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
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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
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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 w^ells.
[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
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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 inlude 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 superstate coupled to a surface acoustic wave
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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
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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
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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.
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[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
(rrns) 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 capillar}- 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.
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[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.
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[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
(rrns) 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 capillar}- 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
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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
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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 microfluidie 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 w?ells. 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 microfluidie 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
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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,
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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. 1Α illustrates a side view of an integrated digital microfluidic and analyte
detection device according to one embodiment.
[0060] Fig. IB illustrates a side view of the integrated digital microfluidic and analyte
detection device according to another embodiment.
[0061] Fig. 2Α illustrates a side view of an integrated digital microfluidic and analyte
detection device according to an embodiment.
[0062] Fig. 2Β illustrates a side view of the integrated digital microfluidic and analyte
detection device according to another embodiment.
[0063] Fig. 3Α illustrates a side view of the device of Fig. 2Α with a liquid droplet being
moved in the device.
[0064] Fig. 3Β illustrate a side view of the device of Fig. 2Β with of droplet being moved in
the device.
[0065] Fig. 4Α illustrates a side view of the device of Fig. 2Α with a droplet containing
particles/beads being moved onto an array of wells.
[0066] Fig. 4Β illustrates a side view of the device of Fig. 2Β with a droplet containing
particles/beads being moved onto an array of wells with a droplet of an immiscible fluid.
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[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. 7Α-7Β 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] Fig. 11Α and Fig. 11Β show a view from the top of a bottom substrate of exemplary
digital microfluidic and analyte detection devices of the present disclosure.
[0074] Figs. 12Α -12D illustrate examples of fabricating the array of wells into the integrated
digital microfluidic and analyte detection device.
[0075] Fig. 13Α 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. 13Β 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. 14Α-14Β 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.
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[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. 21Α-21Ε depicts performance of an immunoassay in a DMF chip, according to
embodiments of the present disclosure.
[0085] Figs. 22Α and 22Β 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. 24Α and 24Β 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.
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[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] Figs. 30A-30D 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.
DFTATFFD DESCRIPTION OF THF INVENTION
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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.
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PET ATT /ΕΤ) DESCRIPTION
[0098] 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
[0099] 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.
[00100] “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.
[00101] 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.
[00102] “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 (KD), the dissociation rate (kd), or
the association rate (ka).
[00103] “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.
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[00104] 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.
[00105] “Bead” and “particle” are used herein interchangeably and refer to a substantially
spherical solid support.
[00106] “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.
[00107] “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-inoil
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.
[00108] 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
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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 highfrequency
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.
[00109] “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.
[00110] “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.
[00111] “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.
[00112] “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
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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.
[00113] “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.
[00114] "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, Ν9-(2-
amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine, N9-(7-deaza-guanine), Ν9-(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.
[00115] “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.
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[00116] “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.
[00117] “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.
[00118] “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
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[00119] “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.
[00120] “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. Patent No. 6,013,785 and U.S. Patent No. 5,696,253.
[00121] “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.
[00122] 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.
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[00123] “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.
[00124] 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.
[00125] “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.
[00126] 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.
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Methods for Analyte Analysis
[00127] 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.
[00128] 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
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more, 30 V or more or 35 V or more. Alternatively, the alternating current can have a frequency
in a radio frequency range.
[00129] 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 fo 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).
[00130] 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.
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[00131] 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.
[00132] 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.
[00133] 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.
[00134] 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
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certain embodiments, method described herein is performed using a Robotics based assay
processing unit.
[00135] 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 nonmechanical
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 nonmechanical
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.
[00136] 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
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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).
[00137] 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 labelspecific
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.
[00138] 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.
[00139] 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.
[00140] 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
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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.
[00141] 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.
[00142] 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
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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.
[00143] 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 fo 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).
[00144] 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
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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.
[00145] 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.
[00146] 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.
[00147] 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.
[00148] 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
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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.
[00149] 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 (“postives”).
The positives are substracted 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 Ρ(χ; μ) as shown below:
P(x;p) = (eW)/x!
where:
e\ A is a constant equal to approximately 2.71828,
μ: ix ghd mean number of successes that occur in a specified region, and
χ: is the tactual number of successes that occur in a specified region.
[00150] 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.
[00151] “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
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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.

In the Claims:
1. A digital microfluidic and analyte detection device, comprising:
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
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,
2. The device according to claim 1, wherein the plurality of electrodes is positioned on a
surface of the first substrate.
3. The device according to claim 1 or claim 2, further comprising a first layer disposed on the
surface of the first substrate and covering the plurality of electrodes.
4. The device of any one of the previous claims, 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.
5. The device of claim 4, wherein the plurality of electrodes and the first layer extend from the
first portion to the second portion of the first substrate.
6. The device of claim 5, wherein the array of wells is positioned in the second portion of the
first substrate.
7. The device according to claim 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.
8. The device of claim 7, wherein the second portion of the second substrate is substantially
transparent to facilitate optical interrogation of the array of wells.
9. The device according to claim 3, further comprising a second layer disposed on a surface
of the first layer.
10. The device according to claim 9, wherein a second layer extends over the first and second
portions of the first substrate.
11. The device according to any one of claims 9-10, wherein the first layer i s a dielectric layer
and the second layer is a hydrophobic layer.
12. The device according to any one of claims 9-11, wherein the array of wells is positioned
in the second layer.
13. The device according to any one of claims 3, wherein the array of wells is positioned in
the first layer.
14. The device according to any one of the previous claims, wherein the array of wells has a
hydrophilic surface.
156
15. The device according to any one of the previous claims, 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.
16. The device according to claim 16, 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.
17. The device according to claim 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.
18. The device according to claim 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.
19. A method of detecting an analyte of interest in a liquid droplet, the method comprising:
(a) providing a first liquid droplet containing an analyte of interest;
(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;
(c) using energy to exert a force to manipulate the first liquid droplet with the second liquid
droplet to create a mixture;
(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; (e) adding
a detectable label to the mixture either before or after moving a portion of the mixture to array
of wells; and
(f) detecting the analyte of interest in the wells.
20. The method of claim 19, wherein the at least one solid support comprises at least one
binding member that specifically binds to the analyte of interest.