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.

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.

As such, there is an interest in integrated devices for performing analyte analysis.

SUMMARY

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.

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.

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.

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.

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.

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.

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.

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. 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

In certain embodiments, generating an electric actuation force includes generating an alternating current. In certain embodiments, the alternating current has a root mean squared (nils) voltage of 10 V or more. In certain other embodiments, the alternating current has a frequency in a radio frequency range.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

Fig. 1A illustrates a side view of an integrated digital microfluidic and analyte detection device according to one embodiment.

Fig. IB illustrates a side view of the integrated digital microfluidic and analyte detection device according to another embodiment.

Fig. 2A illustrates a side view of an integrated digital microfluidic and analyte detection device according to an embodiment.

Fig. 2B illustrates a side view of the integrated digital microfluidic and analyte detection device according to another embodiment.

Fig. 3A illustrates a side view of the device of Fig. 2 A with a liquid droplet being moved in the device.

Fig. 3B illustrate a side view of the device of Fig. 2B with of droplet being moved in the device.

Fig. 4A illustrates a side view of the device of Fig. 2 A with a droplet containing particles/beads being moved onto an array of wells.

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.

Fig. 5 illustrates an aqueous droplet being moved over the array of wells using a hydrophilic capillary region of the device.

Fig. 6 illustrates an aqueous droplet being moved over the array of wells.

Figs. 7A and 7B illustrate various exemplary orientations of the sidewalls of the wells.

Fig. 8 illustrates an example of fabricating a second (e.g., bottom) substrate of the digital microfluidic and analyte detection device.

Fig. 9 illustrates an example of fabricating a first (e.g., top) substrate of the digital microfluidic and analyte detection device.

Fig. 10 illustrates an example of assembling the top and bottom substrates to

manufacture a plurality of digital microfluidic and analyte detection devices.

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.

Figs. 12A -12D illustrate examples of fabricating the array of wells into the integrated digital microfluidic and analyte detection device.

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.

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.

Figs. 14A and 14B illustrate an example of fabricating the sample preparation component and well array component.

Fig. 15 depicts an exemplary method of the present disclosure.

Fig. 16 illustrates an exemplary method for removing beads not located in the wells of the depicted device.

Fig. 17 illustrates another exemplary method for removing beads not located in the wells of the depicted device.

Fig. 18 depicts a schematic of a fabrication process of a low-cost DMF chip.

Fig. 19 depicts a single flexible chip fabricated according to the schematic in Fig. 18.

Fig. 20 depicts actuation of droplets in a DMF chip, according to embodiments of the present disclosure.

Fig. 21, A-E depicts performance of an immunoassay in a DMF chip, according to embodiments of the present disclosure.

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.

Fig 23 shows a schematic diagram of a well design, according to embodiments of the present disclosure.

Figs. 24A and 24B are schematic diagram showing well spacing formats, according to embodiments of the present disclosure.

Fig. 25 are a collection of magnified optical images of the array of wells, according to embodiments of the present disclosure.

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.

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.

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.

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.

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.

Fig. 31, A-F provides a schematic of an analyte detection chip according to one embodiment.

Fig. 32, A-C provides a schematic of an analyte detection chip according to another embodiment.

Fig. 33 provides a schematic of an analyte detection chip according to one embodiment.

Figs. 34A and 34B illustrates side views of an exemplary analyte detection chip.

Fig. 35 illustrates a schematic of a top view of an analyte detection chip according to another embodiment.

Fig. 36 illustrates a schematic of an alternate exemplary analyte detection chip.

Fig. 37 provides a schematic of an exemplary hematology chip.

Figs. 38 and 39 illustrate alternate embodiments of DMF chip with mutiple detection regions.

Fig. 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.

Fig. 41, A and B depict embodiments of a cartridge (Fig. 41A) and an analyte detection device (Fig. 4 IB) that is compatible with the cartridge.

Figs. 42A-42E illustrate cartridges comprising DMF electrodes and optical detection chamber.

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

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.

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.

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.

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

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

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.

"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.

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.

"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 (!¾), or the association rate (ka).

"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.

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.

"Bead" and "particle" are used herein interchangeably and refer to a substantially spherical solid support.

"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.

"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.

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 di electrophoresis 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.

"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.

"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.

"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.

"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.

"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.

"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.

"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 Γ position, such as a ribose, 2'-deoxyribose, or a 2',3'-di-deoxyribose.

"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.

"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.

"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

"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.

"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.

"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.

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.

"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.

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.

"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.

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

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.

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.

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).

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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 P(x; μ) as shown below:

F (x? S) = ((M x ) / 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

x: is the tactual number of successes that occur in a specified region.

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.

"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.

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°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.

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.

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, Cr02, MnAs, MnBi, EuO, NiO/Fe. Examples of ferrimagnetic materials include NiFe204, CoFe204, Fe304 (or FeO Fe203). 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.

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.

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.

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°C-28°C,

e.g., 23°C - 25°C), 37 °C, or 4°C. In certain embodiments, an optional mixing of the sample with the first binding member may be carried out during the contacting step.

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.

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.

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.

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.

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.

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.

In some cases, the limit of detection (e.g., the lowest concentration of an analyte which may be determined in solution) is about 100 fJVI, about 50 fJVI, 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.

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 μΜ, at least about 100 μΜ, at least about 1000 μΜ, at least about 10 mM, at least about 100 mM, at least about 1000 mM, or greater.

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.

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.

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.

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.

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.

Multiplexing

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 eachhave 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.

Exemplary Target Analytes

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.

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.

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.

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); UPV etc.); spores, etc.

A non-limiting list of analytes that may be analyzed by the methods presented herein include Αβ42 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-Ια), transthyretin, Vitamin D-binding Protein, proapoptotic kinase R (PKR) and its phosphorylated PKR (pPKR), CXCL13, IL-12p40, CXCL13, IL-8, Dkk-3 (semen), pl4 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, BARFl, IL-8, carcinoembryonic antigen (CEA), BRAF, CCNI, EGRF, FGF19, FRS2, GREB 1, 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 (TIAMl), N-cadherin, EC39, amphiregulin, dUTPase, secretory gelsolin (pGSN), PSA (prostate specific antigen), thymosin β15, insulin, plasma C-peptide, glycosylated hemoglobin (HBAlc), 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- Al l), 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), IQGAPl (Ras GTPase-activating-like protein IQGAPl), CP

(Ceruloplasmin precursor), and IGLC2 (IGLCl protein), PCDGF/GP88, EGFR, HER2, MUC4, IGF-IR, p27(kipl), 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, FRAPl, RG1, ESR1, ESR2, PGR, CDKN1B, MAP2K1, NEDD4-1, FOX03A, PPP1R1B, PXN, ELA2, CTN B l, 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 (VCAMl) and Von Willebrand factor (VWF),

myeloperoxidase (MPO), ILla, T Fa, 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.

Samples

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.

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 μΐ., about 0.1 μΐ., about 1 \L, about 5 μΐ., about 10 μΐ., about 50 μΐ., about 100 μΐ., 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 μL and about 10 mL, between about 0.01 μL and about 1 mL, between about 0.01 μL and about 100 μL, between about 0.1 μL and about 10 μL, between about 1 \L and about 100 μΐ,, between about 10 μL and about 100 μL, or between about 10 μL and about 75 μL.

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.

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.

CLAIMS

1. An instrument for detection of an analyte in a sample, the instrument comprising: a control unit;

a detection unit; and

a cartridge interface for operable connection to one or more cartridges comprising the sample,

wherein the control unit is configured for controlling activation of an array of electrodes in the cartridge for moving a sample droplet present in the cartridge,

wherein the detection unit is configured to detect:

i) a first analyte related signal from a droplet in a cartridge; and

ii) a second analyte related signal from spatially segregated single molecules in a cartridge and/or from spatially segregated molecules in a cartridge.

2. The instrument of claim 1, wherein the first analyte related signal comprises an electrical signal and the second analyte related signal comprises an optical signal.

3. The instrument of claim 1, wherein the first analyte related signal comprises an optical signal and the second analyte related signal comprises an optical signal.

4. The instrument of any one of claims 1-3, wherein the detection unit is configured to detect the first and second analyte related signals from the same cartridge.

5. The instrument of any one of claims 1-3, wherein the detection unit is configured to detect the first and second analyte related signals from different cartridges.

6. The instrument of claim 1, wherein instrument further comprises a power source and wherein the control unit controls the electric power applied to the one or more electrodes.

7. The instrument of any one claims 1-6, wherein the control unit controls duration of activation of the one or more electrodes.

8. The instrument of any one claims 1-6, wherein the control unit controls the sequence of activation and deactivation of one or more electrodes to facilitate movement of a droplet in the cartridge.

9. The instrument of claim 8, wherein the movement comprises merging a sample droplet with a reagent droplet to generate a merged droplet.

10. The instrument of claim 9, wherein the movement comprises moving the merged droplet or a portion thereof to the detection unit.

11. The instrument of any one of claims 1-10, wherein the detection unit comprises an electrical detection unit for detection of an electrical signal from the cartridge.

12. The instrument of claim 11, wherein the electrical detection unit comprises an electrical circuit connected to the cartridge interface.

13. The instrument of claim 12, wherein the electrical circuit is operably connected to a recorder for recording the electrical signal, wherein the electrical signal is generated by:

an electrochemical species produced from action of an analyte-specific enzyme on the analyte; or

an electrochemical species produced from action of an enzyme on a substrate molecule, wherein the enzyme is conjugated to an antibody that specifically binds to the analyte.

14. The instrument of any one of claims 1-13, wherein the detection unit comprises an optical detection unit.

15. The instrument of claim 14, wherein the optical detection unit is configured for detecting an optical signal from molecules spatially segregated into single wells.

16. The instrument of claim 14 or 15, wherein the optical detection unit is configured for detecting an optical signal from single molecules spatially segregated into single wells.

17. The instalment of any one of claims 14-16, wherein the optical detection unit comprises a detector for one or more of a colorimetric signal, a turbidometric signal, or a fluorescent signal.

18. The instrument of any one of claims 14-17, wherein the optical detection unit comprises an imaging system.

19. The instrument of any one of claims 1-18, wherein the instrument comprises a processor which executes a program with instructions to the control unit to activate and deactivate the one or more electrodes and to operate the detection unit.

20. The instrument of any one of claims 1-18, wherein the instrument is configured to conduct two or more of clinical chemistry, immunoassay, spatial segregation of single molecules, imaging, agglutination assay, and hematology.

21. The instrument of any one of claims 1-20, comprising an electrical detection unit comprising an electrical circuit configured for detecting an electrical signal from the working electrode of a cartridge comprising an electrochemical detection region; and comprising an optical detection unit comprising one or more of: a camera, a microscope, a charge coupled device (CCD), a spectrometer, a complementary metal-oxide-semiconductor (CMOS) detector, a fluorimeter, a colorimeter, and a turbidometer.

22. The instrument of any one of claims 1-21, wherein the electrical detection unit detects an electrical signal selected from the group consisting of current, voltage, impedance, capacitance, charge, conductivity, resistance, or a combination thereof.

23. A cartridge comprising:

a first substrate;

a second substrate;

a gap separating the first 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.

24. The cartridge of claim 23, wherein the working electrode and the reference electrode are both disposed on a surface of the first substrate or the second substrate and wherein at least a portion of the working electrode and the reference electrode is in electrical contact with a droplet disposed in the gap in the cartridge.

25. The cartridge of claim 23, wherein one of the working electrode or the reference electrode is disposed on the surface of the first substrate and the other electrode is disposed on the surface of the second substrate, in a facing configuration, and wherein at least a portion of the working electrode and the reference electrode is in electrical contact with a droplet disposed in the gap in the cartridge.

26. The cartridge of any one of claims 23-24, wherein the plurality of electrodes to generate electrical actuation forces on a liquid droplet is positioned on a surface of the first substrate or the second substrate,

27. The cartridge of any one of claims 23-26, further comprising a first layer covering the plurality of electrodes.

28. The cartridge of claim 27, wherein the first layer comprises a dielectric layer and/or a hydrophobic layer.

29. The cartridge of claim 27 or 28, wherein at least a portion of the working electrode and the reference electrode is not covered by the first layer.

30. The cartridge of any one of claims 27-29, wherein the first layer comprises a photodegradable material and wherein the first layer does not cover a portion of the working electrode and the reference electrode due to removal of the first layer from the portion of the working electrode and the reference electrode by exposure to light.

31 . The cartridge of any one of claims 21-30, wherein the working and reference electrodes are disposed in a capillary channel configured to exert capillary force on a droplet thereby moving the droplet to the capillary channel.

32. A multi-functional cartridge comprising:

a first substrate;

a second substrate;

a gap separating the first substrate from the first substrate;

a plurality of electrodes to generate electrical actuation forces on a liquid droplet; and two or more of:

an electrochemical species sensing region comprising a working electrode and a reference electrode;

an optical detection region comprising an array of wells for single molecule detection; and

an optical detection region that is optically transparent and comprises electrodes for actuating a droplet and is configured for optical interrogation of the droplet.

33. The multi-functional cartridge of claim 32, wherein the cartridge comprises: an electrochemical species sensing region comprising a working electrode and a reference electrode.

34. The multi-functional cartridge of claim 32, wherein the cartridge comprises: an electrochemical species sensing region comprising a working electrode and a reference electrode; and

an optical detection region comprising an array of wells for single molecule detection.

35. The multi-functional cartridge of claim 32, wherein the cartridge comprises: an electrochemical species sensing region comprising a working electrode and a reference electrode; and

an optical detection region that is optically transparent and comprises electrodes for actuating a droplet and is configured for optical interrogation of the droplet.

36. The multi-functional cartridge of claim 32, wherein the cartridge comprises: an optical detection region comprising an array of wells for single molecule detection.

37. The multi-functional cartridge of claim 32, wherein the cartridge comprises: an optical detection region comprising an array of wells for single molecule detection; and

an optical detection region that is optically transparent and comprises electrodes for actuating a droplet and is configured for optical interrogation of the droplet.

38. The cartridge of any one of claims 32-37, further comprising a first layer covering the plurality of electrodes.

39. The cartridge of claim 38, wherein the first layer compri ses a dielectric layer and/or a hydrophobic layer.

40. A system for detection of an analyte in a sample, the system comprising:

an analyte detection instrument; and

one or more analyte detection cartridges,

wherein the analyte detection instrument comprises:

a control unit;

a detection unit; and

a cartridge interface for operable connection to the one or more analyte detection cartridges,

wherein the control unit is configured for controlling activation of one or more electrodes for moving a droplet present in the cartridge,

wherein the detection unit is configured to detect:

i) a first analyte related signal from a droplet in a cartridge; and

ii) a second analyte related signal from spatially segregated single molecules in a cartridge and/or from spatially segregated molecules in a cartridge,

wherein the each one or more analyte detection cartridge comprises:

a plurality of electrodes to generate electrical actuation forces on a liquid droplet in response to the control unit; and

at least one detection region for generating an analyte related signal from a droplet in a cartridge or from spatially segregated single molecules and/or from spatially segregated molecules in a cartridge.

41. The system of claim 40, further comprising reagent reservoirs, wherein the reagent reservoirs are adjacent to the cartridge interface or are located on the cartridge.

42. The system of claim 41 , wherein the instrument is programmed for forming droplets from the reagent reservoirs and for moving the droplets by selectively activating and deactivating the plurality of electrodes.

43. The system of any one of claims 40-42, wherein the instrument is programmed for forming at least a sample droplet from a sample introduced into the cartridge and for moving the sample droplet(s) by selectively activating and deactivating the plurality of electrodes.

44. The system of any one of claims 40-43, wherein the reagent reservoirs comprise a reagent comprising an enzyme specific for the analvte in the sample, wherein the enzyme acts on the analvte to generate a reaction product that generates an electrical or optical signal.

45. The system of claim 41, wherein the reagent further comprises a redox mediator.

46. The system of any one of claims 42-45, wherein the reagent reservoirs comprise a first binding member that selectively binds to the analvte, wherein the first binding member is attached to a bead.

47. The system of claim 46, wherein the first binding member is an antibody.

48. The system of claims 46 or 47, wherein the reagent reservoirs comprise a second binding member that selectively binds to the analvte, wherein the second binding member is attached to an enzyme.

49. The system of claim 48, wherein the second binding member is an antibody.

50. The system of claim 48 or 49, wherein the enzyme acts on a substrate to produce a reaction product associated with an electrical signal or an optical signal .

51. The system of any one of claim 40-44, wherein the cartridge interface comprises an insertion slot for accommodating the cartridge, wherein the cartridge comprises:

(i) an electrical detection region, the electrical detection region comprising: a working electrode and a reference electrode for detecting electrical signal from an electrochemical species generated when an analyte is present in the sample; or

(ii) an optical detection region, the optical detection region comprising an array of wells for accommodating at least one bead for detecting the analyte.

52. The system of claim 51, wherein the instalment comprises a plurality of insertion slots.

53. The system of claim 52, wherein the plurality of insertion slots are positioned on a carousel .

54. The system of any one of claims 40-47, wherein the system comprises programming for selecting a function of the instrument, wherein the function is selected based on type of cartridge operably connected to the cartridge interface of the instrument, wherein the function is selected from a plurality of assays for processing the sample.

55. The system of claim 54, wherein when the cartridge comprises an electrical detection region, the instrument is instructed to process the sample for generating an electrical signal in response to presence of an analyte in the sample and to detect the electrical signal via the electrical detection unit.

56. The system of claim 55, wherein instrument receives an indication of the analyte to be detected and processes the sample based on the type of analyte to be detected.

57. The system of claim 56, wherein the indication is provided by a user via a user interface of the instalment or wherein the indication is provided via a machine-readable indicator present on the cartridge.

58. The system of any one of claims 40-56, wherein when the cartridge comprises an optical detection region, the instrument is instructed to process the sample for generating an optical signal in response to presence of an analyte in the sample and to detect the optical signal via the optical detection unit.

59. The system of any one of claims 40-58, wherein the system is configured for detection of an analvte in a whole blood sample and/or a plasma fraction of a whole blood sample.

60. The system of any one of claims 40-58, wherein the system is configured for detection of an analvte in a plasma fraction of a whole blood sample, wherein the plasma fraction of the whole blood sample is generated using a cartridge comprising a filter for separating the plasma fraction from the whole blood sample.

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 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 any one of claims 65-67, further comprising determining a concentration of the analyte based on the electrical signal.

69. The method of any one of claims 65-67, 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. A method for performing analyte detection using an instrument comprising: providing an analyte detection instnmieiit 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.

72. A method for simultaneously or sequentially performing at least two assays using a single system.

73. The method of claim 72, wherein the at least two assays are selected from the group consisting of:

(a) a clinical chemistry assay and an immunoassay;

(b) two clinical chemistry assays;

(c) two immunoassays;

(d) a clinical chemistry assay and a hematology assay;

(e) an immunoassay and a hematology assay;

(f) two hematology assays;

(g) a clinical chemistry assay, immunoassay and a hematology assay;

(h) three clinical chemistry assays;

(i) three immunoassays;

(j) two clinical chemistry assay and a hematology assay;

(k) two immunoassay and a hematology assay;

(1) two hematology assays and a clinical chemistry assay;

(m) two hematology assays and an immunoassay; and

(n) three hematology assays.

74. The method of claim 72 or 73 wherein the single system is the instrument of any of claims 1 to 22.

75. An instrument for detection of an analyte in a sample, the instrument comprising: a control unit;

a detection unit; and

a cartridge interface for operable connection to one or more cartridges comprising the sample,

wherein the control unit is configured for controlling activation of an array of electrodes in the cartridge for moving a sample droplet present in the cartridge,

wherein the detection unit is configured to detect:

i) a first analyte related signal from a droplet in a cartridge; and

ii) a second analyte related signal from a single molecule in a cartridge.

76. The instrument of claim 75, wherein the first analyte related signal comprises an optical or electrical signal and the second analyte related signal comprises an optical signal.

77. The instrument of claim 75, wherein the first analyte related signal comprises an optical or electrical signal and the second analyte related signal comprises an electrical signal.

78. The instrument of any one of claims 75-77, wherein the detection unit is configured to detect the first and second analyte related signals from the same cartridge.

79. The instrument of any one of claims 75-77, wherein the detection unit is configured to detect the first and second analyte related signals from different cartridges.

80. The instrument of claim 1, wherein instrument further comprises a power source and wherein the control unit controls the electric power applied to the one or more electrodes.

81. The instrument of any one claims 75-80, wherein the control unit controls duration of activation of the one or more electrodes.

82. The instrument of any one claims 75-81, wherein the control unit controls the sequence of activation and deactivation of one or more electrodes to facilitate movement of a droplet in the cartridge.

83. The instrument of claim 82, wherein the movement comprises merging a sample droplet with a reagent droplet to generate a merged droplet.

84. The instrument of claim 83, wherein the movement comprises moving the merged droplet or a portion thereof to the detection unit.

85. The instrument of any one of claims 75-84, wherein the detection unit comprises an electrical detection unit for detection of an electrical signal from the cartridge.

86. The instrument of any one of claims 75-85, wherein the detection unit comprises an optical detection unit for detection of an optical signal from the cartridge.