineered binding member); anti-antibody aptamer; aptamer directed
to antibody-binding protein; absorbed or adsorbed detectable
compound; heme; luciferin; a phosphor; an azido, or alkyne (e.g.,
terminal or non-terminal alkyne) or other click chemistry
participant.
[0223] In certain embodiments, the tag may be chosen to provide a
rate of capture that is sufficiently high to enable a rapid
analysis of a sample. In certain embodiments, the capture rate of
the tag may be about 1 event per 10 seconds, 1 event per 5 seconds,
1 event per second or higher. In certain embodiments, linear
polymer tags, such as, ribose polymers, deoxyribose polymers,
oligonucleotides, DNA, or RNA may be used.
[0224] In certain cases, linear polymer tags, such as, ribose
polymers, deoxyribose polymers, oligonucleotides, DNA, or RNA may
not be used as the capture rate for these tags may be too low for
certain applications. Tags that are hemispherical, spherical or
substantially spherical in shape and thus shorten the assay
duration may be used in applications requiring faster tag counting.
In certain cases, the size of the spherical or hemispherical tag
may be chosen based on the capture rate needed for the assay. For
example, for a higher capture rate, spherical or hemispherical tags
of larger size may be selected. In certain cases, the tag may be
spherical tag, such as, a nanoparticle/nanobead that has a capture
rate about a 10 times, 30 times, 50 times, 100 times, 300 times,
500 times, or a 1000 times faster than capture rate for a linear
tag, such as, a DNA tag, under the same measurement conditions.
[0225] In some embodiments, the tag is may be conjugated to an
antibody, for example, a CPSP antibody conjugate. In some
embodiments, the tag may be conjugated to an antibody with a
spacer, for example, a CPSP antibody conjugate with a spacer. In
some embodiments, the tag may be may be conjugated to an
oligonucleotide and an antibody, for example, a CPSP
oligonucleotide-antibody conjugate. In some embodiments, the tag
may be may be conjugated to an oligonucleotide and an antibody with
a spacer, for example, a CPSP oligonucleotide-antibody conjugate
with spacer. In some embodiments, the tag may be may be conjugated
to an oligonucleotide, for example, a CPSP oligonucleotide
conjugate.
[0226] In certain embodiments methods described herein may include
a specific binding member bound to a detectable label, such as a
signal-producing substance, such as chromagens, fluorescent
compounds, enzymes, chemiluminescent compounds, radioactive
compounds, particles (provided that they have fluorescent
properties) and the like. Examples of labels that include moieties
that produce light, e.g., acridinium compounds, and moieties that
produce fluorescence, e.g., fluorescein.
[0227] Any suitable signal-producing substance known in the art can
be used as a detectable label. For example, the detectable label
can be a radioactive label (such as 3H, 14C, 32P, 33P, 35S, 90Y,
99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm), an enzymatic
label (such as horseradish peroxidase, alkaline peroxidase, glucose
6-phosphate dehydrogenase, and the like (if enzymes are used then a
corresponding enzymatic substrate must also be added)), a
chemiluminescent label (such as acridinium esters, thioesters, or
sulfonamides; luminol, isoluminol, phenanthridinium esters, and the
like), a fluorescent label (such as fluorescein (e.g.,
5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein,
5(6)-carboxyfluorescein, 6-hexachloro-fluorescein,
6-tetrachlorofluorescein, fluorescein isothiocyanate, and the
like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots
(e.g., zinc sulfide-capped cadmium selenide), a thermometric label,
or an immuno-polymerase chain reaction label. An introduction to
labels, labeling procedures and detection of labels is found in
Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd
ed., Springer Verlag, N.Y. (1997), and in Haugland, Handbook of
Fluorescent Probes and Research Chemicals (1996), which is a
combined handbook and catalogue published by Molecular Probes,
Inc., Eugene, Oreg. A fluorescent label can be used in FPIA (see,
e.g., U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093,
and 5,352,803, which are hereby incorporated by reference in their
entireties). An acridinium compound can be used as a detectable
label in a homogeneous chemiluminescent assay (see, e.g., Adamczyk
et al., Bioorg. Med. Chem. Lett. 16: 1324-1328 (2006); Adamczyk et
al., Bioorg. Med. Chem. Lett. 4: 2313-2317 (2004); Adamczyk et al.,
Biorg. Med. Chem. Lett. 14: 3917-3921 (2004); and Adamczyk et al.,
Org. Lett. 5: 3779-3782 (2003)).
[0228] In one aspect, the acridinium compound is an
acridinium-9-carboxamide. Methods for preparing acridinium
9-carboxamides are described in Mattingly, J. Biolumin. Chemilumin.
6: 107-114 (1991); Adamczyk et al., J. Org. Chem. 63: 5636-5639
(1998); Adamczyk et al., Tetrahedron 55: 10899-10914 (1999);
Adamczyk et al., Org. Lett. 1: 779-781 (1999); Adamczyk et al.,
Bioconjugate Chem. 11: 714-724 (2000); Mattingly et al., In
Luminescence Biotechnology: Instruments and Applications; Dyke, K.
V. Ed.; CRC Press: Boca Raton, pp. 77-105 (2002); Adamczyk et al.,
Org. Lett. 5: 3779-3782 (2003); and U.S. Pat. Nos. 5,468,646,
5,543,524 and 5,783,699 (each of which is incorporated herein by
reference in its entirety for its teachings regarding same).
[0229] Another example of an acridinium compound is an
acridinium-9-carboxylate aryl ester. An example of an
acridinium-9-carboxylate aryl ester of formula II is
10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available
from Cayman Chemical, Ann Arbor, Mich.). Methods for preparing
acridinium 9-carboxylate aryl esters are described in McCapra et
al., Photochem. Photobiol. 4: 1111-21 (1965); Razavi et al.,
Luminescence 15: 245-249 (2000); Razavi et al., Luminescence 15:
239-244 (2000); and U.S. Pat. No. 5,241,070 (each of which is
incorporated herein by reference in its entirety for its teachings
regarding same). Such acridinium-9-carboxylate aryl esters are
efficient chemiluminescent indicators for hydrogen peroxide
produced in the oxidation of an analyte by at least one oxidase in
terms of the intensity of the signal and/or the rapidity of the
signal. The course of the chemiluminescent emission for the
acridinium-9-carboxylate aryl ester is completed rapidly, i.e., in
under 1 second, while the acridinium-9-carboxamide chemiluminescent
emission extends over 2 seconds. Acridinium-9-carboxylate aryl
ester, however, loses its chemiluminescent properties in the
presence of protein. Therefore, its use requires the absence of
protein during signal generation and detection. Methods for
separating or removing proteins in the sample are well-known to
those skilled in the art and include, but are not limited to,
ultrafiltration, extraction, precipitation, dialysis,
chromatography, and/or digestion (see, e.g., Wells, High Throughput
Bioanalytical Sample Preparation. Methods and Automation
Strategies, Elsevier (2003)). The amount of protein removed or
separated from the test sample can be about 40%, about 45%, about
50%, about 55%, about 60%, about 65%, about 70%, about 75%, about
80%, about 85%, about 90%, or about 95%. Further details regarding
acridinium-9-carboxylate aryl ester and its use are set forth in
U.S. patent application Ser. No. 11/697,835, filed Apr. 9, 2007.
Acridinium-9-carboxylate aryl esters can be dissolved in any
suitable solvent, such as degassed anhydrous N,N-dimethylformamide
(DMF) or aqueous sodium cholate.
[0230] Cleavable Linker
[0231] The tags used in the methods described herein may be
attached to specific binding member by a generic linker. The
cleavable linker ensures that the tag can be removed. The generic
linker may be a cleavable linker. For example, the tag may be
attached to the second binding member via a cleavable linker. The
complex of the first binding member-analyte-second binding member
may be exposed to a cleavage agent that mediates cleavage of the
cleavable linker. The linker can be cleaved by any suitable method,
including exposure to acids, bases, nucleophiles, electrophiles,
radicals, metals, reducing or oxidizing agents, light, temperature,
enzymes etc. Suitable linkers can be adapted from standard chemical
blocking groups, as disclosed in Greene & Wuts, Protective
Groups in Organic Synthesis, John Wiley & Sons. Further
suitable cleavable linkers used in solid-phase synthesis are
disclosed in Guillier et al. (Chem. Rev. 100:2092-2157, 2000). The
linker may be acid-cleavable, base-cleavable or photocleavable. A
redox reaction may be part of the cleavage scheme. The cleavable
linker may be a charged polymer.
[0232] The linker may be a photocleavable linker, a chemically
cleavable linker, or a thermally cleavable linker. Where the linker
is a photocleavable group, the cleavage agent may be light of
appropriate wavelength that disrupts or cleaves the photocleavable
group. In many embodiments, the wavelength of light used to cleave
the photocleavable linking group ranges from about 180 nm to 400
nm, e.g., from about 250 nm to 400 nm, or from about 300 nm to 400
nm. It is preferable that the light required to activate cleavage
does not affect the other components of the analyte. Suitable
linkers include those based on O-nitrobenzyl compounds and
nitroveratryl compounds. Linkers based on benzoin chemistry can
also be used (Lee et al., J. Org. Chem. 64:3454-3460, 1999).
[0233] Alternatively, where the cleavage linker is a chemically
cleavable group, the cleavage agent may be a chemical agent capable
of cleaving the group. A chemically cleavable linker may be cleaved
by oxidation/reduction-based cleavage, acid-catalyzed cleavage,
base-catalyzed cleavage, or nucleophilic displacement. For example,
where the linking group is a disulfide, dithiothreitol or
betamercaptoethanol may be used to release the tag. In yet other
embodiments where the linking group is a restriction site, the
agent is a catalytic agent, such as an enzyme which may be a
hydrolytic enzyme, a restriction enzyme, or another enzyme that
cleaves the linking group. For example, the restriction enzyme may
be a type I, type II, type IIS, type III and type IV restriction
enzyme.
[0234] In some embodiments, the cleavage linker is an enzymatic
cleavable sequence. In one aspect of any of the embodiments herein,
an enzymatic cleavable sequence is a nucleic acid sequence of 2, 3,
4, 5, 6, 7, 8, 9 or 10 nucleotides in length. In one embodiment,
the enzymatic cleavable sequence comprises a sequence of at least
10 nucleotides. In one embodiment, the enzymatic cleavable sequence
comprises a sequence of between 2 and 20 nucleotides. In one
embodiment, the enzymatic cleavable sequence comprises a sequence
of between 2 and 15 nucleotides. In one embodiment, the enzymatic
cleavable sequence comprises a sequence of between 4 and 10
nucleotides. In one embodiment, the enzymatic cleavable sequence
comprises a sequence of between 4 and 15 nucleotides.
[0235] For example, the cleavable linker may be an acridinium,
ethers such as substituted benzyl ether or derivatives thereof
(e.g., benzylhydryl ether, indanyl ether, etc.) that can be cleaved
by acidic or mild reductive conditions (e.g., hydrogen peroxide to
produce an acridone and a sulfonamide), a charged polymer generated
using P-elimination, where a mild base can serve to release the
product, acetals, including the thio analogs thereof, where
detachment is accomplished by mild acid, particularly in the
presence of a capturing carbonyl compound, photolabile linkages
(e.g., O-nitrobenzoyl, 7-nitroindanyl, 2-nitrobenzhydryl ethers or
esters, etc.), or peptide linkers, which are subject to enzymatic
hydrolysis, particularly where the enzyme recognizes a specific
sequence, such as a peptide for Factor Xa or enterokinase. Examples
of linkers include, but are not limited to, disulfide linkers, acid
labile linkers (including dialkoxybenzyl linkers), Sieber linkers,
indole linkers, t-butyl Sieber linkers, electrophilically cleavable
linkers, nucleophilically cleavable linkers, photocleavable
linkers, cleavage under reductive conditions, oxidative conditions,
cleavage via use of safety-catch linkers, and cleavage by
elimination mechanisms.
[0236] Electrophilically cleaved linkers are typically cleaved by
protons and include cleavages sensitive to acids. Suitable linkers
include the modified benzylic systems such as trityl,
p-alkoxybenzyl esters and p-alkoxybenzyl amides. Other suitable
linkers include tert-butyloxycarbonyl (Boc) groups and the acetal
system. The use of thiophilic metals, such as nickel, silver or
mercury, in the cleavage of thioacetal or other sulphur-containing
protecting groups can also be considered for the preparation of
suitable linker molecules.
[0237] For nucleophilic cleavage, groups such as esters that are
labile in water (i.e., can be cleaved simply at basic pH) and
groups that are labile to non-aqueous nucleophiles, can be used.
Fluoride ions can be used to cleave silicon-oxygen bonds in groups
such as triisopropyl silane (TIPS) or t-butyldimethyl silane
(TBDMS).
[0238] A linker susceptible to reductive cleavage may be used such
as with disulphide bond reduction. Catalytic hydrogenation using
palladium-based catalysts has been used to cleave benzyl and
benzyloxycarbonyl groups.
[0239] Oxidation-based approaches are well known in the art. These
include oxidation of p-alkoxybenzyl groups and the oxidation of
sulphur and selenium linkers. Aqueous iodine to cleave disulphides
and other sulphur or selenium-based linkers may also be used.
[0240] Safety-catch linkers are those that cleave in two steps. In
a preferred system the first step is the generation of a reactive
nucleophilic center followed by a second step involving an
intra-molecular cyclization that results in cleavage. For example,
levulinic ester linkages can be treated with hydrazine or
photochemistry to release an active amine, which can then be
cyclised to cleave an ester elsewhere in the molecule (Burgess et
al., J. Org. Chem. 62:5165-5168, 1997).
[0241] Elimination reactions may also be used. For example, the
base-catalysed elimination of groups such as Fmoc and cyanoethyl,
and palladium-catalysed reductive elimination of allylic systems,
may be used.
Integrated Digital Microfluidic and Analyte Detection Device
[0242] Systems, devices, and method are described herein that
relate to an integrated digital microfluidic and analyte detection
device.
[0243] In certain embodiments, the integrated digital microfluidic
and analyte detection device may have two modules: a sample
preparation module and an analyte detection module. In certain
embodiments, the sample preparation module and the analyte
detection module are separate or separate and adjacent. In certain
embodiments, the sample preparation module and the analyte
detection module are co-located, comingled or interdigitated. The
sample preparation module may include a series or plurality of
electrodes for moving, merging, diluting, mixing, separating
droplets of samples and reagents. The analyte detection module may
include an array of wells in which an analyte related signal is
detected. In certain cases, the detection module may also include
the series or plurality of electrodes for moving a droplet of
prepared sample to the array of wells. In certain embodiments, the
detection module may include an array of wells in a first substrate
(e.g., upper substrate) which is disposed over a second substrate
(e.g., lower substrate) separated by a gap. In these embodiments,
the array of wells is in an upside-down orientation. In certain
embodiments, the detection module may include an array of wells in
a second substrate (e.g., lower substrate) which is disposed below
a first substrate (e.g., upper substrate) separated by a gap. In
such embodiments, the first substrate and the second substrate are
in a facing arrangement. A droplet may be moved (e.g., by
electrical actuation) to the array of wells using electrode(s)
present in the first substrate and/or the second substrate. In
certain embodiments, the array of wells including the region in
between the wells may be hydrophobic. In other embodiments, the
series or plurality of electrodes may be limited to the sample
preparation module and a droplet of prepared sample (and/or a
droplet of immiscible fluid) may be moved to the detection module
using other means.
[0244] In certain embodiments, the sample preparation module may be
used for performing steps of an immunoassay. Any immunoassay format
may be used to generate a detectable signal which signal is
indicative of presence of an analyte of interest in a sample and is
proportional to the amount of the analyte in the sample. Exemplary
immunoassays are described herein.
[0245] In certain cases, the detection module includes the array of
wells that are optically interrogated to measure a signal related
to the amount of analyte present in the sample. The array of wells
may have sub-femtoliter volume, femtoliter volume, sub-nanoliter
volume, nanoliter volume, sub-microliter volume, or microliter
volume. For example the array of wells may be array of femoliter
wells, array of nanoliter wells, or array of microliter wells. In
certain embodiments, the wells in an array may all have
substantially the same volume. The array of wells may have a volume
up to 100 e.g., about 0.1 femtoliter, 1 femtoliter, 10 femtoliter,
25 femtoliter, 50 femtoliter, 100 femtoliter, 0.1 pL, 1 pL, 10 pL,
25 pL, 50 pL, 100 pL, 0.1 nL, 1 nL, 10 nL, 25 nL, 50 nL, 100 nL,
0.1 microliter, 1 microliter, 10 microliter, 25 microliter, 50
microliter, or 100 microliter.
[0246] In certain embodiments, the sample preparation module and
the detection module may both be present on a single base substrate
and both the sample preparation module and the detection module may
include a series or plurality of electrodes for moving liquid
droplets. In certain embodiments, such a device may include a first
substrate and a second substrate, where the second substrate is
positioned over the first substrate and separated from the first
substrate by a gap. The first substrate may include a first portion
(e.g., proximal portion) at which the sample preparation module is
located, where a liquid droplet is introduced into the device, and
a second portion (e.g., distal portion) towards which the liquid
droplet moves, at which second portion the detection module is
located. It will be understood by one skilled in the art that the
use of "proximal" in view of "distal" and "first" in view of
"second" are relative terms and are interchangeable with respect to
each other. In certain embodiments, first portion and the second
portion are separate or separate and adjacent. In certain
embodiments, the first portion and the second portion are
co-located, comingled or interdigitated. The first substrate may
include a series or plurality of electrodes overlayed on an upper
surface of the first substrate and extending from the first portion
to the second portion. The first substrate may include a layer
disposed on the upper surface of the first substrate, covering the
series or plurality of electrodes, and extending from the first
portion to the second portion. The first layer may be made of a
material that is a dielectric and a hydrophobic material. Examples
of a material that is dielectric and hydrophobic include
polytetrafluoroethylene material (e.g., Teflon.RTM.) or a
fluorosurfactant (e.g., FluoroPel.TM.). The first layer may be
deposited in a manner to provide a substantially planar surface. An
array of wells may be positioned in the second portion of the first
substrate and overlying a portion of the series or plurality of
electrodes, and form the detection module. The array of wells may
be positioned in the first layer. In certain embodiments, prior to
or after fabrication of the array of wells in the first layer, a
hydrophilic layer may be disposed over the first layer in the
second portion of the first substrate to provide an array of wells
that have a hydrophilic surface. The space/gap between the first
and second substrates may be filled with air or an immiscible
fluid. In certain embodiments, the space/gap between the first and
second substrates may be filled with air.
[0247] In certain embodiments, the sample preparation module and
the detection module may both be fabricated using a single base
substrate but a series or plurality of electrodes for moving liquid
droplets may only be present only in the sample preparation module.
In such an embodiment, the first substrate may include a series or
plurality of electrodes overlayed on an upper surface of the first
substrate at the first portion of the first substrate, where the
series or plurality of electrodes do not extend to the second
portion of the first substrate. In such embodiments, the the series
or plurality of electrodes are only positioned in the first
portion. A first layer of a dielectric/hydrophobic material (e.g.,
Teflon), as described above, may be disposed on the upper surface
of the first substrate and may cover the series or plurality of
electrodes. In certain embodiments, the first layer may be disposed
only over a first portion of the first substrate. In other
embodiments, the first layer may be disposed over the upper surface
of the first substrate over the first portion as well as the second
portion. An array of wells may be positioned in the first layer in
the second portion of the first substrate, forming the detection
module that does not include a series or plurality of electrodes
present under the array of wells.
[0248] In certain cases, the first layer may be a dielectric layer
and a second layer of a hydrophobic material may be disposed over
the dielectric layer. The array of wells may be positioned in the
hydrophobic layer. Prior to or after fabrication of the array of
wells in the hydrophobic layer, a hydrophilic layer may be disposed
over the hydrophobic layer in the second portion of the first
substrate.
[0249] In certain embodiments, the second substrate may extend over
the first and second portions of the first substrate. In such an
embodiment, the second substrate may be substantially transparent,
at least in region overlaying the array of wells. In other cases,
the second substrate may be disposed in a spaced apart manner over
the first portion of the first substrate and may not be disposed
over the second portion of the first substrate. Thus, in certain
embodiments, the second substrate may be present in the sample
preparation module but not in the detection module.
[0250] In certain cases, the second substrate may include a
conductive layer that forms an electrode. The conductive layer may
be disposed on a lower surface of the second substrate. The
conductive layer may be covered by a first layer made of a
dielectric/hydrophobic material, as described above. In certain
cases, the conductive layer may be covered by a dielectric layer.
The dielectric layer may be covered by a hydrophobic layer. The
conductive layer and any layer(s) covering it may be disposed
across the lower surface of the second substrate or may only be
present on the first portion of the second substrate. In certain
embodiments, the second substrate may extend over the first and
second portions of the first substrate. In such an embodiment, the
second substrate and any layers disposed thereupon (e.g.,
conductive layer, dielectric layer, etc.) may be substantially
transparent, at least in region overlaying the array of wells.
[0251] In other cases, the series or plurality of electrodes on the
first substrate may be configured as co-planar electrodes and the
second substrate may not include an electrode.
[0252] In certain cases, the electrodes present in the first layer
and/or the second layer may be fabricated from a substantially
transparent material, such as indium tin oxide, fluorine doped tin
oxide (FTO), doped zinc oxide, and the like.
[0253] In some embodiments, the sample preparation module and the
detection module may be fabricated on a single base substrate. In
other embodiments, the sample preparation module and the detection
modules may be fabricated on separate substrates that may
subsequently be joined to form an integrated microfluidic and
analyte detection device. In certain embodiments, the first and
second substrates may be spaced apart using a spacer that may be
positioned between the substrates.
[0254] The devices described herein may be planar and may have any
shape, such as, rectangular or square, rectangular or square with
rounded corners, circular, triangular, and the like.
[0255] Droplet-based microfluidics refer to generating and
actuating (such as moving, merging, splitting, etc.) liquid
droplets via active or passive forces. Examples of active forces
include, but are not limited to, electric field. Exemplary active
force techniques include electrowetting, dielectrophoresis,
opto-electrowetting, electrode-mediated, electric-field mediated,
electrostatic actuation, and the like or a combination thereof. In
some examples, the device may actuate liquid droplets across the
upper surface of the first layer (or upper surface of the second
layer, when present) in the gap via droplet-based microfluidics,
such as, electrowetting or via a combination of electrowetting and
continuous fluid flow of the liquid droplets. In other examples,
the device may include micro-channels to deliver liquid droplets
from the sample preparation module to the detection module. In
other examples, the device may rely upon the actuation of liquid
droplets across the surface of the hydrophobic layer in the gap via
droplet based microfluidics. Electrowetting may involve changing
the wetting properties of a surface by applying an electrical field
to the surface, and affecting the surface tension between a liquid
droplet present on the surface and the surface. Continuous fluid
flow may be used to move liquid droplets via an external pressure
source, such as an external mechanical pump or integrated
mechanical micropumps, or a combination of capillary forces and
electrokinetic mechanisms. Examples of passive forces include, but
are not limited to, T-junction and flow focusing methods. Other
examples of passive forces include use of denser immiscible
liquids, such as, heavy oil fluids, which can be coupled to liquid
droplets over the surface of the first substrate and displace the
liquid droplets across the surface. The denser immiscible liquid
may be any liquid that is denser than water and does not mix with
water to an appreciable extent. For example, the immiscible liquid
may be hydrocarbons, halogenated hydrocarbons, polar oil, non-polar
oil, fluorinated oil, chloroform, dichloromethane, tetrahydrofuran,
1-hexanol, etc.
[0256] The space between the first and second substrates may be up
to 1 mm in height, e.g., 0.1 .mu.m, 0.5 .mu.m, 1 .mu.m, 5 .mu.m, 10
.mu.m, 20 .mu.m, 50 .mu.m, 100 .mu.m, 140 .mu.m, 200 .mu.m, 300
.mu.m, 400 .mu.m, 500 .mu.m, 1 .mu.m-500 .mu.m, 100 .mu.m-200
.mu.m, etc. The volume of the droplet generated and moved in the
devices described herein may range from about 10 .mu.l to about 5
picol, such as, 10 .mu.l-1 picol, 7.5 .mu.l-10 picol, 5 .mu.l-1 nL,
2.5 .mu.l-10 nL, or 1 .mu.l-100 nL, 800-200 nL, 10 nL-0.5 .mu.l
e.g., 10 .mu.l, 1 .mu.l, 800 nL, 100 nL, 10 nL, 1 nL, 0.5 nL, 10
picol, or lesser.
[0257] FIG. 1A illustrates an exemplary integrated digital
microfluidic and analyte detection device 10. The device 10
includes a first substrate 11 and a second substrate 12, where the
second substrate 12 is positioned over the first substrate 11 and
separated from the first substrate by a gap 13. As illustrated in
FIG. 1A, the second substrate 12 is the same length as the first
substrate 11. However, in other exemplary devices, the first
substrate 11 and the second substrate 12 may be of different
lengths. The second substrate may or may not include an electrode.
The first substrate 11 includes a first portion 15, where liquid
droplet, such as, a sample droplet, reagent droplet, etc., is
introduced onto the first substrate 11. The first substrate 11
includes a second portion 16, towards which a liquid droplet is
moved. The first portion 15 may also be referred to as the sample
preparation module and the second portion 16 may be referred to as
the analyte detection module. The first substrate 11 includes a
series or plurality of electrodes 17 positioned on the upper
surface of the first substrate 11. A layer 18 of
dielectric/hydrophobic material (e.g., Teflon which is both
dielectric and hydrophobic) is disposed on the upper surface of the
first substrate and covers the series or plurality of electrodes
17. An array of wells 19 is positioned in the layer 18 on the
second portion 16 of the first substrate.
[0258] FIG. 1B illustrates another example of an integrated digital
microfluidic and analyte detection device 20 that includes a first
substrate 21 and a second substrate 22, where the second substrate
22 is positioned over the first substrate 20 and separated from an
upper surface of the first substrate by a gap 23. The first
substrate 21 includes a first portion 25, where a liquid is
introduced onto the first substrate 21, and a second portion 26,
towards which liquid is directed for detection of an analyte
related signal. The first substrate 21 includes a series or
plurality of electrodes 27 positioned on the upper surface of the
first substrate. A layer 28 of dielectric material is positioned on
the upper surface of the first substrate 21 and covers the series
or plurality of electrodes 27. In this exemplary device, the series
or plurality of electrodes 27 is positioned on only the first
portion of the first substrate 21. The second substrate 22 may or
may not include an electrode.
[0259] FIG. 2A illustrates another exemplary integrated digital
microfluidic and analyte detection device 30. The device 30
includes a first substrate 31 and a second substrate 32, where the
second substrate 32 is positioned over the first substrate 31 and
separated from an upper surface of the first substrate by a gap 33.
The first substrate 31 includes a first portion 35, where liquid
droplet, such as, a sample droplet, reagent droplet, etc., is
introduced onto the first substrate 31. The first substrate 31
includes a second portion 36, towards which a liquid droplet is
moved. The first portion may also be referred to as the sample
preparation module and the second portion may be referred to as the
detection module. The first substrate 31 includes a series or
plurality of electrodes 37 positioned on the upper surface of the
first substrate. A layer 38 of dielectric material is disposed on
the upper surface of the first substrate and covers the series or
plurality of electrodes 37. A layer 34 of hydrophobic material is
overlayed on the dielectric layer 38. An array of wells 39 is
positioned in the hydrophobic layer 34 on the second portion of the
first substrate 31. The array of wells may have a hydrophilic or
hydrophobic surface.
[0260] FIG. 2B illustrates another example of an integrated digital
microfluidic and analyte detection device 40 that includes a first
substrate 41 and a second substrate 42, where the second substrate
42 is positioned over the first substrate 40 and separated from an
upper surface of the first substrate by a gap 43. The first
substrate includes a first portion 45, where a liquid is introduced
onto the first substrate 41, and a second portion 46, towards which
liquid is directed for detection of an analyte related signal. The
first substrate 41 includes a series or plurality of electrodes 47
positioned on the upper surface of the first substrate. A layer 48
of dielectric material is positioned on the upper surface of the
first substrate 41 and covers the series or plurality of electrodes
47. In this exemplary device, the series or plurality of electrodes
47 is positioned on only the first portion 45 of the first
substrate 41. The dielectric layer 48 covers the entire upper
surface of the first substrate 41 and the hydrophobic layer 44
covers the entire upper surface of the dielectric layer. An array
of wells 49 is positioned in the hydrophobic layer 44, and the
array of wells 49 are positioned at only a portion of the
hydrophobic layer overlaying the second portion 46 of the first
substrate 41. In this example, the dielectric layer 48 is shown as
extending over the entire upper surface of the first substrate 41.
In other examples, the dielectric layer and the hydrophobic layer
may be limited to the first portion and the wells may be positioned
in a hydrophilic layer positioned on the second portion of the
first substrate.
[0261] In some examples, liquid may be introduced into the gap via
a droplet actuator (not illustrated). In other examples, liquid may
be into the gap via a fluid inlet, port, or channel. Additional
associated components of the device are not illustrated in the
figures. Such figures may include chambers for holding sample, wash
buffers, binding members, enzyme substrates, waste fluid, etc.
Assay reagents may be contained in external reservoirs as part of
the integrated device, where predetermined volumes may be moved
from the reservoir to the device surface when needed for specific
assay steps. Additionally, assay reagents may be deposited on the
device in the form of dried, printed, or lyophilized reagents,
where they may be stored for extended periods of time without loss
of activity. Such dried, printed, or lyophilized reagents may be
rehydrated prior or during analyte analysis.
[0262] In some examples, the first substrate can be made from a
flexible material, such as paper (with ink jet printed electrodes)
or polymers. In other examples, the first substrate can be made
from a non-flexible material, such as for example, printed circuit
board, plastic or glass or silicon. In some examples, the first
substrate is made from a single sheet, which then may undergo
subsequent processing to create the series or plurality of
electrodes. In some examples, multiple series or plurality of
electrodes may be fabricated on a first substrate which may be cut
to form a plurality of first substrates overlayed with a series or
plurality of electrodes. In some examples, the electrodes may be
bonded to the surface of the conducting layer via a general
adhesive agent or solder. The second substrate may be made from any
suitable material including but not limited to a flexible material,
such as paper (with or without ink jet printed electrodes),
polymers, printed circuit board, and the like.
[0263] In some examples, the electrodes are comprised of a metal,
metal mixture or alloy, metal-semiconductor mixture or alloy, or a
conductive polymer. Some examples of metal electrodes include
copper, gold, indium, tin, indium tin oxide, and aluminum. In some
examples, the dielectric layer comprises an insulating material,
which has a low electrical conductivity or is capable of sustaining
a static electrical field. In some examples, the dielectric layer
may be made of porcelain (e.g., a ceramic), polymer or a plastic.
In some examples, the hydrophobic layer may be made of a material
having hydrophobic properties, such as for example Teflon and
generic fluorocarbons. In another example, the hydrophobic material
may be a fluorosurfactant (e.g., FluoroPel). In embodiments
including a hydrophilic layer deposited on the dielectric layer, it
may be a layer of glass, quartz, silica, metallic hydroxide, or
mica.
[0264] One having ordinary skill in the art would appreciate that
the array (e.g., series) of electrodes may include a certain number
of electrodes per unit area of the first substrate, which number
may be increased or decreased based on size of the electrodes and a
presence or absence of inter-digitated electrodes. Electrodes may
be fabricated using a variety of processes including,
photolithography, atomic layer deposition, laser scribing or
etching, laser ablation, flexographic printing and ink-jet printing
of electrodes.
[0265] In some examples, a special mask pattern may be applied to a
conductive layer disposed on an upper surface of the first
substrate followed by laser ablation of the exposed conductive
layer to produce a series or plurality of electrodes on the first
substrate.
[0266] In some examples, the electrical potential generated by the
series or plurality of electrodes transfer liquid droplets formed
on an upper surface of the first layer (or the second layer when
present) covering the series or plurality of electrodes, across the
surface of the digital microfluidic device to be received by the
array of wells. Each electrode may be capable of independently
moving the droplets across the surface of the digital microfluidic
device.
[0267] FIG. 3A illustrates a side view of an exemplary integrated
digital microfluidic and analyte detection device 100 with a liquid
droplet being moved in the gap 170. The device 100 includes a first
substrate 110 and a second substrate 120, where the second
substrate 120 is positioned over the first substrate 110 and
separated from an upper surface of the first substrate by a gap
170. The first substrate 110 includes a first portion 115, where
liquid droplet, such as, a sample droplet, reagent droplet, etc.,
is introduced onto the first substrate 110. The first substrate 110
includes a second portion 130, towards which a liquid droplet is
moved. The first portion may also be referred to as the sample
preparation module and the second portion may be referred to as the
detection module. The first substrate 110 includes a series or
plurality of electrodes 145 positioned on the upper surface of the
first substrate. A layer 150 of dielectric material is disposed on
the upper surface of the first substrate and covers the series or
plurality of electrodes 145. A layer 155 of hydrophobic material is
overlayed on the dielectric layer 150. An array of wells 160 is
positioned in the hydrophobic layer 155 on the second portion of
the first substrate 110. The array of wells may have a hydrophilic
or hydrophobic surface. As illustrated in FIG. 3A, a liquid droplet
is illustrated as being actuated from the first portion 115 to the
second portion 130 containing the array of wells 160. A liquid
droplet 180 containing a plurality of beads or particles 190 is
being moved across the first portion 115 and over to the second
portion 130 via active directional movement using the series or
plurality of electrodes 145. The arrow indicates the direction of
movement of the liquid droplet. Although not shown here,
polarizable oil may be used to move the droplet and seal the wells.
Although beads/particles are illustrated here, the droplet may
include analyte molecules instead of or in addition to the solid
supports.
[0268] FIG. 3B illustrates a side view of an exemplary integrated
digital microfluidic and analyte detection device 101 with a
droplet 180 being moved in the gap 170 from the first portion 115
to the second portion 130 that includes the array of wells 160. The
device 101 includes a first substrate 110 and a second substrate
120, where the second substrate 120 is positioned over the first
substrate 110 and separated from an upper surface of the first
substrate by a gap 170. The first substrate 110 includes a first
portion 115, where liquid droplet, such as, a sample droplet,
reagent droplet, etc., is introduced onto the first substrate 110.
The first substrate 110 includes a second portion 130, towards
which a liquid droplet is moved. The first portion may also be
referred to as the sample preparation module and the second portion
may be referred to as the detection module. The first substrate 110
includes a series or plurality of electrodes 145 positioned on the
upper surface at the first portion 115 of the first substrate. A
layer 150 of dielectric material is disposed on the upper surface
of the first substrate and covers the series or plurality of
electrodes 145. A layer 155 of hydrophobic material is overlayed on
the dielectric layer 150. An array of wells 160 is positioned in
the hydrophobic layer 155 on the second portion of the first
substrate 110. The array of wells may have a hydrophilic or
hydrophobic surface. Movement across the surface of the first
portion of the device is via the electrodes 145 and then the
droplet 180 is moved to the second portion using passive fluid
force, such as capillary movement through capillary element formed
by 191 and 192. In some examples, the capillary element may include
a hydrophilic material for facilitating movement of the aqueous
droplet from the first portion to the second portion in the absence
of an applied electric field generated by the series or plurality
of electrodes. In some examples, a striping of a hydrophobic
material may be disposed next to the hydrophilic capillary space.
The striping of hydrophobic material may be used to move a droplet
of immiscible fluid over to the array of wells in absence of the
digital microfluidics electrodes. Some examples of liquids that may
flow through a hydrophobic capillary element includes heavy oil
fluids, such as fluorinated oils, can be used to facilitate liquid
droplet movement over the array of wells. In other examples, oil
droplets may also be utilized to remove excess droplets.
[0269] In addition to moving aqueous-based fluids, immiscible
fluids, such as organic based immiscible fluids, may also be moved
by electrical-mediated actuation. It is understood that droplet
actuation is correlated with dipole moment and dielectric constant,
which are interrelated, as well as with conductivity. In certain
embodiments, the immiscible liquid may have a molecular dipole
moment greater than about 0.9 D, dielectric constant greater than
about 3 and/or conductivities greater than about 10.sup.-9 S
m.sup.-1. Examples of movable immiscible liquids and
characteristics thereof are discussed in Chatterjee, et al. Lab on
Chip, 6, 199-206 (2006). Examples of use of the immiscible liquid
in the analyte analysis assays disclosed herein include aiding
aqueous droplet movement, displacing aqueous fluid positioned above
the wells, displacing undeposited beads/particles/analyte molecules
from the wells prior to optical interrogation of the wells, sealing
of the wells, and the like. Some examples of organic-based
immiscible fluids that are moveable in the devices disclosed herein
include 1-hexanol, dichloromethane, dibromomethane, THF and
chloroform. Organic-based oils that satisfy the above mentioned
criteria would also be expected to be moveable under similar
conditions. In some embodiments using immiscible fluid droplets,
the gap/space in the device may be filled with air.
[0270] FIG. 4A illustrates a liquid droplet 180 containing beads or
particles 190 that has been moved to the second portion of the
integrated device of FIG. 3A and is positioned over the array of
wells 160. The droplet may be continuously moved over the array of
wells in linear or reciprocating motion or movement and may be
paused over the array of wells. Moving of the droplet and/or
pausing the droplet over the array of wells facilitates the
deposition of the particles or beads 190 into the array of wells
160. The wells are dimensioned to include one bead/particle. In the
device illustrated in FIG. 4A, the droplet is moved over the array
of wells using the series or plurality of electrodes 145. Although
beads/particles are depicted here, droplets contain analyte
molecules may also be moved in a similar manner, and by pausing the
droplet containing the analyte molecules above the wells for a
sufficient period of time to allow for the analyte molecules to
diffuse into the wells before the immiscible fluid seals the wells.
The wells are dimensioned to include one bead/particle. The wells
can also be dimensioned to include one analyte molecule per
well.
[0271] FIG. 4B illustrates a liquid droplet 185 containing beads or
particles 190 that has been moved to the second portion of the
integrated device of FIG. 3B and is positioned over the array of
wells without using a series or plurality of electrodes. In FIG.
4B, a droplet of hydrophobic liquid 195 (such as an immiscible
fluid) is being used to move the liquid droplet over the well array
to facilitate deposition of the beads/particles 190 into the wells
160. The direction of the arrow indicates the direction in which
the droplet 185 is being moved.
[0272] FIG. 5 shows a hydrophobic fluid droplet 62 (e.g.,
polarizable fluid) being moved over the first portion 55 using the
series or plurality of electrodes 57. The depicted device 50
includes a a first substrate 51 and a second substrate 52, where
the second substrate 52 is positioned over the first substrate 51
and separated from an upper surface of the first substrate by a gap
53. The first substrate 51 includes a first portion 55, where
liquid droplet, such as, a sample droplet, reagent droplet, etc.,
is introduced onto the first substrate 51. The first substrate 51
includes a second portion 56, towards which a liquid droplet is
moved. The first substrate 51 includes a series or plurality of
electrodes 57 positioned on the upper surface at the first portion
55 of the first substrate. A layer 58 of dielectric material is
disposed on the upper surface of the first substrate and covers the
series or plurality of electrodes 57. A layer 54 of hydrophobic
material is overlayed on the dielectric layer 58. An array of wells
59 is positioned in the hydrophobic layer 54 on the second portion
of the first substrate 51. The array of wells may have a
hydrophilic or hydrophobic surface. A capillary element 60 is
formed by deposition of two stripes of a hydrophobic material on
the first 51 and second substrates 52. The hydrophobic capillary
facilitates movement of the hydrophobic fluid droplet 62 to the
array of wells 59, in absence of the series or plurality of
electrodes in the second portion 56. In other embodiments, the
capillary element may be formed by deposition of two stripes of a
hydrophilic material on the first 51 and second substrates 52. The
hydrophilic material facilitates movement of an aqueous droplet to
the array of wells 59, in absence of the series or plurality of
electrodes in the second portion 56. In certain embodiments, the
capillary element may include a pair of stripes of hydrophilic
material alternating with a pair of stripes of hydrophobic
material. An aqueous droplet may be directed to the region at which
a pair of hydrophilic stripes is positioned, while a droplet of
immiscible fluid may be directed to the region at which a pair of
hydrophobic stripes is positioned.
[0273] FIG. 6 depicts another embodiment of an integrated digital
microfluidics and analyte detection device. The device 600 includes
a bottom layer 601 over which an array of electrodes 607 is formed.
The array of electrodes is covered by a dielectric layer 608. A
hydrophobic layer 609 is disposed only in the first portion 605 of
the bottom substrate. A hydrophilic layer 610 is disposed on the
second portion 606 of the bottom substrate 601. An array of wells
is located in the second portion in the hydrophilic layer 610. A
top substrate 602 separated from the bottom substrate by a
gap/space 603 is also depicted. The top substrate 602 includes a
dielectric layer 608 disposed on a bottom surface of the top
substrate over the first portion of the bottom substrate. The top
substrate includes a hydrophilic layer 610 disposed on a bottom
surface of the top substrate across from the second portion of the
bottom substrate. An aqueous droplet 611 does not wet the
hydrophobic layer and upon reaching the hydrophilic second portion
the droplet 611 spreads over the array of wells 619, thereby
facilitating movement of the aqueous phase via passive capillary
forces. In a similar manner, the above concept may be reversed to
facilitate wetting and spreading of an organic-based immiscible
fluid over the wells. In this case, the top and bottom substrate on
the second portion can be coated with a hydrophobic
material/coating, thereby allowing an organic-based immiscible
fluid to flow over the wells via passive capillary forces.
[0274] As used herein, digital microfluidics refers to use of a a
series of electrodes to manipulate droplets in a microfluidics
device, e.g., move droplets, split droplets, merge droplets, etc.
in a small space. As used herein, the terms "droplet(s)" and
"fluidic droplet(s)" are used interchangeably to refer to a
discrete volume of liquid that is roughly spherical in shape and is
bounded on at least one side by a wall or substrate of a
microfluidics device. Roughly spherical in the context of the
droplet refers to shapes such as spherical, partially flattened
sphere, e.g., disc shaped, slug shaped, truncated sphere,
ellipsoid, hemispherical, or ovoid. The volume of the droplet in
the devices disclosed herein may range from about 10 .mu.l to about
5 pL, such as, 10 .mu.l-1 pL, 7.5 .mu.l-10 pL, 5 .mu.l-1 nL, 2.5
.mu.l-10 nL, or 1 .mu.l-100 nL, e.g., 10 .mu.l, 5 .mu.l, 1 .mu.l,
800 nL, 500 nL, or lesser.
[0275] In some examples, the array of wells includes a plurality of
individual wells. The array of wells may include a plurality of
wells that may range from 10 to 10.sup.9 in number per 1 mm.sup.2.
In certain cases, an array of about 100,000 to 500,000 wells (e.g.,
femtoliter wells) covering an area approximately 12 mm.sup.2 may be
fabricated. Each well may measure about 4.2 .mu.m wide.times.3.2
.mu.m deep (volume approximately 50 femtoliters), and may be
capable of holding a single bead/particle (about 3 .mu.m diameter).
At this density, the femtoliter wells are spaced at a distance of
approximately 7.4 .mu.m from each other. In some examples, the well
array may be fabricated to have individual wells with a diameter of
10 nm to 10,000 nm.
[0276] The placement of single beads/particles/analyte molecules in
the wells allows for either a digital readout or analog readout.
For example, for a low number of positive wells (<.about.70%
positive) Poisson statistics can be used to quantitate the analyte
concentration in a digital format; for high numbers of positive
wells (>.about.70%) the relative intensities of signal-bearing
wells are compared to the signal intensity generated from a single
bead/particle/analyte molecule, respectively, and used to generate
an analog signal. A digital signal may be used for lower analyte
concentrations, whereas an analog signal may be used for higher
analyte concentrations. A combination of digital and analog
quantitation may be used, which may expand the linear dynamic
range. As used herein, a "positive well" refers to a well that has
a signal related to presence of a bead/particle/analyte molecule,
which signal is above a threshold value. As used herein, a
"negative well" refers to a well that may not have a signal related
to presence of a bead/particle/analyte molecule. In certain
embodiments, the signal from a negative well may be at a background
level, i.e., below a threshold value.
[0277] The wells may be any of a variety of shapes, such as,
cylindrical with a flat bottom surface, cylindrical with a rounded
bottom surface, cubical, cuboidal, frustoconical, inverted
frustoconical, or conical. In certain cases, the wells may include
a sidewall that may be oriented to facilitate the receiving and
retaining of a microbead or microparticle present liquid droplets
that have been moved over the well array. In some examples, the
wells may include a first sidewall and a second sidewall, where the
first sidewall may be opposite the second side wall. In some
examples, the first sidewall is oriented at an obtuse angle with
reference to the bottom of the wells and the second sidewall is
oriented at an acute angle with reference to the bottom of the
wells. The movement of the droplets may be in a direction parallel
to the bottom of the wells and from the first sidewall to the
second sidewall.
[0278] In some examples, the array of wells can be fabricated
through one or more of molding, pressure, heat, or laser, or a
combination thereof. In some examples, the array of wells may be
fabricated using nanoimprint/nanosphere lithography. Other
fabrication methods well known in the art may can also be used.
[0279] FIGS. 7A-7B illustrate several exemplary sidewall
orientations of the wells. As illustrated in FIGS. 7A-B, the wells
comprise a first sidewall opposite to a second sidewall. FIG. 7A
illustrates a vertical cross-section showing individual wells 460
in the array of wells. FIG. 7A illustrates a first sidewall 401 and
a second sidewall 402. The first side wall is at an obtuse angle
with reference to a bottom surface 143 of the well and the second
side wall is at an acute angle with reference to a bottom surface
143 of the well. The arrow illustrates the direction in which a
liquid droplet moves across the array. This orientation of the
sidewalls of the wells facilitates receiving and retaining
beads/particles/analyte molecules 490.
[0280] In FIG. 7B, a top portion 415 of the first sidewall 410 is
oriented at an obtuse angle with reference to a bottom 412 of the
wells and a bottom portion 416 of the first sidewall 410 is
oriented perpendicular to the bottom 412 of the wells, and the
second sidewall 411 is oriented perpendicular to the bottom 412 of
the wells, where movement of liquid droplets is in a direction
parallel to the bottom of the wells and from the first sidewall to
the second sidewall, where the top portion of the first sidewall is
at an opening of the wells.
[0281] The integrated devices described herein may be fabricated by
a number of methods. In certain cases, the methods may involve a
combination of laser ablation, spray coating, roll to roll,
flexographic printing, and nanoimprint lithography (NIL) to
construct the first substrate, series or plurality of electrode,
dielectric layer and hydrophobic layer.
[0282] In some examples, a plurality of rollers may unwind a first
roll to drive the first substrate to a first position. A conductive
material may then be applied to the first substrate. The conductive
material may be patterned into a series or plurality of electrodes.
In some examples, the printer device comprising one or more coating
rollers to apply the at least one of the hydrophobic or the
dielectric material to the at least one electrode pattern on the
first substrate. In some examples, the coating rollers are to apply
an anti-fouling material to the first substrate.
[0283] In some examples, the system further comprises a merger to
align the first substrate with the second substrate. In some
examples, the merger comprises two rollers. Also, some of the
disclosed examples include a curing station to cure the hydrophobic
material or the dielectric material. Some of the disclosed examples
also include a bonding station to bond at least a first portion of
the first substrate with at least a first portion of the second
substrate. The bonded portions include the electrode pattern. The
method also includes associating the first substrate and the second
substrate at a spaced apart distance. The space between the first
and second substrates may be about 0.01 mm to 1 mm in height, e.g.,
0.1 .mu.m, 0.5 .mu.m, 1 .mu.m, 5 .mu.m, 10 .mu.m, 20 .mu.m, 50
.mu.m, 100 .mu.m, 140 .mu.m, 200 .mu.m, 300 .mu.m, 400 .mu.m, 500
.mu.m, 1 .mu.m-500 .mu.m, 100 .mu.m-200 .mu.m, etc.
[0284] In some examples, the method includes embossing the first
substrate to create one or more projections on the first substrate.
In such examples, the projections are to separate the first
substrate and the second substrate at the spaced apart
distance.
[0285] The devices of the present disclosure may be operated
manually or automatically or semiautomatically. In certain cases,
the devices may be operated by a processor that runs a program for
carrying out the steps required for generating an analyte related
signal and detecting the signal. As used hereon, the phrase
"analyte related signal" or "analyte associated signal" refers to a
signal that is indicative of presence of an analyte and is
proportional to the amount of the analyte in a sample. The signal
may be fluorescence, chemiluminescence, colorimetric,
turbidimetric, etc. In certain cases, the read out may be digital,
for example, the number of positive counts (e.g., wells) is
compared to the number of negative counts (e.g., wells) to obtain a
digital count.
[0286] FIG. 8 is a diagram of a first exemplary system or assembly
500 for creating a base substrate of an integrated digital
microfluidics and analyte detection device. The first example
assembly 500 includes a series or a plurality of rollers, including
a first roller 502, a second roller 504, and a third roller 506,
which operate in synchronized rotation to drive a base substrate
508 through the first example assembly 500. The first example
assembly 500 can include rollers in addition to the first through
third rollers 502, 504, 506 to move the base substrate 508 through
the assembly using roll-to-roll techniques. Other examples may use
conveyors, pulleys and/or any other suitable transport
mechanism(s).
[0287] In the first example assembly 500, the first roller 502
rotates to unwind the base substrate 508, which, in some examples,
is a single sheet in a rolled configuration. The base substrate 508
includes a first layer 510 and a second layer 512. In this example,
the first layer 510 comprises a non-conductive flexible substrate
or web, such as for example a plastic, and the second layer 512
includes a conductive material. The conductive material of the
second layer 512 can be, for example, a metal such as gold, silver,
or copper, or a non-metallic conductor, such as a conductive
polymer. In other examples different metal(s) or combination(s) of
metal(s) and/or conductive polymer(s) may be used. In some
examples, the base substrate 508 includes an optional adhesive
layer 513 disposed between the non-conductive first layer 510 and
the conductive second layer 512. As an example, the adhesive layer
513 can comprise chrome, with a layer of gold disposed on top of
the chrome adhesive layer 513 to form the conductive second layer
512. Thus, in the base substrate 508 of FIG. 8, the non-conductive
first layer 510 and the conductive second layer 512 are pre-adhered
to form the base substrate 508 prior to being unwound by the first
roller 502.
[0288] In the example base substrate 508 of FIG. 8, the
non-conductive first layer 510 has a thickness of less than about
500 nm. As will be described below, such a thickness allows for the
base substrate 508 to move through the example first assembly 500
via the plurality of rollers. Also, in some examples, the thickness
of the nonconductive first layer 510 is greater than a thickness of
the conductive second layer 512. As an example, the thickness of
the conductive second layer 512 can be approximately 30 nm. In
other examples, the thickness of the conductive second layer 512 is
less than about 500 nm. In some examples, the thickness of the
non-conductive first layer 510 and/or the conductive second layer
512 is selected based on, for example, the materials of the first
and/or second layers 510, 512 and/or an operational purpose for
which the droplet actuator formed from the base substrate 508 is to
be used.
[0289] The first roller 502 drives the base substrate 508 to a
laser ablation station 514. The laser ablation station 514 includes
a mask 516 containing a master pattern 518 that is to be projected
onto the conductive second layer 512 of the base substrate 508. The
master pattern 518 associated with the mask 516 may be predefined
based on characteristics such as resolution (e.g., number of
electrodes per an area of the base substrate 508 to be ablated),
electrode size, configuration of lines defining the electrode
pattern, inter-digitation of the electrodes, gaps or spacing
between the electrodes, and/or electrical traces for connecting the
electrodes to an instrument, such as, a power source. In some
examples, the characteristics of the master pattern 518 are
selected based on one or more operational uses of the droplet
actuator with which the base substrate 508 is to be associated
(e.g., for use with biological and/or chemical assays). Also, in
some examples, the master pattern 518 is configurable or
reconfigurable to enable the laser ablation station 514 to form
different patterns on the base substrate 508. Additionally or
alternatively, in some examples the mask 516 is replaceable with
one or more alternative masks.
[0290] The laser ablation station 514 includes a lens 520. As the
base substrate 508 encounters the laser ablation station 514 as
result of the rotation of the rollers (e.g., the first roller 502),
a portion 522 of the base substrate 508 passes under or past the
lens 520. The portion 522 may be, for example, a rectangular or
square section of the base substrate 508 having an area less than
the area of the base substrate 508 and including the conductive
second layer 512. The lens 520 images or projects at least a
portion of the master pattern 518 onto the conductive second layer
512 associated with the portion 522. A laser beam 524 is directed
onto the portion 522 via the mask 516 and the lens 520 such that
the laser beam 524 selectively penetrates the conductive second
layer 512 based on the projected master pattern 518. In some
examples, the non-conductive first layer 500 or a portion (e.g., a
fraction of the thickness of the non-conductive first layer 510)
may also be penetrated by the laser beam 524 based on the projected
master pattern 518. The solid portions of the mask 516 block the
laser beam 524, and the open portions of the mask 516 allow the
laser beam 524 to pass through the mask 516 and into contact with
the base substrate 508. The laser beam 524 can be associated with,
for example, an excimer laser.
[0291] As a result of exposure to the laser beam 524, the
irradiated nonconductive first layer 510 of the portion 522 absorbs
energy associated with the laser beam 524. The irradiated
non-conductive first layer 510 undergoes photochemical
dissociation, resulting in a selective breaking up of the
structural bonds of nonconductive first layer 510 and ejection of
fragments of the non-conductive first layer 510 and portions of the
conductive second layer 512 overlaying the irradiated
non-conductive first layer 510 in accordance with the master
pattern 518. In some examples, a depth (e.g., a radiation
intensity) to which the laser beam 524 penetrates the base
substrate 508 is predefined based on a depth (e.g., a thickness) of
the non-conductive first layer 510 and/or the conductive second
layer 512. In some examples, the laser beam 524 penetration depth
is adjustable to change the depth at which the laser beam 524
ablates the conductive second layer 512 as a result of the
fragmentation of the underlying nonconductive first layer 510. In
some examples, this adjustment is dynamic as the example system 500
operates. Also, in some examples, the base substrate 508 undergoes
cleaning after exposure to the laser beam 524 to remove particles
and/or surface contaminants.
[0292] As illustrated in FIG. 8, after exposure to the laser
ablation station 514, the portion 522 of the base substrate 508
includes an electrode array 526. The electrode array 526 is made up
of a plurality of electrodes formed into the conductive second
layer 512. As a result of the exposure to the laser beam 524 and
fragmentation of the non-conductive first layer 510, portions of
the conductive second layer 512 are removed from the base substrate
508. The removed portions associated with the electrode array 526
are based on the master pattern 518. In some examples, the removed
portions match the open portions of the mask 516.
[0293] Returning to FIG. 8, after the portion 522 undergoes laser
ablation at the laser ablation station 514 to form the electrode
array 526, the portion 522 is moved, via rotation of the first
through third rollers 502, 504, 506, to a printer 528. In the first
example assembly 500, the printer 528 includes an apparatus or an
instrument capable of applying at least one layer of material 530
having a hydrophobic and/or a dielectric property to the electrode
array 526. In the first example assembly 500, the printer 528 can
deposit the hydrophobic and/or dielectric material 530 via
deposition techniques including, but not limited to, web-based
coating (e.g., via rollers associated with the printer 528),
slot-die coating, spin coating, chemical vapor deposition, physical
vapor deposition, and/or atomic layer deposition. The printer 528
can also apply other materials in addition to the hydrophobic
and/or dielectric material 530 (e.g., anti-fouling coatings,
anti-coagulants). Also, the printer 528 can apply one or more
layers of the material(s) with different thicknesses and/or
covering different portions of the base substrate 508.
[0294] As described above, in the first example assembly 500, at
least one of the first through third rollers 502, 504, 506 advance
the base substrate 508 to the printer 528 for application of the
hydrophobic and/or dielectric material 530 to the electrode array
526. In some examples, the printer 528 includes a plurality of
registration rollers 531 to facilitate accuracy in feeding and
registration of the base substrate 508 as part of operation of the
printer 528 in applying the hydrophobic and/or dielectric material
530, for example, via roller coating methods.
[0295] In the first example assembly 500, the hydrophobic and/or
dielectric material 530 is applied to the electrode array 526 to
completely or substantially completely insulate the electrode array
526.
[0296] In some examples, the hydrophobic and/or dielectric material
530 is deposited via the printer 528 in substantially liquid form.
To create a structural or treated layer 532 on the base substrate
508 to support a droplet, the portion 522 is moved via the rollers
(e.g., the first through third rollers 502, 504, 506) through a
curing station 534. At the curing station 534, the hydrophobic
and/or dielectric material is treated and/or modified to form the
first treated layer 532. Treating and/or modifying the hydrophobic
and/or dielectric material can include curing the material. For
example, at the curing station 534, heat is applied to facilitate
the hardening of the hydrophobic and/or dielectric material 530. In
some examples, the portion 522 is exposed to an ultraviolet light
to cure the hydrophobic and/or dielectric material 530 and form the
treated layer 532 to insulate the electrode array 526. In other
examples, the curing and/or modification of the hydrophobic and/or
dielectric material is accomplished without heat and/or a photon
source. In some examples, the treated layer 532 supports a droplet
as an electric field is applied (e.g., in connection with electrode
array 526) to manipulate the droplet. For example, during an
electrowetting process, a contact angle of the droplet with respect
to the treated layer 532 changes as a result of an applied voltage,
which affects the surface tension of the droplet on the treated
surface 532. Electrowetting is merely exemplary, the droplet may be
moved using other forces as well.
[0297] After passing through the curing station 534, the portion
522 is prepared to serve as a bottom substrate of a droplet
actuator and/or as a digital microfluidic chip. Because the base
substrate 508 includes the non-conductive first layer 510 bonded
with the conductive second layer 512, as disclosed above,
additional adhesion of, for example the electrode array 526 to the
non-conductive first layer 510 is not required. Such a
configuration increases the efficiency of the preparation of the
base substrate 508 for the droplet actuator by reducing processing
steps. Also, as described above, when the portion 522 is at any one
of the laser ablation station 514, the printer 528, or the curing
station 534, other portions of the base substrate 508 are
concurrently moving through the others of the respective stations
514, 528, 534 of the first example assembly 500. For example, when
the portion 522 is at the curing station 534, the first through
third rollers 502, 504, 506 are continuously, periodically, or
aperidiocally advancing one or more other portions of the base
substrate 508 through, for example, the laser ablation station 514
and/or the printer 528. In such a manner, preparation of the base
substrate 508 for the droplet actuator is achieved via a
substantially continuous, high-speed, automated process.
[0298] After the curing step, a pattern roller is rolled over a
second portion of the base substrate to create an array of wells
540. The array wells 540 may subsequently be coated with a
hydrophilic material (not shown).
[0299] Although the base substrate 508 may be considered as
including successive portions, during some example operations of
the first example assembly 500, the base substrate 508 remains as a
single sheet as the successive portions undergo processing to
create the electrode arrays 526 (e.g., via the electrode pattern)
and receive the coating of hydrophobic and/or dielectric material
530. Thus, to create one or more droplet actuators using the
processed base substrate 508, the base substrate 508, in some
examples, is cut (e.g., diced) to form individual units comprising
the electrode arrays 526, as will be further disclosed below. In
some examples, prior to dicing, the base substrate 508, including
the portion 522, is rewound in a rolled configuration similar to
the initial rolled configuration of the base substrate 508 prior to
being unwound by the first roller 502. Such rewinding may be
accomplished via one or more rollers as part of the roll-to-roll
processing. In such examples, the base substrate 508 may be diced
or otherwise separated at a later time. In other examples, the
rollers (e.g., the second and third rollers 504, 506), advance the
base substrate 508 for merging with a top substrate.
[0300] FIG. 9 illustrates a second example assembly 600 for
creating an example top substrate of a droplet actuator having a
single electrode. The second example assembly 600 includes a series
or a plurality of rollers, including a first roller 602, a second
roller 604, and a third roller 606, which operate in synchronized
rotation to drive a top substrate 608 through the second example
assembly 600. The second example assembly 600 can include rollers
in addition to the first through third rollers 602, 604, 606 to
move the top substrate 608 through the assembly 600.
[0301] In the second example assembly 600, the first roller 602
rotates to unwind the top substrate 608, which, in some examples,
is a sheet in a rolled configuration. The example top substrate 608
of FIG. 9 includes a first layer 610 and a second layer 612. As
with the example base substrate 508, in this example, the example
first layer 610 of the top substrate 608 comprises a non-conductive
material such as, for example, a plastic, and the example second
layer 612 includes a conductive material, such as a metal
including, for example, one or more of gold, chrome, silver, indium
tin oxide, or copper and/or any other suitable metal(s), conductive
polymer(s), or combination(s) of metal(s) and/or conductive
polymer(s). In some examples, the conductive second layer 612 is
adhered to the nonconductive first layer via an adhesive layer
(e.g., chrome).
[0302] In the second example assembly 600, the first through third
rollers 602, 604, 606 rotate to advance the top substrate 612 to a
printer 614. The printer 614 coats the conductive second layer 612
with a hydrophobic and/or dielectric material 616 (e.g. Teflon.RTM.
or parylene C, or a dielectric such as a ceramic). The printer 614
is substantially similar to the printer 528 of the first example
assembly 500 of FIG. 8. For example, the printer 614 can apply the
hydrophobic and/or dielectric material 616 to the top substrate 608
via web-based coating, slot-die coating, spin coating, chemical
vapor deposition, physical vapor deposition, atomic layer
deposition, and/or other deposition techniques. The printer 614 can
include registration rollers 617 to facilitate alignment of the top
substrate 608 with respect to the printer 614 during application of
the hydrophobic and/or dielectric material 616 and/or other coating
materials.
[0303] After receiving the coating of the hydrophobic and/or
dielectric material 616, the second roller 504 and the third roller
506 advance the portion 618 to a curing station 620. As disclosed
in connection with the curing station 534 of FIG. 8, the curing
station 620 of the second example assembly 600 facilitates the
modification (e.g., curing) of the hydrophobic material via heat to
form a treated layer 622. The treated layer 622 insulates the
conductive second layer 612, which serves as the single electrode
of the top substrate 608, by completely or substantially completely
covering the conductive second layer 612. Thus, in coating the
second layer 612 of the portion 618, electrical potential
conducting portion of the top substrate 608 is insulated from a
droplet that may be applied to a droplet actuator that includes the
portion 618.
[0304] After passing through the curing station 620, the portion
618 is prepared to serve as a top substrate of a droplet actuator.
Because the top substrate 608 includes the non-conductive first
layer 610 pre-adhered to the conductive second layer 612,
additional adhesion of, for example, an electrode to the
non-conductive first layer 610 is not required, thereby increasing
the efficiency of the preparation of the top substrate 608 for the
droplet actuator.
[0305] In the second example assembly 600, the first through third
rollers 602, 604, 606 rotate to advance the top substrate 608 such
that portions of the top substrate pass through one of the printer
614 or the curing station 620 in substantially continuous, periodic
and/or aperiodic succession as part of the roll-to-roll operation
of the second example assembly 60. Thus, although the second
example assembly 600 is described in association with the portion
618, it is to be understood that successive portions of the top
substrate 608 are prepared in substantially the manner as the
portion 618 as a result of rotation of the first through third
rollers 602, 604, 606. In such as manner, the top substrate 308 is
provided with a treated layer 622 along the length of the top
substrate 608.
[0306] In the example top substrate 608, the conductive second
layer 612 serves an electrode. However, in some examples, the
conductive second layer 612 undergoes laser ablation to form one or
more electrode arrays. In such examples, the second example
assembly 600 includes a laser ablation station. Thus, prior to
receiving the hydrophobic material 616, the top substrate 608 is
exposed to a laser beam, which creates an electrode pattern in the
irradiated conductive second layer 612. Also, in some examples, the
electrode array is not formed on/in the base substrate but only
on/in the top substrate 608.
[0307] During operation of the second example assembly 600, the top
substrate remains single sheet as successive portions of the top
substrate 608 are coated with the hydrophobic material 616. As part
of the fabrication of one or more droplet actuators, the top
substrate 608 is aligned with the base substrate. In some examples,
after passing through the curing station 620, the top substrate is
rewound into a rolled configuration via one or more rollers. In
such examples, the finished roll may be diced or otherwise cut
and/or separated into individual units that are aligned at a spaced
apart distance and bonded with individual diced units of the base
substrate to create a droplet actuator.
[0308] In other examples, after passing through the curing station
620, the rollers (e.g., the first through third rollers 602, 604,
606) continue to advance the top substrate 608 to merge the top
substrate 608 with the base substrate via automated roll-to-roll
processing. In such examples, to prepare the top substrate 608 for
alignment with the base substrate 508, the first through third
rollers 602, 604, 606 rotate so as to reverse the orientation of
the top substrate relative to the base substrate such that the
treated layer of the base substrate faces the treated layer 622 of
the top substrate 608 when the base substrate 508 and the top
substrate 608 are aligned in parallel configuration.
[0309] As show in FIG. 10, the third example assembly 650 includes
a third roller 656 and a fourth roller 608 that form a pair of
merging rollers to which the base substrate 508 and the top
substrate 608 are fed via the respective first roller 652 and the
second roller 654 of the third example assembly 650. As each of the
merging rollers 656, 658 rotates, the base substrate 658 and the
top substrate 658 are aligned in a parallel configuration at a
predetermined spaced apart distance, or gap.
[0310] The example third assembly 650 includes a bonding station
664. The bonding station 664 joins, or bonds, the base substrate
508 and the top substrate 608 as part of fabricating the droplet
actuator. For example, at the bonding station 664, one or more
adhesives may be selectively applied to a predefined portion of the
base substrate 508 and/or the top substrate 608 (e.g., a portion of
the base substrate 508 and/or the top substrate 608 defining a
perimeter of the resulting droplet actuator) to create a bond
between the base substrate 508 and the top substrate 608 while
preserving the gap 662. In some examples, bonding the substrates
508, 608 at the bonding station 664 including forming the gap 662
(e.g., in advance of applying the adhesive).
[0311] Examples of adhesive(s) that may be used at the bonding
station 664 include epoxies, foils, tapes, and/or ultraviolet
curable adhesives. In some examples, layers of polymers such as
SU-8 and/or polydimethylsiloxane (PDMS) are applied to the base
substrate 508 and/or the top substrate 608 to bond the substrates.
Also, in some examples, the bonding station 664 provides for curing
of the adhesive(s) via, for example, ultraviolet light. The bonding
station 664 may apply one more methods involving, for example, heat
(e.g. thermal bonding), pressure, curing, etc. to bond the base
substrate 658 and the top substrate 608.
[0312] In the example third assembly 650, the merged portion 660
can be selectively cut, diced or otherwise separated to form one or
more droplet actuators, as substantially represented in FIG. 10 by
the merged portion 660. The example third assembly 650 includes a
dicing station 666. The dicing station 666 can be, for example, a
cutting device, a splitter, or more generally, an instrument to
divide the continuous merged portion 660 into discrete units
corresponding to individual droplet actuators. The merged portion
660 may be cut into individual droplet actuators based on, for
example, the electrode pattern such that each droplet actuator
includes a footprint of the electrode array and the other
electrodes that are formed via the electrode pattern.
[0313] FIG. 11A depicts a top view of the bottom substrate 70 on
which an array of electrodes is present in the first portion 73 and
second portion 74. The bottom substrate 72, after step 71 of
fabrication of an array of wells on the second portion, is shown.
FIG. 11B depicts a top view of a bottom substrate 80 with an array
of electrodes disposed only in the first portion 83. The bottom
substrate 82 is depicted after the step 81 in which an array of
wells is formed in the second portion 84.
[0314] The well array may be fabricated onto the
dielectric/hydrophobic layer, hydrophobic layer (if present), or
hydrophilic layer (if present). One exemplary method for
fabricating a well array onto the hydrophobic layer of the first
substrate uses thermal or ultraviolet nanoimprint lithography. FIG.
12A illustrates one exemplary method for fabricating a well array
by utilizing a flat nanoimprint mold 770 to apply sufficient
pressure to the hydrophobic layer 750 at the second portion of the
first substrate 710 in order to form the well array 760 pattern. In
this example, the nanoimprint stamper may be a flat stamping
element whose stamping contours correspond to the upper surface of
the second layer.
[0315] FIG. 12B illustrates another exemplary method in which a
nanoimprint roller 775 may be utilized to apply the pattern of well
arrays to the hydrophobic layer of the second portion of the first
substrate. The nanoimprint roller may imprint the pattern onto the
hydrophobic layer 750 of the first substrate 710 by advancing the
roller 775 in one direction. As the roller advances in the one
direction, the roller leaves behind an imprint of a pattern of the
well array 760 that corresponds to the imprint pattern on the
roller. In one example, the roller 775 rolls in a counter
clock-wise direction as the roller 775 imprints pattern onto the
hydrophobic layer 750 of the first substrate 710. It is understood
that the roller or stamper may be changed to form wells of suitable
volume, for example, a femtolitre roller or stamper may be used for
forming femtoliter wells.
[0316] FIG. 12C illustrates another exemplary method of forming a
pattern of well arrays to the hydrophobic layer of the second
portion of the first substrate. In this example, a laser may be
applied to ablate the upper surface of the hydrophobic layer 750.
The laser ablation step can produce a well array 760 pattern on the
second layer. Some examples of suitable lasers for ablating the
second layer include parameters with femtosecond and picosecond
lasers. In some examples, the laser ablation step includes use of a
special mask to define the well array pattern required. In some
examples, the laser 775 utilizes a focusing element 777 (e.g.,
lens) to accurately target and ablate the pattern. In some
examples, following the laser ablation step, the well array may be
coated with a dielectric and/or hydrophobic layer.
[0317] FIG. 12D illustrates yet another example of forming a
pattern of well arrays 760 onto the dielectric layer 740 of the
second portion of the first substrate 710. As illustrated in FIG.
12D, the method utilizes roll-to-roll fabrication to separately
fabricate microfluidic component and the well array. In one
example, a first roll 725 contains a microfluidic component, which
includes the first substrate 710, where the first substrate
comprises a series or plurality of electrodes 745, and a dielectric
layer 740 disposed over the upper surface of the first substrate
and covering the series or plurality of electrodes 745. A second
roll 780 contains a substrate 750 with the pattern of well array
760 already included on the substrate. In some examples, the
pattern of well array 760 previously included on the substrate 750
can be applied through thermal or UV nanoimprint lithography. In
other examples, the pattern of well array can be previously
included on the substrate through laser ablation. As illustrated in
FIG. 12D, the imprinted second roll 780 may also include a
hydrophobic coating imprinted onto the substrate of the well array.
The separate rolls are unwound via rollers 705 and 708, and then
subject to a lamination process where the two films may be
laminated together by overlying the well substrate over the
microfluidic component substrate to form a stacked configuration of
the well array and microfluidic components.
[0318] As described herein, "roll-to-roll" may include the
equivalent term "reel-to-reel" and operates by moving a substrate
through various components at high speeds, including, for example,
rates of meters per second. Roll-to-roll assemblies facilitate the
unwinding of a rolled substrate, the advancement of the substrate
through the components, and the rewinding of the processed
substrate into a roll.
[0319] As previously noted, the detection module formed by the
second portions of the first and second substrates is used for
detecting an analyte related signal. In some examples, detection of
the analyte or biological sample of interest may occur through
optical signal detection. For example, shining an excitation light
(e.g., laser) in order to measure the signal intensity result. In
other examples, the analyte desired may be detected by measuring an
optical signal emanating from each well chamber and quantified by
quantifying the result. For example, the number of positive counts
(e.g., wells) is compared to the number of negative counts (e.g.,
wells) via digital analysis. A variety of signals from the wells of
the device may be detected. Exemplary signals include fluorescence,
chemiluminescence, colorimetric, turbidimetric, etc.
[0320] The devices described herein may be used to generate an
analyte related signal and quantitate the signal. Exemplary method
is depicted in FIG. 15. The device in FIG. 15 includes a top
substrate 80 with an array of electrodes 81. The top substrate is
positioned in a spaced apart manner from the bottom substrate 82
which includes an array of wells 83 in a second portion of the
device. A droplet 84 containing particles or beads or analyte
molecules (not shown) may be moved to the array of wells 83 using
the electrodes 81. After a sufficient period of time to allow the
particles or beads or analyte molecules to move into the wells, the
droplet 84 may be moved to a waste chamber/absorption pad and the
like. A droplet of buffer 85 may then be moved to the array of
wells to remove any particles or beads not deposited into the
wells. In some cases, the buffer droplet may push the droplet 84
over to the waste chamber. A droplet of immiscible fluid 86 may be
moved over the array of wells and seal the wells. Any excess
droplet 86 may be removed prior to optically interrogating the
wells.
[0321] FIG. 16 depicts a method in which the digital microfluidics
electrodes (e.g. electrode 145) position the droplet 180 containing
particles/beads or analyte molecules 190 over the array of wells
160. After a period of time sufficient for deposition of
particles/beads/analyte molecules into the wells, the droplet is
displaced by a droplet of immiscible liquid 195 (or an immiscible
liquid as explained herein). The droplet of immiscible liquid
functions to move droplet 180 with any bead/particles/analyte
molecules not deposited into the wells away from the wells and to
cover the wells.
[0322] FIG. 17 depicts another method for removing any beads not
deposited into wells. In FIG. 17, many beads 190 are remaining over
the wells after removal of the droplet containing the beads. These
beads are washed away using an aqueous droplet. 185 After removal
of the aqueous droplet, the array of wells contains the deposited
beads. An immiscible fluid 195 is then moved over the array of
wells to seal the wells.
[0323] A number of forces may be utilized to facilitate the
movement of particles/beads from a droplet positioned over the
array of wells into the wells. Such forces include gravity,
electrical force, magnetic force, etc. Permanent magnets or
electromagnets may be used as source of magnetic force. In certain
embodiments, the magnets are not located on the integrated
microfluidic and detection chip. Analyte molecules may be deposited
into the wells via diffusion.
[0324] Variations on Methods and on Use of the Device
[0325] The disclosed methods of determining the presence or amount
of analyte of interest present in a sample, and the use of the
microfluidics device, may be as described above. The methods and
use of the disclosed microfluidics device may also be adapted in
view of other methods for analyzing analytes. Examples of
well-known variations include, but are not limited to, immunoassay,
such as sandwich immunoassay (e.g., monoclonal-polyclonal sandwich
immunoassays, including enzyme detection (enzyme immunoassay (EIA)
or enzyme-linked immunosorbent assay (ELISA), competitive
inhibition immunoassay (e.g., forward and reverse), enzyme
multiplied immunoassay technique (EMIT), particle-enhanced
turbidimetric inhibition immunoassay (PETINIA), homogeneous enzyme
immunoassay (HEIA), a competitive binding assay, bioluminescence
resonance energy transfer (BRET), one-step antibody detection
assay, homogeneous assay, heterogeneous assay, capture on the fly
assay, etc. In some instances, the descriptions below may overlap
the method described above; in others, the descriptions below may
provide alternates.
[0326] Immunoassay
[0327] The analyte of interest, and/or peptides or fragments
thereof, may be analyzed using an immunoassay. Any immunoassay may
be utilized. The immunoassay may be an enzyme-linked immunoassay
(ELISA), a competitive inhibition assay, such as forward or reverse
competitive inhibition assays, or a competitive binding assay, for
example. In some embodiments, a detectable label (e.g., such as one
or more fluorescent labels one or more tags attached by a cleavable
linker (which can be cleaved chemically or by photocleavage)) is
attached to the capture antibody and/or the detection antibody.
[0328] A heterogeneous format may be used. For example, after the
test sample is obtained from a subject, a first mixture is
prepared. The mixture contains the test sample being assessed for
analyte of interest and a first specific binding partner, wherein
the first specific binding partner and any analyte of interest
contained in the test sample to form a first specific binding
partner-analyte of interest complex. Preferably, the first specific
binding partner is an anti-analyte of interest antibody or a
fragment thereof. The order in which the test sample and the first
specific binding partner are added to form the mixture is not
critical. Preferably, the first specific binding partner is
immobilized on a solid phase. The solid phase used in the
immunoassay (for the first specific binding partner and,
optionally, the second specific binding partner) can be any solid
phase known in the art, such as, but not limited to, a magnetic
particle, a bead, a nanobead, a microbead, a nanoparticle, a
microparticle, a membrane, a scaffolding molecule, a film, a filter
paper, a disc, or a chip (e.g., a microfluidic chip).
[0329] After the mixture containing the first specific binding
partner-analyte of interest complex is formed, any unbound analyte
of interest is removed from the complex using any technique known
in the art. For example, the unbound analyte of interest can be
removed by washing. Desirably, however, the first specific binding
partner is present in excess of any analyte of interest present in
the test sample, such that all analyte of interest that is present
in the test sample is bound by the first specific binding
partner.
[0330] After any unbound analyte of interest is removed, a second
specific binding partner is added to the mixture to form a first
specific binding partner-analyte of interest-second specific
binding partner complex. The second specific binding partner is
preferably an anti-analyte of interest (such as an antibody) that
binds to an epitope on analyte of interest that differs from the
epitope on analyte of interest bound by the first specific binding
partner. Moreover, also preferably, the second specific binding
partner is labeled with or contains a detectable label (e.g., a
detectable label, a tag attached by a cleavable linker, etc.).
[0331] The use of immobilized antibodies or fragments thereof may
be incorporated into the immunoassay. The antibodies may be
immobilized onto a variety of supports, such as magnetic or
chromatographic matrix particles, latex particles or modified
surface latex particles, polymer or polymer film, plastic or
plastic film, planar substrate, a microfluidic surface, pieces of a
solid substrate material, and the like.
[0332] Sandwich Immunoassay
[0333] A sandwich immunoassay measures the amount of antigen
between two layers of antibodies (i.e., a capture antibody (i.e.,
at least one capture antibody) and a detection antibody (i.e. at
least one detection antibody)). The capture antibody and the
detection antibody bind to different epitopes on the antigen, e.g.,
analyte of interest. Desirably, binding of the capture antibody to
an epitope does not interfere with binding of the detection
antibody to an epitope. Either monoclonal or polyclonal antibodies
may be used as the capture and detection antibodies in the sandwich
immunoassay.
[0334] Generally, at least two antibodies are employed to separate
and quantify analyte of interest in a test sample. More
specifically, the at least two antibodies bind to certain epitopes
of analyte of interest or an analyte of interest fragment forming
an immune complex which is referred to as a "sandwich". One or more
antibodies can be used to capture the analyte of interest in the
test sample (these antibodies are frequently referred to as a
"capture" antibody or "capture" antibodies), and one or more
antibodies with a detectable label (e.g., a fluorescent label, a
tag attached by a cleavable linker, etc.) that also bind the
analyte of interest (these antibodies are frequently referred to as
the "detection" antibody or "detection" antibodies) can be used to
complete the sandwich. In some embodiments, an aptamer may be used
as the second binding member. In a sandwich assay, the binding of
an antibody to its epitope desirably is not diminished by the
binding of any other antibody in the assay to its respective
epitope. In other words, antibodies are selected so that the one or
more first antibodies brought into contact with a test sample
suspected of containing analyte of interest do not bind to all or
part of an epitope recognized by the second or subsequent
antibodies, thereby interfering with the ability of the one or more
second detection antibodies to bind to the analyte of interest.
[0335] In one embodiment, a test sample suspected of containing
analyte of interest can be contacted with at least one capture
antibody (or antibodies) and at least one detection antibodies
either simultaneously or sequentially. In the sandwich assay
format, a test sample suspected of containing analyte of interest
(such as a membrane-associated analyte of interest, a soluble
analyte of interest, fragments of membrane-associated analyte of
interest, fragments of soluble analyte of interest, variants of
analyte of interest (membrane-associated or soluble analyte of
interest) or any combinations thereof)) is first brought into
contact with the at least one capture antibody that specifically
binds to a particular epitope under conditions which allow the
formation of an antibody-analyte of interest complex. If more than
one capture antibody is used, a multiple capture antibody-analyte
of interest complex is formed. In a sandwich assay, the antibodies,
preferably, the at least one capture antibody, are used in molar
excess amounts of the maximum amount of analyte of interest or the
analyte of interest fragment expected in the test sample.
[0336] Optionally, prior to contacting the test sample with the at
least one first capture antibody, the at least one capture antibody
can be bound to a solid support which facilitates the separation
the antibody-analyte of interest complex from the test sample. Any
solid support known in the art can be used, including but not
limited to, solid supports made out of polymeric materials in the
form of planar substrates or beads, and the like. The antibody (or
antibodies) can be bound to the solid support by adsorption, by
covalent bonding using a chemical coupling agent or by other means
known in the art, provided that such binding does not interfere
with the ability of the antibody to bind analyte of interest or
analyte of interest fragment. Moreover, if necessary, the solid
support can be derivatized to allow reactivity with various
functional groups on the antibody. Such derivatization requires the
use of certain coupling agents such as, but not limited to, maleic
anhydride, N-hydroxysuccinimide, azido, alkynyl, and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
[0337] After the test sample suspected of containing analyte of
interest is brought into contact with the at least one capture
antibody, the test sample is incubated in order to allow for the
formation of a capture antibody (or capture antibodies)-analyte of
interest complex. The incubation can be carried out at a pH of from
about 4.5 to about 10.0, at a temperature of from about 2.degree.
C. to about 45.degree. C., and for a period from at least about one
(1) minute to about eighteen (18) hours, from about 2-6 minutes, or
from about 3-4 minutes.
[0338] After formation of the capture antibody (antibodies)-analyte
of interest complex, the complex is then contacted with at least
one detection antibody (under conditions which allow for the
formation of a capture antibody (antibodies)-analyte of
interest-detection antibody (antibodies) complex). If the capture
antibody-analyte of interest complex is contacted with more than
one detection antibody, then a capture antibody
(antibodies)-analyte of interest-detection antibody (antibodies)
detection complex is formed. As with the capture antibody, when the
at least one detection (and subsequent) antibody is brought into
contact with the capture antibody-analyte of interest complex, a
period of incubation under conditions similar to those described
above is required for the formation of the capture antibody
(antibodies)-analyte of interest-detection antibody (antibodies)
complex. Preferably, at least one detection antibody contains a
detectable label (e.g., a fluorescent label, a tag attached by a
cleavable linker, etc.). The detectable label can be bound to the
at least one detection antibody prior to, simultaneously with or
after the formation of the capture antibody (antibodies)-analyte of
interest-detection antibody (antibodies) complex. Any detectable
label known in the art can be used, e.g., a fluorescent label, a
cleavable linker as discussed herein, and others known in the
art.
[0339] The order in which the test sample and the specific binding
partner(s) are added to form the mixture for assay is not critical.
If the first specific binding partner is detectably labeled (e.g.,
a fluorescent label, a tag attached with a cleavable linker, etc.),
then detectably-labeled first specific binding partner-analyte of
interest complexes form. Alternatively, if a second specific
binding partner is used and the second specific binding partner is
detectably labeled (e.g., a fluorescent label, a tag attached with
a cleavable linker, etc.), then detectably-labeled complexes of
first specific binding partner-analyte of interest-second specific
binding partner form. Any unbound specific binding partner, whether
labeled or unlabeled, can be removed from the mixture using any
technique known in the art, such as washing.
[0340] Next, signal, indicative of the presence of analyte of
interest or a fragment thereof is generated. Based on the
parameters of the signal generated, the amount of analyte of
interest in the sample can be quantified. Optionally, a standard
curve can be generated using serial dilutions or solutions of known
concentrations of analyte of interest by mass spectroscopy,
gravimetric methods, and other techniques known in the art.
[0341] Provided herein are methods for measuring or detecting an
analyte of interest present in a biological sample. In such
methods, a sample droplet containing the target analyte of interest
may be merged with a droplet containing beads (such as magnetic
beads) on which a first specific binding partner that specifically
binds to the target analyte of interest present in the sample is
attached. Merging creates a single droplet which may be incubated
for a time sufficient to allow binding of the first specific
binding partner to an analyte of interest present in the sample
droplet. Optionally, the single droplet may be agitated to
facilitate mixing of the sample with the first specific binding
partner. Mixing may be achieved by moving the single droplet back
and forth, moving the single droplet around over a plurality of
electrodes, splitting a droplet and then merging the droplets, or
using SAWs, and the like. Next, the single droplet may be subjected
to a magnetic force to retain the beads at a location in the device
while the droplet may be moved away to a waste chamber or pad and
replaced with a droplet containing a second binding member. The
second specific binding partner may be detectably labeled. The
label may be any label that can be optically detected. For example,
the label may be a fluorescent label. An optional wash step may be
performed, prior to adding the second binding member, by moving a
droplet of wash buffer to the location at which the beads are
retained using the force, e.g., magnetic. The beads may or may not
be resuspended in the wash buffer. If magnetic beads are used, a a
magnetic force can be applied to the magnetic beads and the wash
buffer is transported to a waste location. After a period of time
sufficient for the second specific binding partner to bind the
analyte of interest bound to the first binding member, the droplet
containing the second specific binding partner may be moved away
while the beads are retained at the location. The beads may be
washed using a droplet of wash buffer. Following the wash step, a
droplet containing the labeled beads which has a complex of the
first binding member, analyte of interest and the second binding
partner may be moved over to the detection module (such as by
removal of the magnetic force if magnetic beads are used). As
explained herein, the immunoassay may be carried out in the sample
preparation module. The labeled beads may be allowed to settle into
the array of wells in the detection module. The beads may settle
using gravitational force or by applying electric or magnetic
force. Following a wash step to remove any beads not located inside
the wells, the wells may be sealed by using a hydrophobic
liquid.
[0342] Forward Competitive Inhibition
[0343] In a forward competitive format, an aliquot of labeled
analyte of interest (e.g., analyte having a fluorescent label, a
tag attached with a cleavable linker, etc.) of a known
concentration is used to compete with analyte of interest in a test
sample for binding to analyte of interest antibody.
[0344] In a forward competition assay, an immobilized specific
binding partner (such as an antibody) can either be sequentially or
simultaneously contacted with the test sample and a labeled analyte
of interest, analyte of interest fragment or analyte of interest
variant thereof. The analyte of interest peptide, analyte of
interest fragment or analyte of interest variant can be labeled
with any detectable label, including a detectable label comprised
of tag attached with a cleavable linker. In this assay, the
antibody can be immobilized on to a solid support. Alternatively,
the antibody can be coupled to an antibody, such as an antispecies
antibody, that has been immobilized on a solid support, such as a
microparticle or planar substrate.
[0345] Provided herein are methods for measuring or detecting an
analyte of interest present in a biological sample. In such
methods, a sample droplet containing the target analyte of interest
may be merged with a droplet containing magnetic beads on which a
first specific binding partner that specifically binds to the
target analyte of interest present in the sample is attached and
analyte labeled with a detectable label (such as a fluorescent
label). Optionally, the single droplet may be agitated to
facilitate mixing of the sample with the first specific binding
partner and the labeled analyte. Mixing may be achieved by moving
the single droplet back and forth, moving the single droplet around
over a plurality of electrodes, splitting a droplet and then
merging the droplets, or using SAWs, and the like. Next, the single
droplet may be subjected to a force (such as a magnetic force) to
retain the beads at a location in the device while the droplet may
be moved away to a waste chamber or pad and replaced with a droplet
containing a second binding member. An optional wash step may be
performed by moving a droplet of wash buffer to the location at
which the beads are retained using the magnetic force. The beads
may or may not be resuspended in the wash buffer; a force is
applied to the beads (such as a magnetic force if magnetic beads
are used) and the wash buffer is transported to a waste location.
After a period of time sufficient for the first specific binding
partner to bind to the analyte of interest, the droplet may be
moved away while the beads are retained at the location. Following
the optional wash step, a droplet containing the labeled beads
which has a complex of the first binding member and analyte of
interest may be moved over to the detection module (such as by
removing a magnetic force if magnetic beads are used). As explained
herein, the immunoassay may be carried out in the sample
preparation module. The labeled beads may be allowed to settle into
the array of wells in the detection module. The beads may settle
using gravitational force or by applying a force, e.g., electric or
magnetic. Following a wash step to remove any beads not located
inside the wells, the wells may be sealed by using a hydrophobic
liquid.
[0346] The labeled analyte of interest, the test sample and the
antibody are incubated under conditions similar to those described
above in connection with the sandwich assay format. Two different
species of antibody-analyte of interest complexes may then be
generated. Specifically, one of the antibody-analyte of interest
complexes generated contains a detectable label (e.g., a
fluorescent label, etc.) while the other antibody-analyte of
interest complex does not contain a detectable label. The
antibody-analyte of interest complex can be, but does not have to
be, separated from the remainder of the test sample prior to
quantification of the detectable label. Regardless of whether the
antibody-analyte of interest complex is separated from the
remainder of the test sample, the amount of detectable label in the
antibody-analyte of interest complex is then quantified. The
concentration of analyte of interest (such as membrane-associated
analyte of interest, soluble analyte of interest, fragments of
soluble analyte of interest, variants of analyte of interest
(membrane-associated or soluble analyte of interest) or any
combinations thereof) in the test sample can then be determined,
e.g., as described above.
[0347] Reverse Competition Assay
[0348] In a reverse competition assay, an immobilized analyte of
interest can either be sequentially or simultaneously contacted
with a test sample and at least one labeled antibody.
[0349] Provided herein are methods for measuring or detecting an
analyte of interest present in a biological sample. In such
methods, a sample droplet containing the target analyte of interest
may be merged with a droplet containing a first specific binding
partner that specifically binds to the target analyte of interest
present in the sample and is labeled with a detectable label (such
as a fluorescent label, enzymatic label, etc.) and magnetic beads
to which the analyte of interest is attached. Merging creates a
single droplet which may be incubated for a time sufficient to
allow binding of the first specific binding partner to an analyte
of interest present in the sample droplet. Optionally, the single
droplet may be agitated to facilitate mixing of the sample with the
first specific binding partner. Mixing may be achieved by moving
the single droplet back and forth, moving the single droplet around
over a plurality of electrodes, splitting a droplet and then
merging the droplets, or using SAWs, and the like. Next, the single
droplet may be subjected to a magnetic force to retain the beads at
a location in the device while the droplet may be moved away to a
waste chamber or pad and replaced with a droplet containing a
second binding member. An optional wash step may be performed by
moving a droplet of wash buffer to the location at which the beads
are retained using the magnetic force. The beads may or may not be
resuspended in the wash buffer; a magnetic force is applied to the
magnetic beads and the wash buffer is transported to a waste
location. After a period of time sufficient for the first specific
binding partner to bind the analyte of interest bound, the magnetic
force may be removed and a droplet containing the labeled beads
which has a complex of the first binding member, analyte of
interest may be moved over to the detection module. As explained
herein, the immunoassay may be carried out in the sample
preparation module. The labeled beads may be allowed to settle into
the array of wells in the detection module. The beads may settle
using gravitational force or by applying electric or magnetic
force. Following a wash step to remove any beads not located inside
the wells, the wells may be sealed by using a hydrophobic
liquid.
[0350] The analyte of interest can be bound to a solid support,
such as the solid supports discussed above in connection with the
sandwich assay format.
[0351] The immobilized analyte of interest, test sample and at
least one labeled antibody are incubated under conditions similar
to those described above in connection with the sandwich assay
format. Two different species analyte of interest-antibody
complexes are then generated. Specifically, one of the analyte of
interest-antibody complexes generated is immobilized and contains a
detectable label (e.g., a fluorescent label, etc.) while the other
analyte of interest-antibody complex is not immobilized and
contains a detectable label. The non-immobilized analyte of
interest-antibody complex and the remainder of the test sample are
removed from the presence of the immobilized analyte of
interest-antibody complex through techniques known in the art, such
as washing. Once the non-immobilized analyte of interest antibody
complex is removed, the amount of detectable label in the
immobilized analyte of interest-antibody complex is then quantified
following cleavage of the tag. The concentration of analyte of
interest in the test sample can then be determined by comparing the
quantity of detectable label as described above.
[0352] One-Step Immunoassay or "Capture on the Fly"
[0353] In a capture on the fly immunoassay, a solid substrate is
pre-coated with an immobilization agent. The capture agent, the
analyte and the detection agent are added to the solid substrate
together, followed by a wash step prior to detection. The capture
agent can bind the analyte and comprises a ligand for an
immobilization agent. The capture agent and the detection agents
may be antibodies or any other moiety capable of capture or
detection as described herein or known in the art. The ligand may
comprise a peptide tag and an immobilization agent may comprise an
anti-peptide tag antibody. Alternately, the ligand and the
immobilization agent may be any pair of agents capable of binding
together so as to be employed for a capture on the fly assay (e.g.,
specific binding pair, and others such as are known in the art).
More than one analyte may be measured. In some embodiments, the
solid substrate may be coated with an antigen and the analyte to be
analyzed is an antibody.
[0354] In certain other embodiments, in a one-step immunoassay or
"capture on the fly", a solid support (such as a microparticle)
pre-coated with an immobilization agent (such as biotin,
streptavidin, etc.) and at least a first specific binding member
and a second specific binding member (which function as capture and
detection reagents, respectively) are used. The first specific
binding member comprises a ligand for the immobilization agent (for
example, if the immobilization agent on the solid support is
strepativdin, the ligand on the first specific binding member may
be biotin) and also binds to the analyte of interest. The second
specific binding member comprises a detectable label and binds to
an analyte of interest. The solid support and the first and second
specific binding members may be added to a test sample (either
sequentially or simultaneously). The ligand on the first specific
binding member binds to the immobilization agent on the solid
support to form a solid support/first specific binding member
complex. Any analyte of interest present in the sample binds to the
solid support/first specific binding member complex to form a solid
support/first specific binding member/analyte complex. The second
specific binding member binds to the solid support/first specific
binding member/analyte complex and the detectable label is
detected. An optional wash step may be employed before the
detection. In certain embodiments, in a one-step assay more than
one analyte may be measured. In certain other embodiments, more
than two specific binding members can be employed. In certain other
embodiments, multiple detectable labels can be added. In certain
other embodiments, multiple analytes of interest can be
detected.
[0355] The use of a capture on the fly assay can be done in a
variety of formats as described herein, and known in the art. For
example the format can be a sandwich assay such as described above,
but alternately can be a competition assay, can employ a single
specific binding member, or use other variations such as are
known.
[0356] Combination Assays (Co-Coating of Microparticles with
Ag/Ab)
[0357] In a combination assay, a solid substrate, such as a
microparticle is co-coated with an antigen and an antibody to
capture an antibody and an antigen from a sample, respectively. The
solid support may be co-coated with two or more different antigens
to capture two or more different antibodies from a sample. The
solid support may be co-coated with two or more different
antibodies to capture two or more different antigens from a
sample.
[0358] Additionally, the methods described herein may use blocking
agents to prevent either specific or non-specific binding reactions
(e.g., HAMA concern) among assay compounds. Once the agent (and
optionally, any controls) is immobilized on the support, the
remaining binding sites of the agent may be blocked on the support.
Any suitable blocking reagent known to those of ordinary skill in
the art may be used. For example, bovine serum albumin ("BSA"),
phosphate buffered saline ("PBS") solutions of casein in PBS,
Tween20.TM. (Sigma Chemical Company, St. Louis, Mo.), or other
suitable surfactant, as well as other blocking reagents, may be
employed.
[0359] As is apparent from the present disclosure, the methods and
devices disclosed herein, including variations, may be used for
diagnosing a disease, disorder or condition in a subject suspected
of having the disease, disorder, or condition. For example, the
sample analysis may be useful for detecting a disease marker, such
as, a cancer marker, a marker for a cardiac condition, a toxin, a
pathogen, such as, a virus, a bacteria, or a portion thereof. The
methods and devices also may be used for measuring analyte present
in a biological sample. The methods and devices also may be used in
blood screening assays to detect a target analyte. The blood
screening assays may be used to screen a blood supply.
[0360] Surface Acoustic Wave Device, System, and Methods
[0361] Systems, device, and methods related to an integrated
surface acoustic wave (SAW) sample preparation and analyte
detection device are provided by the subject disclosure.
[0362] In one example, the device includes a sample preparation
component, e.g., a substrate with a surface that allows for liquid
or fluids to propagate across the surface thereof via manipulation
by acoustic forces. In the same example, the device includes an
analyte detection component configured to receive the propagated
liquid and perform analyte detection on the received liquid.
[0363] "Surface acoustic waves (SAW)" and grammatical equivalents
thereof as used herein refer generally to propagating acoustic
waves in a direction along a surface. "Traveling surface acoustic
waves" (TSAWs) enable coupling of surface acoustic waves into a
liquid. In some examples, the coupling may be in the form of
penetration or leaking of the surface acoustic waves into the
liquid. In some examples, the surface acoustic waves are Raleigh
waves. Propagation of the surface acoustic waves can be performed
by streaming the surface acoustic waves through a liquid.
Propagation of surface acoustic waves may be conducted in a variety
of different ways and by using different materials, including
generating an electrical potential by a transducer, such as a
series or plurality of electrodes.
[0364] The electrodes may be patterned onto a planar substrate. In
some examples, the planar substrate may be a piezoelectric layer.
In some examples, the electrodes may be fabricated onto the
piezoelectric layer using standard lithography and lift off/wet
etching processes. The structure of the electrodes, spacing between
electrodes, the number of electrodes (i.e., resolution) on the
substrate may vary. In some examples, interdigitated (IDT)
transducers or electrodes are used. In some examples, the sample
preparation component may include a liquid. In some examples, there
may be multiple layers. The different layers may have different
arrangement or configuration of scattering structures for
scattering surface acoustic waves. As a result, liquid droplet
movement across the different layers may differ due to the varied
scattering structures present.
[0365] In some examples, SAW are propagated when a single
transducer or electrode is activated. In other examples, a
plurality (e.g., pair) of electrodes fabricated on the substrate
surface may generate two traveling SAWs propagating towards each
other. In some examples, SAW displacement is activated when a radio
frequency (RF) range is applied to the electrodes. Upon being
activated, the electrodes or transducers emit an electric potential
across the surface of the substrate, where the substrate is
subjected to mechanical stress. Examples of mechanical stress are
continuous contraction and expansion of the surface of the
substrate. As a result of this continuous deformation of the
substrate, surface acoustic waves are propagated across the
surface.
[0366] Surface acoustic waves can be measured according to
amplitude and frequency. Therefore, the frequency and amplitude of
the electric potential generated by the electrodes is responsible
for the amplitude and frequency of SAW.
[0367] Propagation of SAW may be in a linear direction. In some
examples, SAW may propagate across the longitudinal length of the
substrate surface. In other examples, SAW may propagate across the
width of the substrate surface. In other examples, propagation of
SAW may be in a non-linear direction and motion. Because fluid is a
dissipative system, the response to harmonic forcing via SAW may
not necessarily be harmonic.
[0368] When a TSAW contacts liquid, the liquid absorbs part of the
SAW's energy and may refract it in the form of longitudinal waves.
Absorption of the refracted acoustic energy induces fluid flow or
propagation across the surface of the substrate. When a surface
acoustic wave is propagated along the surface of the sample
preparation component, the SAW may come into contact with the
liquid. As a result of the liquid interacting with SAW, results in
the SAW being transferred into the liquid. SAWs manipulate fluid by
means of "contact free manipulation", which is meant the liquids
are propagated to the detection component by the acoustic waves
leaking or penetrating into the fluid. As a result, there is a
minimization of outside contamination of the biological sample or
analyte.
[0369] In some examples, exemplary driving fluid actions includes
pumping, mixing, jetting, etc. As a result, the liquid is
propagated along the surface of the sample preparation
component.
[0370] In some examples, the liquid can be dispensed as a droplet
to be actuated onto the surface of the sample preparation component
prior to the activation of the SAW electrodes. Droplet actuation
can be used for positioning droplets and dispensing droplets onto
the sample preparation component.
[0371] In other examples, instead of liquid droplet-based
microfluidics, a SAW driven pump may be used to pump liquid onto
the open surface. In some examples, fluid may be pumped through
enclosed channels.
[0372] The liquid may be any test sample containing or suspected of
containing any analyte of interest. As used herein, "analyte",
"target analyte", "analyte of interest" refer to the analyte being
measured in the methods and devices disclosed herein. The liquid
droplets may also refer to particles or beads in an aqueous
solution. Samples may include biological fluid samples such as, for
example, blood, plasma, serum, saliva, sweat, etc.
[0373] In some examples, the liquid can be disposed as a single
particle. In other examples, the liquid can be disposed as a group
of particles (e.g., thousands of particles). The liquid droplets
may vary according to a wide range of length scales, size (nm to
mm), as well as shape.
[0374] The propagation of surface acoustic waves may also be
affected by the presence of phononic structures patterned onto the
surface of the sample preparation platform. These phononic
structures may control the propagation of the sound acoustic waves.
For example, the phononic structures may control the direction,
movement, velocity of the SAW; thus, providing enhanced
functionality. The phononic structures may be fabricated onto the
substrate using standard lithography, lift off/wet etching
processes, embossing/nanoimprint lithography, and micromachining,
pressure, heat, and laser modification of the substrate to form
these phononic structures. These phononic structures may assume a
variety of shapes and sizes as well. In some examples, the phononic
structures may be pillars, cones, or holes that form a lattice
within the substrate.
[0375] Surface Acoustic Waves Sample Preparation Component
[0376] "Sample preparation component" and grammatical equivalents
thereof as used herein refer to a generally planar surface on which
the liquid droplets are initially dispersed upon and where steps of
immunoassay as described herein may be carried out. In some
examples, the substrate may be made of materials with high acoustic
reflection.
[0377] In some examples, the sample preparation component includes
a superstrate coupled to a substrate. In some examples, the
superstrate is removably coupled to the substrate. In other
examples, the superstrate is permanently coupled to the substrate.
Some examples include making the substrate from a polymer-based or
paper-material. The polymer-based substrate may be treated with a
hydrophobic coating or fabrication may add a hydrophobic layer over
the polymer-based substrate or with another substrate such that the
substrate is impermeable to aqueous fluid.
[0378] In some examples, the sample preparation component may also
include an assay reagent included on the superstrate. The sample
preparation component further includes a superstrate coupled to a
substrate.
[0379] In yet another example, the sample preparation component may
include a series of scattering structures included on the
superstrate. Examples of the scattering structures may include
phononic structures, which are described in greater detail
below.
[0380] In some examples, the substrate may be a piezoelectric
material. The piezoelectric layer may be made from a composite
layer, such as single crystal lithium niobate (LiNbO3). The
superstrate may further include a series or plurality of electrodes
or transducer. In some examples, surface acoustic waves generated
by the electrodes or IDT may also be coupled into the
superstrate.
[0381] In some examples, the superstrate may be made from a variety
of materials, such as plastics (e.g., PET, PC, etc.).
[0382] In some examples, the superstrate may be fabricated of a
material with a relatively high electromechanical coupling
coefficient. In some examples, electrodes may be fabricated onto
piezoelectric materials. In one example, LiNbO3 may be used as a
substrate to pattern electrodes in SAW microfluidic applications.
In another example, silicon may be used as a substrate material to
pattern electrodes. Other examples of material applicable for
fabricating a SAW-generating substrate include polycrystalline
material, microcrystalline material, nanocrystalline material,
amorphous material or a composite material. Other examples of
material applicable for fabricating a SAW-generating substrate
include ferroelectrical material, pyroelectric material,
piezoelectric material or magnetostrictive material.
[0383] As described herein, the substrate is a material capable of
generating surface acoustic waves and propagating acoustic
waves.
[0384] In addition to the analyte or biological sample to be
analyzed, the sample preparation component may also include buffer
or wash fluids. In some examples, these buffer or wash fluids may
facilitate the propagation of liquids across the sample preparation
component and onto the detection component. In other instances,
these fluids may be used to wash away any remaining liquid or
biological samples once they have being positioned into the well
array. Examples of such fluids include air, inert gases,
hydrophobic liquids, hydrophilic liquids, oils, organic-based
solvents, and high-density aqueous solutions. In certain cases, the
device may be filled with a filler fluid which may be air, inert
gases, hydrophobic liquids, hydrophilic liquids, oils,
organic-based solvents, and high-density aqueous solutions.
[0385] In some examples, SAW induced fluidic movement can be
visualized by introducing small dyes or particles into the liquid
droplet.
[0386] The sample preparation surface has a surface on which the
liquid may be propagated along the surface. The surface of the
sample preparation surface may be any convenient surface in planar
or non-planar conformation. The surface may be coated with a
hydrophobic material to facilitate movement of the liquid along the
surface. In some examples, the hydrophobic material may include
octadecyltrichlorosilane (OTS). In other examples, the surface may
be patterned to facilitate liquid movement.
[0387] In some examples, the substrate of the sample preparation
surface may be elastic or flexible. The substrate on which the
surface is formed upon may be elastic so that the surface is able
to deform so as to facilitate the propagation of surface acoustic
waves across the surface.
[0388] In certain embodiments, the surface of the substrate may
include microfluidic channels to facilitate propagating fluid. In
other embodiments, a microfluidic channel is included internal of
the substrate to transmit fluid into the substrate.
[0389] In some examples, a cover seal may be provided over the
upper surface of the substrate of the sample preparation component.
In certain instances, the cover seal may prevent contamination of
the liquid contents of the surface. In other instances, the cover
seal may be a liquid impermeable layer. In other instances, the
cover seal may be made from a flexible material such as plastics,
silicon, or other type of rubber. In other instances, the cover
seal may be made from a non-flexible material such as a glass or
other non-flexible material. In some examples, the cover seal may
be impenetrable to heat, ultraviolet light, or other
electromagnetic radiation to prevent deformation of either the
surface or liquid contents present on the surface.
[0390] In some examples, a suitable spacer may be positioned
between the substrate and the cover seal. By "suitable spacer" as
used herein, refers to an element positioned between the substrate
of the sample preparation component and the cover seal. In some
examples, the suitable spacer may facilitate liquid droplets to
move between the surface and the cover seal. In other examples, the
suitable spacer may reduce coupling between the traveling surface
acoustic waves and the surface.
[0391] In the first example sample preparation component, the first
substrate incorporates a material with a relatively high
electromechanical coupling coefficient and having a flexible and
deformable surface. For example, the first substrate may be a
piezoelectric material or silicon.
[0392] In some examples, electrodes are arranged on the surface or
embedded within the piezoelectric layer. The term "electrodes", as
used in this context, refers to electric circuit including a
electrode, a series or plurality of electrodes (e.g., more than
one), a transducer. The electrode may also be patterned into the
piezoelectric layer. In some examples, the electrodes may be
fabricated onto the substrate using standard lithography and lift
off/wet etching processes. The structure of the electrodes, spacing
between electrodes, the number of electrodes (i.e., resolution) on
the substrate may vary. In some examples, interdigitated (IDT)
transducers or electrodes are used. IDT is defined as a combination
of a series or plurality of electrodes and a piezoelectric layer on
which the series or plurality of electrodes are included on. In
some examples the transducer electrode structures are formed onto
the piezoelectric layer. In other examples, the transducer
electrode structures are embedded within the piezoelectric
layer.
[0393] In some examples, surface acoustic waves are propagated when
a single transducer or electrode is activated. In other examples, a
plurality (e.g., pair) of electrodes fabricated on the substrate
surface may generate two traveling surface acoustic waves
propagating towards each other. In some examples, surface acoustic
waves displacement is activated when a radio frequency (RF) range
is applied to the electrodes. Upon being activated, the electrodes
or transducers emit an electric potential across the surface of the
substrate, where the material is subjected to mechanical stress.
Examples of mechanical stress are continuous contraction and
expansion of the surface of the substrate. As a result of this
continuous deformation of the substrate, surface acoustic waves are
propagated across the surface.
[0394] In some examples, wavelength of surface acoustic waves is
dependent upon the pitch of the transducer (IDT) or series or
plurality of electrodes.
[0395] In one example, the sample preparation component may include
a series of phononic structure that are included on the surface of
the superstrate. The phononic structures may control the
propagation of the acoustic waves. For example, the phononic
structures may control the direction, movement, velocity of the
surface acoustic waves. The phononic structures may assume a
variety of shapes and sizes as well. In some examples, the phononic
structures may be pillars, cones, or holes that form a lattice
within the substrate. The pattern of phononic structures on the
surface of the superstrate may be predefined based on
characteristics such as resolution (e.g., number of electrodes per
area on the surface), electrode size, inter-digitation of the
electrodes, and/or gaps or spacing between the electrodes. In some
examples, characteristics of the pattern are selected based on one
or more operational uses of the droplet actuator with which the SAW
sample prep component is to be associated (e.g., for use with
biological and/or chemical assays). In other configurations, the
pattern of electrodes may be reconfigurable to enable different
patterns to suit different applications. In some examples, an
increase in the size or dimensions of the series or plurality of
electrodes or each individual electrode may also reduce the amount
of hydrophobic material applied between adjacent electrodes. Thus,
the features of the electrode pattern may maximize the surface area
of the SAW platform. Furthermore, increased inter-digitation of the
series or plurality of electrodes/transducers facilitates the ease
with which liquid is propagated across the surface via manipulation
of their electrical potentials.
[0396] In the first example sample preparation component,
hydrophobic material may be applied to the series or plurality of
electrodes and surface of the substrate to make the superstrate
impermeable to aqueous solutions. As a result of the hydrophobic
material, a liquid actuated through a droplet or fluid pump is in a
beaded configuration forming a contact angle with the hydrophobic
layer of the surface of the substrate. In operation, SAW acoustic
waves propagate across the surface coupling to the liquid, for
example by penetrating or leaking into the liquid. The amplitude or
frequency of the SAW acoustic wave may control the resulting
frequency and motion of the moving liquid.
[0397] In certain embodiments, the surface acoustic waves propagate
along the surface of the substrate and are then coupled into the
superstrate. Thereafter, the surface acoustic waves continue to
propagate and are guided by phononic structures that may be formed
in the superstrate.
[0398] In some examples, where SAW acoustic wave are generated by
two or more electrodes, it may result in controlling the direction
of the liquid that is coupled to the resulting surface acoustic
waves. The direction of the propagating liquid may be in a linear
direction or non-linear direction. In some examples, the
propagation of the liquid droplet may be in a rolling motion. In
other examples, propagation of the liquid droplet may be in a
sliding motion across the surface. In some examples, where there is
a lack of phononic structures on the surface, propagation of the
SAW and propagation of the resulting liquid droplet are in the same
direction. In other examples, where there is a presence of phononic
structures on the surface, propagation of SAW and propagation of
resulting liquid droplets are in opposing directions or different
directions.
[0399] In some examples, the hydrophobic material is a
polytetrafluoroethylene material (e.g., Teflon.RTM.) or a
fluorosurfactant (e.g., FluoroPel.TM.) applied to the surface of
the superstrate.
[0400] Analyte Detection Component
[0401] In some embodiments, the analyte detection component may
include an array of wells in which molecules, particles, beads, or
cells may be isolated for analyte or biological sample detection
purpose. TSAWs (traveling surface acoustic waves) generate acoustic
streaming over the surface are across the fluid channels to push
fluid (either droplets or cells) towards the well array.
[0402] The shape and geometry of the wells may vary according to
the type of procedure or application required. In some examples,
the wells may vary between being deep chambers to shallow chambers.
The wells may be any of a variety of shapes, such as, cylindrical
with a flat bottom surface, cylindrical with a rounded bottom
surface, cubical, cuboidal, frustoconical, inverted frustoconical,
or conical. In certain cases, the wells may include a sidewall that
may be oriented to facilitate the receiving and retaining of a
microbead or microparticle present in liquid droplets that have
been moved over the well array. In some examples, the wells may
include a first sidewall and a second sidewall, where the first
sidewall may be opposite the second side wall. In some examples,
the first sidewall is oriented at an obtuse angle with reference to
the bottom of the wells and the second sidewall is oriented at an
acute angle with reference to the bottom of the wells. The movement
of the droplets may be in a direction parallel to the bottom of the
wells and from the first sidewall to the second sidewall. The array
of wells may have sub-femtoliter volume, femtoliter volume,
sub-nanolitre volume, nanolitre volume, sub-microliter volume, or
microliter volume. For example the array of wells may be array of
femtoliter wells, array of nanoliter wells, or array of microliter
wells. In certain embodiments, the wells in an array may all have
substantially the same volume. The array of wells may have a volume
up to 100 .mu.l, e.g., about 0.1 femtoliter, 1 femtoliter, 10
femtoliter, 25 femtoliter, 50 femtoliter, 100 femtoliter, 0.5 pL, 1
pL, 10 pL, 25 pL, 50 pL, 100 pL, 0.1 nL, 1 nL, 10 nL, 25 nL, 50 nL,
500 nL, 0.1 microliter, 1 microliter, 10 microliter, 25 microliter,
50 microliter, or 100 microliter.
[0403] In certain cases, the sample preparation component and the
analyte detection component may be fabricated from a single planar
surface using, for example, a continuous web-fed manufacturing
process. In such an example, the sample preparation component and
the digital analyte detection component may be positioned adjacent
to each other.
[0404] In some examples, the sample preparation component may
include a sample inlet. By "sample inlet" as used herein, refers to
a tubular member, channel, or pipe for introducing liquid to the
sample preparation component. For example, the sample inlet may
introduce a biological sample onto the surface of the substrate. In
other example, the sample inlet may introduce a biological sample
internally within the substrate.
[0405] In other examples, the sample preparation component and the
digital analyte detection component may be positioned over one
another in a stacked configuration, separated by a space for
droplet manipulation. In the example of the sample preparation
component being positioned over the analyte detection component in
a stacked configuration or vice versa (the analyte detection
component being positioned over the sample preparation component),
an inlet or channel may be positioned between the two components.
The inlet or channel may direct a sample or analyte between the two
components.
[0406] Phononic structures may be fabricated or included on the
superstrate of the sample preparation component. In certain cases,
the phononic structures are imprinted or embossed onto the
superstrate. In such examples, the embossing or imprinting of the
phononic structures is in a single step. In other examples, it may
be multiple steps. Imprinting or embossing of phononic structures
may be through the combination of an application of pressure, heat,
or ultraviolet light in the presence of a mold, mask, or pattern.
In one example, pressure elicited from a mold onto the superstrate
may induce deformation of the a surface of the superstrate.
[0407] After the phononic structures are included on the
superstrate, it may be cured for a sufficient period of time to
allow for hardening or deformation of the phononic structures. In
addition, the phononic structures may be subject to reagents that
modify the physical properties of the phononic structures.
[0408] In some examples, the reagents for analyte detection may be
printed during fabrication of the integrated sample preparation and
analyte detection device in a dehydrated form. Rehydration of the
reagents occurs through use of a sample or buffer.
[0409] In some examples, the array of wells includes individual
well chambers, with each well chamber having a first end and a
second end. In one example, the first end of the well may be open,
while the second end of the well is closed. In other examples, both
the first end of the wells and the second end of the well chambers
are closed. Closure of the first end of the well chambers may be
through both a permanent closure mechanism and a temporary closure
mechanism. By "permanent" as used herein is meant that the closure
mechanism is intended to remain a fixture of the chamber of the
well. By "temporary" as used herein is meant that the closure
mechanism can be removed without affecting the structure,
integrity, or rigidity of the closure mechanism. In some aspects,
the closure of the well chamber first end may be through a
combination of a permanent and a temporary closure mechanism. In
one example, the temporary closure mechanism may be a liquid, such
as an oil fluid, that can fill the first end of the well chamber.
In certain examples, the oil drop may fill the first well end after
an analyte, biological sample, or analyte related detectable label
has been previously deposited into the well. In other examples, the
oil drops may be closure of the first end of the well regardless of
the presence of an analyte or biological sample within the
well.
[0410] The array of wells has a pattern of well chambers (e.g., the
formation of wells in the array) suitable for receiving a plurality
of labels, beads, labeled beads, tags, and the like. The pattern of
the array of the wells may vary according to resolution and spacing
between well chambers.
[0411] In some examples, the pattern of the well array can be
fabricated using nanoimprint lithography. In other examples, the
pattern of the well array can be fabricated through a combination
of any one of molding, pressure, heat, or laser.
[0412] The size of the well array may vary. In some examples, the
well array may be fabricated to have individual well chambers with
a diameter of 100 nm and with a periodicity of 500 nm.
[0413] In some examples the well array may be substantially as
described in the section related to digital microfluidics and
detection module.
[0414] In some examples, detection of the analyte or biological
sample of interest may occur through optical signal detection. For
example, shining an excitation light (e.g., laser) in order to
measure the signal intensity result. In other examples, the analyte
desired may be detected by measuring an optical signal emanating
from each well chamber and quantified by quantifying the result.
For example, the number of positive counts (e.g., wells) is
compared to the number of negative counts (e.g., wells) to obtain a
digital count. Alternately or in addition, a signal correlated to
analyte concentration may be measured (analog quantitiation). A
variety of signals from the wells of the device may be detected.
Exemplary signals include fluorescence, chemiluminescence,
colorimetric, turbidimetric, etc.
[0415] Adjacent Configuration of Sample Preparation and Analyte
Detection Device
[0416] In some embodiments, the array of wells is positioned on the
same superstrate as the sample preparation component. In some
examples, the superstrate and the array of wells may be positioned
on a first substrate. The first substrate may be divided into a
first portion at which droplets to be analyzed are initially
disposed and a second portion towards which the droplets are moved
for analyte detection. The superstrate may be present on the first
portion of the first substrate and the array of wells may be
positioned on a second portion of the first substrate. As such the
superstrate which forms the sample preparation component and the
array of wells which form the analyte detection component may be
directly adjacent. As used herein, the term "directly adjacent"
refers to there being a lack of object separating or dividing the
sample prep component and the array of wells. In examples, where
the sample prep component and array of wells are directly adjacent
to each other, the propagation of the liquid droplets across the
surface of the sample prep component is seamlessly transitioned
onto the surface of the array of wells. In other examples, the
array of wells is positioned indirectly adjacent to the sample prep
component. As used herein, the term "indirectly adjacent" refers to
there being an object or element separating or dividing the sample
prep component.
[0417] In some examples, to facilitate liquid movement and improve
position accuracy of the droplets into the individual well
chambers, the substrate surface of the sample preparation component
may be patterned or coated with a hydrophilic material. In other
examples, reagents such as oils and emulsions may be used to seal
the well arrays.
[0418] FIG. 13A illustrates a side view of a sample preparation
component positioned adjacent to an analyte detection component. As
shown in FIG. 13A, the sample preparation component includes a
superstrate 810. The superstrate 810 includes a series of phononic
structures 830. The size, shape, and dimensions of the phononic
structures may vary. As shown in FIG. 13A, the sample preparation
component is positioned to be directly adjacent to the analyte
detection component comprising an array of wells 860. Where these
components are positioned adjacent to each other, liquid propagated
across the surface of the superstrate 810 can be collected into
individual well chambers on the well array 860. In this particular
example, a sample inlet channel 840 is positioned between the
superstrate 810 and the cover 870. The superstrate 810 and the
cover 870 are separated by space/gap 850 defining a space where
liquid droplets are manipulated (e.g., merged, split, agitated,
etc.). However, in other examples, a sample inlet channel is not
included. The size, dimensions, and variations of the sample inlet
channel may vary. For example, the sample inlet channel may
introduce a fluid onto the surface of the superstrate 810. In other
examples, the sample inlet channel may introduce a fluid internally
within the superstrate 810.
[0419] In some examples, a cover seal may be provided over the
surface of the sample preparation component. In certain instances,
the cover seal may prevent contamination of the liquid contents of
the surface. In other instances, the cover seal is a liquid
impermeable layer. In other instances, the cover seal is made from
a flexible material such as plastics, silicon, or other type of
rubber. In other instances, the cover seal is made from a
non-flexible material such as a glass or other non-flexible
material. In some examples, the cover seal may be impenetrable to
heat, ultraviolet light, or other electromagnetic radiation to
prevent deformation of either the surface or liquid contents
present on the surface of the sample preparation component.
[0420] In some examples, a heat sink may be provided in order to
dissipate the heat generated by generation of surface acoustic
waves across the surface of the substrate.
[0421] Stacked Configuration of Sample Preparation and Analyte
Detection Device
[0422] In some embodiments, the array of wells (detection
component) is positioned over the sample preparation component
separated by a space where the droplets are manipulated. In some
examples, an inlet or channel may be positioned between the two
components. The inlet or channel may direct a sample or analyte
between the two components.
[0423] In some examples, the well array may be imprinted or
embossed onto a first substrate and the phononic structure may be
present on a superstrate positioned in a spaced apart manner from
the first substrate. The superstrate may be supported by a second
substrate.
[0424] In some examples, the step of coupling the first substrate
that includes the array of wells with the superstrate may be
facilitated with the use of a bonding agent, adhesive agent, tapes,
glues, soldering, or other affixing agent capable of coupling the
array of wells to the superstrate. In other examples, the step of
coupling the array of wells onto the phononic structures of the
sample prep component may be achieved through use of mechanical
fasteners, fixers, bolts, and other mechanical components such as
latches. In other examples, the step of coupling the array of wells
onto the phononic structures of the sample prep component may occur
through setting and positioning the array of wells over the
phononic structures of the sample prep component. In some examples,
the phononic structures of the substrate may be in parallel
orientation to the well array component.
[0425] The spacing between the phononic structures of the
superstrate and the well array may vary according to the type of
application to be performed, the size of the liquid droplet being
actuated onto the surface of the substrate, the size, shape and
arrangement of phononic structures, the size of the sample
channel/inlet, and the amplitude of the surface acoustic waves
propagating across the surface.
[0426] FIG. 13B illustrates a side view of a stacked configuration
of a superstrate and well array component. As shown in FIG. 13B,
the superstrate 810 includes a series of phononic structures 830.
The phononic structures 830 are arranged in an array of repeating
structural elements. The size, shape, and dimensions of the
phononic structures may vary. In this example, an array of wells
860 is also present. In this example, the array of wells 860 is
positioned directly over the superstrate. As illustrated in FIG.
13B, the opening of the wells may be directly opposite the phononic
structures. In this particular example, a sample inlet channel 840
is positioned between the well array and the superstrate. However,
in other examples, a sample inlet channel is not included. The
size, dimensions, and variations of the sample inlet channel may
vary. For example, the sample inlet channel may introduce a fluid
onto the surface of the superstrate 810. In other examples, the
sample inlet channel may introduce a fluid internally within the
superstrate 810. The substrate 820 that includes the array of wells
860 is positioned in a spaced apart manner from the superstrate 810
and is separated from the superstrate 810 by a gap/space 850.
[0427] The array of wells as shown in FIGS. 13A-B can vary in size
and/or shape. For example, the well array can be substantially
shallow or deep. The resolution of the well array is affected by
the spacing between each well chamber. For example, minimal spacing
between the well chambers allows for a greater number of wells to
collect a greater number of analytes or biological samples. In some
examples, well array may be formed via ablating the substrate. The
pattern of the well array may be formed by using a special pattern
or special mask, and subjecting the mask to laser ablation.
[0428] Fabricating Surface Acoustic Wave Sample Preparation and
Detection Device
[0429] FIGS. 14A-14B illustrate exemplary methods for separately
fabricating the SAW devices disclosed in the foregoing sections.
FIG. 14A illustrates that the sample preparation component and well
array component are positioned adjacent to each other by
fabricating the phononic structures and the array of wells on a
single base substrate. A superstrate (e.g., see FIG. 13A,
superstrate 810) is placed on an assembly line 900. Propagation of
the superstrate along the assembly line 900 is facilitated by a
conveyer belt-like mechanism utilizing a series of rollers. A roll
914 of the superstrate is unspooled and is subjected to an
embossing unit 910, which subjects the material to intense heat,
pressure, or ultraviolet light in order to form phononic structures
on the superstrate or embedded within the superstrate using a mold.
The array of wells is created using laser ablation 924. Thereafter,
the superstrate passes through a plurality of rollers to a surface
treatment component 920, which modifies properties of the
superstrate. Thereafter, the superstrate passes through an inkjet
printer 930 that deposits assay reagents on the superstrate. In
some examples, the resulting structures may be subject to a curing
step. In other examples, the resulting structures may be subjected
to surface treatment to modify their physical properties, for
example, incorporating functionalized reagents required for assay
protocols. A cover (e.g., FIG. 13A, cover 870) is then laminated
940 onto the superstrate. The cover may be provided as a roll 905
which is unspooled and moved using rollers. Prior to placing the
cover on the superstrate, a suitable spacer is placed between the
superstrate and the cover to enable liquid droplets to move between
the two surfaces. The assembled structure may be diced 950 to
generate individual devices.
[0430] FIG. 14B illustrates an exemplary method for fabricating the
device depicted in FIG. 13B. A roll 914 of superstrate (e.g., see
FIG. 13B, superstrate 810) is subjected to a fabrication process
using an embossing unit 910, which subjects the superstrate to
intense heat, pressure, or ultraviolet light in order to form
repeating structural elements of phononic structures in the
presence of a mold. Thereafter, the superstrate passes through a
surface treatment component 920 to modify properties of the
superstrate surface. Thereafter, the superstrate passes through an
inkjet printer 930, to deposit assay reagents in situ. To form the
detection module comprising an array of wells, a roll 906 of a
first substrate (e.g., see FIG. 13B, substrate 820) is subjected to
laser ablation 924. At the lamination unit 940, both the
superstrate and the first substrate containing well array are
combined together and subsequently bonded in a spaced apart
configuration. As a result, the superstrate and the substrate are
aligned vertically within a stack configuration. Thereafter, the
stacked substrates are subject to a dicing component 950, for
example, to generate individual devices.
[0431] The devices and systems and method described herein that
propagate droplet actuation may also include a variety of other
forces that affect droplet actuation. For example, movement of the
droplets across the surfaces may include electric field-mediated
forces, electrostatic actuation (such as electrical actuation),
electrowetting, dielectrophoresis, electric field gradients or
electrode-mediated forces. In embodiments where a combination of
surface acoustic waves and digital microarray electrodes are used
for droplet manipulation the SAW devices described herein may
include a series or plurality of electrodes.
[0432] The integrated devices disclosed herein may be used to
prepare a variety of samples, such as biological sample, for
detection of an analyte of interest. In certain cases, the device
may be used for carrying out digital immunoassay and detect
presence or absence of particles/beads that are correlated to the
presence or absence of an analyte.
[0433] Kits and Cartridges
[0434] Also provided herein is a kit for use in performing the
above-described methods with or without the disclosed device. The
kit may include instructions for analyzing the analyte with the
disclosed device. Instructions included in the kit may be affixed
to packaging material or may be included as a package insert. The
instructions may be written or printed materials, but are not
limited to such. Any medium capable of storing such instructions
and communicating them to an end user is contemplated by this
disclosure. Such media include, but are not limited to, electronic
storage media (e.g., magnetic discs, tapes, cartridges, chips),
optical media (e.g., CD ROM), and the like. As used herein,
"instructions" may include the address of an internet site that
provides the instructions.
[0435] The kit may include a cartridge that includes a
microfluidics module with a built-in analyte detection, as
described above. In some embodiments, the microfluidics and analyte
detection may be separate components for reversible integration
together or may be fully or irreversibly integrated in a cartridge.
The cartridge may be disposable. The cartridge may include one or
more reagents useful for practicing the methods disclosed above.
The cartridge may include one or more containers holding the
reagents, as one or more separate compositions, or, optionally, as
admixture where the compatibility of the reagents will allow. The
cartridge may also include other material(s) that may be desirable
from a user standpoint, such as buffer(s), a diluent(s), a
standard(s) (e.g., calibrators and controls), and/or any other
material useful in sample processing, washing, or conducting any
other step of the assay. The cartridge may include one or more of
the specific binding members described above.
[0436] Alternatively or additionally, the kit may comprise a
calibrator or control, e.g., purified, and optionally lyophilized
analyte of interest or in liquid, gel or other forms on the
cartridge or separately, and/or at least one container (e.g., tube,
microtiter plates or strips) for use with the device and methods
described above, and/or a buffer, such as an assay buffer or a wash
buffer, either one of which can be provided as a concentrated
solution. In some embodiments, the kit comprises all components,
i.e., reagents, standards, buffers, diluents, etc., which are
necessary to perform the assay. The instructions also can include
instructions for generating a standard curve.
[0437] The kit may further comprise reference standards for
quantifying the analyte of interest. The reference standards may be
employed to establish standard curves for interpolation and/or
extrapolation of the analyte of interest concentrations. The kit
may include reference standards that vary in terms of concentration
level. For example, the kit may include one or more reference
standards with either a high concentration level, a medium
concentration level, or a low concentration level. In terms of
ranges of concentrations for the reference standard, this can be
optimized per the assay. Exemplary concentration ranges for the
reference standards include but are not limited to, for example:
about 10 fg/mL, about 20 fg/mL, about 50 fg/mL, about 75 fg/mL,
about 100 fg/mL, about 150 fg/mL, about 200 fg/mL, about 250 fg/mL,
about 500 fg/mL, about 750 fg/mL, about 1000 fg/mL, about 10 pg/mL,
about 20 pg/mL, about 50 pg/mL, about 75 pg/mL, about 100 pg/mL,
about 150 pg/mL, about 200 pg/mL, about 250 pg/mL, about 500 pg/mL,
about 750 pg/mL, about 1 ng/mL, about 5 ng/mL, about 10 ng/mL,
about 12.5 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL,
about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL,
about 60 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL,
about 90 ng/mL, about 95 ng/mL, about 100 ng/mL, about 125 ng/mL,
about 150 ng/mL, about 165 ng/mL, about 175 ng/mL, about 200 ng/mL,
about 225 ng/mL, about 250 ng/mL, about 275 ng/mL, about 300 ng/mL,
about 400 ng/mL, about 425 ng/mL, about 450 ng/mL, about 465 ng/mL,
about 475 ng/mL, about 500 ng/mL, about 525 ng/mL, about 550 ng/mL,
about 575 ng/mL, about 600 ng/mL, about 700 ng/mL, about 725 ng/mL,
about 750 ng/mL, about 765 ng/mL, about 775 ng/mL, about 800 ng/mL,
about 825 ng/mL, about 850 ng/mL, about 875 ng/mL, about 900 ng/mL,
about 925 ng/mL, about 950 ng/mL, about 975 ng/mL, about 1000
ng/mL, about 2 .mu.g/mL, about 3 .mu.g/mL, about 4 .mu.g/mL, about
5 .mu.g/mL, about 6 .mu.g/mL, about 7 .mu.g/mL, about 8 .mu.g/mL,
about 9 .mu.g/mL, about 10 .mu.g/mL, about 20 .mu.g/mL, about 30
.mu.g/mL, about 40 .mu.g/mL, about 50 .mu.g/mL, about 60 .mu.g/mL,
about 70 .mu.g/mL, about 80 .mu.g/mL, about 90 .mu.g/mL, about 100
.mu.g/mL, about 200 .mu.g/mL, about 300 .mu.g/mL, about 400
.mu.g/mL, about 500 .mu.g/mL, about 600 .mu.g/mL, about 700
.mu.g/mL, about 800 .mu.g/mL, about 900 .mu.g/mL, about 1000
.mu.g/mL, about 2000 .mu.g/mL, about 3000 .mu.g/mL, about 4000
.mu.g/mL, about 5000 .mu.g/mL, about 6000 .mu.g/mL, about 7000
.mu.g/mL, about 8000 .mu.g/mL, about 9000 .mu.g/mL, or about 10000
.mu.g/mL.
[0438] Any specific binding members, which are provided in the kit
may incorporate a label, such as a fluorophore, enzyme, dendrimer,
bead, nanoparticle, nanobead, microparticle, microbead, polymer,
protein, biotin/avidin label, or the like, or the kit can include
reagents for labeling the specific binding members or reagents for
detecting the specific binding members and/or for labeling the
analytes or reagents for detecting the analyte. If desired, the kit
can contain one or more different tags or labels. The kit may also
include components to elicit cleavage, such as a cleavage mediated
reagent. For example, a cleavage mediate reagent may include a
reducing agent, such as dithiothreitol (DTT) or
tris(2-carboxyethyl)phosphine) TCEP. The specific binding members,
calibrators, and/or controls can be provided in separate containers
or pre-dispensed into an appropriate assay format or cartridge.
[0439] The kit may include one or more specific binding members,
for example, to detect one or more target analytes in the sample in
a multiplexing assay. The number of different types of specific
binding members in the kit may range widely depending on the
intended use of the kit. The number of specific binding members in
the kit may range from 1 to about 10, or higher. For example, the
kit may include 1 to 10 specific binding members, 1 to 9 specific
binding members, 1 to 8 specific binding members, 1 to 7 specific
binding members, 1 to 6 specific binding members, 1 to 5 specific
binding members, 1 to 4 specific binding members, 1 to 3 specific
binding members, 1 to 2 specific binding members, 2 to 10 specific
binding members, 2 to 9 specific binding members, 2 to 8 specific
binding members, 2 to 7 specific binding members, 2 to 6 specific
binding members, 2 to 5 specific binding members, 2 to 4 specific
binding members, 3 to 10 specific binding members, 3 to 9 specific
binding members, 3 to 8 specific binding members, 3 to 7 specific
binding members, 3 to 6 specific binding members, 3 to 5 specific
binding members, 3 to 4 specific binding members, 4 to 10 specific
binding members, 4 to 9 specific binding members, 4 to 8 specific
binding members, 4 to 7 specific binding members, 4 to 6 specific
binding members, 5 to 10 specific binding members, 5 to 9 specific
binding members, 5 to 8 specific binding members, 5 to 7 specific
binding members, 5 to 6 specific binding members, 6 to 10 specific
binding members, 6 to 9 specific binding members, 6 to 8 specific
binding members, 6 to 7 specific binding members, 7 to 10 specific
binding members, 7 to 9 specific binding members, 7 to 8 specific
binding members, 8 to 10 specific binding members, 8 to 9 specific
binding members, or 9 to 10 specific binding members. Each of the
one or more specific binding members may bind to a different target
analyte and each specific binding member may be labeled with a
different detectable label. For example, the kit may include a
first specific binding member binds to a first target analyte, a
second specific binding member binds to a second target analyte, a
third specific binding member binds to a third target analyte, etc.
and the first specific binding member is labeled with a first
detectable label, the second specific binding member is labeled
with a second detectable label, the third specific binding member
is labeled with a third detectable label, etc. In addition to the
one or more specific binding member, the kits may further comprise
one or more additional assay components, such as suitable buffer
media, and the like. Finally, the kits may comprise instructions
for using the specific binding members in methods of analyte
detection according to the subject invention, where these
instructions for use may be present on the kit packaging and/or on
a package insert.
[0440] Optionally, the kit includes quality control components (for
example, sensitivity panels, calibrators, and positive controls).
Preparation of quality control reagents is well-known in the art
and is described on insert sheets for a variety of immunodiagnostic
products. Sensitivity panel members optionally are used to
establish assay performance characteristics, and further optionally
are useful indicators of the integrity of the kit reagents, and the
standardization of assays.
[0441] The kit can also optionally include other reagents required
to conduct a diagnostic assay or facilitate quality control
evaluations, such as buffers, salts, enzymes, enzyme co-factors,
substrates, detection reagents, and the like. Other components,
such as buffers and solutions for the isolation and/or treatment of
a test sample (e.g., pretreatment reagents), also can be included
in the kit. The kit can additionally include one or more other
controls. One or more of the components of the kit can be
lyophilized, in which case the kit can further comprise reagents
suitable for the reconstitution of the lyophilized components. One
or more of the components may be in liquid form.
[0442] The various components of the kit optionally are provided in
suitable containers as necessary. The kit further can include
containers for holding or storing a sample (e.g., a container or
cartridge for a urine, saliva, plasma, cerebrospinal fluid, or
serum sample, or appropriate container for storing, transporting or
processing tissue so as to create a tissue aspirate). Where
appropriate, the kit optionally also can contain reaction vessels,
mixing vessels, and other components that facilitate the
preparation of reagents or the test sample. The kit can also
include one or more sample collection/acquisition instruments for
assisting with obtaining a test sample, such as various blood
collection/transfer devices such as microsampling devices,
micro-needles, or other minimally invasive pain-free blood
collection methods; blood collection tube(s); lancets; capillary
blood collection tubes; other single fingertip-prick blood
collection methods; buccal swabs, nasal/throat swabs; 16-gauge or
other size needle, circular blade for punch biopsy (e.g., 1-8 mm,
or other appropriate size), surgical knife or laser (e.g.,
particularly hand-held), syringes, sterile container, or canula,
for obtaining, storing or aspirating tissue samples; or the like.
The kit can include one or more instruments for assisting with
joint aspiration, cone biopsies, punch biopsies, fine-needle
aspiration biopsies, image-guided percutaneous needle aspiration
biopsy, bronchoaveolar lavage, endoscopic biopsies, and laproscopic
biopsies.
[0443] If the tag or detectable label is or includes at least one
acridinium compound, the kit can comprise at least one
acridinium-9-carboxamide, at least one acridinium-9-carboxylate
aryl ester, or any combination thereof. If the tag or detectable
label is or includes at least one acridinium compound, the kit also
can comprise a source of hydrogen peroxide, such as a buffer,
solution, and/or at least one basic solution. If desired, the kit
can contain a solid phase, such as a magnetic particle, bead,
membrane, scaffolding molecule, film, filter paper, disc, or
chip.
[0444] If desired, the kit can further comprise one or more
components, alone or in further combination with instructions, for
assaying the test sample for another analyte, which can be a
biomarker, such as a biomarker of a disease state or disorder, such
as infectious disease, cardiac disease, metabolic disease, thyroid
disease, etc.
[0445] The present invention has multiple aspects, illustrated by
the non-limiting examples provided herein.
[0446] Integrated DMF-Electochemical/Electrical/Optical Detection
Chip, Device, and System
[0447] As noted in the foregoing sections, an analyte detection
device configured to operate an analyte detection chip to prepare a
test sample and to detect an analyte related signal from the
prepared test sample in the analyte detection chip is disclosed.
The analyte detection chip may include a digital microfluidics
(DMF) region and an analyte detection region which may overlap or
may be spatially separated. In certain embodiments, the analyte
detection region may include electrodes for detection of an
electrochemical species generated when the analyte is present in
the sample. In other embodiments, the analyte detection region may
be configured for detection of a light signal generated when the
analyte is present in the sample. The DMF region may be used to
transfer a droplet for analysis to a region where the droplet will
be analysed optically or electrically. Optical detection may be
colorimetric detection, turbidometric detection, fluorescent
detection, and/or image analysis. Image analysis may include a
detection of an optical signal from the analyte detection
cartridge. Optical signal may be a light signal, such as a
colorimetric, turbidometric, or fluorescent signal. Optical signal
may be a combination of an image and a colorimeteric or fluorescent
signal detection, such as, those utilized for detection of wells
with spatially segregated beads and for determining the fraction of
beads on which an analyte molecule is captured. Electrochemical
detection may involve amperometry, coulometry, potentiometry,
voltametery, impedance, or a combination thereof. In some
embodiments, an instrument of the present disclosure operates one
or more of a cartridge comprising DMF electrodes and array of
wells; a cartridge comprising DMF electrodes and electrodes for
electrochemical sensing; and/or a cartridge comprising DMF
electrodes and an an optically interrogatable region for detecting
an optical signal. In other embodiments, an instrument of the
present disclosure operates a multi-functional cartridge (e.g., a
cartridge containing detection region for clinical chemistry, e.g.,
detection of electrochecmical species or chromogenic reaction
products and a detection region containing an array of wells for
spatially segregating portions of a droplet).
[0448] In certain cases, clinical chemistry may involve detection
of electrochemical species or chromogenic substrate produced by
action of an enzyme on a substrate. For example, the substrate may
be an analyte present in a sample and the enzyme may be specific
for the analyte and may generate an electrochemical species or a
colored reaction product by acting on the substrate. In other
cases, clinical chemistry may involve capturing the analyte using a
first binding member to generate a first complex comprising the
analyte and the first binding member; contacting the complex with a
second binding member, that binds to the analyte, to generate a
second complex comprising the analyte, the first binding member,
and the second binding member. The second binding member is
conjugated to an enzyme that generates a electrochemical species or
chromogenic reaction product upon exposure to a suitable
substrate.
[0449] The phrases "analyte detection chip," "analyte detection
cartridge," and the terms "chip" and "cartridge" are used
interchangeably herein to refer to a disposable or reuseable sample
processing device compatible with the analyte detection instruments
disclosed herein. The analyte detection instrument disclosed herein
is also referred to as analyte detection device that is used to
process a sample in the chips provided here. In certain
embodiments, the analyte detection chip may include a first
substrate and a second substrate, where the second substrate is
positioned over the first substrate and separated from the first
substrate by a gap. The first or the second substrate may include a
plurality of DMF electrodes. The plurality of DMF electrodes may be
a series of electrodes that are individually controllable for
activation and deactivation. The plurality of DMF electrodes may be
overlayed with an insulating material to electrically isolate the
DMF electrodes. In certain embodiments, the space/gap between the
first and second substrates may be filled with air or with an inert
fluid, such as oil. In certain embodiments, the DMF electrodes may
be arranged as described in the preceding sections herein. In
exemplary embodiments, a series of DMF electrodes may be disposed
on the first substrate and a single electrode disposed on the
second substrate in a facing configuration with the series of
electrodes on the first substrate. The series of electrodes and the
single electrode may be covered with an insulating layer. In other
cases; the series or plurality of electrodes on the first substrate
may be configured as co-planar electrodes and the second substrate
may not include an electrode. Various configurations of DMF
electrodes are described in the preceding sections describing an
integrated microfluidics and analyte detection device comprising an
array of wells. Any of these configurations of DMF electrodes can
be present in the additional cartridges disclosed here.
[0450] As described in the preceding sections, the electrodes
present in the first layer and/or the second layer may be
fabricated from a substantially transparent material, such as
indium tin oxide, fluorine doped tin oxide (FTO), doped zinc oxide,
and the like. In addition one or both substrates may be
substantially transparent to facilitate optical interrogation.
[0451] The analyte detection device may contain an optical,
electrochemical, and/or electrical means for detecting an optical
signal, electrochemical, electrical signal in an analyte detection
chip inserted into the device. In addition the analyte detection
device includes means for operating the DMF electrodes present in
the analyte detection chips. The analyte detection device disclosed
herein may include one or a plurality of interfaces for interacting
with the cartridge disclosed herein. In certain cases, the
cartridge interface may be an insertion slot. In other cases, the
interface may be a recess for accepting the cartridge and may be
enclosed by a door or lid. The analyte detection device may include
a single interface which may be compatible with a plurality of
analyte detection chips. For example, an insertion slot may be
compatible with an analyte detection chip that detects an
electrochemical signal, an analyte detection chip that detects an
optical signal, and/or analyte detection chip that detects an
electrical signal. In certain embodiments, the analyte detection
device may be configured for operating a plurality of analyte
detection chips simulatenously, for example, for detecting the same
analyte in different samples using multiple chips or for
simultaneously detecting multiple different analytes in the same
sample using multiple different chips. In such embodiments, the
device may include a plurality of interfaces, such as, insertion
areas.
[0452] The analyte detection chips of the present disclosure may
optionally include a plasma separation component. In certain
embodiments, the plasma separation component may include a filter
that captures cells present in a whole blood sample, allowing
plasma to filter through and be available for processing into a
sample droplet(s) for analysis. In other embodiments, the plasma
separation component may be a fluidic separation element.
Embodiments of analyte detection chips are disclosed below. Any of
the analyte detection chips described below may optionally include
a plasma separation component. In certain cases, the plasma
separation component may be a commercially available membrane. In
certain embodiments, a commercially available membrane such as
those available from International Point of Care, Inc. (e.g.,
Primecare.TM. Hydrophilic Asymetric Membranes) or from Pall
Corporation (e.g., Vivid.TM. Plasma Separation Membrane) may be
used for separating plasma. In certain cases, the membrane may be
integrated into the cartridges of the present disclosure. In other
embodiments, the chips, instruments, and systems of the present
disclosure may be configured to detect an analyte in a whole blood
sample.
[0453] i. DMF-Electrochemical Detection Cartridge
[0454] In certain embodiments, the cartridge disclosed herein
include a DMF region and an analyte detection region which may
overlap or be spatially segregated. The DMF region may be used to
transfer a droplet for analysis to a detection region where the
droplet will be analysed electrochemically. Electrochemical
analysis is performed by utilizing a working electrode that detects
an electrical signal generated by a electroactive species generated
by the presence of an analyte in the sample. The detected
electrical signal may be quantitated to determine the presence or
concentration of the analyte in the sample as the electrical signal
is proportional to the amount of analyte present in the sample.
Electrochemical detection may involve amperometry, coulometry,
potentiometry, voltametery, impedance, or a combination
thereof.
[0455] In certain embodiments, the electrochemical species may be
generated by action of an analyte-specific enzyme on the analyte.
In other embodiments, the electrochemical species may be generated
by action of an enzyme on a substrate. In such embodiments, the
enzyme is not specific to the analyte. Rather, the enzyme is
conjugated to a binding member that specifically binds to the
analyte. In certain embodiments, redox mediators may be included in
order to amplify the electrical signal generated by the
electrochemical species. Analyte specific enzymes and redox
mediators are well known and may be selected based on the desired
sensitivity and/or specificity.
[0456] Electrodes for detection of an electrochemical species may
be provided in numerous configurations. Such electrodes may be
separate from the DMF electrodes or may be DMF electrodes that have
been modified into electrodes for electrochemical sensing.
Exemplary configurations of analyte detection chips containing DMF
electrodes and electrodes for electrochemical sensing are further
described below.
[0457] FIG. 31A-31F provide a schematic of electrodes present in a
chip of the present disclosure. FIG. 31A depicts the DMF electrodes
310 that are used to transfer a droplet to a sensor area 311 of the
chip. The sensor area 311 includes a working electrode 312 and a
reference electrode 313. FIG. 31B depicts a droplet 314 positioned
on the sensor area 311. FIG. 31C illustrates the sensor area 311,
working electrode 312 and reference electrode 313, where the
electrodes are semicircular and are disposed in a co-planar
configuration. While not shown here, one of the electrodes may be
placed in a facing configuration with the other electrode. In such
an embodiment, the sensor area for electrochemical detection may
include a gap separating the working and reference electrodes where
the electrodes are brought into electrical connection upon
translocation of a droplet into the sensor area. The working and
reference electrodes are connected to contact pads 316 and 317 via
leads 315. The contact pads are operably connected to the device
that operates the chip. Additional configurations of electrodes for
electrochemical detection of an analyte of interest are shown in
FIGS. 31 D-31E. In FIG. 31D, the working electrode 312 is a
circular while the reference electrode 313 is arc-shaped and is
concentric with the working electrode and encircles the working
electrode. In FIG. 31E, the sensor area includes three
electrodes--a working electrode 312, a reference electrode 313, and
a counter electrode 318. The droplet and the electrodes are sized
such that the droplet is in contact with both working and reference
electrodes (and counter electrode, if present). FIG. 31F depicts
the relative sizes of a droplet and a working electrode and that
the size and shape of the electrode(s) is configured to conform to
the droplet size. It is noted that in this embodiment, the
reference electrode is present in a facing configuration to the
working electrode. The working electrode 313 has a first diameter
(A) that is smaller than the droplet 314 which has a second
diameter B. The first diameter A may be about 50 .mu.m-1.9 mm. The
second diameter B may be about 100 .mu.m-2 mm. Other ratios of the
electrode diameter to the droplet diameter may also be used in the
chips of the present disclosure. In embodiments, where the working
and reference electrodes (and the counter electrode, if present)
are in a coplanar configuration, the total area of the electrodes
(including any gaps between the electrodes) may be sized to conform
to the droplet diameter (see FIG. 31B).
[0458] In certain embodiments, the electrochemical sensors, such as
those depicted in FIGS. 31A-31F, may be on a surface opposite the
DMF surface, such as on the a single top electrode. In this way,
the DMF electrodes can cause a sample droplet to be moved to be in
contact with an electrochemical sensor wherein the sample droplet
can be interrogated while also be in contact with a DMF
electrode.
[0459] FIGS. 32A-32C depict analyte detection chips that include
DMF electrodes (310) where a DMF electrode is modified into a
sensing electrode (310A, 310B, or 310C) suitable for
electrochemical detection. In FIG. 32A, a DMF electrode 310A is
modified by creating an opening in an insulating layer disposed
over the DMF electrodes, the opening provides an area for contact
between a droplet and the modified DMF electrode 310A for
electrochemical sensing. In FIG. 32B, a modified DMF electrode 310B
includes multiple pin-hole openings in the insulating layers
covering the DMF electrodes for contact between a droplet and the
modified DMF electrode. In FIG. 32C, the DMF electrodes are covered
with an insulating layer that is removable by exposure to light.
One or more DMF electrodes may be exposed to light 311 to remove
the insulating layer thereby creating a modified DMF electrode that
is not covered by the insulating layer and can thus contact a
droplet. In FIG. 32C, the electrode 310C is exposed to light to
remove the light sensitive insulating layer and expose the
electrode. A DMF electrode disposed in a facing configuration to
the DMF electrodes 310 may also be exposed to provide a reference
electrode. For example, a DMF electrode may be modified to include
an opening or multiple openings at an area in a facing
configuration with electrodes 310A, 310B, or 310C.
[0460] In another embodiment, the DMF electrodes may be disposed on
a first substrate and at least one of the electrodes for
electrochemical sensing may be disposed on a second substrate. In
some embodiments, the DMF electrodes may be disposed on a first
substrate and the working and reference electrodes for
electrochemical sensing may be disposed on the second
substrate.
[0461] In certain embodiments, the DMF-electrochemical chip may
include a capillary region and electrodes for electrochemical
sensing may be disposed in the capillary region. The capillary
region may facilitate movement of a droplet into the capillary
region of electrochemical sensing.
[0462] In certain embodiments, the analyte detection chips of the
present disclosure may include a sensing region as disclosed in
U.S. Pat. No. 5,200,051. As described in U.S. Pat. No. 5,200,051, a
sensing electrode useful for determining the presence and/or
concentration of analytes of interest was provided. The sensing
electrode detects electrochemical species generated in response to
the analyte by action of an enzyme on the analyte. Sensing
electrode is also referred to as working electrode. As is known in
the literature, the generation of the electrochemical species may
involve use of a redox mediator. Further, the enzyme and/or redox
mediator may be present in a reagent mixture localized at the
sensing electrode. In other cases, the enzyme and/or redox mediator
may be introduced into the chip using DMF electrodes to transport a
droplet containing the enzyme and/or redox mediator from a depot
connected to the chip.
[0463] In other embodiments, an immunoassay may be utilized.
Briefly, in an exemplary immunoassay, the analyte may be captured
by a first binding member (e.g., a receptor, an aptamer, or an
antibody) that binds to the analyte. After, an optional wash step,
a second antibody that binds to the analyte may be used to create a
complex. A second binding member (e.g., an antibody or aptamer) may
be conjugated to an enzyme, which enzyme may act on a substrate to
generate an electrochemical species detected by the working
electrode. In certain cases, the enzyme may hydrolyze the
substrate. This hydrolyzed substrate can then undergo reactions
which produce changes in the concentration of electroactive species
(e.g., dioxygen and hydrogen peroxide) which are electrochemically
detected with the analyte detection chips of the present
disclosure. Such immunoassays are also exemplified by an alkaline
phosphatase that is conjugated to a second binding member. Alkaline
phosphatase reacts with the substrate (5-bromo-4-chloro-3-indoxyl
phosphate) to produce changes in the concentration of electroactive
species (dioxygen and hydrogen peroxide) which are
electrochemically detected with the DMF-electrochemical detection
chip. Both sandwich and competitive assays can be effected using
the procedures described in U.S. Pat. No. 5,200,051. In these
assays, in addition to the DMF electrode, a working (or sensing)
electrode and optional reference electrode may be included. A
bioactive layer may be immobilized on the working electrode, which
bioactive layer includes a first specific binding member (e.g., a
receptor or an antibody) that binds to an analyte of interest. In
other embodiments, the sample may be processed using the DMF
electrodes and transported to the working/reference electrodes for
detection of electrochemical species.
[0464] In an embodiment of the present disclosure, the analyte
detection chip may be used to prepare a droplet that includes the
electrochemical species. For example, the steps of mixing a sample
droplet with a droplet containing an enzyme that acts on the
analyte to create electrochemical species may be conducted by the
DMF electrodes of the chip and the droplet (or a portion thereof)
containing the electrochemical species moved to working and
reference electrodes for detection and optionally measurement of
the electrochemical species.
[0465] In other embodiments, the DMF electrodes may perform the
steps of mixing a sample droplet with a droplet containing a first
binding member (e.g., receptor or antibody) conjugated to a
magnetic bead. The resulting droplet may be mixed with another
droplet containing a second antibody conjugated to an enzyme. The
resulting droplet may then be mixed with a buffer droplet to wash
away any unbound second antibody and the droplet mixed with a
droplet containing a substrate for the enzyme and the resulting
droplet moved to the working/reference electrode for detection of
electrochemical species generated by the action of the enzyme on
the substrate. In such embodiments, since the working electrode
does not need to be functionalized by attachment of a binding
member (e.g., a receptor, aptamer, or antibody that binds to the
analyte), the same analyte detection chip can be used for detecting
different types of analytes by simply loading droplets containing
the binding member specific for the analyte being
detected/measured. Any immunoassay format such as those described
in the preceding sections may be used. DMF electrodes may be
utilized for conducting sample preparation for immunoassay, such
as, in the manner described in the preceding sections.
[0466] Similar advantages are realized by the disclosed analyte
detection chip where the analyte is directly detected by action of
an enzyme. For example, instead of localizing the enzyme (and
additional reagents, such as, redox mediator) on the
working/sensing electrode, the droplet containing the reagents may
be mixed with the sample droplet and the resulting droplet moved to
the working/sensing electrode for detection of the electrochemical
species generated by the enzyme when the analyte is present in the
sample. As such, the same chip can be used to detect different
analytes by simply loading droplets containing the enzyme that acts
on the analyte being detected/measured. For example, enzymes such
as glucose oxidase or dehydrogenase may be used for detection of
glucose; lactate dehydrogenase for detection of lactate; creatinine
amidohydrolase, creatinase, or creatine kinase for detection of
creatine; and the like. In some examples, the glucose dehydrogenase
may be nicotinamide dinucleotide glucose dehydrogenase (NAD-GDH),
pyrrole quinoline quinone glucose dehydrogenase (PQQ-GDH) or
flavin-adenine dinucleotide glucose dehydrogenase (FAD-GDH). In
other examples, the analyte may be beta-hydroxybutyrate (ketone)
and the enzyme may be hydroxybutyrate dehydrogenase.
[0467] The size and shape of the electrodes required for detection
of the electrochemical species (e.g., working and reference
electrodes) can be determined empirically or can be based on the
literature. For example, the electrodes may be similar to those
disclosed in U.S. Pat. No. 5,200,051, which is herein incorporated
by reference in its entirety. The material of the electrodes may be
any material conducive to electrochemical sensing. Exemplary
electrode materials include carbon, platinum, gold, silver,
rhodium, iridium, ruthenium, mercury, palladium, and osmium. In
certain cases, the working electrode may be a made from silver and
the reference electrode may be silver/silver halide (e.g. silver
chloride).
[0468] In certain embodiments, the working electrode (and optional
reference electrode) may be covered with a selectively permeable
layer. The selectively permable layer may substantially exclude
molecules with a molecular weight of about 120 kDa or more while
allowing the free permeation of molecules with a molecular weight
of about 50 kDa or less.
[0469] In certain embodiments, interfering electroactive species
having a molecular weight above a desired threshold (e.g., above
120 kDa) may effectively be excluded from interacting with the
working electrode surface by employing a selectively permeable
silane layer described in U.S. Pat. No. 5,200,051. Such a
permselective layer, however, allows lower molecular weight
electroactive species, like dioxygen and hydrogen peroxide, to
undergo a redox reaction with the underlying electrode surface.
Such a perselective layer may be especially useful in amperometric
measurement.
[0470] In a potentiometric measurement, a polymeric material having
functional groups and chemical properties conducive to the further
incorporation of certain ionophoric compounds may be used as a
semipermeable ion-sensitive film which is established on the
working electrode of the analyte detection chip. The development of
a potential at the electrode-film interface depends on the charge
density, established at equilibrium, of some preselected ionic
species. The identity of such ionic species is determined by the
choice of the ionophore incorporated in the semipermeable film. An
enzyme which is, in turn, immobilized in the biolayers described
herein catalyzes the conversion of a particular analyte, present in
the sample, to the preselected ionic species. As noted herein, the
enzyme may not be immobilized in the biolayers but rather brought
in proximity to the analyte by the DMF electrodes transporting a
droplet containing the enzyme to a sample droplet and fusion of the
two droplets.
[0471] In another aspect, the analyte detection chips of the
present disclosure may include DMF electrodes that are used for
transportation and optional processing of a sample droplet and
modified DMF electrodes that are used for detection of an analytes,
such as, ions, e.g., Na.sup.2+, K.sup.+, Ca.sup.2+, and the like.
For detection of ions in a sample, the modified DMF electrodes may
be covered with an ion-selective membrane instead of the
ion-impermeable insulating layer that covers the DMF
electrodes.
[0472] Redox Mediators
[0473] Representative examples of redox mediators that may be
present in a chip of the present disclosure or introduced into a
chip of the present disclosure via a droplet, include
organometallic redox species such as metallocenes including
ferrocene or inorganic redox species such as hexacyanoferrate
(III), ruthenium hexamine, etc. Additional suitable electron
transfer agents usable as redox mediators in the sensors of the
present invention are osmium transition metal complexes with one or
more ligands, each ligand having a nitrogen-containing heterocycle
such as 2,2'-bipyridine, 1,10-phenanthroline, 1-methyl, 2-pyridyl
biimidazole, or derivatives thereof. The electron transfer agents
may also have one or more ligands covalently bound in a polymer,
each ligand having at least one nitrogen-containing heterocycle,
such as pyridine, imidazole, or derivatives thereof. One example of
an electron transfer agent includes (a) a polymer or copolymer
having pyridine or imidazole functional groups and (b) osmium
cations complexed with two ligands, each ligand containing
2,2'-bipyridine, 1,10-phenanthroline, or derivatives thereof, the
two ligands not necessarily being the same. Some derivatives of
2,2'-bipyridine for complexation with the osmium cation include but
are not limited to 4,4'-dimethyl-2,2'-bipyridine and mono-, di-,
and polyalkoxy-2,2'-bipyridines, including
4,4'-dimethoxy-2,2'-bipyridine. Derivatives of 1,10-phenanthroline
for complexation with the osmium cation include but are not limited
to 4,7-dimethyl-1,10-phenanthroline and mono, di-, and
polyalkoxy-1,10-phenanthrolines, such as
4,7-dimethoxy-1,10-phenanthroline. Polymers for complexation with
the osmium cation include but are not limited to polymers and
copolymers of poly(1-vinyl imidazole) (referred to as "PVI") and
poly(4-vinyl pyridine) (referred to as "PVP"). Suitable copolymer
substituents of poly(1-vinyl imidazole) include acrylonitrile,
acrylamide, and substituted or quaternized N-vinyl imidazole, e.g.,
electron transfer agents with osmium complexed to a polymer or
copolymer of poly(1-vinyl imidazole). Embodiments may employ
electron transfer agents having a redox potential ranging from
about -200 mV to about +200 mV versus the standard calomel
electrode (SCE).
[0474] Enzymes
[0475] The enzymes used in conjunction with the analyte detection
chips of the present disclosure may be selected based upon the
analyte being detected or the substrate being utilized (e.g., in an
immunoassay). Non-limiting examples of enzymes include one or more
of glucose oxidase, glucose dehydrogenase, NADH oxidase, uricase,
urease, creatininase, sarcosine oxidase, creatinase, creatine
kinase, creatine amidohydrolase, cholesterol esterase, cholesterol
oxidase, glycerol kinase, hexokinase, glycerol-3-phosphate oxidase,
lactate dehydrogenase, alkaline phosphatase, alanine transaminase,
aspartate transaminase, amylase, lipase, esterase, gamma-glutamyl
transpeptidase, L-glutamate oxidase, pyruvate oxidase, diaphorase,
bilirubin oxidase, and their mixtures.
[0476] ii. DMF-Optical Chips
[0477] In certain embodiments, the analyte detection chips may be
used to generate an optical signal indicating presence of an
analyte in a sample being assayed by the chips. The optical signal
may be, for example, a colorimetric signal, turbidometric signal,
and/or a fluorescent signal. The magnitude of the optical signal
may be proportional to the amount of analyte and may be used to
determine the presence or concentration of the analyte in the
sample.
[0478] In certain embodiments, at least one of the substrates of
the analyte detection chip may be transparent to facilitate
detection of optical signal. In addition, the DMF electrodes may be
transparent.
[0479] The DMF electrodes may be used to process a sample droplet
for generation of an optical signal indicative of presence of the
analyte in the sample. The optical signal may generated by action
of an enzyme on a substrate. Any assay format may be utilized for
generation of an optical signal, such as, colorimetric assay (e.g.,
detect a chromogenic reaction product produced by action of an
analyte specific enzyme), immunoassay, sandwich immunoassay (e.g.,
monoclonal-polyclonal sandwich immunoassays, including enzyme
detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent
assay (ELISA), competitive inhibition immunoassay (e.g., forward
and reverse), enzyme multiplied immunoassay technique (EMIT),
particle-enhanced turbidimetric inhibition immunoassay (PETINIA),
homogeneous enzyme immunoassay (HEIA), a competitive binding assay,
bioluminescence resonance energy transfer (BRET), one-step antibody
detection assay, homogeneous assay, heterogeneous assay, capture on
the fly assay, etc. These and other assay formats are described in
detail in the preceding sections.
[0480] Cartridges provided herein may include a DMF region and a
region containing a plurality of wells for spatially separating
molecules for optical analysis. Such cartridges may be used for
nucleic acid testing (NAT). For example, the DMF region may process
a sample by amplifying a target nucleic acid that may be present in
the sample. In some embodiments, NAT may assay multiple different
target nucleic acids that may be present in the sample.
[0481] Optical signals that may be measured include fluorescence,
chemiluminescence, colorimetric, turbidimetric, etc. In certain
embodiments, optical signals may be detected using a
spectrophotometer. For example, an optical signal may be detected
as described in Anal Bioanal Chem (2015) 407:7467-7475, which is
herein incorporated by reference in its entirety. In this
technique, a custom manifold aligns optical fibres with a digital
microfluidic chip, allowing optical measurements to be made in the
plane of the device. Because of the greater width vs. thickness of
a droplet on-device, the in-plane alignment of this technique
allows it to outperform the sensitivity of vertical absorbance
measurements on digital microfluidic (DMF) devices. In other
embodiments, the optical signal may be measured at a plane
perpendicular to the chip.
[0482] In certain embodiments, the DMF-optical cartridge may
include a built-in or a separate component for illuminating a
droplet in the cartridge. A built-in or a separate component may
also be used for detecting light from the illuminated droplet. For
example, a waveguide may be used to illuminating a droplet in the
cartridge. A waveguide may also be used for detecting an optical
signal from the droplet. In certain cases, a region of the
DMF-optical cartridge may be manufactured from a waveguide
material. In certain cases, one or both substrates of the
DMF-optical cartridge may be a waveguide. Any suitable waveguide
that can propagate light with minimal loss may be used in such
cartridges.
[0483] The optical signal generation may involve illuminating a
droplet with a light source and measuring the light from the
droplet using a detector, such as, a spectrometer or a CMOS
detector.
[0484] An exemplary chip for optical detection of a signal
generated by action of an enzyme is illustrated in FIG. 7 of Anal
Bioanal Chem (2015) 407:7467-7475. FIG. 7 is reproduced herein as
FIG. 33. As shown in FIG. 33, the DMF chip includes a PPM disc 331
on which an analyte is absorbed. The DMF chip was used to extract
the analyte fluorescein from PPM disc in the reaction zone and
moved to the detection zone and positioned adjacent an optical
fiber. Laser was used to excite the fluorescein and its emission
was measured using the optical fiber.
[0485] In certain embodiments, the DMF-optical chip may not be
configured with an optical fiber. In these embodiments, the droplet
may be interrogated from a vertical direction and either the
reflected light, emitted light or absorbance from the droplet
measured.
[0486] iii. DMF-Imaging Chip
[0487] Also provided herein are DMF chips that are configured for
image analysis. The DMF chip may include an array of electrodes
(individually or collectively energizable) on a first substrate
which electrodes are covered with an insulating layer. The first
substrate may be spaced apart from a second substrate. In certain
cases, the second substrate may include a ground electrode in a
facing configuration to the array of electrodes.
[0488] The two substrates are separated by a gap. In certain cases,
the substrates are separated by a narrow gap of about 5 .mu.m or
less, such as 1 .mu.m. In certain cases, a portion of the DMF chip
may include a region where the substrates are separated by a narrow
gap of about 5 .mu.m or less, such as 1 .mu.m. For example, the gap
between the substrates in a region where the sample is introduced
may be relatively wider (about 100 .mu.m), while the gap where the
sample droplet (or a processed sample droplet) is to be imaged in
narrower. The smaller gap height results in creation of a monolayer
of particles that can be imaged and analyzed, therefore making
analysis of single particles more straightforward.
[0489] FIG. 34A shows a possible representation where red blood
cells (RBC) are represented by the ellipses. The gap height
restricts RBCs from forming multiple layers within the gap. The
image sensor located above the DMF chip is used to collect optical
data for analysis. In this embodiment, the illumination is
co-located with the image sensor. The top substrate must be
optically clear for illumination and imaging. Out of plane from the
DMF chip is an imaging detector that is used to collect optical
data for analysis. The imager technology can include, but not
limited to CMOS and CCD technologies.
[0490] FIG. 34B depicts a DMF-imaging chip where a portion of the
chip includes substrates separated by a larger gap which is
connected to a capillary flow region having a gap dimensioned to
disperse the particles into a single layer. The DMF portion of the
chip is useful to actively control fluid flow, mix fluids, move or
separate particles to different active reagent areas on the chip or
other actions that are useful for analytical operations (dilutions,
etc.) whereas the capillary flow region is a channel that creates
the particle monolayer by the flow gap transition from a droplet
present on the DMF portion of the chip to the narrow capillary gap
due to capillary forces. This allows for materials present in
droplets to be analyzed via an imaging detector that is positioned
and focused on the capillary flow region. The DMF portion of the
chip controls fluids and the capillary flow region creates an
analytical region for colorimetric, absorbance, transmission,
fluorescence particle counting and imaging (such as cells). The
counting and imaging in the capillary channel can be done with
either a static position of the particles or as the particles flow
through the channel.
[0491] The gap within the DMF region in the chip of FIG. 34B does
not need to be constrained to a gap height for monlayer of
particulate matter, rather, since the imaging detector is focused
on the capillary flow region, the gap of the capillary flow region
is held at a value that prevents multiple layers from forming. The
imaging detector assembly can have multiple depths of fields and
movement of the lenses to focus on the contents within the
capillary channel or accommodate different capillary heights.
[0492] FIGS. 42A and 42B depict embodiments of integrated sample
preparation and sample analysis cartridges. A schematic of a
cartridge 1000 with a DMF element and an imaging chamber is
provided in FIG. 42A. A first substrate 1001 including DMF
electrodes 1003 is disposed in a spaced apart manner from a second
substrate 1002. The space between the first and second substrates
varies such that a first region of the cartridge includes a first
chamber having a height h.sub.1 and a second region includes a
second chamber having a height h.sub.2. As depicted in FIG. 42A,
h.sub.1 is larger than h.sub.2. In certain embodiments, h.sub.1 may
range from 20-200 .mu.m (microns), e.g., 50-200 microns, 75-200
microns, 100-200 microns, 125-200 microns, 100-175 microns, e.g,
150 microns and h.sub.2 may range from 2-10 .mu.m (microns), e.g.,
2-8 microns, 2-6 microns, 3-6 microns, e.g., 4 microns. Aspects of
the disclosed cartridge 1000 include embodiments where the first
chamber is configured for actuating a sample droplet, e.g., a blood
droplet to move the blood droplet in the first chamber such that
the droplet contacts reagents disposed in the first chamber,
thereby facilitating processing of the sample and preparation for
subsequent analysis in the second chamber. For example, the first
chamber may include reagents for staining of cells present in a
blood sample to facilitate cell detection/counting, complete blood
count and/or other hematology measurements, e.g., staining,
counting, and/or morphological analysis of bacteria, RBCs, WBCs,
and/or platelets, etc. As disclosed herein, the DMF electrodes may
be operated to move the sample droplet to a region in the first
chamber having a reagent (e.g., a staining reagent, such as, a dye
that binds to nucleic acid, e.g., acridine orange, ethidium
bromide, TOTO, TO-PRO, or SYTOX) disposed in a dry form or in form
of a droplet. The sample droplet may be mixed with the reagent to
provide uniform distribution of the reagent in the sample droplet.
Mixing may be performed by splitting and merging the sample droplet
till at least 80% of the staining reagent is uniformly distributed
within the sample droplet. The second chamber may be transparent at
least in an imaging region 1004 to facilitate optical analysis of a
sample transposed into the second chamber from the first chamber.
As shown, the second chamber may be configured to facilitate
distribution of cells present in the sample as a monolayer,
avoiding overlapping cells which tend to introduce error in optical
analysis of the cells. The second substrate 1002 may be include a
first planar region and a second planar region separated by a
sloping region that introduces a shoulder or a step element 1006
for changing the height of the plane of the first planar region
with reference to the second planar region. The two-tiered second
substrate is disposed over the first substrate that is
substantially planar to provide the cartridge that includes the two
chambers of different heights. As discussed herein, the sample may
be moved from the first chamber into the second chamber by
capillary action, DMF electrodes, SAW, or other methods.
[0493] FIG. 42B provides a schematic of an embodiment of a
cartridge 2000 comprising a first chamber 2001 defined by a first
substrate 2002 and a second substrate 2003, spaced apart by a
spacer having a height h.sub.3. The cartridge 2000 also includes a
second chamber 2004 defined by a third substrate 2006 and a fourth
substrate 2005 spaced apart by beads 2007 having a height h.sub.4.
The first substrate is depicted with DMF electrodes 2008 although
the DMF electrodes may be present on the second substrate or on
both substrates, as described herein. Similar to the cartridge in
FIG. 42A, the first chamber has a height that is larger than that
of the second chamber. In certain embodiments, h.sub.3 may range
from 20-200 .mu.m (microns), e.g., 50-200 microns, 75-200 microns,
100-200 microns, 125-200 microns, 100-175 microns, e.g, 150 microns
and h.sub.4 may range from 2-10 .mu.m (microns), e.g., 2-8 microns,
2-6 microns, 3-6 microns, e.g, 4 microns. The cartridge depicted in
FIG. 42B includes polystyrene beads dispersed between the third and
fourth substrates for defining a uniform height in the second
chamber for facilitating distribution of cells as a monolayer. At
least a portion of the second chamber may be transparent to
facilitate interrogation by an optical device 2010. Similar to the
cartridge in FIG. 42A, the first chamber actuates the sample 2012
for preparation for analysis (e.g., by mixing with a staining
reagent) in the second chamber. Cells 2015 dispersed in a monolayer
in the second chamber are also depicted. The optical device may be
positioned to interrogate the sample through the third or the
fourth substrate. In certain embodiments, the first 2002 and third
2006 substrates may be formed from a single substrate such that the
cartridge has a common bottom substrate. The first and second
chambers may be configured to allow for a sample to move from the
first chamber to the second chamber utilizing capillary action, DMF
electrodes, SAW, or other methods.
[0494] Additional configurations for the DMF chamber and the
imaging chamber include embodiments depicted in FIGS. 42C-42E. The
cartridge may include a DMF chamber that includes DMF electrodes
for sample preparation (e.g., mixing a sample droplet with a
staining reagent) operably connected to an imaging chamber. As
noted in the descriptions for FIGS. 42A and 42B, the height
(h.sub.1) of the DMF chamber may range from 20-200 .mu.m (microns),
e.g., 50-200 microns, 75-200 microns, 100-200 microns, 125-200
microns, 100-175 microns, e.g, 150 microns and the height (h.sub.2)
of the imaging chamber may range from 2-10 .mu.m (microns), e.g.,
2-8 microns, 2-6 microns, 3-6 microns, e.g., 4 microns. FIG. 42C
depicts a cartridge 1100a comprising a DMF chamber defined by a
first substrate 1101 disposed over a second substrate 1102. The DMF
electrodes are not illustrated and may be present on the first
and/or second substrate. The second substrate 1102 extends to the
imaging chamber which is defined by the second substrate 1102 and
third substrate 1104. A spacer 1103 defines distal end of the DMF
chamber. The spacer 1103 may contact a lower surface of the first
substrate 1101 and an upper surface of substrate 1104. FIG. 42D
depicts a cartridge 1100b in which the imaging chamber is operably
connected to the DMF chamber via a two-part spacer 1103a-1103b,
where a first part of the spacer (1103a) is disposed between a
first substrate 1101 and a third substrate 1104 and a second part
of the spacer (1103b) is disposed between a second substrate 1102
and a fourth substrate 1105. FIG. 42E depicts a cartridge 1100c in
which the imaging chamber is disposed in the distal region of the
DMF chamber. The DMF chamber is defined by substrates 1101 and
1102. The imaging chamber is defined by the substrate 1101 and
substrate 1104. The spacer 1103 supports the substrate 1104.
[0495] As noted herein, the DMF chamber may be reversibly coupled
to the analyte detection region (electrochemical detection,
electrical detection, optical detection, etc.) to form an
integrated or semi integrated cartridge. The coupling of the DMF
chamber and the analyte detection region may be performed as
described herein.
[0496] In certain embodiments, the cartridge may include reagents
for analysis of a blood sample and may be configured as disclosed
in U.S. Pat. Nos. 6,004,821 or 8,367,012, which are herein
incorporated by reference in their entirety. In certain
embodiments, the DMF electrodes and the chamber for sample
preparation may be configured as disclosed in WO2016/161400,
WO2016/161402, or US2015/0298124, which are herein incorporated by
reference in their entirety. It is understood that instead of or in
addition to the DMF electrodes, the cartridges may be configured
for actuating sample droplets by SAW.
[0497] iv. DMF Chip with Multiple Detection Regions
[0498] Also provided herein is a DMF chip that allows for multiple
analyses and techniques to be utilized on a single DMF chip. FIG.
35 shows a DMF chip layout where specific detection zones have been
created, not utilizing a singular detection technology, but rather,
creating zones that are configured for the detection technology
itself. For example, the zones 350a may be created and configured
to allow for electrochemical detection; the zones 350b are specific
to imaging analysis, and zones 350c are for absorbance based
measurements.
[0499] The chip depicted in FIG. 35 provides a compact DMF chip on
which multiple typical diagnostic tests utilizing different
detection technologies can be carried out. For example, hematology
measurements typically rely upon imaging analyses, whereas clinical
chemistry or immunoassay measurements typically rely upon
electrochemical or optical based detections. Using this chip, a
user can utilize a single blood collection across multiple
diagnostic analytic devices, thus greatly simplifying the
diagnostic process and time to result for users.
[0500] FIG. 36 illustrates a DMF chip layout comprised of imaging,
photon and electrochemical sensing. The DMF configuration can be
any combination and/or number of sensing zones depending on the
diagnostic requirements. Imaging sensors such as CMOS technology
may be utilized. Where higher sensitivity is required CCD or
enhanced CCD (eCCD) can be used. CMOS detectors are versatile such
that they can be used as electrochemical sensor with the proper
coatings. Where high sensitivity photon sensing is required (listed
from highest to lowest) Photomultiplier Tubes (PMT) or Avalanche
Photodiode Detectors (APD) or photodiodes can be used. Illumination
may be achieved by using Light Emitting Diodes (LED) or solid state
type lasers. The illumination configurations shown, addresses both
brightfield and fluorescence excitation. The dichroic or beam
splitter reflects the fluorescent excitation wavelengths and
transmits emission wavelengths. Bandpass wavelengths in the
dichroic will allow for transmitted brightfield wavelengths to be
transmitted to the sensor. Dichroic optical component is not needed
for transmitted light analysis, only fluorescence. Additional
excitation and emission filters may be required for the different
assays and can be included in the respective optical paths. An
analyte detection instrument compatible with such a chip may be
configured to include means for detecting an optical signal and an
electrical signal. For example, the analyte detection instrument
may include imaging sensors such as CMOS, CCD or enhanced CCD
(eCCD) camera, PMT, APD. In addition, the analyte detection
instrument may include means for sample illumination such as LED,
lasers, and the like.
[0501] An embodiment of a DMF chip with multiple detection regions
is shown in FIG. 37. The chip in FIG. 37 includes a sample
acquisition port through which a 20-80 .mu.l whole blood sample is
loaded onto the chip and immediately transferred to the
re-suspension area where the sample can reside up to 1/2 hour or
more. The re-suspension area is used to re-suspend the blood before
it is distributed to the 5 aliquots which are 0.5-1 .mu.l in
volume. Re-suspension is achieved by reciprocating the blood sample
such that the fluid path allows for total inversion of the blood,
fluid path >2.times. the length of the blood slug.
[0502] The re-suspended blood is transferred and divided into
several aliquots. Individually, each aliquot is transferred to a
sensing/imaging areas passing through a reagent section to stain,
sphere or lyse the cells. Mixing is accomplished by reciprocating
motion of the aliquot in this region. The diagram illustrates the
different reagents and imaging areas for hematology measurements.
All reagents in the consumable are dried. Other coatings in the
fluid path provide hydrophobic and hydrophilic surfaces for
maneuvering the liquid. Alternative configurations may be included
for conducting electrochemical sensing and optical sensing.
Platelets (PLT), Reticulocytes (RETC) and Nucleated Red Blood Cells
(NRBC) can be imaged in the same imaging areas as Red Blood Cells
(RBC) and White Blood Cells (WBC). DMF may be used for collecting
the sample in the acquisition port, re-suspending the sample,
optionally include the aliquot partioning, and stain the entire
sample. Such chips may be used for assaying blood agglutination,
for example, determining blood type.
[0503] FIGS. 38 and 39 illustrate DMF chips capable of
electrochemical and optical detection. A 20-80 .mu.l whole blood
sample is acquired from a patient at the fill port and immediately
transferred to the re-suspension area where the sample can reside
up to 1/2 hour or longer. The re-suspension area is used to
re-suspend the blood before it is distributed. Re-suspension is
achieved by reciprocating the blood sample such that the fluid path
allows for total inversion of the blood, fluid path >2.times.
the length of the blood slug. The sample is transferred and divided
into two aliquots; one for plasma separation and the other for
whole blood analysis. Plasma separation can be achieved with a
separation medium; i.e. filtering, or by fluidic methods utilizing
the capabilities of DMF. The consumable utilizes the same imaging
sensor layout as the hematology construct in FIG. 37. Each imaging
area has a corresponding area for reagent mixing. All reagents in
the "Staining & Mixing Areas" are dried. To provide sample
wash, reagent packs for wash may be added to the fluidics design
(FIG. 39).
[0504] v. Analyte Detection Device
[0505] As noted herein, an analyte detection device that includes a
cartridge interface for interacting with the analyte detection
chips are provided. In certain embodiments, the analyte detection
device may be compatible with only one type of analyte detection
chip. Such an analyte detection device may include a single
cartridge interface, e.g, a single insertion slot and may operate
on a single chip inserted into the slot. In other embodiments, the
analyte detection device may include a plurality of cartridge
interface, e.g, insertion slots that may be used to operate a
plurality of chips (of the same type, loaded with different
samples). In yet other embodiments, the analyte detection
instrument may include a single slot in which a single analyte
detection chip may be inserted. However, the analyte detection
device may be a multi-functional instrument or a universal
instrument that can operate upon a plurality of different types of
analyte detection chips, e.g, two or more of DMF-electrochemical
detection chip; DMF-optical detection chip (DMF-nanowell detection
chip, including a DMF-NAT detection chip; DMF-imaging chip, where
the DMF and the optical detection regions are adjacent, partially
integrated, or fully integrated as described herein);
DMF-electrical chip; and DMF chip with multiple detection regions.
In some embodiments, the universal instrument may include a
separate insertion slot for each different analyte detection chip.
In other embodiments, the universal instrument may have a single
insertion slot that is compatible with the different types of
analyte detection chips. A multi-functional or an universal analyte
detection instrument may include optical detection and electrical
detection unit.
[0506] The analyte detection device may include a power source and
circuits for actuating the DMF electrodes. Depending upon the
analyte detection chip that the device operates upon, the analyte
detection device may include circuits for detecting electrical
signals from the working electrode (for electrochemical detection);
optical detection which may include sensors for detecting light
signals and camera(s) for imaging; and combinations thereof. The
analyte detection device may include a memory or may be operably
connected to a memory storing instructions for operation of the
analyte detection chips. In certain cases, the devices may be
operated by a processor that runs a program for carrying out the
steps required for generating an analyte related signal and
detecting the signal. The analyte detection device may also include
algorithm(s) to calculate a concentration of the analyte based on
the detected electrical or light signal.
[0507] The analyte detection devices disclosed herein may also be
configured to operate upon DMF-nanowell cartridge, such as those
disclosed in PCT/US2016/025785. In certain embodiments, the DMF
part of the cartridges provided here (e.g., DMF-electrochemical
detection chips, DMF-optical detection chips and the like) may be
configured and formed as disclosed in PCT/US2016/025785 or
PCT/US2016/025787.
[0508] FIGS. 40A and 40B depict an analyte detection device.
Analyte detection device 410a in FIG. 40A is compatible with a
single type of analyte detection chip. The device 410a in FIG. 40A
may include a single interface, such as a single slot 411a for a
single type of analyte detection chip. In certain cases, the device
410a in FIG. 40A may include multiple interfaces, such as slots
(411a, 411b, 4111c, 411d) that each accept the same type of analyte
detection chip. The device 410a in FIG. 40A may be used for
simultaneously analyzing multiple samples for presence of an
analyte. In certain embodiments, the device in FIG. 40A may include
a housing that includes processor 413 which is operably connected
to a memory that contains programming for using the detection
chips. The slot 411a and additional slots (if present) may all be
contained in the housing. In other embodiments, the housing may
only include the processor and may optionally include a screen or a
monitor and hardware and software sufficient for connecting to and
operating a separate device comprising one or more slots, such as,
slots 411a-411d. Thus, in some embodiments, the operating system of
the device may be physically separable from the slots into which
cartridges are placed.
[0509] The device 410b in FIG. 40B includes multiple slots, 412a,
412b, 412c, and 412d. Slot 412a is compatible with a
DMF-electrochemical detection chip. Slot 412b is compatible with a
DMF-optical detection chip. Slot 412c and 412d are compatible with
a DMF-nanopore and a DMF-nanowell chip, respectively. The devices
410a and 410b also include a processor 413 which is operably
connected to a memory that contains programming for using the
detection chips. Similar to the device 410a, device 410b may
include a housing containing the processor 413, an optional screen
or a monitor and the slots 412a-412d or the housing may not include
the slots 412a-412d, which may be present in a separate device(s)
connected to the device 410b.
[0510] FIG. 40C depicts an analyte detection chip compatible with
the analyte detection device shown in FIGS. 40A and 40B. In certain
embodiments, the devices and systems described herein may include a
cartridge adaptor (s) that can be utilized to adapt a single slot
to different types of cartridges. For example, a cartridge adapter
1 may include a first interface for connecting to a slot 1 and a
second interface for connecting to a cartridge 1, a cartridge
adapter 2 may include a first interface for connecting to a slot 1
and a second interface for connecting to a cartridge 2. Cartridge
adapters and cartridges compatible with the cartridge adaptors are
depicted in FIGS. 40D and 40E. FIG. 40D illustrates a cartridge
adapter 5812a that includes a first interface comprising pins 5810a
and 5810b compatible with a slot present in a device that either
includes a processor or is connectable (physically or wirelessly)
to a processor with instructions for performing the steps required
for preparing a sample in the DMF region of a cartridge and/or
analyzing the prepared sample (e.g., detecting analyte related
signal). The second interface of the cartridge adapter 5812a
includes a port 5811 that mates with pin 5813 present on a
cartridge 5814 (e.g., an immunoassay cartridge). FIG. 40E
illustrates a cartridge adapter 5812b that is compatible with the
same slot that was compatible with cartridge adapter 5812a due to
presence of pins 5810a and 5810b. However, the second interface of
the cartridge adapter 5812b includes a cavity that accommodates and
is connectable to cartridge 5815 (e.g., a hematology cartridge) but
not to cartridge 5814. Thus, a cartridge adaptor may be used to
adapt a slot to connect with multiple different types of
cartridges.
[0511] FIG. 41A depicts an analyte detection chip that can be used
for conducting the analysis of a sample according to the methods
described herein. The analyte detection chip includes an opening in
a distal region which opening provides an inlet (marked with an
arrow in FIG. 41A) for introducing a sample into the analyte
detection chip. As noted herein, in certain cases, the sample may
be a whole blood sample. In certain embodiments, the sample may be
pipetted into the opening of the analyte detection chip. In other
embodiments, a sample droplet may be directly loaded into the
analyte detection chip from a lanced area of the skin, such as, a
finger tip. The distal region of the analyte detection chip may
include elements for processing the sample and/or transferring the
sample to appropriate regions in the analyte detection chip for
detection of one or more analytes present in the sample. The
proximal region of the analyte detection chip is insertable into an
analyte detection device for sample analysis. In certain cases, the
analyte detection chip may include a cover, where a part of the
cover at the proximal region of the chip is moveable to expose the
interior of the proximal region. The movable portion of the cover
may be hingedly attached to the cover of the chip and may be
pivoted up to expose the interior of the chip. In other cases, the
movable portion of the cover may be slidable towards the distal
region to expose the interior of the chip at the proximal region.
In certain cases, the analyte detection chip may be compatible with
the analyte detection device shown in FIG. 41B. As noted herein,
the chip may include nanopores and/or nanowells. In addition, the
chip may include electrodes, e. g., an array of electrodes for
digital microfluidics.
[0512] FIG. 41B depicts an analyte detection device compatible with
the analyte detection chips as described herein. For example, the
analyte detection device is compatible with the analyte detection
chip shown in FIG. 41A. The analyte detection device of FIG. 41B
includes a single insertion slot (indicated by an arrow) into which
at least a proximal region of an analyte detection chip is
inserted. In certain cases, the entire or substantially the entire
chip is inserted into the insertion slot. In some cases, this
analyte detection device may also be configured to include multiple
interfaces, such as multiple insertion slots. As depicted in FIG.
41B, the analyte detection device has an ideal size for a benchtop
device with the height in the range of about 12 inches.
[0513] The chips, devices, and systems disclosed herein provide
many advantages in the field of sample analysis. These chips and
devices are highly reliable even for small sample volumes and are
low cost alternatives to other sample analysis devices. In addition
to the small footprint of the device, these devices are easy to use
and can be used to perform multiple core lab tests, including
immunoassay and/or clinical chemistry. As explained herein, the
disclosed chips, device, and systems provide high sensitivity which
enables analysis of small sample volumes. Furthermore, the
configuration of the chip and the device requires minimal user
input and enables a minimally trained user to operate the device
and chip for analyzing a sample. The chips and devices of the
present disclosure have no or minimal moving parts which also
reduce manufacturing and/or maintenance costs and while increasing
life of the device.
[0514] The analyte detection instrument may include imaging sensors
such as CMOS, CCD or enhanced CCD (eCCD) camera, PMT, APD. In
addition, the analyte detection instrument may include means for
sample illumination such as LED, lasers, and the like. An analyte
detection instrument may also include electrical circuits for
operating a DMF chip and for operating a
DMF-electrochemical/electrical chip.
[0515] In some embodiments, analyte detection may require a certain
level of sensitivity. Depending upon the desired sensitivity, a
DMF-optical chip (e.g., a DMF-well array chip) or a
DMF-electrochemical or DMF-electrical chip may be utilized. In yet
other assays for analyte detection a DMF-imaging chip for example,
where a droplet present on the DMF electrodes is optically
interrogated (e.g, using a spectrophotometer) may be used.
[0516] vi. Analyte Detection Systems
[0517] Also disclosed herein are systems comprising the analyte
detection chips and analyte detection devices compatible with the
chips. As noted in this disclosure, the instrument can perform
multiple assays using a single multi-functional chip or using
different chips. For example, the instrument can detect electrical
signals (such as those from an electrochemical species in contact
with the working and reference electrodes in a DMF-electrochemical
chip or from a molecule traversing a nanopore in a DMF-nanopore
chip) and optical signals including imaging an array of wells on a
DMF-well array chip, detecting analyte related signals from an
array of wells, and/or imaging a droplet on a DMF-imaging chip. As
noted herein, in a DMF-electrochemical chip, the DMF electrodes may
be adjacent to the working and reference electrodes on a single
substrate or may the working and reference electrodes may be
disposed in a capillary fluidically connected to the region of the
chip where the DMF electrodes are disposed. In some cases, the
location of the array of wells on a DMF-optical chip with reference
to the array of DMF electrodes may be as described in the foregoing
sections.
[0518] The systems of the present disclosure may be programmed for
performing a menu of tests for analysis of analyte(s) in a sample.
For example, the instrument may detect the type of chip placed in
the instrument and may select the assay to be performed on the
chip. The instrument may activate and deactivate the DMF electrodes
to process a sample droplet(s) and generate a droplet that can be
interrogated electrically or optically. For example, the instrument
may detect electrochemical species in the droplet and/or optically
active molecules in the droplet (e.g., chromogenic molecules,
fluorescent molecules and the like). In addition, the instrument
may separate the droplet into smaller portions, e.g., by dividing
the droplet across an array of wells (e.g., such that a single
analyte is present in each well) and optically interrogate the
wells.
[0519] The systems may further include memory with instructions
that are executed on a processor included in the system (for
example, included in the device) for controlling the DMF electrodes
and for controlling the electrodes used for electrochemical
detection or for optical detection, and the like.
[0520] In certain embodiments, the analyte detection systems of the
present disclosure may include an analyte detection device that
includes a processor for executing a program with instructions for
first activating the DMF electrodes for movement of sample
droplets/buffer droplets/reagent droplets and the like. The
instructions may further include deactivating the DMF electrodes
and measuring electrical signals from a working electrode for
detecting electrochemical species generated in response to presence
of an analyte in the sample. The system may further include
algorithms for normalizing the signal recorded from the chips, for
example, to remove noise prior to determining concentration of the
analyte. The algorithms may include a calibration curve to assist
in determining analyte concentration.
[0521] The systems disclosed herein may be used to process a sample
droplet for generation of an electrical signal (e.g., from an
electrochemical species) and/or an optical signal indicative of
presence of the analyte in the sample. The electrical and/or
optical signal may generated by action of an enzyme on a substrate.
A sample may be processed utilizing any assay format described in
this disclosure. For example, the sample may be processed for
generation of an optical signal. In certain cases, the assay may be
a colorimetric assay (e.g., generate a chromogenic reaction product
by action of an analyte specific enzyme), immunoassay, sandwich
immunoassay (e.g., monoclonal-polyclonal sandwich immunoassays,
including enzyme detection (enzyme immunoassay (EIA) or
enzyme-linked immunosorbent assay (ELISA), competitive inhibition
immunoassay (e.g., forward and reverse), enzyme multiplied
immunoassay technique (EMIT), particle-enhanced turbidimetric
inhibition immunoassay (PETINIA), homogeneous enzyme immunoassay
(HEIA), a competitive binding assay, bioluminescence resonance
energy transfer (BRET), one-step antibody detection assay,
homogeneous assay, heterogeneous assay, capture on the fly assay,
etc. These and additional exemplary assay formats are described in
detail in the preceding sections.
[0522] The systems of the present disclosure may be used in a
method for electrochemical detection of an analyte in a sample. The
method may include (a) introducing the sample into a cartridge, the
cartridge comprising: a first substrate; a second substrate; a gap
separating the first substrate from the second substrate; a
plurality of electrodes to generate electrical actuation forces on
a liquid droplet; and an electrochemical species sensing region
comprising a working electrode and a reference electrode; (b)
actuating the plurality of electrodes to provide a first liquid
droplet comprising the analyte; (c) actuating the plurality of
electrodes to provide a second liquid droplet comprising an enzyme
specific for the analyte; (d) actuating the plurality of electrodes
to merge the first and second droplets to create a mixture; (e)
actuating the plurality of electrodes to move all or a portion of
the mixture to the electrochemical sensing region; (f) detecting,
via the working and reference electrodes, an electrical signal of
an electrochemical species generated by action of the enzyme on the
analyte.
[0523] In some cases, the second liquid droplet may also include a
redox mediator. In some cases, the system may determine a
concentration of the analyte based on the electrical signal. In
some cases, the electrochemical sensing region is located in a
capillary region in the cartridge.
[0524] In certain embodiments, the method may include (a)
introducing the sample into a cartridge, the cartridge comprising:
a first substrate; a second substrate; a gap separating the second
substrate from the first substrate; a plurality of electrodes to
generate electrical actuation forces on a liquid droplet; and an
electrochemical species sensing region comprising a working
electrode and a reference electrode; (b) actuating the plurality of
electrodes to provide a first liquid droplet comprising the
analyte; (c) actuating the plurality of electrodes to provide a
second liquid droplet comprising a solid substrate comprising a
first binding member that specifically binds to the analyte; (d)
actuating the plurality of electrodes to merge the first and second
droplets to create a mixture; (e) actuating the plurality of
electrodes to merge all or a portion of the mixture with a third
liquid droplet comprising a second binding member that specifically
binds to the analyte; (f) holding the solid substrate in place
while actuating the plurality of electrodes to remove any unbound
analyte and/or second binding member; (g) actuating the plurality
of electrodes to contact the solid substrate with a substrate
molecule for the enzyme conjugated to the second binding member;
and (h) detecting, via the working and reference electrodes, an
electrical signal of an electrochemical species generated by action
of the enzyme on the substrate molecule.
[0525] In some cases, the method may include moving a liquid
droplet comprising the solid second substrate from step (f) to the
electrochemical sensing region prior to steps (g) and (h). In other
cases, the method may include moving a liquid droplet comprising
the solid second substrate and enzyme substrate from step (g) to
the electrochemical sensing region.
[0526] In some cases, the second liquid droplet may also include a
redox mediator. In some cases, the system may determine a
concentration of the analyte based on the electrical signal. In
some cases, the electrochemical sensing region is located in a
capillary region in the cartridge.
[0527] As noted herein, the systems and instruments may perform two
or more separate assays using a multifunctional cartridge(s) or
using multiple separate single assay cartridge. In certain cases,
the system also performs a method that includes conducting an
immunoassay on a sample, using a single cartridge (e.g., configured
for clinical chemistry and with an array of well) or a different
cartridge. The method may involve spatially segregating single
molecules and optically detecting the segregated single molecules
to detect presence of an analyte in the sample.
[0528] In certain embodiments a method for performing analyte
detection using an instrument is disclosed. The method may include
providing an analyte detection instrument comprising a cartridge
interface for operable connection to the one or more analyte
detection cartridges; providing a plurality of cartridges having a
plurality of electrodes to generate electrical actuation forces on
a liquid droplet: interfacing a first cartridge with the instrument
and detecting an analyte related signal from a droplet in a
cartridge; and interfacing a second cartridge with the instrument
and detecting an analyte related signal from spatially segregated
single molecules and/or from spatially segregated molecules in a
cartridge.
[0529] In certain cases, a system may include programming that
allows it to interface with a plurality of instruments, where each
of the pluralities of instruments conducts a plurality of assays,
where the plurality of instruments are different or same. For
example, a system as depicted in FIG. 43A may include a processor
413 operably connected to a plurality of devices 411, the plurality
of devices may each include at least one slot (411a-411d) in which
a cartridge may be inserted and operated upon by the processor. The
devices 411 may be identical and may provide means for increasing
the number of samples that can be processed simultaneously. In
certain embodiments, the system may include programming or may be
upgraded to include programming that allows it to interface with
additional or alternate instruments as they become available. A
system as depicted in FIG. 43B may include a processor 413 operably
connected to a plurality of different devices 414a-414d, the
plurality of devices may each include at least one slot (412a-411d)
in which a different type of cartridge may be inserted and operated
upon by the processor. For example, the first device may be
configured for conducting an immunoassay, the second device may be
configured for conducting an electrochemical assay, the third
device may be configured for conducting a hematology assay, and the
like. In such an embodiment, the device and the cartridge
compatible with the device may include means for sample preparation
and detection of an analyte related signal (for sample analysis).
The programming executed by the processor 413 may include
instructions that are communicated to the devices for preforming
the steps required for conducting an assay, such as, actuating DMF
electrodes or generating SAW for sample preparation and controlling
detection modules, such as, camera, microscope, electrochemical
sensors, etc., for detecting a signal from the prepared sample. The
system may additionally include algorithm for analyzing collected
data prior to providing assay results. The system may be equipped
for wireless communication to provide assay results on a remote
device connected wirelessly to the system. In certain cases, the
remote device may receive the results in real time. In certain
cases, a printer may also be connected to the system to provide a
printout of the assay results.
[0530] The modularity of the system depicted in FIGS. 43A and 43B
allows for adding to or removing from the functionality of the
system to provide flexibility to the consumer, e.g., at
point-of-care facilities.
[0531] Analytes
[0532] A non-limiting list of analytes that may be analyzed by the
methods, chips, instruments and methods presented herein include
molecules present in a biological sample, such as, a blood sample
(or a portion thereof, e.g., serum or plasma). Exemplary analytes
of interest include nucleic acids, one or more of low density
lipoprotein (LDL), high density lipoprotein (HDL), cholesterol,
triglycerides, glucose, hemoglobin (Hb), HbA1c, albumin,
microalbumin, total protein, sodium (Na.sup.+), potassium
(K.sup.+), chloride (Cl.sup.-), carbon dioxide, oxygen, creatinine,
calcium (Ca.sup.2+), blood urea nitrogen (BUN), pH, lactate, ketone
bodies, alanine aminotransferase (ALT), aspartate aminotransferase
(AST), alkaline phosphatase (ALP), bilirubin, ferritin, alcohol
(blood alcohol), amphetamine, methamphetamine, cannabis, opiates,
barbiturates, benzodiazepine, tricyclic acid, cocaine, and
phencyclidine (PCP). Additional analytes that may be detected and
optionally measured using the
DMF-electrochemical/electrical/optical detection chips disclosed
herein include one or more of the analytes detected in the
preceeding sections. Further examples of analytes that may be
detected and measured using the methods, devices, and systems of
the present disclosure include blood cells, flu virus,
streptococcal bacteria, raus sarcoma virus, adenovirus,
mononucleosis, tuberculosis, B-HCG, HIV, HCV, HBV, syphilis,
herpes, troponin, BNP, CK-MB, myoglobin, D-dimer, PSA, TSH, T3, T4,
FSH, LH, estradiol, testosterone, vitamin D, B12, and H.
Pylori.
[0533] The sample in which the analyte is being detected may be any
sample disclosed herein, such as, a sample of blood, solid tissue,
another body fluid, such as, urine, sputum, saliva, cerebrospinal
fluid, as well as, environmental samples, such as, water, soil,
food samples and the like.
EXAMPLES
Example 1
[0534] Fabrication of Low-Cost DMF Chip
[0535] Low-cost flexible DMF chips were fabricated using
roll-to-roll (R2R) flexographic printing combined with a wet
lift-off process for electrode patterning. A schematic of the
fabrication process is depicted in FIG. 18. A roll of Melinex ST506
polyethylene terephthalate (PET) 5.0 mil substrate (1) was used as
the starting material for DMF electrode printing. A layer of yellow
ink (Sun Chemical) was flexo-printed (2) on the PET substrate using
a 1.14 mm thick printing plate (Flint MCO3) at a rate of 10
m/minute using an ink transfer volume of 3.8 ml/m.sup.2 on an
Anilox roller assembly. A negative image of the DMF electrode
pattern results from the flexo printing step (3). Prior to metal
deposition, the ink was dried two times in a hot air oven
(2.times.100.degree. C.). An EVA R2R Metal Evaporator was used to
deposit a layer of silver metal onto the printed PET substrate to
form a uniform coating of silver at a thickness of 80 nm (4). The
metalized ink-film substrate (5) was subjected to a wet lift-off
process using a combination of acetone plus ultrasound in a
sonication bath at a speed of 1 m/minute (6). This
chemical/physical treatment allows the silver-ink layer to
dissolve, while keeping the silver-only layer intact. Removal of
the ink-silver layer resulted in a DMF printed electrode pattern
consisting of 80 actuation electrodes (2.25.times.2.25 mm) with
either 50 or 140 .mu.m electrode gap spacing (7). As a QC check, a
total of 80-90 random chips from a single roll were visually
inspected for electrode gap spacing and connector lead width
variation. Typical yields of chips, determined to have acceptable
gap specifications, were close to 100%. A single fabricated
flexible chip is depicted in FIG. 19. The fabricated flexible chip
measures 3''.times.2'' and includes electrodes, reservoirs, contact
pads and leads.
[0536] A dielectric coating was applied to the electrodes and
reservoirs by using either rotary screen printing or Gravure
printing. For rotary screen printing, Henkel EDAC PF-455B was used
as a dielectric coating by printing with a Gallus NF (400 L) screen
at a printing speed of 2 m/minute and a UV curing rate of 50%.
Typical dielectric thickness was 10-15 .mu.m. For Gravure printing,
cylinders were designed to print a high-viscosity dielectric ink,
such as IPD-350 (Inkron), at a speed of 2 m/minute using an ink
volume of 50 ml/m2. Typical dielectric thickness for Gravure
printing was 7-8 .mu.m. A final hydrophobic layer was printed using
either Millidyne Avalon 87 or Cytonix Fluoropel PFC 804 UC coating
with Gravure cylinders (140-180 L) and a printing speed of 8
m/minute, followed by four successive oven drying steps
(4.times.140.degree. C.). Typical hydrophobic thickness was 40-100
nm.
[0537] Alternatively, for small batches of individual chips, the
dielectric and hydrophobic coatings may be applied using chemical
vapor deposition (CVD) and spin coating, respectively.
Example 2
[0538] Functional Testing of Low-Cost DMF Chip
[0539] A 3''.times.2'' PET-based DMF bottom chip manufactured as
outlined in Example 1 above was tested for actuation capability.
FIG. 20 depicts a 3''.times.2'' PET-based DMF chip (1) over which a
0.7 mm thick glass substrate (3) is positioned. The glass substrate
(3) includes a transparent indium tin oxide (ITO) electrode on a
lower surface of the glass substrate and a Teflon coating over the
ITO electrode. The DMF chip includes 80 silver actuation electrodes
with a straight edge electrode design and a 50 .mu.m gap between
electrodes, along with 8 buffer reservoirs (see Example 1
above).
[0540] The bottom electrodes were coated with a layer of dielectric
Parylene-C (6-7 .mu.m thick) and a final coating of Teflon (50 nm
thick) by CVD and spin-coating, respectively. Approximately 50
.mu.l of PBS buffer with 0.1% surfactant (2) was pipetted into four
adjacent reservoirs on the bottom DMF chip. Droplet sizes ranged
from 700-1,500 nl (one or two droplets) and were checked for both
vertical and horizontal lateral movement (4), in addition to
circular sweep patterns necessary for mixing. Droplet actuation was
achieved using a voltage of 90 Vrms. Approximately 90% of the
actuation electrodes on the chip were tested and found to be fully
functional.
Example 3
[0541] TSH Immunoassay on Low-Cost DMF Chip
[0542] The 3'.times.2'' PET-based DMF chip overlayed with the glass
substrate as described in Example 2 above, was tested for its
ability to carry out a thyroid stimulating hormone (TSH)
immunoassay, using chemiluminescence detection. Mock samples
included TSH calibrator material spiked into TBS buffer containing
a blocking agent and a surfactant. Three samples were tested 31 0,
4, 40 .mu.IU/ml. 2 .mu.l of anti-beta TSH capture antibody, coated
on 5 .mu.m magnetic microparticles (3.times.108 particles/ml), was
dispensed from the microparticle reservoir into the middle of the
DMF electrode array. The magnetic microparticles were separated
from the buffer by engaging a neodymium magnet bar under the DMF
chip (FIG. 21A) (3 in..times.1/2 in..times.1/4 in. thick, relative
permeability .mu.r=1.05, remnant field strength Br=1.32 T). 5 .mu.l
of sample was moved to the microparticle slug, followed by mixing
the microparticle suspension (FIG. 21B) over a four-electrode
square configuration for 5 minutes. The microparticles were
separated from the sample by the magnet, and the supernatant was
moved to a waste reservoir (FIGS. 21C and 21D). 2 .mu.l of 1
.mu.g/ml anti-TSH detection antibody conjugated to horseradish
peroxidase (HRP) was moved to the microparticle slug and mixed for
2 minutes. The microparticles were separated by the magnet, and the
supernatant was moved to the waste reservoir. The microparticles
containing the immunoassay sandwich complex were washed a total of
four times with 4.times.2 .mu.l of PBS wash buffer containing 0.1%
surfactant. Wash buffer from each wash step was moved to waste
after the step was completed. Chemiluminescent substrate consisted
of 1 .mu.l of SuperSignal H.sub.2O.sub.2 and 1 .mu.l luminol
(ThermoFisher Scientific), which was moved to the microparticle
slug, followed by mixing for 6 minutes. Chemiluminescent signal was
measured at 427 nm emission (347 nm excitation) using an integrated
Hamamatsu H10682-110 PMT with a 5 V DC source. A dose-response
curve was plotted against relative luminescence (see FIG. 21E).
Example 4
[0543] Fabrication and Design of DMF Top Electrode Chips and Well
Array
[0544] Top Electrode Design:
[0545] The low-cost flexible DMF top electrode chips containing the
well array were fabricated using roll-to-roll (R2R) gravure
printing and UV imprinting. With reference to FIG. 22, the basic
design (1) incorporated two sets of well arrays printed on a
flexible substrate of polyethylene terephthalate (PET) that was
used as the top electrode for the DMF chip. The design consisted of
100 nm thick layer of indium-tin oxide (ITO)(3) printed on Melinex
ST504 PET substrate (2) (Solutia, OC50 ST504). A coating of
PEDOT:PSS primer (4) was used to improve adhesion of the UV
embossing resist (5), which contained the final well arrays.
[0546] Roll-to-Roll Fabrication of Top Electrode:
[0547] FIG. 22B shows a schematic of a fabrication process for the
DMF top electrode. Gravure printing (8) was used to coat a 20 nm
thick primer layer of PEDOT:PSS (7) (Clevios VP AI4083), diluted
with isopropanol to reduce the viscosity, on 5-mil Melinex ST504
PET substrate (6). The roll size was 250 m.times.200 mm.times.125
.mu.m; printing speed was 10 m/minute. The resulting primer-coated
ITO electrode (9) was transferred to a second R2R printing line
where a layer of UV resist (10) was applied with gravure coating
(11) to form the precursor material for UV embossing (13). Contact
with the nanoarray mold, followed by UV curing, produced the final
R2R top electrode film (14), ready to be cut.
[0548] Well Design:
[0549] A well design is shown in FIG. 23. The top chip containing
the ITO common electrode was designed to be cut into
3''.times.1.4'' strips (15), each containing two nano-dimensioned
well arrays (16) positioned to align with two exterior actuation
electrodes on the bottom chip (17). Placement of the top electrode
over the bottom chip gave the final DMF chip assembly (18). Each
array contained approximately 60,000 wells (245.times.245).
[0550] With reference to FIGS. 24A and 24B, two different well
spacing formats were used in the initial design--hexagonal (19) and
straight rows (20). In addition, two shim designs were used to
print two different well sizes (21) 4.2 .mu.m wide.times.3.0 .mu.m
deep with a pitch of 8.0 .mu.m; 4.5 .mu.m.times.3.2 .mu.m with a
pitch of 8.0 .mu.m. Varying the well size and geometric spacing was
done in order to optimize for subsequent microparticle loading of
2.7 .mu.m diameter beads. Post-fab QC was conducted on various R2R
runs to check for proper well spacing, size and integrity. If
deformed wells were observed or the well spacing and/or size was
incorrect, a new shim was manufactured and the array was
re-printed. FIG. 25 shows optical images at 200.times.
magnification for hexagonal (22) and straight row (23) arrays.
Example 5
[0551] Assembly of DMF Top Electrode Chips Containing a Well
Array
[0552] DMF Plastic Chip Assembly:
[0553] FIG. 26 schematically describes assembly of the integrated
DMF-well device from DMF top electrode chip and a nano-dimensioned
well array, as described in Example 4 above. The DMF chip is
assembled by placing 2.times.2 pieces of 90 .mu.m double-sided tape
(24) (3M) on opposite sides of the bottom DMF chip (25). The top
electrode, containing the embedded arrays (26), is centered on top
of the bottom chip so that the position of the two arrays aligns
with the two underlying actuation electrodes. The final assembled
chip (27) has a gap height of 180 .mu.m (2.times.90 .mu.m
tape).
Example 6
[0554] TSH Immunoassay on Low-Cost DMF-Well Integrated Chip
[0555] An immunoassay for TSH is described using a combination of
DMF, micro-dimensioned well array and digital detection of a
fluorescent substrate (FIGS. 27A-27G). A DMF chip (top and bottom
electrodes) is pre-loaded with immunoassay (IA) reagents, as shown
in (1) (FIG. 27A). The assay is carried out on the DMF chip using
air as the filler fluid. The sample may be a biological sample such
as whole blood, serum, plasma, urine, sputum, interstitial fluid,
or similar matrix. The capture microparticles consists of a
suspension of solid-phase magnetic microparticles coated with
anti-beta TSH antibodies at a density of 2.times.107-2-108
particles/ml. Approximately 1-2 .mu.l of sample is moved onto the
DMF chip and combined with 1-2 .mu.l of microparticles, followed by
mixing within Zone 1 (2) of the DMF chip (FIG. 27B). Zone 1
consists of 16 DMF electrodes reserved for combining, mixing and
washing of the sample to form a capture complex of TSH on the
magnetic microparticles. Incubation time can range from 1-10
minutes, followed by 1-3 washes of 1-2 .mu.l wash buffer (PBS, 0.1%
surfactant) from wash buffer 1 reservoir. Supernatant is removed
from the IA complex by engaging a magnet below the DMF chip and
moving the supernatant to waste reservoir 1.
[0556] The microparticle slug containing the captured TSH antigen
on solid phase is resuspended in 1-2 .mu.l of anti-beta TSH
detection conjugate antibody labeled with biotin
(streptavidin-.beta.-galactosidase complex, at a concentration of
40 pM in PBS). The mixture is allowed to incubate, with mixing over
4.times.4 electrodes, in Zone 2 on the DMF electrode array (3) for
1-10 minutes (FIG. 27C). The magnet is engaged to capture the
magnetic microparticles, and the supernatant is removed to
reservoir waste 1. The slug is washed 1-3 times with 1-2 .mu.l wash
buffer (PBS, 0.1% surfactant) from wash buffer 2 reservoir.
Supernatant is removed from the IA complex by engaging a magnet
below the DMF chip and moving the supernatant to waste reservoir 2.
The microparticle slug, containing the immunoassay sandwich
complex, is resuspended by moving 1-2 .mu.l of 100 .mu.M detection
substrate (4) (resorufin-.beta.-D-galactopyranoside, RGP) (FIG.
27D). The mixture is incubated for 1-3 minutes to allow for the
enzymatic turnover of RGP--a fluorescent product produced from the
enzymatic turnover of RGP by .beta.-galactosidase.
[0557] The mixture is moved (5), as shown in FIG. 27E, to a spot on
the DMF chip that contains an array of femtoliter wells (6), either
on the bottom or top substrate. The size of the femtoliter well
size is slightly larger than the size of the microparticle being
used in the assay. The number of wells may range from
1,000-2,000,000. The microparticles are deposited in the wells by
using either gravity (passive loading) or a magnet (active
loading). Excess supernatant is moved to waste-3 reservoir.
[0558] The femtoliter wells are sealed by moving 1-5 .mu.l of a
polarizable immiscible fluid (i.e. organic solvent, oil, etc.) to
the array position (7, 8), as shown in FIGS. 27F and 27G, using
electrowetting on dielectric (EWOD), dielectrophoresis (DEP)
force(s), or surface acoustic waves (SAW) thereby sealing the
wells. Some examples of suitable polarizable immiscible fluids
include silicone oil, fluorosilicone oil, mineral oil, 1-hexanol,
THF, m-dichlorobenzene, chloroform, and the like (S. Fan, et al.,
Lab On Chip, 9, 1236, 2009; D. Chatterjee, et al., Lab On Chip, 6,
199, 2006). The filler fluid for the entire assay is air.
[0559] The number of total particles in the wells is determined by
white light illumination with a wide field microscope/CCD camera,
followed by imaging at 574 nm/615 nm excitation/emission (exposure
time=3-10 seconds) for determining the number of beads containing a
detection label. The final TSH concentration is determined from a
standard curve run with TSH calibrators. Digital quantitation is
determined by using the Poisson equation and the ratio of positive
to negative beads.
Example 7
[0560] Nanodimensioned Well Top Loading with Polarizable Fluid
[0561] General Immunoassay Format:
[0562] The 3'.times.2'' PET-based DMF chip can be used to run an
ELISA-based sandwich immunoassay, coupled with digital fluorescence
detection in the well array. With reference to FIG. 28, a sample
(3), containing a specific antigen to be analyzed, is mixed with
magnetic microparticles (2) coated with a capture antibody (1) and
mixed to allow for immunocapture of the desired antigen. After
washing, the captured antigen (4) is mixed with a second detection
antibody (5) labeled with a detection moiety (6). The bead mixture,
containing the sandwich immunoassay complex (7), is washed again to
remove unbound detection antibody. The microparticles are loaded in
the top substrate of the well array by moving the aqueous droplet
to the array and applying a magnet to pull the beads into the
wells. The wells are sealed by moving a droplet of polarizable
immiscible fluid over the wells using DMF forces. A CCD camera
images the array to determine the number of positive and negative
microparticles. The sample is quantitated using Poisson statistics.
All immunoassay processing steps are carried out on a DMF chip
using air as the filler fluid.
[0563] TSH Immunoassay--DMF:
[0564] One-two .mu.l of anti-TSH capture antibody, coated on 2.7
.mu.m magnetic microparticles (3.times.108 particles/ml), is
dispensed from the microparticle reservoir on the DMF chip into the
middle of the DMF electrode array. The magnetic microparticles are
separated from the buffer by engaging a magnet, located under the
DMF chip, and moving the supernatant to the waste reservoir. One
.mu.l of an aqueous sample is pulled from a DMF sample reservoir
and moved to the microparticle slug, followed by a mixing step
where the droplet is moved over several electrodes for 1-5 minutes.
The microparticles are separated from the sample by applying the
bottom magnet, followed by removal of the supernatant to a waste
reservoir. One-two .mu.l of anti-TSH detection antibody (0.5
.mu.g/ml) conjugated to .beta.-galactosidase (.beta.-gal) is moved
to the microparticle slug and mixed for 2-5 minutes. The
microparticles are separated using the bottom magnet and the
supernatant is moved to the waste reservoir. The microparticles
containing the immunoassay sandwich complex are washed a total of
four times with 4.times.2 .mu.l of PBS wash buffer containing 0.1%
surfactant. Wash buffer from each wash step is moved to waste after
the step is completed. One .mu.l of 100 .mu.M
resorufin-.beta.-D-galactopyranoside (RGP) is taken from the RGP
reservoir and moved to the microparticle slug, followed by mixing
for 15-30 seconds. The beads are now ready for deposition into the
well array.
[0565] TSH Immunoassay--Digital Array Detection:
[0566] As shown in FIG. 29, the basic components for DMF-directed
top loading of the microparticles into the array includes the
bottom PET-based electrode chip with 80-nm thick silver electrodes
(8) (electrode gap <100 .mu.m, 2.25 mm.times.2.25 mm), 5-10
.mu.m thick dielectric/hydrophobic layer (9), the top PET-based ITO
electrode (10) chip containing the array of wells (14) (configured
to hold no more than one microparticle) and an aqueous droplet (11)
containing 2.7 .mu.m magnetic microparticles (12). The filler fluid
is air (13). The gap height between the top and bottom electrodes
is approximately 180 .mu.m (from 2 pieces of 90 .mu.m double-sided
tape).
[0567] Transport and sealing is accomplished by using a combination
of DMF forces (EWOD, DEP, and/or electromediated force) to move
aqueous and immiscible fluids, such as silicone oil. It has been
previously shown that different driving voltages are required to
move both aqueous and oil droplets on the same DMF chip (S-K Fan,
et al., Lab on Chip, 9, 1236, 2009).
[0568] After addition of the fluorescent substrate RGP to the
aqueous droplet containing the immunoassay complex (FIG. 30A), the
droplet is moved to an electrode positioned below a well array
containing approx. 60,000 wells (245.times.245 array; 4.2 .mu.m
diameter; 3.0 .mu.m depth; 8.0 .mu.m pitch) using a voltage of
20-50 Vrms (1 KHz). A top magnet (15) is engaged to promote
efficient loading of the microparticles into the wells (FIG. 30B);
total deposition time is 30-60 seconds. The aqueous droplet is
moved away as the top magnet is dis-engaged, leaving behind a thin
layer of deposited and surface-bound beads (FIG. 30C). A droplet of
silicone oil is moved from a reservoir using a voltage of 200-300 V
(DC) and moved to the electrode positioned under the array (FIG.
30D), thereby washing away any surface-bound microparticles, while
sealing microparticles contained in the wells. The fluorescence
generated from enzymatic turnover of RGP to resorufin is monitored
by a CCD camera at 574/615 nm (excitation/emission). The ratio of
"on" microparticles to "off" microparticles is determined. The TSH
concentration in the sample is determined by interpolation from a
TSH calibration curve.
[0569] Finally, although the various aspects and features of the
invention have been described with respect to various embodiments
and specific examples herein, all of which may be made or carried
out conventionally, it will be understood that the invention is
entitled to protection within the full scope of the appended
claims.
[0570] It is understood that the foregoing detailed description and
accompanying examples are merely illustrative and are not to be
taken as limitations upon the scope of the invention, which is
defined solely by the appended claims and their equivalents.
[0571] Various changes and modifications to the disclosed
embodiments will be apparent to those skilled in the art. Such
changes and modifications, including without limitation those
relating to the chemical structures, substituents, derivatives,
intermediates, syntheses, compositions, formulations, or methods of
use of the invention, may be made without departing from the spirit
and scope thereof.
For reasons of completeness, various aspects of the invention are
set out in the following numbered clauses:
[0572] 1. A digital microfluidic and analyte detection device,
comprising:
[0573] a first substrate and a second substrate, wherein the second
substrate is separated from the first substrate by a gap, the first
substrate comprising a plurality of electrodes to generate
electrical actuation forces on a liquid droplet; and
[0574] an array of wells dimensioned to hold a portion of the
liquid droplet, wherein at least a portion of the array of wells is
positioned between one or more of the plurality of electrodes and
the gap.
[0575] 2. The device according to clause 1, wherein the plurality
of electrodes is positioned on a surface of the first
substrate.
[0576] 3. The device according to clause 1 or clause 2, further
comprising a first layer disposed on the surface of the first
substrate and covering the plurality of electrodes.
[0577] 4. The device of any one of the previous clauses, wherein
the first substrate comprises a first portion at which the liquid
droplet is introduced and a second portion toward which a liquid
droplet is moved.
[0578] 5. The device of clause 4, wherein the plurality of
electrodes and the first layer extend from the first portion to the
second portion of the first substrate.
[0579] 6. The device of clause 5, wherein the array of wells is
positioned in the second portion of the first substrate.
[0580] 7. The device according to clause 4, wherein the second
substrate comprises a first portion and a second portion, wherein
the first portion is in facing arrangement with the first portion
of the first substrate and the second portion is in facing
arrangement with the array of wells.
[0581] 8. The device of clause 7, wherein the second portion of the
second substrate is substantially transparent to facilitate optical
interrogation of the array of wells.
[0582] 9. The device according to clause 3, further comprising a
second layer disposed on a surface of the first layer.
[0583] 10. The device according to clause 9, wherein a second layer
extends over the first and second portions of the first
substrate.
[0584] 11. The device according to any one of clauses 9-10, wherein
the first layer is a dielectric layer and the second layer is a
hydrophobic layer.
[0585] 12. The device according to any one of clauses 9-11, wherein
the array of wells is positioned in the second layer.
[0586] 13. The device according to any one of clauses 3, wherein
the array of wells is positioned in the first layer.
[0587] 14. The device according to any one of the previous clauses,
wherein the array of wells has a hydrophilic surface.
[0588] 15. The device according to any one of the previous clauses,
wherein the array of wells comprise a sidewall that is oriented to
facilitate receiving and retaining of beads or particles present in
droplets moved over the well array.
[0589] 16. The device according to clause 15, wherein the array of
wells comprise a first sidewall opposite to a second side wall,
wherein the first sidewall is oriented at an obtuse angle with
reference to a bottom of the wells, and wherein the second sidewall
is oriented at an acute angle with reference to the bottom of the
wells, wherein movement of droplets is in a direction parallel to
the bottom of the wells and from the first sidewall to the second
sidewall.
[0590] 17. The device according to clause 15, wherein the array of
wells have a frustoconical shape with a narrower part of the
frustoconical shape providing an opening of the array of wells.
[0591] 18. The device according to clause 15, wherein the array of
wells comprise a first sidewall opposite to a second side wall,
wherein a top portion of the first sidewall is oriented at an
obtuse angle with reference to a bottom of the wells and a bottom
portion of the sidewall is oriented perpendicular to the bottom of
the wells, and wherein the second sidewall is oriented
perpendicular with reference to the bottom of the wells, wherein
the movement of droplets is in a direction parallel to the bottom
of the wells and from the first sidewall to the second sidewall,
wherein the top portion of the first side wall is at an opening of
the wells.
[0592] 19. A digital microfluidic and analyte detection device,
comprising:
[0593] a first substrate and a second substrate defining the
device, wherein the second substrate is separated from the first
substrate by a gap, wherein the device comprises a first portion
and a second portion; and
[0594] the first portion comprising a plurality of electrodes to
actuate combining of a first liquid droplet containing an analyte
of interest from a biological sample and a second liquid droplet
containing at least one bead; and
[0595] the second portion comprising an array of wells dimensioned
to hold a portion of the liquid droplet.
[0596] 20, The device according to clause 19, wherein the plurality
of electrodes are only positioned in the first portion of the
device.
[0597] 21. The device according to clause 19 or clause 20, wherein
the plurality of electrodes is positioned on a surface of the first
substrate.
[0598] 22. The device according to any one of clauses 19-21,
further comprising a first layer disposed on the surface of the
first substrate and covering the plurality of electrodes.
[0599] 23. The device of any new of clauses 19-22, wherein the
first substrate comprises a first portion at which the liquid
droplet is introduced and a second portion toward which a liquid
droplet is moved.
[0600] 24. The device of clause 23, wherein the plurality of
electrodes and e first layer extend from the first portion to the
second portion of the first substrate.
[0601] 25. The device of clause 24, wherein the array of wells is
positioned in the second portion of the first substrate.
[0602] 26. The device according to clause 23, wherein the second
substrate comprises a first portion and a second portion, wherein
the first portion is in facing arrangement with the first portion
of the first substrate and the second portion is in facing
arrangement with the array of wells,
[0603] 27. The device of clause 26, wherein the second portion of
the second substrate is substantially transparent to facilitate
optical interrogation of the array of wells.
[0604] 28. The device according to any one of clauses 19-27,
wherein the plurality of electrodes are configured to move a
droplet placed in the gap towards the second portion of the device,
the device comprising a capillary portion fluidically connecting
the first portion to the second portion, wherein the capillary
comprises a hydrophilic material to facilitate movement of the
droplet from the first portion to the second portion via the
capillary portion in absence of an electric force.
[0605] 29. The device according to any one of clauses 22, wherein a
second layer is disposed on an upper surface of the first
layer.
[0606] 30. The device according to clause 29, wherein a second
layer extends over the first substrate.
[0607] 31. The device according to any one of clauses 29-30,
wherein the first layer is a dielectric layer and the second layer
is a hydrophobic layer.
[0608] 32. The device according to any one of clauses 29-31,
wherein the plurality of wells is positioned in the second
layer.
[0609] 33. The device according to clauses 22, wherein the array of
wells is positioned in the first layer.
[0610] 34. The device according to any one of clauses 19-33,
wherein the array of wells has a hydrophilic surface.
[0611] 35. The device according to any one of clauses 19-34,
wherein the wells comprise a sidewall that is oriented to
facilitate receiving and retaining of nanobeads or nanoparticles
present in droplets moved over the well array.
[0612] 36. The device according to clause 35, wherein the wells
comprise a first sidewall opposite to a second side wall, wherein
the first sidewall is oriented at an obtuse angle with reference to
a bottom of the wells, and wherein the second sidewall is oriented
at an acute angle with reference to the bottom of the wells,
wherein the movement of droplets is in a direction parallel to the
bottom of the wells and from the first sidewall to the second
sidewall.
[0613] 37. The device according to clause 36, wherein the wells
have a frustoconical shape with the narrower part of the
frustoconical shape providing the opening of the wells.
[0614] 38. The device according to clause 35, wherein the wells
comprise a first sidewall opposite to a second side wall, wherein a
top portion of the first sidewall is oriented at an obtuse angle
with reference to a bottom of the wells and a bottom portion of the
sidewall is oriented perpendicular to the bottom of the wells, and
wherein the second sidewall is oriented perpendicular to the bottom
of the wells, wherein the movement of droplets is in a direction
parallel to the bottom of the wells and from the first sidewall to
the second sidewall, wherein the top portion of the first side wall
is at an opening of the wells.
[0615] 39. A surface acoustic wave microfluidic and analyte
detection device, comprising:
[0616] a first substrate and a second substrate, wherein the second
substrate is separated from the first substrate by a gap, wherein
the device comprises a first portion and a second portion,
[0617] the first portion comprising a superstrate coupled to a
surface acoustic wave generating component; and
[0618] the second portion co p sing a plurality of wells positioned
on the first substrate or the second substrate.
[0619] 40. The device according to clause 39 lie superstrate
includes phononic structures on an upper surface of the
superstrate.
[0620] 41. The device according to clause 39 or clause 40, wherein
the superstrate overlays a piezoelectric crystal layer.
[0621] 42. The device according to any one of clauses 39-40,
wherein the second substrate is substantially transparent.
[0622] 43. A surface acoustic wave microfluidic and analyte
detection device, comprising:
[0623] a first substrate and a second substrate, wherein the second
substrate is separated from the first substrate by a gap,
[0624] the first substrate comprising a plurality of wells, and
[0625] the second substrate comprising phononic structure, wherein
the plurality of wells and the phononic structures are located
across to each other.
[0626] 44. The device according to clause 43, wherein the second
substrate is a supers rate.
[0627] 45. The device according to clause 43, wherein a superstrate
is disposed on the second substrate and the phononic structure are
located on the superstrate.
[0628] 46. The device according to any one of clauses 43-45,
wherein the first substrate, second substrate and superstrate are
substantially transparent.
[0629] 47. A method of detecting an analyte of interest in a liquid
droplet, the method comprising:
[0630] (a) providing a first liquid droplet containing an analyte
of interest;
[0631] (b) providing a second liquid droplet containing at least
one solid support which contains a specific binding member that
binds to the analyte of interest;
[0632] (c) using energy to exert a force to manipulate the first
liquid droplet with the second liquid droplet to create a
mixture;
[0633] (d) moving all or at least a portion of the mixture to an
array of wells, wherein one or more wells of the array is of
sufficient size to accommodate the at least one solid support;
[0634] (e) adding a detectable label to the mixture either before
or after moving a portion of the mixture to array of wells; and
[0635] (f) detecting the analyte of interest in the wells.
[0636] 48. The method of clause 47, wherein the at least one solid
support comprises at least one binding member that specifically
binds to the analyte of interest.
[0637] 49. The method of any one of clauses 47 or 48, further
comprising adding a detectable label to the mixture before the
moving at least a portion of the mixture to the array of wells.
[0638] 50. The method of any one of clauses 47 or 48, further
comprising adding a detectable label to the mixture after the
moving at least a portion of the mixture to the array of wells.
[0639] 51. The method of any one of clauses 47 to 50, wherein the
detectable label comprises at least one binding member that
specifically binds to the analyte of interest.
[0640] 52. The method of any one of clauses 47 to 51, wherein the
detectable label comprises a chromagen, a fluorescent compound, an
enzyme, a chemiluminescent compound or a radioactive compound.
[0641] 53. The method of any one of clauses 47 to 52, wherein the
binding member is a receptor or an antibody.
[0642] 54. The method of any one of clauses 47 to 53, wherein the
energy is an electric actuation force or acoustic force.
[0643] 55. The method of clause 54, wherein the electric actuation
force is droplet actuation, electrophoresis, electrowetting,
dielectrophoresis, electrostatic actuation, electric field
mediated, electrode mediated, capillary force, chromatography,
centrifugation, or aspiration.
[0644] 56. The method clause 54, wherein the acoustic force is
surface acoustic wave.
[0645] 57. The method of any one of clauses 47 to 56, wherein the
first liquid droplet is a polarizable liquid, the second liquid
droplet is a polarizable liquid, the mixture is a polarizable
liquid or both the first liquid droplet and second liquid droplet
are each polarizable polarizable liquids.
[0646] 58. The method of any one of clauses 47 to 55, further
comprising manipulating the at least a portion of the mixture over
the array of wells using an electric actuation force.
[0647] 59. The method of any one of clauses 47 to 58, further
comprising manipulating the at least a portion of the mixture over
the array of wells using a capillary element configured to
facilitate movement of the mixture to the array of wells.
[0648] 60. The method of any one of clauses 47 to 59, wherein the
solid supports are magnetic solid supports.
[0649] 61. The method of clause 60, wherein an electric actuation
force and the magnetic field are applied from opposite directions
relative to the at least a portion of the mixture.
[0650] 62. The method of any one of clauses 47 to 61, further
comprising mixing the mixture by moving the mixture back and forth,
moving the mixture in a circular pattern, splitting the mixture
into two or more submixtures and merging the submixtures.
[0651] 63. The method of any one of clauses 47 to 62, wherein the
mixture is an aqueous liquid.
[0652] 64. The method of any one of clauses 47 to 62, wherein the
mixture is an immiscible liquid.
[0653] 65. The method of any one of clauses 47 to 64, wherein the
liquid droplet is a hydrophobic liquid droplet.
[0654] 66. The method of any one of clauses 47 to 65, wherein the
array of wells has a hydrophilic surface.
[0655] 67. The method of any one of clauses 47 to 65, wherein the
array of wells has a hydrophobic surface.
[0656] 68. The method of any one of clauses 47 to 67, wherein the
method is performed in an device comprising a first and second
substrate and at least one substrate comprises a hydrophilic
surface.
[0657] 69. The method of any one of clauses 47 to 67, wherein the
method is performed in an device comprising a first and second
substrate and at least one substrate comprises a hydrophobic
surface.
[0658] 70. The method of any one of clauses 47 to 69, further
comprising generating an electric actuation force with a plurality
of electrodes to move the mixture to the array of wells to load the
wells.
[0659] 71. The method of any one of clauses 47 to 70, wherein one
or more wells of the array are loaded with at least one solid
support.
[0660] 72. The method of clause 71, wherein the loading comprises
applying a magnetic field to facilitate movement of at least one
solid support into the one or more wells of the array.
[0661] 73. The method of clause 72, further comprising removing any
solid supports that are not loaded into a well of the array after
the loading.
[0662] 74. The method of clause 73, wherein the removing comprises
generating an electric actuation force with the plurality of
electrodes to move a polarizable fluid droplet to the array of
wells to move the at least a portion of the mixture to a distance
from the array of wells.
[0663] 75. The method of clause 74, wherein the removing comprises
generating an electric actuation force with the plurality of
electrodes to move an aqueous washing droplet across the array of
wells.
[0664] 76, The method of clause 75, wherein generating an electric
actuation force comprises generating an alternating current.
[0665] 77. The method of clause 75, wherein the alternating current
has a root mean squared voltage of 10 V or more.
[0666] 78. The method of any one of clauses 75 or 76, wherein the
alternating current has a frequency in a radio frequency range.
[0667] 79. The method of any one of the clauses 47 to 78, wherein
the method is performed using a microfluidics device, digital
microfluidics device (DMF), a surface acoustic wave based
microfluidic device (SAW), an integrated DMF and analyte detection
device, an integrated SAW and analyte detection device, or robotics
based assay processing unit.
[0668] 80. A method of detecting an analyte of interest in a liquid
droplet, the method comprising:
[0669] (a) providing a first liquid droplet containing an analyte
of interest;
[0670] (b) providing a second liquid droplet containing a
detectable label which contains a specific binding member that
binds to the analyte of interest;
[0671] (c) using energy to exert a force to manipulate the first
liquid droplet and the second liquid droplet to create a
mixture;
[0672] (d) moving all or at least a portion of the mixture to an
array of wells; and
[0673] (e) detecting the analyte of interest in the wells.
[0674] 81. The method of clause 80, wherein the detectable label
comprises a chromagen, a fluorescent compound, an enzyme, a
chemiluminescent compound or a radioactive compound.
[0675] 82. The method of any of clauses 80 to 81, wherein the
energy is an electric actuation force or acoustic force.
[0676] 83. The method of clause 82, wherein the electric actuation
force is droplet actuation, electrophoresis, electrowetting,
dielectrophoresis, electrostatic actuation, electric field
mediated, electrode mediated, capillary force, chromatography,
centrifugation, or aspiration.
[0677] 84. The method clause 83, wherein the acoustic force is
surface acoustic wave.
[0678] 85. The method of clauses 80 to 83, wherein the first liquid
droplet is a polarizable immiscible liquid, the second liquid
droplet is a polarizable liquid, the mixture is a polarizable
liquid or both the first liquid droplet and second liquid droplet
are each polarizable liquids.
[0679] 86. The method of any one of clauses 80 to 83 and 85,
further comprising manipulating the mixture over the array of wells
using an electric actuation force.
[0680] 87. The method of any one of clauses 80 to 86, further
comprising manipulating the mixture over the array of wells using a
capillary element configured to facilitate movement of the mixture
to the array of wells.
[0681] 88. The method of any one of clauses 80 to 87, further
comprising mixing the mixture by moving the mixture back and forth,
moving the mixture in a circular pattern, splitting the mixture
into two or more submixtures and merging the submixtures.
[0682] 89. The method of any one of clauses 80 to 88, wherein the
mixture is an aqueous liquid.
[0683] 90. The method of any one of clauses 80 to 88, wherein the
mixture is an immiscible liquid.
[0684] 91. The method of any one of clauses 80 to 90, wherein the
liquid droplet is a hydrophobic liquid droplet.
[0685] 92. The method of any one of clauses 80 to 91, wherein the
array of wells has a hydrophilic surface.
[0686] 93. The method of any one of clauses 80 to 91, wherein the
array of wells has a hydrophobic surface.
[0687] 94. The method of any one of clauses 80 to 93, wherein the
method is performed in an device comprising a first and second
substrate and at least one substrate comprises a hydrophilic
surface.
[0688] 95. The method of any one of clauses 80 to 93, wherein the
method is performed in an device comprising a first and second
substrate and at least one substrate comprises a hydrophobic
surface.
[0689] 96. The method of any one of clauses 80 to 95, further
comprising generating an electric actuation force with a plurality
of electrodes to move the mixture to the array of wells to load the
wells.
[0690] 97. The method of any one of clauses 80 to 96, wherein one
or more wells of the array are loaded with at least one detectable
label.
[0691] 98. The method of clause 97, further comprising removing any
detectable labels that are not loaded into a well of the array
after the loading.
[0692] 99. The method of clause 98, wherein the removing comprises
generating an electric actuation force with the plurality of
electrodes to move a polarizable fluid droplet to the array of
wells to move the at least a portion of the mixture to a distance
from the array of wells.
[0693] 100. The method of clause 99, wherein the removing comprises
generating an electric actuation force with the plurality of
electrodes to move an aqueous washing droplet across the array of
wells.
[0694] 101, The method of clause 100, wherein generating an
electric actuation force comprises generating an alternating
current.
[0695] 102. The method of clause 101, wherein the alternating
current has a root mean squared (rms) voltage of 10 V or more.
[0696] 103. The method of any one of clauses 100 or 101, wherein
the alternating current has a frequency in a radio frequency
range.
[0697] 104. The method of any one of the clauses 80 to 103, wherein
the method is performed using a microfluidics device, digital
microfluidics device (DMF), a surface acoustic wave based
microfluidic device (SAW), an integrated DMF and analyte detection
device, an integrated SAW and analyte detection device, or robotics
based assay processing unit.
[0698] 105. A method of measuring an analyte of interest in a
liquid droplet, the method comprising:
[0699] (a) providing a first liquid droplet containing an analyte
of interest;
[0700] (b) providing a second liquid droplet containing at least
one solid support which contains a specific binding member that
binds to the analyte of interest;
[0701] (c) using energy to exert a force to manipulate the first
liquid droplet with the second liquid to create a mixture;
[0702] (d) moving all or at least a portion of the mixture to an
array of wells, wherein one or more wells of the array is of
sufficient size to accommodate the at least one solid support;
[0703] (e) adding a detectable label to the mixture either before
or after moving a portion of the mixture to array of wells; and
[0704] (f) measuring the detectable label in the wells.
[0705] 106. The method of clause 105, wherein the at least one
solid support comprises at least one binding member that
specifically binds to the analyte of interest.
[0706] 107. The method of any one of clauses 105 or 106, further
comprising adding a detectable label to the mixture before the
moving at least a portion of the mixture to the array of wells.
[0707] 108. The method of any one of clauses 105 or 106, further
comprising adding a detectable label to the mixture after the
moving at least a portion of the mixture to the array of wells
[0708] 109. The method of any one of clauses 105 to 108, wherein
the detectable label comprises at least one binding member that
specifically binds to the analyte of interest.
[0709] 110. The method of any one of clauses 105 to 109, wherein
the detectable label comprises a chromagen, a fluorescent compound,
an enzyme, a chemiluminescent compound or a radioactive
compound.
[0710] 111. The method of any one of clauses 105 to 109, wherein
the binding member is a receptor or an antibody.
[0711] 112. The method of any one of clauses 105 to 109, wherein
the energy is an electric actuation force or acoustic force.
[0712] 113. The method of clause 112, wherein the electric
actuation force is droplet actuation, electrophoresis,
electrowetting, dielectrophoresis, electrostatic actuation,
electric field mediated, electrode mediated, capillary force,
chromatography, centrifugation, or aspiration.
[0713] 114. The method clause 112, wherein the acoustic force is
surface acoustic wave.
[0714] 115. The method of clauses 105 to 114, wherein the first
liquid droplet is a polarizable liquid, the second liquid droplet
is a polarizable liquid, the mixture is a polarizable liquid or
both the first liquid droplet and second liquid droplet are each
polarizable liquids.
[0715] 116. The method of any one of clauses 105 to 112 and 115,
further comprising manipulating the mixture over the array of wells
using an electric actuation force.
[0716] 117. The method of any one of clauses 105 to 116, further
comprising manipulating the mixture over the array of wells using a
capillary element configured to facilitate movement of the mixture
to the array of wells.
[0717] 118. The method of any one of clauses 105 to 117, wherein
the solid supports are magnetic solid supports.
[0718] 119. The method of clause 118, wherein an electric actuation
force and the magnetic field are applied from opposite directions
relative to the at least a portion of the mixture.
[0719] 120, The method of any one of clauses 105 to 119, further
comprising mixing the mixture by moving the mixture back and forth,
moving the mixture in a circular pattern, splitting the mixture
into two or more submixtures and merging the submixtures.
[0720] 121. The method of any one of clauses 105 to 120, wherein
the mixture is an aqueous liquid.
[0721] 122. The method of any one of clauses 105 to 121, wherein
the mixture is an immiscible liquid.
[0722] 123. The method of any one of clauses 105 to 122, wherein
the liquid droplet is a hydrophobic liquid droplet.
[0723] 124. The method of any one of clauses 105 to 123, wherein
the array of wells has a hydrophilic surface.
[0724] 125. The method of any one of clauses 105 to 123, wherein
the array of wells has a hydrophobic surface.
[0725] 126. The method of any one of clauses 105 to 124, wherein
the method is performed in an device comprising a first and second
substrate and at least one substrate comprises a hydrophilic
surface.
[0726] 127. The method of any one of clauses 105 to 124, wherein
the method is performed in an device comprising a first and second
substrate and at least one substrate comprises a hydrophobic
surface.
[0727] 128. The method of any one of clauses 105 to 128, further
comprising generating an electric actuation force with a plurality
of electrodes to move the mixture to the array of wells to load the
wells.
[0728] 129. The method of any one of clauses 105 to 128, wherein
one or more wells of the array are loaded with at least one solid
support.
[0729] 130. The method of clause 129, wherein the loading comprises
applying a magnetic field to facilitate movement of at least one
solid support into the one or more wells of the array.
[0730] 131. The method of clause 130, further comprising removing
any solid supports that are not loaded into a well of the array
after the loading.
[0731] 132. The method of clause 131, wherein the removing
comprises generating an electric actuation force with the plurality
of electrodes to move a polarizable fluid droplet to the array of
wells to move the at least a portion of the mixture to a distance
from the array of wells.
[0732] 133. The method of clause 132, wherein the removing
comprises generating an electric actuation force with the plurality
of electrodes to move an aqueous washing droplet across the array
of wells.
[0733] 134, The method of clause 133, wherein generating an
electric actuation force comprises generating an alternating
current.
[0734] 135. The method of clause 134, wherein the alternating
current has a root mean squared (rms) voltage of 10 V or more.
[0735] 136. The method of any one of clauses 134 or 135, wherein
the alternating current has a frequency in a radio frequency
range.
[0736] 137. The method of any one of clauses 105 to 136, wherein
the method is performed using a microfluidics device, digital
microfluidics device (DMF), a surface acoustic wave based
microfluidic device (SAW), an integrated DMF and analyte detection
device, an integrated SAW and analyte detection device, or robotics
based assay processing unit.
[0737] 138. The method of any one of clauses 105 to 137, wherein
the measuring involves determining the total number of solid
supports in the wells of an array.
[0738] 139. The method of clause 138, wherein the measuring
involves determining the number of solid supports in the wells of
the array that contain the detectable label.
[0739] 140. The method of clause 139, wherein the measuring
involves subtracting the number of solid supports that contain a
detectable label from the total number of solid supports in the
wells of the array to determine the number of solid supports in the
wells of the array that do not contain any detectable label.
[0740] 141. The method of clause 140, determining the ratio of
solid supports that contain a detectable label to the number of
solid supports that do not contain any detectable label.
[0741] 142. A method of loading wells with particles,
comprising:
[0742] (a) generating an electric field with a plurality of
electrodes to move a liquid droplet containing microparticles to an
array of wells, wherein one or more wells of the array of wells is
of sufficient size to have loaded therein a particle;
[0743] (b) loading one or more wells with a particle; and
[0744] (c) generating an electric field with the plurality of
electrodes to move a polarizable fluid droplet to the array of
wells to seal the array of wells.
[0745] 143. The method of clause 142, further comprising
manipulating liquid droplet over the array of wells using the
electric field.
[0746] 144. The method of any one of clauses 142 or 143, further
comprising manipulating the liquid droplet over the array of wells
using a capillary element configured to facilitate movement of the
liquid droplet to the array of wells.
[0747] 45. The method of any one of clauses 142 to 144, wherein the
particle is a magnetic bead.
[0748] 146. The method of clause 142, wherein the loading comprises
applying a magnetic field to facilitate movement of the one or more
magnetic beads into the one or more wells of the array.
[0749] 147. The method of any one of clauses 142 to 146, wherein
the array of wells has a hydrophilic surface.
[0750] 148. The method of any one of clauses 142 to 146, wherein
the array of wells has a hydrophobic surface.
[0751] 149. The method of any one of clauses 142 to 148, wherein
generating an electric field comprises generating an alternating
current.
[0752] 150. The method of clause 149, wherein the alternating
current has a root mean squared (rms) voltage of 10 V or more.
[0753] 151, The method of any of clauses 149 or 159, wherein the
alternating current has a frequency in a radio frequency range.
[0754] 152. A method of forming a digital microfluidic and analyte
detection device, comprising:
[0755] unwinding a first roll comprising a first substrate to
position a first portion of the first substrate at a first
position;
[0756] forming a plurality of electrodes on the first portion of
the first substrate at the first position; and
[0757] forming an array of wells on a second portion of the first
substrate at a second position.
[0758] 153, The method of clause 152, further comprising:
[0759] unwinding the first roll to position the second portion
adjacent the first portion of the first substrate at the second
position prior to forming the array of wells.
[0760] 154. The method of clause 152 of clause 153, further
comprising:
[0761] unwinding a second roll comprising a second substrate to
position a third portion of the third substrate at a third
position; and
[0762] bonding the second substrate with the first substrate at the
third position in a manner sufficient to position the second
substrate spaced apart from the first substrate.
[0763] 155. A method of forming an integrated digital microfluidic
and analyte detection device, comprising:
[0764] unwinding a first roll comprising a first substrate to
position a first portion of the first substrate at a first
position;
[0765] forming a plurality of electrodes on the first portion of
the first substrate at the first position;
[0766] unwinding a second roll comprising a second substrate to
position a second portion of the second substrate at a second
position;
[0767] forming an array of wells on the second portion at the
second position; and
[0768] bonding the second substrate with the first substrate in a
manner sufficient to: [0769] position the second substrate spaced
apart from the first substrate; and [0770] position the second
portion above the first portion, or above a third portion adjacent
the first portion of the first substrate, [0771] wherein the array
of wells faces the first substrate.
[0772] 156. The method of any one of clauses 152 to 155, wherein
the forming the array of wells comprises using thermal or
ultraviolet nanoimprint lithography, nanoimprint roller, laser
ablation, or by bonding a prefabricated substrate comprising an
array of wells onto the first portion of the first substrate.
[0773] 157. The method of any one of clauses 152 to 156, further
comprising subjecting the first substrate to intense heat,
pressure, or ultraviolet light to form phononic structures on or
within the first substrate using a mold.
[0774] 158. The method of any one of clauses 152 to 157, further
comprising applying a hydrophobic and/or a dielectric material on
electrodes of the series using a printer device.
[0775] 159. The method of clause 158, wherein the hydrophobic
and/or dielectric material comprises a curing material.
[0776] 160. The method of clause 159, further comprising applying
heat or ultraviolet light to cure the applied hydrophobic and/or
dielectric material.
[0777] 161. The method of any one of clauses 152 to 160, further
comprising dicing the first and second substrates to generate a
bonded substrates comprising the first and second portions.
[0778] 162. A method of detecting an analyte of interest in a
liquid droplet, the method comprising:
[0779] (a) providing a first liquid droplet comprising an analyte
of interest;
[0780] (b) providing a second liquid droplet comprising a specific
binding member and a labeled analyte, wherein the binding member is
immobilized on at least one solid support, the specific binding
member specifically binds to the analyte of interest, and the
labeled analyte is an analyte of interest labeled with a detectable
label;
[0781] (c) using energy to exert a force to manipulate the first
liquid droplet with the second liquid droplet to create a mixture;
and
[0782] (d) moving all or at least a portion of the mixture to an
array of wells, wherein one or more wells of the array is of
sufficient size to accommodate the at least one solid support;
[0783] 163. A method of detecting an analyte of interest in a
liquid droplet, the method comprising:
[0784] (a) providing a first liquid droplet comprising an analyte
of interest;
[0785] (b) providing a second liquid droplet comprising an
immobilized analyte and at least one specific binding member,
wherein the immobilized analyte is an analyte of interest
immobilized on at least one solid support, the at least one
specific binding member specifically binds to the analyte of
interest, and the at least one specific binding member is labeled
with a detectable label;
[0786] (c) using energy to exert a force to manipulate the first
liquid droplet with the second liquid droplet to create a
mixture;
[0787] (d) moving all or at least a portion of the mixture to an
array of wells, wherein one or more wells of the array is of
sufficient size to accommodate the at least one solid support;
and
[0788] (e) detecting the analyte of interest in the wells.
[0789] 164. A method of detecting an analyte of interest in a
liquid droplet, the method comprising:
[0790] (a) providing a first liquid droplet containing an analyte
of interest;
[0791] (b) providing a second liquid droplet containing at least
one solid support which contains a specific binding member that
binds to the analyte of interest;
[0792] wherein the first and second liquid droplets are provided
into a digital microfluidic and analyte detection device,
comprising: [0793] a first substrate and a second substrate,
wherein the second substrate is separated from the first substrate
by a gap, the first substrate comprising a plurality of electrodes
to generate electrical actuation forces on a liquid droplet; and
[0794] an array of wells dimensioned to hold a portion of the
liquid droplet, wherein at least a portion of the array of wells is
positioned between one or more of the plurality of electrodes and
the gap;
[0795] (c) using the electrical actuation forces to manipulate the
first liquid droplet with the second liquid droplet to create a
mixture;
[0796] (d) moving all or at least a portion of the mixture to the
array of wells, wherein one or more wells of the array is of
sufficient size to accommodate the at least one solid support;
[0797] (e) adding a detectable label to the mixture either before
or after moving a portion of the mixture to array of wells; and
[0798] (f) detecting the analyte of interest in the wells.
[0799] 165. The method of clause 164, wherein the at least one
solid support comprises at least one binding member that
specifically binds to the analyte of interest.
[0800] 166. The method of any one of clauses 164 or 165, further
comprising adding a detectable label to the mixture before the
moving at least a portion of the mixture to the array of wells.
[0801] 167, The method of any one of clauses 164 or 166, further
comprising adding a detectable label to the mixture after the
moving at least a portion of the mixture to the array of wells.
[0802] 165, The method of any one of clauses 164 to 167, wherein
the detectable label comprises at least one binding member that
specifically binds to the analyte of interest.
[0803] 169. The method of any one of clauses 164 to 168, wherein
the detectable label comprises a chromagen, a fluorescent compound,
an enzyme, a chemiluminescent compound or a radioactive
compound.
[0804] 170. The method of any one of clauses 164 to 168, wherein
the binding member is a receptor or an antibody.
[0805] 171. The method of any one of clauses 164 to 170, wherein
the electric actuation forces are droplet actuation,
electrophoresis, electrowetting, di electrophoresis, electrostatic
actuation, electric field mediated, electrode mediated, capillary
force, chromatography, centrifugation, or aspiration.
[0806] 172. The method of any one of clauses 164 to 171, wherein
the first liquid droplet is a polarizable liquid, the second liquid
droplet is a polarizable liquid, the mixture is a polarizable
liquid or both the first liquid droplet and second liquid droplet
are each polarizable liquids.
[0807] 173. The method of any one of clauses 164 to 171, further
comprising manipulating the mixture over the array of wells using
an electric actuation force.
[0808] 174. The method of any one of clauses 164 to 173, further
comprising manipulating the mixture over the array of wells using a
capillary element configured to facilitate movement of the mixture
to the array of wells.
[0809] 175. The method of any one of clauses 164 to 174, wherein
the solid supports are magnetic solid supports.
[0810] 176, The method of clause 175, wherein the electric
actuation forces and the magnetic field are applied from opposite
directions relative to the at least a portion of the mixture.
[0811] 177. The method of any one of clauses 164 to 176, further
comprising mixing the mixture by moving the mixture back and forth,
moving the mixture in a circular pattern, splitting the mixture
into two or more submixtures and merging the submixtures.
[0812] 178. The method of any one of clauses 164 to 176, wherein
the mixture is an aqueous liquid.
[0813] 179. The method of any one of clauses 164 to 176, wherein
the mixture is an immiscible liquid.
[0814] 180. The method of any one of clauses 164 to 179, wherein
the liquid droplet is a hydrophobic liquid droplet.
[0815] 181. The method of any one of clauses 164 to 180, wherein
the array of wells has a hydrophilic surface.
[0816] 182. The method of any one of clauses 164 to 180, wherein
the array of wells has a hydrophobic surface.
[0817] 183. The method of any one of clauses 164 to 182, wherein
the substrate comprises a hydrophilic surface.
[0818] 184. The method of any one of clauses 164 to 182, wherein
the substrate comprises a hydrophobic surface.
[0819] 185. The method of any one of clauses 164 to 184, further
comprising generating an electric actuation force with a plurality
of electrodes to move the mixture to the array of wells total load
the wells.
[0820] 186. The method of any one of clauses 164 to 185, wherein
one or more wells of the array are loaded with at least one solid
support.
[0821] 187. The method of clause 186, wherein the loading comprises
applying a magnetic field to facilitate movement of at least one
solid support into the one or more wells of the array.
[0822] 188, The method of clause 187, further comprising removing
any solid supports that are not loaded into a well of the array
after the loading.
[0823] 189. The method of clause 188, wherein the removing
comprises generating an electric actuation force with the plurality
of electrodes to move a polarizable fluid droplet to the array of
wells to move the at least a portion of the mixture to a distance
from the array of wells.
[0824] 190. The method of clause 189, wherein the removing
comprises generating an electric actuation force with the
electrodes to move an aqueous washing droplet across the array of
wells.
[0825] 191. The method of clause 190, wherein generating an
electric actuation force comprises generating an alternating
current.
[0826] 192, The method of clause 191, wherein the alternating
current has a root mean squared (rims) voltage of 10 V or more.
[0827] 193. The method of any one of clauses 191 or 192, wherein
the alternating current has a frequency in a radio frequency
range.
[0828] 194. A method of detecting an analyte of interest in a
liquid droplet, the method comprising:
[0829] (a) providing a first liquid droplet containing an analyte
of interest;
[0830] (b) providing a second liquid droplet containing at least
one solid support which contains a specific binding member that
binds to the analyte of interest;
[0831] wherein the first and second liquid droplets are provided
into a surface acoustic wave microfluidic and analyte detection
device, comprising: [0832] a first substrate and a second
substrate, wherein the second substrate is separated from the first
substrate by a gap, wherein the device comprises a first portion
and a second portion, [0833] the first portion comprising a
superstrate coupled to a surface acoustic wave generating
component; and [0834] the second portion comprising a plurality of
wells positioned on the first substrate or the second
substrate,
[0835] (c) using surface acoustic forces to manipulate the first
liquid droplet with the second liquid droplet to create a
mixture;
[0836] (d) moving all or at least a portion of the mixture to the
array of wells, wherein one or more wells of the array is of
sufficient size to accommodate the at least one solid support;
[0837] (e) adding a detectable label to the mixture either before
or after moving a portion of the mixture to array of wells; and
[0838] (f) detecting the analyte of interest in the wells.
[0839] 195. The method of clause 194, wherein the at least one
solid support comprises at least one binding member that
specifically binds to the analyte of interest.
[0840] 196. The method of any one of clauses 194 or 195, further
comprising adding a detectable label to the mixture before the
moving at least a portion of the mixture to the array of wells.
[0841] 197. The method of any one of clauses 194 or 196, further
comprising adding a detectable label to the mixture after the
moving at least a portion of the mixture to the array of wells.
[0842] 198. The method of any one of clauses 194 to 197, wherein
the detectable label comprises at least one binding member that
specifically binds to the analyte of interest.
[0843] 199. The method of any one of clauses 194 to 198, wherein
the detectable label comprises a chromagen, a fluorescent compound,
an enzyme, a chemiluminescent compound or a radioactive
compound.
[0844] 200, The method of any one of clauses 194 to 199, wherein
the binding member is a receptor or an antibody.
[0845] 201. The method of any one of clauses 194 to 200, wherein
the first liquid droplet is a polarizable liquid, the second liquid
droplet is a polarizable liquid, the mixture is a polarizable
liquid or both the first liquid droplet and second liquid droplet
are each polarizable liquids.
[0846] 202. The method of any one of clauses 194 to 201, further
comprising manipulating the mixture over the array of wells using a
capillary element configured to facilitate movement of the mixture
to the array of wells.
[0847] 203. The method of any one of clauses 194 to 202, wherein
the solid supports are magnetic solid supports.
[0848] 204. The method of clause 70 wherein the method further
comprises sealing the array of wells with a hydrophobic liquid.
[0849] 205. The method of clause 96, wherein the method further
comprises sealing the array of wells with a hydrophobic liquid.
[0850] 206. The method of clause 185, wherein the method further
comprises sealing the array of wells with a hydrophobic liquid.
* * * * *