[0001] This application claims priority to United States Provisional Application Serial No. 62/404,722, filed on October 5, 2016, and United States Provisional Application Serial No. 62/424,996, filed on November 21, 2016, the disclosures of which applications are herein incorporated by reference.

TECHNICAL FIELD

[0002] This disclosure relates to methods, devices, and systems for analyte analysis using an analyte detection device, e.g., operably coupled with a microfluidic device.

BACKGROUND

[0003] Methods and devices that can accurately analyze analyte(s) of interest in a sample are essential for diagnostics, prognostics, environmental assessment, food safety, detection of chemical or biological warfare agents and the like. Such methods and devices not only need to be accurate, precise and sensitive but are also advantageous when a minute sample is to be analyzed quickly and with minimal instrumentation. As such, there in an interest in methods and devices with improved sample analysis capabilities.

SUMMARY

[0004] 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.

[0005] The method for analysis of an analyte in a sample may involve contacting the sample with a first binding member, where the first binding member is immobilized on a solid support and where the first binding member specifically binds to the analyte; contacting the solid support with a second binding member, where the second binding member specifically binds to the analyte and wherein the second binding member includes a cleavable tag attached thereto; removing second binding member not bound to the analyte bound to the first binding member; cleaving the tag attached to the second binding member that is bound to the analyte bound to the first binding member; translocating the cleaved tag through or across a nanopore in a layer; determining the number of tags translocating through the layer; determining concentration of the analyte in the sample based on the number of tags translocating through the layer. In certain embodiments, the concentration of the analyte may be determined by counting the number of tags translocating through the layer per unit time. In other embodiments, the concentration of the analyte may be determined by determining the time at which the number of tags translocating through the layer reaches a threshold or by setting a period of time and counting cumulative number of counts in the set period of time.

[0006] In another embodiment, the method may include combining the sample containing the target analyte with a known amount of the target analyte or a competitor molecule, where the target analyte (combined with the sample) or the competitor molecule are attached to a tag via a cleavable linker to produce a tagged analyte or tagged competitor molecule, respectively, and the tagged analyte or tagged competitor molecule compete with the target analyte for binding to a first binding member. The method may further include contacting the combined sample with the first binding member, where the first binding member is immobilized on a solid support and where the first binding member specifically binds to the target analyte (and to the tagged analyte or tagged competitor molecule); contacting the solid support with buffer for an optional washing step; cleaving the tag attached to the tagged analyte or tagged competitor that is bound to the first binding member immobilized on the solid support; translocating the cleaved tag through or across a nanopore in a layer; determining the number of tags translocating through the layer; determining concentration of the analyte in the sample based on the number of tags translocating through the layer. In certain embodiments, the concentration of the analyte may be determined by counting the number of tags translocating through the layer per unit time. In other embodiments, the concentration of the analyte may be determined by determining the time at which the number of tags translocating through the layer reaches a threshold or by setting a period of time and counting cumulative number of counts in the set period of time. In this embodiment, the number of tags translocated through the nanopore or the time at which the number of tags translocating through the layer reaches a threshold may be inversely correlated to the concentration of the analyte in the sample. For example, the lower count or the longer the time period for reaching a threshold, the higher the concentration of the target analyte in the sample.

[0007] In one aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample. The method comprising contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds to the analyte; contacting the analyte with a second binding member, wherein the second binding member specifically binds to the analyte and wherein the second binding member comprises a cleavable tag attached thereto; removing second binding member not bound to the analyte bound to the first binding member; cleaving the tag attached to the second binding member that is bound to the analyte bound to the first binding member; translocating the cleaved tag through or across one or more nanopores in a layer; and assessing the tag translocating through the layer, wherein measuring the number of tags translocating through the layer measures the amount of analyte present in the sample, or wherein detecting tags translocating through the layer detects that the analyte is present in the sample. In some embodiments, measuring the tags translocating through the layer is assessed, wherein the number of tags translocating through the layer measures the amount of analyte present in the sample. In some embodiments, detecting the tags translocating through the layer is assessed, wherein detecting tags translocating through the layer detects that the analyte is present in the sample.

[0008] In one aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample. The method comprising contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds to the analyte; contacting the analyte with a second binding member, wherein the second binding member specifically binds to the analyte and wherein the second binding member comprises an aptamer; removing aptamer not bound to the analyte bound to the solid substrate; dissociating the aptamer bound to the analyte and translocating the dissociated aptamer through or across one or more nanopores in a layer; and assessing the aptamer translocating through the layer, wherein measuring the number of aptamers translocating through the layer measures the amount of analyte present in the sample, or wherein detecting aptamers translocating through the layer detects that the analyte is present in the sample. In some embodiments, measuring the aptamers translocating through the layer is assessed, wherein the number of aptamers translocating through the layer measures the amount of analyte present in the sample. In some embodiments, detecting the aptamers translocating through the layer is assessed, wherein detecting tags translocating through the layer detects that the analyte is present in the sample.

[0009] In one aspect, the present invention relates to an integrated digital microfluidics nanopore device comprising a bottom substrate, comprising an array of electrodes; a top substrate spaced apart from the bottom substrate; and a nanopore layer disposed in between the bottom and top substrates. The device includes a proximal portion and a distal portion and the nanopore layer is disposed in the distal portion. The array of electrodes in the proximal portion is configured to generate a droplet. The array of electrodes are configured to position the droplet across the nanopore layer such that the droplet is split by the nanopore layer into a first portion and a second portion, wherein at least two electrodes of the array of electrodes are positioned across the nanopore layer, where the two electrodes form an anode and a cathode and operate to drive current through a nanopore in the nanopore layer when a liquid droplet is positioned across the nanopore layer.

[0010] In one aspect, the present invention relates to an integrated digital microfluidics nanopore device comprising a bottom substrate, comprising an array of electrodes; a top substrate spaced apart from the bottom substrate and comprising an electrode; and a nanopore layer disposed in between the bottom and top substrates. The device includes a proximal portion and a distal portion and the nanopore layer is disposed in the distal portion. The array of electrodes and the electrode in the proximal portion are configured to generate a droplet. The array of electrodes and the electrode are configured to position the droplet across the nanopore layer such that the nanopore layer splits the droplet into a first portion and a second portion, wherein at least one electrode of the array of electrodes is in contact with the first portion of a droplet positioned across the nanopore layer and the electrode in the top substrate is positioned to contact the second portion of the droplet positioned across the nanopore layer, where the two electrodes form an anode and a cathode and operate to drive current through a nanopore in the nanopore layer when a liquid droplet is positioned across the nanopore layer.

[0011] In one aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample. The method comprising contacting the sample with a binding member, wherein the binding member is immobilized on a solid support and wherein the binding member specifically binds to the analyte; contacting the sample, which may contain analyte bound to the binding member, with a labeled analyte, wherein the labeled analyte is labeled with a cleavable tag; removing labeled analyte not bound to the binding member;

cleaving the tag attached to the labeled analyte that is bound to the binding member;

translocating the cleaved tag through or across one or more nanopores in a layer; and assessing the tag translocating through the layer, wherein measuring the number of tags translocating through the layer measures the amount of analyte present in the sample, or detecting tags translocating through the layer detects that the analyte is present in the sample. In some embodiments, measuring the tags translocating through the layer is assessed, wherein the number of tags translocating through the layer measures the amount of analyte present in the

sample. In some embodiments, detecting the tags translocating through the layer is assessed, wherein detecting tags translocating through the layer detects that the analyte is present in the sample.

[0012] In one aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample. The method comprising contacting the sample with a binding member, wherein binding member is immobilized on a solid support and wherein binding member specifically binds to the analyte; contacting the sample, which may contain analyte bound to the binding member, with a labeled analyte, wherein the labeled analyte comprises an aptamer; removing labeled analyte not bound to the binding member; dissociating the aptamer bound to the labeled analyte that is bound to the binding member and translocating the dissociated aptamer through or across one or more nanopores in a layer; and assessing the aptamer translocating through the layer, wherein measuring the number of aptamers

translocating through the layer measures the amount of analyte present in the sample, or detecting aptamers translocating through the layer detects that the analyte is present in the sample. In some embodiments, measuring the aptamers translocating through the layer is assessed, wherein the number of aptamers translocating through the layer measures the amount of analyte present in the sample. In some embodiments, detecting the aptamers translocating through the layer is assessed, wherein detecting tags translocating through the layer detects that the analyte is present in the sample.

[0013] In one aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample. The method comprising contacting the sample with a binding member, wherein the binding member specifically binds to the analyte, and the binding member is labeled with a cleavable tag; contacting the sample, which may contain analyte bound to the binding member, with an immobilized analyte, wherein the immobilized analyte is immobilized on a solid support; removing binding member not bound to the immobilized analyte; cleaving the tag attached to the binding member that is bound to the immobilized analyte; translocating the cleaved tag through or across one or more nanopores in a layer; and assessing the tag translocating through the layer, wherein measuring the number of tags translocating through the layer measures the amount of analyte present in the sample, or detecting tags translocating through the layer detects that the analyte is present in the sample. In some embodiments, measuring the tags translocating through the layer is assessed, wherein the number of tags translocating through the layer measures the amount of analyte present in the

sample. In some embodiments, the tags translocating through the layer is assessed, wherein detecting tags translocating through the layer detects that the analyte is present in the sample.

[0014] In one aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample. The method comprises contacting the sample with a binding member, wherein the binding member specifically binds to the analyte, and the binding member comprises an aptamer; contacting the sample, which may contain analyte bound to the binding member, with a immobilized analyte, wherein the immobilized analyte is immobilized on a solid support; removing binding member not bound to the immobilized analyte;

dissociating the aptamer bound to the binding member that is bound to the immobilized analyte and translocating the dissociated aptamer through or across one or more nanopores in a layer; and assessing the aptamer translocating through the layer, wherein measuring the number of aptamers translocating through the layer measures the amount of analyte present in the sample, or detecting aptamers translocating through the layer detects that the analyte is present in the sample. In some embodiments, measuring the aptamers translocating through the layer is assessed, wherein the number of aptamers translocating through the layer measures the amount of analyte present in the sample. In some embodiments, detecting the aptamers translocating through the layer is assessed, wherein detecting tags translocating through the layer detects that the analyte is present in the sample.

[0015] In certain aspects, the tag may be an anionic polymer, a cationic polymer, or a nanoparticle. In certain cases, the tag may include an anionic polymer, such as, an

oligonucleotide polymer. In certain cases, the oligonucleotide polymer may be a

deoxyribonucleic acid or a ribonucleic acid. In certain cases, the oligonucleotide polymer may be a DNA aptamer or a RNA aptamer, where the aptamer does not bind to the analyte. In exemplary cases, the tag may include a nanoparticle which may be a positively charged nanoparticle or a negatively charged nanoparticle.

[0016] In certain embodiments, the tag may be spherical tag, such as, a dendrimer, a bead, a nanoparticle, e.g., a nanobead, and the like. In certain embodiments, the tag may not be linear or substantially linear or elongate in shape, such as, a polymer of ribose or deoxyribose units, an oligonucleotide, and a nucleic acid, for example, DNA or RNA.

[0017] In certain cases, the first and the second binding members may be aptamers, antibodies or receptors. For example, the first binding member may be a receptor and the second binding member may be an antibody or the first binding member may be an antibody and the second binding member may be a receptor. In certain instances, the first binding member may be a first antibody and the second binding member may be a second antibody.

[0018] In certain instances, the tag may be negatively charged and the translocating may include applying a positive potential across the layer thereby translocating the tag across the layer.

[0019] In certain instances, the tag may be positively charged and the translocating may include applying a negative potential across the layer thereby translocating the tag across the layer.

[0020] In other embodiments, the tag may be a nucleic acid and the tag may be hybridized to an oligonucleotide that includes a sequence complementary to sequence of the tag prior to the translocating.

[0021] In another embodiment, a method for measuring an analyte present in a biological sample by using an aptamer as the second binding member is provided. For example, the method may include contacting the sample with a first binding member, where the first binding member is immobilized on a solid support and where the first binding member specifically binds to the analyte; contacting the analyte with a second binding member, wherein the second binding member specifically binds to the analyte and wherein the second binding member comprises an aptamer; removing aptamer not bound to the analyte bound to the solid substrate; dissociating the aptamer from the analyte that is bound to the solid substrate and translocating the dissociated aptamer through nanopore(s) in a layer; determining the number of aptamers translocating through the layer; measuring the analyte in the sample based on the number of aptamers translocating through the layer. In this embodiment, the second binding member is not attached to a tag as the second binding member is directly detected by the nanopore(s).

[0022] The aptamer may be a DNA aptamer or a RNA aptamer. The first binding member may be an antibody. In certain instance, the analyte may be a ligand and the first binding member may be a receptor.

[0023] Also disclosed herein are methods for simultaneously analyzing multiple different analytes in a sample, for example, the method may include analysis of a first and a second analyte; a first, a second, and a third analyte; and so on. In certain cases, the method for analysis of plurality of different analytes in a sample may include contacting the sample with a plurality of different first binding members, where a first binding member of the different first binding members binds specifically to a first analyte of the plurality of the different analytes, a second binding member of the different first binding members binds specifically to a second analyte of the plurality of the different analytes, and so on. The method may further include contacting the different analytes with a plurality of second binding members, where a first binding member of the plurality of second binding members binds to the first analyte, a second binding member of the plurality of second binding members binds to the second analyte, and so on. In certain instances, each of the plurality of different second binding members may include a tag that is distinct or distinguishable from each other (e.g., each of the different second binding members has a different tag). For example, the first binding member of the plurality of the second binding members may include a first tag, the second binding member of the plurality of the second binding members may include a second tag, and so on, where the first and second tags are distinguishable from each other. Distinguishing the tags can be done using any suitable method, e.g., based on the nature or characteristic properties of the tags.

[0024] The method may further include removing unbound second binding members;

cleaving the tags attached to the plurality of second binding members bound to the analytes; translocating the tags through nanopores in a layer; determining the number of each of the tags translocating through the layer; measuring the plurality of different analytes in the sample based on the number of each of the tags translocating through the layer. In certain embodiments, the concentration of the analyte may be determined by counting the number of tags translocating through the layer per unit time. In other embodiments, the concentration of the analyte may be determined by determining the time at which the number of tags translocating through the layer reaches a threshold. As noted herein, in certain cases, the second binding members may be a plurality of aptamers and these aptamers are not attached to a tag as the aptamers are counted. In these embodiments, the aptamers may be dissociated from the analyte prior to translocating through or across a nanopore(s).

[0025] In certain cases, the different tags, such as the different aptamers, may be

distinguishable from each other via nanopore force spectroscopy, optical means or electrical means or a combination thereof.

[0026] Also provided herein are kits, systems and devices for carrying out the disclosed methods. The kits, systems and devices may be used to perform analyte analysis in an automated or a semi-automated manner and optionally may include disposable/consumable components that are utilized for analyte analysis. Automated and semi-automated devices may utilize microfluidics. Exemplary microfluidics include digital microfluidics (DMF), surface acoustic wave (SAW) microfluidics, droplet based microfluidic device, and the like. Exemplary microfluidics also include a fully integrated DMF and nanopore device, or a fully integrated SAW and nanopore device. In certain cases, the device for carrying out the disclosed methods may be a digital microfluidics device used in conjunction with a nanopore device. In other embodiments, the device for carrying out the disclosed methods may be an integrated digital microfluidics nanopore device. These devices may be single-use devices or may be reusable (used multiple times for analyte analysis). The digital microfluidic and nanopore devices described herein may provide miniaturized, low cost analyte analysis and may be fabricated using low cost technologies.

[0027] Also disclosed herein is an integrated digital microfluidics nanopore device comprising a microfluidics module and a nanopore module; the microfluidics module, comprising an array of electrodes spaced apart from a single electrode sized to overlap with at least a portion of the array of electrodes, where the array of electrodes and the single electrode transport at least one droplet of fluid to a transfer electrode in the array of electrodes, wherein the transfer electrode is positioned at an interface that operatively couples the microfluidics module and the nanopore module; the nanopore module comprising a first microchannel positioned on a first surface of a first substrate; a second microchannel positioned on a first surface of a second substrate; wherein the first surface of the first substrate is in contact with the first surface of the second substrate thereby enclosing the first microchannel and the second microchannel to provide a first capillary channel and a second capillary channel, respectively, wherein at least the first capillary channel extends to the interface between the microfluidics module and the nanopore module and is adjacent to the transfer electrode, and is positioned to receive a fluid droplet positioned on the transfer electrode; wherein the first capillary channel intersects with the second capillary channel, wherein a nanopore layer is positioned in between the first and second substrates at the location where the first and the second capillary channels intersect.

[0028] In certain embodiments, the array of electrodes may comprise a first and a second transfer electrodes each of which transfer electrodes are configured to position a fluid droplet over a surface of the transfer electrodes, wherein the first capillary channel extends to the interface between the microfluidics module and the nanopore module, is adjacent to the first

transfer electrode and is positioned to receive a fluid droplet located on the first transfer electrode and wherein the second capillary extends to the interface between the microfluidics module and the nanopore module, is adjacent to the second transfer electrode and is positioned to receive a fluid droplet located on the second transfer electrode.

[0029] In certain embodiments, the second capillary channel may not extend to the interface and may not be connected to the electrodes of the microfluidics module and may be connected to a vent or a reservoir on one or both ends of the second capillary. In certain cases, the second capillary is connected to a first reservoir at one end and a second reservoir at the other end.

[0030] In certain embodiments, the first reservoir and/or the second reservoir comprises a fluid to be positioned across from the first capillary channel at the intersection which fluid facilitates operation of the nanopore layer to drive current through a nanopore of the nanopore layer. In certain embodiments, the first capillary channel and/or the second capillary channel varies in cross sectional width across a length of the capillary such that the width decreases at the intersection compared to the width on either sides of the intersection.

[0031] In some embodiments, the first capillary comprises a first pair of electrodes and the second capillary comprises a second pair of electrodes, wherein the first pair of electrodes is positioned in the first capillary channel and flank the nanopore in the nanopore layer and wherein second pair of electrodes is positioned in the second capillary channel and flank the nanopore in the nanopore layer. The droplets may be droplets comprising a molecule to be detected and/or counted by transporting through the nanopore in the nanopore layer.

[0032] In certain embodiments, the fluid droplets have different compositions and are a first droplet and a second droplet, the first droplet comprising a molecule to be detected and/or counted by transporting across the nanopore layer through the nanopore and the second droplet comprising a conductive fluid lacking the molecule, where the conductive fluid facilitates transport of the molecule across the nanopore layer via the nanopore.

[0033] In certain embodiments, the first capillary channel comprises a first electrode positioned proximal to the nanopore layer and the second capillary channel comprising a second electrode positioned proximal to the nanopore layer, wherein each of the first and second electrodes are exposed in the capillary channels such that they are in contact with a fluid present in the capillary channels and wherein the first and second electrodes operate to drive current through a nanopore in the nanopore layer when a liquid is positioned across the nanopore layer in the first and second capillary channels.

[0034] In certain embodiments, the transfer electrode and the first capillary channel are on substantially the same plane, and wherein the fluid droplet is aligned with an opening of the first capillary channel.

[0035] In some embodiments, the transfer electrode is at a plane higher than the first capillary channel and wherein the device is configured with a vertical port for transferring the fluid droplet down to an opening of the first capillary channel.

[0036] In a particular embodiment, the first surface of the first substrate comprises a first area on which the array of electrodes is disposed and a second area in which the first microchannel is formed, wherein the array of electrodes is on a plane higher than the plane at which the first microchannel is formed.

[0037] In some embodiments, the second substrate comprises a notch at a side edge located at the interface, wherein the notch is aligned over the first capillary channel and provides a vertical port for transport of a droplet located at the transfer electrode to the opening of the first capillary channel.

[0038] In some cases, the single electrode extends over the transfer electrode and is in bi-planar configuration with the transfer electrode and wherein the single electrode and the transfer electrode operate to move the fluid droplet to the transfer electrode.

[0039] In other cases, the single electrode extends over the transfer electrodes and is in bi-planar configuration with the transfer electrodes and wherein the single electrode and the transfer electrodes operate to move the fluid droplets to the transfer electrodes.

[0040] In certain embodiments, the single electrode does not extend over the transfer electrode and is not in bi-planar configuration with the transfer electrode, wherein the fluid droplet is moved to the transfer electrode by using coplanar electrodes.

[0041] In certain embodiments, the single electrode does not extend over the transfer electrodes and is not in bi-planar configuration with the transfer electrodes, wherein the fluid droplets are moved to the transfer electrodes by using coplanar electrodes.

[0042] Thus, using the devices, kits, systems and methods as described herein, analyte present in a biological sample can be measured, and a patient can be diagnosed.

[0043] In another aspect, the present invention relates to a method of measuring or detecting an analyte present in a biological sample comprising (a) contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds to the analyte, (b) contacting the analyte with a second binding member, wherein the second binding member specifically binds to the analyte and wherein the second binding member comprises a cleavable tag attached thereto, (c) removing second binding member not bound to the analyte bound to the first binding member, (d) cleaving the tag attached to the second binding member bound to the analyte bound to the first binding member, (e) translocating the tag through one or more nanopores in a layer, and (f) assessing the tag translocating through the layer, wherein measuring the number of tags translocating through the layer measures the amount of analyte present in the sample, or wherein detecting tags translocating through the layer detects that the analyte is present in the sample.

[0044] In another aspect, the present invention relates to a method of measuring or detecting an analyte of interest present in a biological sample comprising (a) contacting the sample with a solid support, a first specific binding member, and a second specific binding member, wherein the solid support comprises an immobilization agent, the first specific binding member comprises a ligand for the immobilization agent and the first specific binding member specifically binds the analyte of interest, the second specific binding member comprises a cleavable tag, and the second specific binding member specifically binds the analyte of interest, wherein a solid support/first specific binding member/analyte of interest/second specific binding member complex is formed, (b) removing second specific binding member not bound to the solid support/first specific binding member/analyte/second specific binding member complex, (c) cleaving the tag attached to the labeled analyte bound to the second specific binding member in the solid support/first specific binding member/analyte of interest/second specific binding member complex, (d) translocating the tag through one or more nanopores in a layer, and (e) assessing the tags translocating through the layer, wherein measuring the number of tags translocating through the layer measures the amount of analyte present in the sample, or wherein detecting tags translocating through the layer detects that the analyte is present in the sample.

[0045] In another aspect, the present invention relates to a method of measuring or detecting an analyte present in a biological sample comprising (a) contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds to the analyte, (b) contacting the analyte with a second binding member, wherein the second binding member specifically binds to the analyte and wherein the second binding member comprises an aptamer, (c) removing aptamer not bound to the analyte bound to the solid substrate, (d) dissociating the aptamer bound to the analyte, (e) translocating the dissociated aptamer through one or more nanopores in a layer, and (f) assessing the aptamer translocating through the layer, wherein measuring the number of aptamers translocating through the layer measures the amount of analyte present in the sample, or wherein detecting aptamers translocating through the layer detects that the analyte is present in a the sample.

[0046] In one aspect, the present invention relates to an integrated digital microfluidics nanopore device comprising: a first substrate, comprising an array of electrodes; a second substrate spaced apart from the first substrate; and a nanopore layer disposed between the first and second substrates, wherein the array of electrodes are configured to position the droplet across the nanopore layer such that the droplet is split by the nanopore layer into a first portion and a second portion, wherein at least two electrodes of the array of electrodes are positioned across the nanopore layer, where the two electrodes form an anode and a cathode and operate to drive current through a nanopore in the nanopore layer when a liquid droplet is positioned across the nanopore layer.

[0047] In yet another aspect, the present invention relates to an integrated digital

microfluidics nanopore device comprising: a first substrate, comprising an array of electrodes; a second substrate spaced apart from the first substrate; and a nanopore layer disposed between the first and second substrates, wherein the array of electrodes are configured to position a droplet across the nanopore layer such that the nanopore layer splits the droplet into a first portion and a second portion, wherein at least one electrode of the array of electrodes is in contact with the first portion of a droplet positioned across the nanopore layer and the electrode in the second substrate is positioned to contact the second portion of the droplet positioned across the nanopore layer, where the two electrodes form an anode and a cathode and operate to drive current through a nanopore in the nanopore layer when a liquid droplet is positioned across the nanopore layer.

[0048] In yet another aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample comprising: (a) contacting the sample with a binding member, wherein the binding member is immobilized on a solid support and wherein the binding member specifically binds to the analyte, (b) contacting the sample with a labeled analyte, wherein the labeled analyte is labeled with a cleavable tag, (c) removing labeled analyte not bound to the binding member, (d) cleaving the tag attached to the labeled analyte bound to the binding member, (e) translocating the tag through one or more nanopores in a layer, and (f) assessing the tags translocating through the layer, wherein measuring the number of tags translocating through the layer measures the amount of analyte present in the sample, or wherein detecting tags translocating through the layer detects that the analyte is present in the sample.

[0049] In yet another aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample, the method comprising: (a) contacting the sample with a binding member, wherein binding member is immobilized on a solid support and wherein binding member specifically binds to the analyte, (b) contacting the sample with a labeled analyte, wherein the labeled analyte comprises an aptamer; (c) removing labeled analyte not bound to the binding member, (d) dissociating the aptamer bound to the labeled analyte and translocating the dissociated aptamer through one or more nanopores in a layer, and (e) assessing the aptamer translocating through the layer, wherein measuring the number of aptamers translocating through the layer measures the amount of analyte present in the sample, or wherein detecting aptamers translocating through the layer detects that the analyte is present in the sample.

[0050] In yet another aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample comprising: (a) contacting the sample with a binding member, wherein the binding member specifically binds to the analyte, and the binding member is labeled with a cleavable tag, (b) contacting the sample with a immobilized analyte, wherein the immobilized analyte is immobilized on a solid support, (c) removing binding member not bound to the immobilized analyte, (d) cleaving the tag attached to the binding member bound to the immobilized analyte, (e) translocating the tag through one or more nanopores in a layer, and (f) assessing the tag translocating through the layer, wherein measuring the number of tags translocating through the layer measures the amount of analyte present in the sample, or wherein detecting tags translocating through the layer detects that the analyte is present in the sample.

[0051] In yet another aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample comprising: (a) contacting the sample with a binding member, wherein the binding member specifically binds to the analyte, and the binding member comprises an aptamer, (b) contacting the sample with a immobilized analyte, wherein the immobilized analyte is immobilized on a solid support, (c) removing binding member not bound to the immobilized analyte, (d) dissociating the aptamer bound to the binding member bound to the immobilized analyte and translocating the dissociated aptamer through one or more nanopores in a layer, and (e) assessing the aptamer translocating through the layer, wherein measuring the number of aptamers translocating through the layer measures the amount of analyte present in the sample, or wherein detecting aptamers translocating through the layer detects that the analyte is present in the sample.

[0052] In yet another aspect, the present invention relates to an integrated digital

microfluidics nanopore device comprising a microfluidics module and a nanopore module; the microfluidics module comprising an array of electrodes, wherein the array of electrodes transports at least one droplet of fluid to a first transfer position in the array of electrodes, wherein the first transfer position is at an interface between the microfluidics module and the nanopore module; the nanopore module comprising: a first capillary channel; and a second capillary channel; wherein at least the first capillary channel extends to the interface and is adjacent to the first transfer position, and is positioned to receive a fluid droplet positioned at the first transfer position; wherein the first capillary channel intersects with the second capillary channel, wherein a nanopore layer is positioned in between the first and second capillary channels at the location where the first and the second capillary channels intersect.

[0053] In yet another aspect, the present invention relates to a method for measuring an analyte present in a biological sample comprising: (a) contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds to the analyte, (b) contacting the analyte with a second binding member, wherein the second binding member specifically binds to the analyte and wherein the second binding member comprises a cleavable tag attached thereto, (c) removing second binding member not bound to the analyte bound to the first binding member, (d) cleaving the tag attached to the second binding member bound to the analyte bound to the first binding member, (e) translocating the tag through one or more nanopores in a layer, and (f) assessing the tag translocating through the layer, wherein each tag translocating through the layer is a translocation event, wherein measuring the number of translocation events measures the amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined by: i) counting the number of translocation events during a set period of time and

correlating the number of translocation events to a control; ii) measuring the amount of time for a set number of translocation events to occur and correlating to a control; or iii) measuring the average time between translocation events to occur and correlating to a control, wherein the control is a reference standard comprising a calibration curve, standard addition, or digital polymerase chain reaction, wherein the standard curve in subsection i) is determined by measuring the number of translocation events for control concentrations of analyte during a set period of time; wherein the standard curve in subsection ii) is determined by measuring the time it takes for a set number of translocation events to occur for control concentrations of analyte; and wherein the standard curve in subsection iii) is determined by measuring the average time between translocation events to occur for control concentrations of analyte.

[0054] In yet another aspect, the present invention relates to a method for measuring an analyte present in a biological sample comprising: (a) contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds to the analyte, (b) contacting the analyte with a second binding member, wherein the second binding member specifically binds to the analyte and wherein the second binding member comprises an aptamer, (c) removing aptamer not bound to the analyte bound to the solid substrate, (d) dissociating the aptamer bound to the analyte, and (e) translocating the dissociated aptamer through one or more nanopores in a layer; and (f) assessing the aptamer translocating through the layer, wherein each aptamer translocating through the layer is a translocation event, wherein measuring the number of translocation events measures the amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined by: i) counting the number of translocation events during a set period of time and correlating the number of translocation events to a control; ii) measuring the amount of time for a set number of translocation events to occur and correlating to a control; or iii) measuring the average time between translocation events to occur and correlating to a control, wherein the control is a reference standard comprising a calibration curve, standard addition, or digital polymerase chain reaction, wherein the standard curve in subsection i) is determined by measuring the number of translocation events for control concentrations of analyte during a set period of time; wherein the standard curve in subsection ii) is determined by measuring the time it takes for a set number of translocation events to occur for control concentrations of analyte; and wherein the standard curve in subsection iii) is determined by measuring the average time between translocation events to occur for control concentrations of analyte.

[0055] In yet another aspect, the present invention relates to method for measuring an analyte present in a biological sample comprising: (a) contacting the sample with a binding member, wherein the binding member is immobilized on a solid support and wherein the binding member specifically binds to the analyte, (b) contacting the sample with a labeled analyte, wherein the labeled analyte is labeled with a cleavable tag, (c) removing labeled analyte not bound to the binding member, (d) cleaving the tag attached to the labeled analyte bound to the binding member, (e) translocating the tag through one or more nanopores in a layer, and (f) assessing the tags translocating through the layer, wherein each tag translocating through the layer is a translocation event, wherein measuring the number of translocation events measures the amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined by: i) counting the number of translocation events during a set period of time and correlating the number of translocation events to a control; ii) measuring the amount of time for a set number of translocation events to occur and correlating to a control; or iii) measuring the average time between translocation events to occur and correlating to a control, wherein the control is a reference standard comprising a calibration curve, standard addition, or digital polymerase chain reaction, wherein the standard curve in subsection i) is determined by measuring the number of translocation events for control concentrations of analyte during a set period of time; wherein the standard curve in subsection ii) is determined by measuring the time it takes for a set number of translocation events to occur for control concentrations of analyte; and wherein the standard curve in subsection iii) is determined by measuring the average time between translocation events to occur for control concentrations of analyte.

[0056] In another aspect, the present invention relates to a method for measuring an analyte present in a biological sample comprising: (a) contacting the sample with a binding member, wherein binding member is immobilized on a solid support and wherein binding member specifically binds to the analyte, (b) contacting the sample with a labeled analyte, wherein the labeled analyte comprises an aptamer, (c) removing labeled analyte not bound to the binding member, (d) dissociating the aptamer bound to the labeled analyte and translocating the dissociated aptamer through one or more nanopores in a layer, and (e) assessing the aptamer translocating through the layer, wherein each aptamer translocating through the layer is a translocation event, wherein measuring the number of translocation events measures the amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined by: i) counting the number of translocation events during a set period of time and correlating the number of translocation events to a control; ii) measuring the amount of time for a set number of translocation events to occur and correlating to a control; or iii) measuring the average time between translocation events to occur and correlating to a control, wherein the control is a reference standard comprising a calibration curve, standard addition, or digital polymerase chain reaction, wherein the standard curve in subsection i) is determined by measuring the number of translocation events for control concentrations of analyte during a set period of time; wherein the standard curve in subsection ii) is determined by measuring the time it takes for a set number of translocation events to occur for control concentrations of analyte; and wherein the standard curve in subsection iii) is determined by measuring the average time between translocation events to occur for control concentrations of analyte.

[0057] In yet another aspect, the present invention relates to a method for measuring an analyte present in a biological sample comprising: (a) contacting the sample with a binding member, wherein the binding member specifically binds to the analyte, and the binding member is labeled with a cleavable tag, (b) contacting the sample with a immobilized analyte, wherein the immobilized analyte is immobilized on a solid support, (c) removing binding member not bound to the immobilized analyte, (d) cleaving the tag attached to the binding member bound to the immobilized analyte, (e) translocating the tag through one or more nanopores in a layer, and (f) assessing the tag translocating through the layer, wherein each tag translocating through the layer is a translocation event, wherein measuring the number of translocation events measures the amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined by: i) counting the number of translocation events during a set period of time and correlating the number of translocation events to a control; ii) measuring the amount of time for a set number of translocation events to occur and correlating to a control; or iii) measuring the average time between translocation events to occur and correlating to a control, wherein the control is a reference standard comprising a calibration curve, standard addition, or digital polymerase chain reaction, wherein the standard curve in subsection i) is determined by measuring the number of translocation events for control concentrations of analyte during a set period of time; wherein the standard curve in subsection ii) is determined by measuring the time it takes for a set number of translocation events to occur for control concentrations of analyte; and wherein the standard curve in subsection iii) is determined by measuring the average time between translocation events to occur for control concentrations of analyte.

[0058] In yet another aspect, the present invention relates to a method for measuring an analyte present in a biological sample comprising: (a) contacting the sample with a binding member, wherein the binding member specifically binds to the analyte, and the binding member comprises an aptamer, (b) contacting the sample with a immobilized analyte, wherein the immobilized analyte is immobilized on a solid support, (c) removing binding member not bound to the immobilized analyte, (d) dissociating the aptamer bound to the binding member bound to the immobilized analyte and translocating the dissociated aptamer through one or more nanopores in a layer, and (e) assessing the aptamer translocating through the layer, wherein each aptamer translocating through the layer is a translocation event, wherein measuring the number of translocation events measures the amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined by: i) counting the number of

translocation events during a set period of time and correlating the number of translocation events to a control; ii) measuring the amount of time for a set number of translocation events to occur and correlating to a control; or iii) measuring the average time between translocation events to occur and correlating to a control, wherein the control is a reference standard comprising a calibration curve, standard addition, or digital polymerase chain reaction, wherein the standard curve in subsection i) is determined by measuring the number of translocation events for control concentrations of analyte during a set period of time; wherein the standard curve in subsection ii) is determined by measuring the time it takes for a set number of translocation events to occur for control concentrations of analyte; and wherein the standard curve in subsection iii) is determined by measuring the average time between translocation events to occur for control concentrations of analyte.

[0059] In yet another aspect, the present invention relates to a method for measuring or detecting an analyte present in a biological sample comprising: (a) contacting the sample with a binding member, wherein the binding member is immobilized on a solid support, the binding member comprises a cleavable tag attached thereto, and the binding member specifically binds to the analyte, (b) removing binding member not bound to the analyte, (c) cleaving the tag attached to the binding member bound to the analyte, (d) translocating the tag through one or more nanopores in a layer, and (e) assessing the tag translocating through the layer, wherein each tag translocating through the layer is a translocation event, wherein measuring the number of translocation events measures the amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined by: i) counting the number of

translocation events during a set period of time and correlating the number of translocation events to a control; ii) measuring the amount of time for a set number of translocation events to occur and correlating to a control; or iii) measuring the average time between translocation events to occur and correlating to a control, wherein the control is a reference comprising a calibration curve, standard addition, or digital polymerase chain reaction.

[0060] In yet another aspect, the present invention relates to an integrated digital

microfluidics nanopore-enabled device comprising: a microfluidics module and a nanopore-enabled module; the microfluidics module, comprising an array of electrodes spaced apart from a single electrode sized to overlap with at least a portion of the array of electrodes, where the array of electrodes and the single electrode transport at least one droplet of fluid to a transfer electrode in the array of electrodes, wherein the transfer electrode is positioned at an interface between the microfluidics module and the nanopore-enabled module; the nanopore-enabled module comprising: a first microchannel positioned on a first surface of a first substrate; a second microchannel positioned on a first surface of a second substrate; wherein the first surface of the first substrate is in contact with the first surface of the second substrate thereby enclosing the first microchannel and the second microchannel to provide a first capillary channel and a second capillary channel, respectively, wherein at least the first capillary channel extends to the interface between the microfluidics module and the nanopore-enabled module and is adjacent to the transfer electrode, and is positioned to receive a fluid droplet positioned on the transfer electrode; wherein the first capillary channel intersects with the second capillary channel, wherein a layer is positioned in between the first and second substrates at the location where the first and the second capillary channels intersect, wherein the layer is devoid of a nanopore and separates an ionic liquid present in the first and second capillary channels, wherein the first and second capillary channels are in electrical connection with electrodes for driving a voltage from the first to the second capillary channel or vice versa for creating a nanopore in the layer at the intersection of the first and second capillary channels.

[0061] In yet another aspect, the present invention relates to a method for generating a nanopore in an integrated digital microfluidics nanopore-enabled device, the method

comprising: providing an integrated digital microfluidics nanopore-enabled device as previously described herein; applying a voltage in the first and second capillary channels to drive current through the layer; measuring conductance across the layer; terminating application of voltage upon detection of a conductance indicative of generation of a nanopore in the layer.

[0062] In yet another aspect, the present invention relates to an integrated digital

microfluidics nanopore device comprising: a first substrate comprising an array of electrodes; a second substrate spaced apart from the first substrate; an opening in the first or second substrate in fluid communication with a nanopore layer comprising a nanopore; and a pair of electrodes configured to apply an electric field through the nanopore, wherein the array of electrodes are configured to transport at least one droplet of fluid to the opening.

[0063] In yet another aspect, the present invention relates to a pair of integrated digital microfluidics nanopore devices comprising: a first integrated digital microfluidics nanopore device described previously herein, wherein the single electrode is a first single electrode, and the capillary channel is a first capillary channel; and a second integrated digital microfluidics nanopore device comprising: a third substrate, comprising a fifth side and a sixth side opposite the fifth side, wherein the fifth side comprises an array of electrodes; a fourth substrate spaced apart from the third substrate, wherein the fourth substrate comprises a seventh side facing the fifth side of the third substrate and a eight side opposite the seventh side, wherein the seventh side comprises a second single electrode and wherein the nanopore layer is disposed on the eight side, wherein the fourth substrate comprises a second capillary channel extending from the seventh side to the eight side of the fourth substrate, wherein the nanopore layer is positioned over an opening of the capillary channel, wherein the nanopore layer is interposed between the second substrate and the fourth substrate such that the nanopore provides an electroosmotic conduit between the first capillary channel and the second capillary channel, wherein the pair of detection electrodes comprises a second detection electrode that is the second single electrode.

[0064] In yet another aspect, the present invention relates to an integrated digital

microfluidics nanopore-enabled device comprising: a first substrate, comprising a first side and a second side opposite the first side, wherein the first side comprises an array of electrodes; a second substrate spaced apart from the first substrate, wherein the second substrate comprises a third side facing the first side of the first substrate and a fourth side opposite the third side; a nanopore-enabled layer devoid of a nanopore and disposed on an external side of the device, wherein the external side is selected from the second side or the fourth side, wherein one of the first or second substrates comprising the external side comprises a capillary channel extending from the first side to the second side of the first substrate, or the third side to the fourth side of the second substrate, wherein the nanopore-enabled layer is positioned over an opening of the capillary channel; and a pair of electrodes configured to apply an electric field across the nanopore-enabled layer, wherein the array of electrodes are configured to transport at least one droplet of fluid to the capillary channel.

[0065] In yet another aspect, the present invention relates to a method for generating a nanopore in an integrated digital microfluidics nanopore-enabled device comprising: providing an integrated digital microfluidics nanopore-enabled device described previously herein;

submerging both sides of the nanopore-enabled layer in an ionic liquid such that the ionic liquid on each side of the layer is in electrical contact with either one of the pair of detection electrodes; applying a voltage between the pair of detection electrodes to drive current through the layer; measuring conductance across the layer; terminating application of voltage upon detection of a conductance indicative of generation of a nanopore in the layer.

[0066] In another aspect, the present invention relates to a composition comprising a binding member, a tag and a spacer.

[0067] In yet another aspect, the present invention relates to an integrated digital

microfluidics nanopore device comprising: a first substrate, comprising an array of electrodes; a second substrate spaced apart from the first substrate; and a nanopore layer having a first surface and a second surface disposed between the first and second substrates, wherein the array of electrodes are configured to position a first droplet at the first surface of the nanopore layer, wherein at least two electrodes of the array of electrodes are positioned across the nanopore layer, where the two electrodes form an anode and a cathode and operate to drive current through a nanopore in the nanopore layer when a liquid droplet is at the first surface of the nanopore layer.

[0068] In yet another aspect, the present invention relates to an integrated digital

microfluidics nanopore device comprising a microfluidics module and a nanopore module; the microfluidics module comprising an array of electrodes, where the array of electrodes transport at least one droplet of fluid to a transfer position in the array of electrodes, wherein the transfer position is at an interface between the microfluidics module and the nanopore module; the nanopore module comprising: a first capillary channel extending from the transfer position to a nanopore layer.

[0069] In yet another aspect, the present invention relates to an integrated digital

microfluidics nanopore device comprising: a first substrate, comprising an array of electrodes; a second substrate spaced apart from the first substrate; a first nanopore layer having one or more nanopores therein; a second nanopore layer having one or more nanopores therein; and at least

two electrodes for creating an electric field to drive tags through a nanopore in the first and second nanopore layers.

[0070] In yet another aspect, the present invention relates to a kit comprising any of the aforementioned devices for use in any of the aforementioned methods.

[0071] In yet another aspect, the present invention relates to a method of using any of the aforementioned devices for measuring or detecting an analyte present in a biological sample or for diagnosing a patient or screening a blood supply.

[0072] In yet another aspect, the present invention relates to a method for measuring or detecting an analyte of interest present in a biological sample comprising (a) contacting the sample with a solid support, a binding member, and a labeled analyte that is labeled with a cleavable tag, wherein the solid support comprises an immobilization agent, the binding member comprises a ligand for the immobilization agent, and the binding member specifically binds the analyte of interest to form either a solid support/binding member/analyte of interest complex or a solid support/binding member/labeled analyte complex; (b) removing labeled analyte not bound to the binding member in the solid support/binding member/labeled analyte complex; (c) cleaving the tag attached to the labeled analyte bound to the binding member in the solid support/binding member/labeled analyte complex; (d) translocating the tag through one or more nanopores in a layer; and (e) assessing the tags translocating through the layer, wherein measuring the number of tags translocating through the layer measures the amount of analyte present in the sample, or wherein detecting tags translocating through the layer detects that the analyte is present in the sample.

[0073] In yet another aspect, the present invention relates to a method for measuring or detecting an analyte of interest present in a biological sample comprising (a) contacting the sample with a solid support, a binding member, and exogenous analyte, wherein the solid support comprises an immobilization agent, the exogenous analyte comprises a ligand for the immobilization agent and binds the solid support so as to form a solid support/immobilized analyte complex, and the binding member comprises a cleavable tag and specifically binds the analyte of interest to form either a solid support/analyte of interest/binding member complex or a solid support /immobilized analyte/binding member complex; (b) removing binding member not bound in either the solid support/immobilized analyte/ binding member complex or the solid support/analyte of interest/binding member complex; (c) cleaving the tag attached to the binding member in the solid support/immobilized analyte/ binding member complex; (d) translocating the tag through one or more nanopores in a layer; and (e) assessing the tags translocating through the layer, wherein measuring the number of tags translocating through the layer measures the amount of analyte present in the sample, or wherein detecting tags translocating through the layer detects that the analyte is present in the sample.

[0074] In yet another aspect, the present invention relates to a method for measuring or detecting an analyte of interest present in a biological sample, the method comprising (a) contacting the sample with a solid support, a binding member, and a labeled analyte that is labeled with a cleavable tag, wherein the solid support comprises an immobilization agent, the binding member comprises a ligand for the immobilization agent, and the binding member specifically binds the analyte of interest so as to form either a solid support/binding

member/analyte of interest complex or a solid support/binding member/labeled analyte complex; (b) removing labeled analyte not bound to the binding member in the solid support/binding member/labeled analyte complex; (c) cleaving the tag attached to the labeled analyte bound to the binding member in the solid support/binding member/labeled analyte complex; (d) translocating the tag through one or more nanopores in a layer; and (e) assessing the tags translocating through the layer, wherein measuring the number of tags translocating through the layer measures the amount of analyte present in the sample, or wherein detecting tags translocating through the layer detects that the analyte is present in the sample.

[0075] In yet another aspect, the present invention relates to a method for measuring or detecting an analyte of interest present in a biological sample, the method comprising (a) contacting the sample with a solid support, a binding member, and exogenous analyte, wherein the solid support comprises an immobilization agent, the exogenous analyte comprises a ligand for the immobilization agent and binds the solid support so as to form a solid

support/immobilized analyte complex, and the binding member comprises a cleavable tag and specifically binds the analyte of interest so as to form either a solid support/analyte of interest/binding member complex or a solid support /immobilized analyte/binding member complex; (b) removing binding member not bound in either the solid support/immobilized analyte/ binding member complex or the solid support/analyte of interest/binding member complex; (c) cleaving the tag attached to the binding member in the solid support/immobilized analyte/binding member complex; (d) translocating the tag through one or more nanopores in a layer; and (e) assessing the tags translocating through the layer, wherein measuring the number of tags translocating through the layer measures the amount of analyte present in the sample, or wherein detecting tags translocating through the layer detects that the analyte is present in the sample.

[0076] In yet another aspect, the present invention relates to a method for measuring or detecting an analyte of interest present in a biological sample, the method comprising (a) contacting the sample with a solid support, a binding member, and a labeled analyte that is labeled with an aptamer, wherein the solid support comprises an immobilization agent, the binding member comprises a ligand for the immobilization agent, and the binding member specifically binds the analyte of interest so as to form either a solid support/binding

member/analyte of interest complex or a solid support/binding member/labeled analyte complex; (b) removing labeled analyte not bound to the binding member in the solid support/binding member/labeled analyte complex; (c) dissociating the aptamer attached to the labeled analyte bound to the binding member in the solid support/binding member/labeled analyte complex; (d) translocating the dissociated aptamer through one or more nanopores in a layer; and (e) assessing the aptamer translocating through the layer, wherein measuring the number of aptamers translocating through the layer measures the amount of analyte present in the sample, or wherein detecting aptamers translocating through the layer detects that the analyte is present in the sample.

[0077] In yet another aspect, the present invention relates to a method for measuring or detecting an analyte of interest present in a biological sample, the method comprising (a) contacting the sample with a solid support, a binding member, and exogenous analyte, wherein the solid support comprises an immobilization agent, the exogenous analyte comprises a ligand for the immobilization agent and binds the solid support so as to form a solid

support/immobilized analyte complex, and the binding member comprises an aptamer and specifically binds the analyte of interest so as to form either a solid support/analyte of interest/binding member complex or a solid support /immobilized analyte/binding member complex; (b) removing binding member not bound in either the solid support/immobilized analyte/ binding member complex or the solid support/analyte of interest/binding member complex; (c) dissociating the aptamer bound to the binding member in the solid

support/immobilized analyte/binding member complex; (d) translocating the tag through one or more nanopores in a layer; and (e) assessing the tags translocating through the layer, wherein measuring the number of tags translocating through the layer measures the amount of analyte present in the sample, or wherein detecting tags translocating through the layer detects that the analyte is present in the sample.

[0078] Also provided herein are instruments that are programmed to operate a cartridge that includes an array of electrodes for actuating a droplet and further includes an electrochemical species sensing region. The instrument may be used to analyse a sample in a cartridge that includes an array of electrodes for actuating a droplet and further includes a nanopore layer for detecting translocation of a molecule through nanopore. An instrument configured to operate a first cartridge that includes an array of electrodes for actuating a droplet and further includes an electrochemical species sensing region and a second cartridge that includes an array of electrodes for actuating a droplet and further includes a nanopore layer for detecting

translocation of a molecule through nanopore is disclosed. An instrument configured to operate a cartridge that includes an array of electrodes for actuating a droplet, an electrochemical species sensing region, and a nanopore layer for detecting translocation of a molecule through nanopore is disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The details of the subject matter set forth herein, both as to its structure and operation, may be apparent by study of the accompanying figures, in which like reference numerals refer to like parts. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the subject matter. Moreover, all illustrations are intended to convey concepts, where relative sizes, shapes and other detailed attributes may be illustrated schematically rather than literally or precisely.

[0080] Fig.1A and Fig.1B depict a microfluidics device 10 used in conjunction with a nanopore device 15.

[0081] Fig.2A and Fig.2B depict a schematic of a reversibly integrated device having a microfluidics module 20 combined with a nanopore module 30 via a channel 40. Figs.2C-2L depict schematics of exemplary integrated devices in which a microfluidics module is fluidically connected to a nanopore module. The nanopore module includes a nanopore in a layer physically separating two microfluidic channels at a location where the two microfluidic channels intersect.

[0082] Fig.3 illustrates an exemplary integrated device which includes a microfluidics module 300 and a nanopore module 325.

[0083] Fig.4 provides an integrated device 400 in which the digital microfluidics modules includes a built-in nanopore module.

[0084] Fig.5A shows a top view of an integrated device. Fig.5B shows a side view of the integrated device of Fig.5A.

[0085] Fig.6 depicts an exemplary device and method of the present disclosure.

[0086] Fig.7 depicts an exemplary device and method of the present disclosure.

[0087] Fig.8 depicts a side view of an exemplary integrated device of the present disclosure.

[0088] Fig.9 depicts an exemplary system of the present disclosure.

[0089] Fig.10 depicts a schematic of a fabrication process of a low-cost DMF chip.

[0090] Fig.11 depicts a single flexible DMF chip fabricated according to the schematic in Fig.10.

[0091] Fig.12 depicts actuation of droplets in a DMF chip, according to embodiments of the present disclosure.

[0092] Fig.13, A-E depict performance of an immunoassay in a DMF chip, according to embodiments of the present disclosure.

[0093] Figs.14A-14C depict fabrication and design of a nanopore module, according to embodiments of the present disclosure.

[0094] Fig.15A shows a plot of leakage current measured in real-time. Fig.15B depicts a current-voltage (I-V) curve for a nanopore.

[0095] Figs.16A-16C show filling of a capillary channel in an integrated DMF-nanopore module device, according to embodiments of the present disclosure.

[0096] Fig.17 shows a schematic diagram for droplet transfer between modules in an integrated DMF-nanopore module device, according to embodiments of the present disclosure.

[0097] Fig.18 shows a schematic diagram of a nanopore module design, according to embodiments of the present disclosure.

[0098] Fig.19 shows a schematic diagram of an integrated DMF-nanopore module device adapted to perform droplet transfer between the modules by passive transport, according to embodiments of the present disclosure.

[0099] Fig.20 shows a schematic diagram of an integrated DMF-nanopore module device adapted to perform droplet transfer between the modules by passive transport, according to embodiments of the present disclosure.

[00100] Fig.21 is a schematic diagram of a silicon microfluidic device containing silicon microchannels that allow passive movement of a liquid droplet by passive transport, according to embodiments of the present disclosure.

[00101] Fig.22 is an image of a silicon microchannel of a silicon microfluidic device that allows passive movement of a liquid droplet by passive transport, according to embodiments of the present disclosure.

[00102] Fig.23A and Fig.23B show a schematic of a fabrication method for an integrated nanopore sensor, according to embodiments of the present disclosure.

[00103] Figs.24A-24C display the scatter plot (level duration versus level of blockage) for plots obtained using showing translocation events through: (Fig.24A) nanopores comprised of regular double stranded DNA (“dsDNA”); (Fig.24B) nanopores comprised of DBCO-modified dsDNA; and (Fig.24C) nanopores comprised of dsDNA stars.

[00104] Fig.25 shows a schematic of the thiol-mediated chemical cleavage.

[00105] Fig.26A and Fig.26B show a schematic of photocleavage experiments performed on magnetic microparticles.

[00106] Fig.27 shows a schematic of the reagent placement on the DMF chip.

[00107] Fig.28 displays a bar chart of sample versus nanopore flux (DMF cleavage) in sec-1.

[00108] Fig.29 displays the means by which a threshold for digital signal counting is determined.

[00109] Figs.30A-30C show current blockages over different time periods for three standards of 94 nM (Fig.30A), 182 nM (Fig.30B), and 266 nM (Fig.30C).

[00110] Fig.31 shows a dose-response curve of number of events over a fixed amount of time (5 min).

[00111] Fig.32 shows a dose-response curve of time required for fixed number of events. [00112] Fig.33 shows a dose-response curve of events per unit time.

[00113] Fig.34 shows a dose-response curve of events per unit time using Seq31-SS-biotin.

[00114] Fig.35 shows a schematic diagram of a nanopore chamber design in a silicon nanopore module, according to embodiments of the present disclosure.

[00115] Fig.36 shows a table listing the physical parameters used for COMSOL electrical field simulations in a nanopore chamber of a silicon nanopore module, according to

embodiments of the present disclosure.

[00116] Fig.37 is a collection of images showing simulation results for counter ion concentration gradients near a nanopore in a silicon nanopore module, according to

embodiments of the present disclosure.

[00117] Fig.38 is a graph showing the effects of the diameter of a SiO2 via made over a nanopore membrane with a nanopore on the electroosmotic flow through the nanopore, according to embodiments of the present disclosure.

[00118] Fig.39 is a graph showing the effects of the diameter of a SiO2 via made over a nanopore membrane with a nanopore on the conductance through the nanopore, according to embodiments of the present disclosure.

[00119] Fig.40 shows a schematic diagram of an integrated DMF-nanopore module device with the nanopore module positioned on one side of the DMF module, according to

embodiments of the present disclosure.

[00120] Fig.41 is a collection of images showing movement of liquid from a DMF module through a hole in a DMF module substrate by capillary force, according to embodiments of the present disclosure.

[00121] Fig.42 is a collection of images showing an integrated DMF-nanopore module device with the nanopore module positioned on one side of the DMF module and electrodes configured for nanopore fabrication, according to embodiments of the present disclosure.

[00122] Fig.43 is a schematic diagram of an integrated DMF-nanopore module device with the nanopore module positioned on one side of the DMF module, according to embodiments of the present disclosure.

[00123] Fig.44 is a schematic diagram of an integrated DMF-nanopore module device with the nanopore module positioned between two DMF modules, according to embodiments of the present disclosure.

[00124] Fig.45 is a graph showing fabrication of a nanopore in a nanopore membrane (a transmission electron microscope (TEM) window) by applying a voltage across the nanopore membrane, and as evidenced by dielectric breakdown, according to embodiments of the present disclosure.

[00125] Fig.46A and Fig.46B are a collection of graphs showing current-voltage (I-V) curves of a nanopore formed in a membrane, before and after a conditioning process, according to embodiments of the present disclosure.

[00126] Fig.47 shows a scatter plot of the averages of ratios plotted between counting label average diameter and nanopore size to the SNR (signal to noise ratio).

[00127] Fig.48, A-F provides a schematic of an analyte detection chip according to one embodiment.

[00128] Fig.49, A-C provides a schematic of an analyte detection chip according to another embodiment.

[00129] Fig.50 provides a schematic of an analyte detection chip according to one

embodiment.

[00130] Figs.51A and 51B illustrate side views of an exemplary analyte detection chip.

[00131] Figs.52A-52E illustrate cartridges comprising DMF electrodes and optical detection chamber.

[00132] Fig.53 illustrates a schematic of a top view of an analyte detection chip according to another embodiment.

[00133] Fig.54 illustrates a schematic of an alternate exemplary analyte detection chip.

[00134] Fig.55 provides a schematic of an exemplary hematology chip.

[00135] Figs.56 and 57 illustrate alternate embodiments of DMF chip with mutiple detection regions.

[00136] Fig.58, 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. Fig.58D and 58E illustrate cartridge adapters that allow insertion of different types of cartridges into the same slot.

[00137] Fig.59, A and B depict embodiments of a cartridge (A) and an analyte detection device (B) that is compatible with the cartridge.

[00138] Figs.60A and 60B illustrate exemplary analyte detection systems with a plurality of instruments for conducting a plurality of assays.

DETAILED DESCRIPTION

[00139] 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.

1. Definitions

[00140] 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.

[00141] “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.

[00142] 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.

[00143] “Affinity” and“binding affinity” as used interchangeably herein refer to the tendency or strength of binding of the binding member to the analyte. For example, the binding affinity may be represented by the equilibrium dissociation constant (KD), the dissociation rate (kd), or the association rate (ka).

[00144] “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.

[00145] “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.

[00146] “Bead” and“particle” are used herein interchangeably and refer to a substantially spherical solid support.

[00147] “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.

[00148] “Control” as used herein refers to a reference standard for an analyte such as is known or accepted in the art, or determined empirically using acceptable means such as are commonly employed. A“reference standard” is a standardized substance which is used as a measurement base for a similar substance. For example, there are documented reference standards published in the U.S. Pharmacopeial Convention (USP–NF), Food Chemicals Codex, and Dietary Supplements Compendium (all of which are available at http://www.usp.org), and other well-known sources. Methods for standardizing references are described in the literature.

Also well-known are means for quantifying the amounts of analyte present by use of a calibration curve for analyte or by comparison to an alternate reference standard. A standard curve can be generated using serial dilutions or solutions of known concentrations of analyte, by mass spectroscopy, gravimetric methods, and by other techniques known in the art. Alternate reference standards that have been described in the literature include standard addition (also known as the method of standard addition), or digital polymerase chain reaction.

[00149] “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.

[00150] Digital microfluidics operates on discrete volumes of fluids that can be manipulated by binary electrical signals. By using discrete unit-volume droplets, a microfluidic operation may be defined as a set of repeated basic operations, i.e., moving one unit of fluid over one unit of distance. Droplets may be formed using surface tension properties of the liquid. Actuation of a droplet is based on the presence of electrostatic forces generated by electrodes placed beneath the bottom surface on which the droplet is located. Different types of electrostatic forces can be used to control the shape and motion of the droplets. One technique that can be used to create the foregoing electrostatic forces is based on dielectrophoresis which relies on the difference of electrical permittivities between the droplet and surrounding medium and may utilize high-frequency AC electric fields. Another technique that can be used to create the foregoing electrostatic forces is based on electrowetting, which relies on the dependence of surface tension between a liquid droplet present on a surface and the surface on the electric field applied to the surface.

[00151] “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.

[00152] “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.

[00153] “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.

[00154] “Label” or“detectable label” as used interchangeably herein refers to a tag attached to a specific binding member or analyte by a cleavable linker.

[00155] “Nanoparticle(s)” and“nanobead(s)” are used interchangeably herein and refer to a nanobead or nanoparticle sized to translocate through or across a nanopore used for counting the number of nanobeads/nanoparticles traversing through it.

[00156] "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.

[00157] “Nucleoside” refers to a compound consisting of a purine, deazapurine, or pyrimidine nucleobase, e.g., adenine, guanine, cytosine, uracil, thymine, 7- deazaadenine, 7-deazaguanosine, that is linked to the anomeric carbon of a pentose sugar at the 1' position, such as a ribose, 2'-deoxyribose, or a 2',3'-di-deoxyribose.

[00158] “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.

[00159] “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.

What is claimed is:

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:

(a) a tag translocating through a pore of a nanopore layer in a cartridge; or (b) an analyte-specific binding member translocating through a pore of a nanopore layer 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 the electrical signal.

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

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

6. The instrument of claim 1, wherein the tag is cleaved from a second binding member that specifically binds to a complexcomprising a first binding member and the analyte; and wherein the analyte-specific binding member is an aptamer.

7. The instrument of any one claims 1-6, wherein the control unit controls duration of activation of the array of electrodes and the electric power applied to the array of electrodes.

8. The instrument of any one claims 1-6, wherein the control unit controls the sequence of activation and deactivation of array of 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 a detection region of a cartridge for interrogation by 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 comprises a detector for one or more of a colorimetric signal, a turbidometric signal, or a fluorescent signal.

16. The instrument of any one of claims 14-15, wherein the optical detection unit comprises an imaging system for imaging the droplet.

17. The instrument of any one of claims 1-16, wherein the instrument comprises a processor which executes a program with instructions to the control unit to activate and de-activate the array of electrodes and to operate the detection unit.

18. The instrument of any one of claims 1-17, wherein the instrument is configured to conduct two or more of clinical chemistry, immunoassay, cleavage of a tag for translocation through a nanopore layer, dissociation of an analyte-specific binding member from the analyte, translocation of tag/ analyte-specific binding member thorugh a pore of a nanopore layer, imaging, agglutination assay, and hematology.

19. The instrument of any one of claims 1-18, 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 an electrical circuit for detecting change in electrical signal at a nanopore in a nanopore layer present in a cartridge comprising an electrical detection region.

20. The instrument of any one of claims 1-18, 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.

21. A cartridge comprising:

a first substrate;

a second substrate;

a gap separating the first substrate from the first substrate;

an array 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.

22. The cartridge of claim 21, 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.

23. The cartridge of claim 22, 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.

24. The cartridge of any one of claims 21-22, 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.

25. The cartridge of any one of claims 21-24, further comprising a first layer covering the plurality of electrodes.

26. The cartridge of claim 25, wherein the first layer comprises a dielectric layer and/or a hydrophobic layer.

27. The cartridge of claim 25 or 26, wherein at least a portion of the working electrode and the reference electrode is not covered by the first layer.

28. The cartridge of any one of claims 25-27, 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.

29. The cartridge of any one of claims 23-28, 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.

30. 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 electrical detection region comprising a nanopore layer comprising a nanopore; 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.

31. The multi-functional cartridge of claim 30, wherein the cartridge comprises: an electrochemical species sensing region comprising a working electrode and a reference electrode; and

an electrical detection region comprising a nanopore layer comprising a nanopore.

32. The multi-functional cartridge of claim 30, wherein the cartridge comprises: an electrochemical species sensing region comprising a working electrode and a reference electrode.

33. The multi-functional cartridge of claim 30, 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.

34. The multi-functional cartridge of claim 30, wherein the cartridge comprises: an electrical detection region comprising a nanopore layer comprising a nanopore.

35. The multi-functional cartridge of claim 30, wherein the cartridge comprises:

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 cartridge of any one of claims 30-35, further comprising a first layer covering the plurality of electrodes.

37. The cartridge of claim 36, wherein the first layer comprises a dielectric layer and/or a hydrophobic layer.

38. 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 a plurality of electrodes in a cartridge 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 a tag or an analyte-specific binding member translocating through a pore of a nanopore layer 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

a detection region for generating an analyte related signal from a droplet in the cartridge; and/or

a detection region for generating an analyte related signal from a tag or an analyte-specific binding member translocating through a pore of a nanopore layer in the cartridge.

39. The system of claim 38, further comprising reagent reservoirs, wherein the reagent reservoirs are adjacent to the cartridge interface or are located on the cartridge.

40. The system of claim 39, 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.

41. The system of any one of claims 38-40, 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.

42. The system of any one of claims 38-41, wherein the reagent reservoirs comprise a reagent comprising an enzyme specific for the analyte in the sample, wherein the enzyme acts on the analyte to generate a reaction product that generates an electrical or optical signal.

43. The system of claim 38, wherein the reagent further comprises a redox mediator.

44. The system of any one of claims 40-43, wherein the reagent reservoirs comprise a first binding member that selectively binds to the analyte, wherein the first binding member is attached to a bead.

45. The system of claim 44, wherein the first binding member is an antibody.

46. The system of claims 44 or 45, wherein the reagent reservoirs comprise a second binding member that selectively binds to the analyte, wherein the second binding member is attached to an enzyme.

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

48. The system of claim 46 or 47, wherein the enzyme acts on a substrate to produce a reaction product associated with an electrical signal or an optical signal.

49. The system of any one of claim 38-42, wherein the cartridge interface comprises an insertion slot for accommodating the cartridge, wherein the cartridge comprises:

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

a nanopore layer disposed between the first and second substrates, wherein the plurality of electrodes are configured for translocating a tag or an analyte-specific binding member across the nanopore layer and wherein the electrical detection unit detects the translocation.

50. The system of claim 49, wherein the instrument comprises a plurality of insertion slots.

51. The system of claim 50, wherein the plurality of insertion slots are positioned on a carousel.

52. The system of any one of claims 38-45, 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.

53. The system of claim 52, 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.

54. The system of claim 53, wherein instrument receives an indication of the analyte to be detected and processes the sample based on the type of analyte to be detected.

55. The system of claim 54, wherein the indication is provided by a user via a user interface of the instrument or wherein the indication is provided via a machine-readable indicator present on the cartridge.

56. The system of any one of claims 38-54, 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.

57. The system of any one of claims 38-56, wherein the system is configured for detection of an analyte in a whole blood sample and/or a plasma fraction of a whole blood sample.

58. The system of any one of claims 38-56, wherein the system is configured for detection of an analyte 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.

59. 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 first substrate; a plurality of electrodes to generate electrical actuation forces on a liquid droplet; and an electrochemical species sensing region comprising a working electrode and a reference electrode;

(b) actuating the plurality of electrodes to provide a first liquid droplet comprising the analyte;

(c) actuating the plurality of electrodes to provide a second liquid droplet comprising 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.

60. The method of claim 59, wherein the second liquid droplet comprises a redox mediator.

61. The method of claim 59, further comprising determining a concentration of the analyte based on the electrical signal.

62. The method of claim 59, wherein the electrochemical sensing region is located in a capillary region.

63. 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 first substrate; a plurality of electrodes to generate electrical actuation forces on a liquid droplet; and an electrochemical species sensing region comprising a working electrode and a reference electrode;

(b) actuating the plurality of electrodes to provide a first liquid droplet comprising the analyte;

(c) actuating the plurality of electrodes to provide a second liquid droplet comprising a solid substrate comprising a first binding member that specifically binds to the analyte;

(d) actuating the plurality of electrodes to merge the first and second droplets to create a mixture;

(e) actuating the plurality of electrodes to merge all or a portion of the mixture with a third liquid droplet comprising a second binding member that specifically binds to the analyte;

(f) holding the solid substrate in place while actuating the plurality of electrodes to remove any unbound analyte and/or second binding member;

(g) actuating the plurality of electrodes to contact the solid substrate with a substrate molecule for the enzyme conjugated to the second binding member; and

(h) detecting, via the working and reference electrodes, an electrical signal of an electrochemical species generated by action of the enzyme on the substrate molecule.

64. The method of claim 63, 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).

65. The method of claim 63, wherein the method comprises moving a liquid droplet comprising the solid second substrate and enzyme substrate from step (g) to the electrochemical sensing region.

66. The method of any one of claims 63-65, further comprising determining a concentration of the analyte based on the electrical signal.

67. The method of any one of claims 63-65, further comprising conducting an immunoassay on a sample, using a single cartridge or a different cartridge.

68. The method of any one of claims 63-67, wherein the method further comprises using the plurality of electrodes to generate a droplet comprising a tag or an analyte-specific binding member indicative of presence of analyte in the sample and positioning the droplet at the nanopore layer for assaying the electrical signal generated by translocation of the tag or the analyte-specific binding member through a nanopore of the nanopore layer.

69. A method for performing analyte detection using an instrument comprising: providing an analyte detection instrument comprising a cartridge interface for operable connection to the one or more analyte detection cartridges;

providing a plurality of cartridges having a plurality of electrodes to generate electrical actuation forces on a liquid droplet:

interfacing a first cartridge with the instrument and detecting an analyte related signal from a droplet in a cartridge; and

interfacing a second cartridge with the instrument and detecting an analyte related signal from a tag/analyte-specific binding member translocating through a pore of a nanopore layer in the cartridge.

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

71. The method of claim 70, 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;

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

(m) two hematology assays and an immunoassay; and

(n) three hematology assays.

72. The method of claim 70 or 71 wherein the single system is the instrument of any of claims 1 to 22.

73. 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.

74. The instrument of claim 73, wherein the first analyte related signal comprises an optical or electrical signal and the second analyte related signal comprises an optical signal.

75. The instrument of claim 73, wherein the first analyte related signal comprises an optical or electrical signal and the second analyte related signal comprises an electrical signal.

76. The instrument of any one of claims 73-75, wherein the detection unit is configured to detect the first and second analyte related signals from the same cartridge.

77. The instrument of any one of claims 73-75, wherein the detection unit is configured to detect the first and second analyte related signals from different cartridges.

78. The instrument of claim 73, wherein instrument further comprises a power source and wherein the control unit controls the electric power applied to the one or more electrodes.

79. The instrument of any one claims 73-78, wherein the control unit controls duration of activation of the one or more electrodes.

80. The instrument of any one claims 75-79, 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.

81. The instrument of claim 80, wherein the movement comprises merging a sample droplet with a reagent droplet to generate a merged droplet.

82. The instrument of claim 81, wherein the movement comprises moving the merged droplet or a portion thereof to the detection unit.

83. The instrument of any one of claims 73-82, wherein the detection unit comprises an electrical detection unit for detection of an electrical signal from the cartridge.

84. The instrument of any one of claims 73-82, wherein the detection unit comprises an optical detection unit for detection of an optical signal from the cartridge.