Sensitivity enhancement via non-equilibrium label-free sensing

Abstract

The disclosure relates generally to methods for increasing the sensitivity of detecting a target analyte in a sample via a sensor or electrode with a target-binding molecule conjugated on its surface. The disclosure provides methods of inducing a non-equilibrium state in an electrical double layer or reducing potential decay in the electrical double layer to enhance sensitivity of target analyte detection, e.g., in label-free detection of targets.

Description

Attorney Docket No. : 002806-000143WOPTSENSITIVITY ENHANCEMENT VIA NON-EQUILIBRIUM LABEL-FREE SENSINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119 of U.S. Provisional Application No. 63 / 695,513 filed September 17, 2024, the contents of which are incorporated herein by reference in their entirety.TECHNICAL FIELD

[0002] The disclosure relates generally to methods for enhancing sensitivity of target analyte detection with substrates, electrodes, and sensors comprising a target-binding molecule on a surface thereof.BACKGROUND

[0003] Electrochemical sensors play a pivotal role in detecting and quantifying various analytes, contributing to fields such as healthcare monitoring, point-of-care diagnostics, environmental monitoring, food safety analysis, agriculture, water, and biodefense. However, the sensitivity of these sensors can be compromised due to the formation of the electrical double layer (EDL) at the electrode-electrolyte interface. The EDL, a result of the accumulation of charged species near the electrode surface, induces charge screening effects, hindering the accurate recognition of antibody-antigen interaction and subsequently diminishing the sensitivity. This issue becomes particularly pronounced when dealing with low-concentration analytes or in environments with complex matrices, where the interference from ions exacerbates the charge screening effects.

[0004] In conventional sensors, potential decay has occurred in the EDL, which is derived from linearized Poisson-Boltzmann model that illustrates screening as an equilibrium balance between diffusion and drift. The Debye length (XD) is the characteristic distance over which potential decay occurs in electrolyte solutions. It is considerably shorter than typical biomolecular receptors, which is inherent limitations for all electrical sensors. To overcome these limitations, there is an increasing demand for approaches that can tackle the sensitivity challenges arising from potential decay in the EDL.

[0005] The present disclosure addresses this need.14936-2288-6248 1Attorney Docket No. : 002806-000143WQPTSUMMARY

[0006] The demand for point-of-care testing is rapidly growing and electrical sensor offers quick diagnostic testing. However, most of them suffers from charge screening effects in electrical double layer (EDL), where all charges are masked, making it challenging to detect target analytes. Presented herein are methods to increase the sensitivity by disrupting double layer from reaching equilibrium state and more reducing charge screening environment. The technology described herein generates non-equilibrium condition that is desirable for sensitive recognition of targets in label-free sensing.

[0007] The technology described herein represents a universally applicable strategy for enhancing performance in biosensors where an interface between electrodes and electrolyte exists. By generating a non-equilibrium state during signal measurement without altering the sensing layer, sensitivity can be improved.

[0008] In general, various aspects described herein relate to methods of increasing sensitivity of electrodes and sensors analyte response and detection characteristics in label-free detection methods.

[0009] In one aspect provided herein is a method for detecting a target analyte in a sample. The method comprises contacting a sample suspected of comprising a target analyte with a sensor comprising a conductive surface comprising a target binding ligand capable of binding with the target analyte thereon; inducing a non-equilibrium state in an electrical double layer (EDL) of the conductive surface and / or of the target analyte; and measuring an electrochemical signal from the conductive surface, and wherein a change in the electrochemical signal indicates binding of the target analyte with the target binding ligand. It is noted that the inducing a non-equilibrium state in an EDL and the measuring an electrochemical signal are carried out simultaneously. In some embodiments, the inducing a non-equilibrium state in an EDL is performed after the target analyte binds to the target binding ligand. In some embodiments, a means for inducing a non-equilibrium state in an EDL is not applied prior to the measuring of an electrochemical signal.

[0010] Without wishing to be bound by a theory, embodiments of the various aspects described herein allow label-free detection of target analytes. As used herein, the term “label- free” describes a detection method wherein the detectability of an analyte is not dependent upon the presence or absence of a detectable label. In other words, no further signal enhancers are required in order to detect the binding of the target analyte with the target-binding molecule.

[0011] In some embodiments, the method further comprises washing the sample and sensor prior to the inducing a non-equilibrium state in the EDL of the conductive surface and / or of the24936-2288-6248 1Attorney Docket No. : 002806-000143WQPT target analyte, increases the overlap between the EDL of the conductive surface and EDL of the target analyte, and / or reduces / inhibits a potential decay in the EDL of the conductive surface and / or of the target analyte. In some embodiments, the method comprises washing the sample and sensor and adding a buffer prior to the inducing a non-equilibrium state in the EDL of the conductive surface and / or of the target analyte, increases the overlap between the EDL of the conductive surface and EDL of the target analyte, and / or reduces / inhibits a potential decay in the EDL of the conductive surface and / or of the target analyte.

[0012] Without wishing to be bound by a theory, the inducing a non-equilibrium state in the EDL of the conductive surface and / or of the target analyte, increases the overlap between the EDL of the conductive surface and EDL of the target analyte, and / or reduces / inhibits a potential decay in the EDL of the conductive surface and / or of the target analyte. Stated in another way, the EDL of the conductive surface and the EDL of the target analyte are overlap more when the non-equilibrium state is induced relative to when a non-equilibrium state is not induced. The term “electric double layer” as used herein means an electrically neutral boundary layer formed at the interface between a solid material (e.g., conductive substrate) and a liquid material (e.g., sample comprising an analyte of interest or an electrolyte solution). Without wishing to be bound by a theory, as the surface of the solid material attracts positive (or negative) ions in the liquid material so as to be positively (or negatively) charged, the charges in the liquid material are redistributed based on the Coulomb's law so that the level of negative (or positive) ions increases in the liquid material at the interface with the solid material, thereby forming the EDL.

[0013] As known in the art, the thickness of the EDL is the Debye-Huckel length (i.e., KX). It is reciprocally proportional to the square root of the ion concentration C. In aqueous solutions it is typically on the scale of a few nanometers and the thickness decreases with increasing concentration of the electrolyte. Accordingly, the EDL can have a thickness of from few nanometers to one micrometer. For example, the EDL can have a thickness of from about 0.5 nm to about 10 nm. In some embodiments, the electric double layer can have a thickness of from about 0.5 nm to about 75 nm, e.g., from about 0.5 nm to about 5 nm, from about 0.5 nm to about 4 nm, from about 0.5 nm to about 3 nm, from about 0.5 nm to about 2.5 nm, from about 0.5 nm to about 2 nm, or from about 0.5 nm to about 1.5 nm. In some embodiments, the electric double layer can have a thickness of about 0.5 nm, about 1 nm, about 1.5 nm, about 2 nm, about 2.5 nm, about 3 nm, about 3.5 nm, about 4 nm, about 4.5 nm, about 5 nm, about 5.5 nm, about 6 nm, about 6.5 nm, about 7 nm, about 7.5 nm, about 8 nm, about 8.5 nm, about 9 nm, about 9.5 nm, or about 10 nm.34936-2288-6248 1Attorney Docket No. : 002806-000143WQPT

[0014] In some embodiments, the conductive surface is a surface of an electrode. For example, the conductive surface is a surface of a primary, working, electrode. In some embodiments, the primary electrode comprises counter and reference electrodes.

[0015] In some embodiments, the sensor further comprises a secondary electrode. In some embodiments, the secondary electrode is referred to as a non-working electrode. In some embodiments, the secondary, non-working, electrode is used to induce a non-equilibrium state. It is noted that the primary, working, electrode and the secondary, non-working electrode, can be arranged in any desired configuration and / or orientation. For example, the primary, working, and the secondary, non-working, electrode can be in coplanar or offset configuration. In addition, the primary, working, and the secondary, non-working, electrode can be arranged in any geometrical configuration, such as radial, parallel, interdigitated, ring, sandwich and the like. In some embodiments, the secondary, non-working, electrode is coplanar to the primary, working, electrode. In some embodiments, the secondary, non-working, electrode is not coplanar to the primary, working, electrode. In some embodiments, the secondary, nonworking, electrode is positioned on either side of the primary, working, electrode. In some embodiments, the secondary, non-working, electrode is positioned in a top-down configuration to the primary, working, electrode, e.g., the secondary, non-working, electrode is above the primary, working, electrode or the secondary, non-working, electrode is below the primary, working, electrode. In some embodiments, the secondary, non-working, electrode is in a radial configuration to the primary, working, electrode. In some embodiments, the primary, working, electrode and secondary, non-working, electrode are in a non-coplanar configuration.

[0016] In some embodiments, the method comprises incubating the sample with the sensor for a period of time prior to inducing a non-equilibrium state. Generally, the sample is allowed to be in contact with the sensor for a period of time sufficient for the target analyte to bind the target binding ligand. In some embodiments, the method comprises incubating the sample with the sensor for a few seconds to a few hours prior to inducing a non-equilibrium state. For example, the incubation of the sample with the sensor can be from about 5 second to about 5 hours. In some embodiments, the sample can be incubated with the sensor for a period of from about 5 seconds to about 1 hour. For example, the sample can be incubated with the sensor for a period of from about 10 seconds to about 45 minutes, from about 10 seconds to about 30 minutes, from about 15 seconds to about 25 minutes, from about 20 seconds to about 20 minutes, from about 25 seconds to about 15 minutes, from about 30 seconds to about 10 minutes, or from about 45 seconds to about 5 minutes. In some embodiments, the incubation of the sample with the sensor is from about 5 minutes to about 5 hours, e.g., from about 1044936-2288-6248 1Attorney Docket No. : 002806-000143WQPT minutes to about 4 hours, from about 15 minutes to about 3 hours, from about 20 minutes to about 2.5 hours, from about 25 minutes to about 2 hours, from about 30 minutes to about 1.5 hours. In some embodiments, the incubation of the sample with the sensor is for about 1 hour, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hours, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, or about 5 hours. In some embodiments, the incubation of the sample with the sensor for a period of time prior to inducing a non-equilibrium state is for a period of time sufficient for target analyte binding to the target binding ligand.

[0017] In some embodiments, the inducing a non-equilibrium state in an EDL of the conductive surface and / or of the target analyte, and / or inhibiting / reducing the potential decay in the EDL of the conductive surface and / or of the target analyte, or increasing the overlap between the EDL of the conductive surface and EDL of the target analyte comprises applying voltage. In some embodiments, the voltage applied is AC voltage, DC voltage, pulse voltage, or sweep potential. In some embodiments, the voltage is applied to the primary, working, electrode. In some embodiments, the voltage is applied to the secondary, non-working, electrode. It is noted that the applied voltage provides a sufficiently strong electric field to liberate H+from H2O2, but not strong enough to cause electrolysis of water, as electrolysis of water may lead to a decrease in the signal -to-noise ratio.

[0018] In some embodiments, the voltage applied is AC voltage. In some embodiments, the AC voltage has a frequency of about 1 Hz to about 200,000 Hz, about 5 Hz to about 100,000 Hz, about 10 Hz to about 50,000 Hz, about 20 Hz to 10,000 Hz, about 40 Hz to about 5,000 Hz, about 50 Hz to about 1,000 Hz, about 75 Hz to about 500 Hz, or about 100 Hz to about 250 Hz. In some embodiments the AC voltage has a frequency of 100 Hz.

[0019] In some embodiments, the AC voltage has a potential of about 1 mV to about 2,000 mV, about 5 mV to about 1,000 mV, about 10 mV to about 750 mV, about 50 mV to 500 mV, about 100 mV to about 400 mV, about 150 mV to about 350 mV, or about 200 mV to about 300 mV. In some embodiments the AC voltage has a potential of 100 mV.

[0020] In some embodiments, the electrochemical signal measured is an electrochemical property. For example, the electrochemical property can be impedance, capacitance, current, voltammetry, reactance, and / or resistance. In some embodiments, a change in the electrochemical signal indicates binding of the target analyte with the target binding ligand. In some embodiments, the electrochemical signal measures is an electrochemical signal from the conductive surface.54936-2288-6248 1Attorney Docket No. : 002806-000143WQPT

[0021] In some embodiments, measuring the electrochemical signal comprises Electrochemical Impedance Spectroscopy (EIS), Potentiodynamic technique, Linear polarization, Voltammetric methods, Amperometric methods, and / or Coulometric methods. In some embodiments, measuring the electrochemical signal comprises EIS.

[0022] In some embodiments, the sensor comprises a conductive substrate, a target-binding molecule immobilized on a surface of the conductive substrate. It is noted that the targetbinding molecule can be immobilized on the surface of the conductive substrate covalently or non-covalently. For example, the target-binding molecule can be covalently linked, e.g., via a linker, to the surface of the conductive substrate. In some embodiments, the target-binding molecule can be covalently linked, e.g., via a linker, to a component of the polymeric coating layer, e.g., the target-binding molecule can be covalently linked, e.g., via a linker, to the conductive surface.

[0023] In some embodiments, the target-biding molecule is overlaid with a polymeric coating layer. In other words, in some embodiments, the conductive surface does not comprise a coating layer, e.g., a polymeric coating layer on a surface thereof. Thus, in some embodiments, the sensor comprises a conductive substrate, a target-binding molecule immobilized on a surface of the conductive substrate, the target-biding molecule is overlaid with a polymeric coating layer, and at least a non-target binding portion of the target-binding molecule is covered by or embedded within the polymeric coating layer. It is noted that the target-binding molecule can be immobilized on the surface of the conductive substrate covalently or non-covalently. For example, the target-binding molecule can be covalently linked, e.g., via a linker, to the surface of the conductive substrate. In some embodiments, the target-binding molecule can be covalently linked, e.g., via a linker, to a component of the polymeric coating layer, e.g., the target-binding molecule can be covalently linked, e.g., via a linker, to the polymeric coating layer.

[0024] In some embodiments, a thickness of the polymeric coating layer is such that at least a portion of the target-binding portion or site of the target-binding molecule is in the EDL.

[0025] In some embodiments of the various aspects described herein, a target-binding portion or site of the target-binding molecule is not covered by or embedded within the polymeric coating layer. In other words, the polymeric coating layer does not cover a targetbinding portion or site of the target-binding molecule. For example, a target-binding portion or site of the target-binding molecule can be exposed for contact outside the polymeric coating layer with an analyte.64936-2288-6248 1Attorney Docket No. : 002806-000143WQPT

[0026] In some embodiments, a target-binding portion or site of the target-binding molecule is at a surface of the polymeric coating layer. It is noted that, surface of the polymeric coating can be surface of a pore in the polymeric coating layer. In some embodiments, a targetbinding portion or site of the target-binding molecule is at a surface of the polymeric coating layer and said surface of the polymeric coating layer is not a surface of a pore in the polymeric coating layer.

[0027] The polymeric coating layer can be porous or non-porous. Accordingly, in some embodiments of any one of the aspects described herein, the polymeric coating layer is substantially non-porous. As used herein, the term “non-porous” means the polymeric coating layer has at least 90% of its theoretical density. For example, a non-porous polymeric coating layer has a porosity of about 10% or less. In some embodiments, the non-porous polymeric coating layer has a porosity of about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3% about 2.5%, about 2%, about 1.5%, about 1%, about 0.75%, about 0.5% or lower. It is noted that porosity is inversely proportional to density of the material. The porosity of the polymeric coating layer can be determined via the ASTM F1359, ASTM F739 and / or ASTM D4284-83 standard.

[0028] In some embodiments of any one of the aspects described herein, a thickness of the polymeric coating layer is less than or equal to a distance of a target-binding portion or site of the target-binding molecule from the surface on which the target-binding molecule is present. For example, a thickness of the polymeric coating layer is about 95% (e.g., about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%) or less of a distance between a target-binding portion or site of the target-binding molecule and the surface of the conductive substrate on which the target-binding molecule is present, e.g., when the target-binding portion or site is furthest away from the surface. In some embodiments, the polymeric coating layer has thickness that is from about 5% to about 95% (e.g., from about 5% to about 90%, from about 10 % to about 85%, or from about 15% to about 80%) of a distance between a target-binding portion or site of the target-binding molecule and the surface of the conductive substrate on which the target-binding molecule is present, e.g., when the target-binding portion or site is furthest away from the surface.

[0029] In some embodiments, a thickness of the polymeric coating layer is about 95% (e.g., about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%) or less of a length of the target-binding molecule in its fully extended conformation.74936-2288-6248 1Attorney Docket No. : 002806-000143WQPTIt is noted that when the target-binding molecule is linked to the surface via a linker, the length of the target-binding molecule in its fully extended conformation includes the length of the linker. In some embodiments, the polymeric coating layer has thickness that is from about 5% to about 95% (e.g., from about 5% to about 90%, from about 10 % to about 85%, or from about 15% to about 80%) of a length of the target-binding molecule in its fully extended conformation.

[0030] Without wishing to be bound by a theory, inventors have discovered inter alia that thicker polymeric coating layers provide better sensitivity in detecting target analytes. Thus, in some embodiments, the thickness of the polymeric coating layer is at least about 1 nm. For example, the polymeric coating layer has a thickness of about 1.5 nm, about 2 nm, about 2.5 nm, about 3 nm, about 2.5 nm, about 4 nm, about 4.5 nm, about 5 nm, about 5.5 nm, about 6 nm, about 6.5 nm, about 7 nm, about 7.5 nm, about 8 nm, about 8.5 nm, about 9 nm, about 9.5 nm or more.

[0031] In some embodiments, the thickness of the polymeric coating layer is about 20 nm or less. For example, the polymeric coating layer has a thickness of about 19.5 nm, about 19 nm, about 18.5 nm, about 18 nm, about 17.5 nm, about 17 nm, about 16.5 nm, about 16 nm, about 15.5 nm, about 15 nm, about 14.5 nm, about 14 nm, about 13.5 nm, about 13 nm, about 12.5 nm, about 12 nm, about 11.5, about 11 nm, about 10.5 nm, about 10 nm, about 9.5 nm, about 9 nm, about 8.5 nm, about 8 nm, about 7.5 nm, about 7 nm, about 6.5 nm, about 6 nm, about 5.5 nm, about 5 nm, about 4.5 nm, about 4 nm, about 3.5 nm, about 3 nm, about 2.5 nm, about 2 nm, about 1.5 or less. In some embodiments, the polymeric coating layer has a thickness of from about 1 nm to about 10 nm, e.g., from about 1 nm to about 9.5 nm.

[0032] Generally, the polymeric coating layer covers at least about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 97%, about 99%) or more of the surface of the conductive substrate. For example, the polymeric coating layer completely covers, i.e., 100% of the surface of the conductive substrate.

[0033] Without wishing to be bound by a theory, the polymeric coating layer reduces or inhibits mobility of ions in a liquid solution to the surface of conductive substrate when the electrode is disposed in said liquid solution. For example, the polymeric coating layer can reduce or inhibit direct contact between ions in a liquid solution and the surface of the conductive substrate when the electrode is disposed in said liquid solution. In other words, the polymeric coating layer can act as an insulator or insulating layer between the targetbinding portion or site of the target-binding molecule and the surface of the conductive substrate.84936-2288-6248 1Attorney Docket No. : 002806-000143WOPT

[0034] In some embodiments, the polymeric coating layer is not a molecularly imprinted polymer or layer.

[0035] In some embodiments of any one of the aspects described herein, the primary, working, electrode comprises: (i) a conductive substrate (e.g., an electrically conductive substrate); (ii) a target-binding molecule immobilized on a surface of the conductive substrate, and the target-binding molecule capable of binding with a target analyte of interest; and (iii) and a polymeric coating layer on said surface of the conductive substrate, and wherein at least a portion (e.g., a non-target binding portion) of the target-binding molecule is embedded within the polymeric coating layer. In some embodiments of the electrode, the non-target binding portion and the target-binding site of the target-binding molecule are embedded within the polymeric layer.

[0036] In some embodiments of any one of the aspects described herein, the target-binding molecule is covalently conjugated with the surface. For example, the target-binding molecule is covalently conjugated with the surface via a linker. In some embodiments of any one of the aspects described herein, the target-binding molecule is covalently conjugated with the surface and the target-binding molecule and the polymeric coating layer are not covalently linked to each other. Target-binding molecule can be a peptide, polypeptide, a nucleic acid (e.g., oligonucleotide or polynucleotide) or a small molecule. Some exemplary target-binding molecules include, but are not limited to, receptors, ligand for a receptor, antibodies, antigen binding fragment of an antibody, antigens, enzymes, aptamers, affimers, and nucleic acids. In some embodiments of any one of the aspects described herein, the target-binding molecule is an antibody or an antigen binding fragment of an antibody.

[0037] In some embodiments of any one of the aspects described herein, the target-binding molecule and the polymer in the polymeric coating layer are not covalently linked to each other. In some embodiments, the target-binding molecule and the polymer in the polymeric coating layer are covalently linked to each other, e.g., via a linker.

[0038] Generally, the polymeric coating layer comprises a polymer. For example, the polymeric coating layer comprises an electropolymerized polymer, photopolymerized polymer, or auto-polymerized polymer. In some embodiments, the polymeric coating layer comprises an electropolymerized polymer. In some embodiments, the polymeric coating layer comprises an auto-polymerized polymer. In some embodiments, the polymeric coating layer comprises a photopolymerized polymer.

[0039] In some embodiments of any one of the aspects described herein, the polymeric coating layer comprises a polymer selected from the group consisting of polysaccharides,94936-2288-6248 1Attorney Docket No. : 002806-000143WQPT polydopamines, poly-p-phenylenes, polypyrroles, coumarin based polymers, polyhydroxyethylmethacrylates, polyindolyenes, polyanilines, polythiophenes, poly(ethylenedioxy) thiophene, poly(heteroaromatic vinylenes), polyvinylphosphates, poly-p- phenylene sulfides, poly(2,5-thienylenes), poly-o-phenylenes, poly(o-phenylene diamines), polysaccharides, polyacetylenes, porphyrins, phthalocyanins, fullerenes, polyparaphenylenes, polyphenylenevinylenes, polyfluorenes, polypyridines, polycarbazoles, polypyridinevinylenes, polyarylvinylenes, poly (p-phenylmethylvinylenes), derivatives and co-polymers thereof, and any combination thereof. For example, the polymeric coating layer comprises polydopamine, chitosan, or scopoletin (6-methoxy-7-hydroxy coumarin). In some embodiments of any one of the aspects described herein, the polymeric coating layer comprises electropolymerized polydopamine. In some embodiments, the polymeric coating layer comprises autopolymerized chitosan. In some embodiments, the polymeric coating layer comprises electropolymerized chitosan. In some embodiments, the polymeric coating layer comprises electropolymerized scopoletin.

[0040] In some embodiments of any one of the aspects described herein, the polymeric coating layer further comprises an antifouling material. Some exemplary antifouling materials include, but are not limited to, ethanolamine (ETA), hyaluronic acid (HA), and poly vinyl alcohol (PVA).

[0041] In some embodiments of any one of the aspects described herein, the polymeric coating layer further comprises a conductive element. In some other embodiments, of any one of the aspects described herein, the polymeric coating layer does not comprise a conductive element.

[0042] In some embodiments of any one of the aspects described herein, the polymeric coating layer comprises a redox active material. Some, exemplary redox active materials include, but are not limited to, metallocenes, metallocence derivatives, quinones, polycyclic aromatic hydrocarbons, redox-active organic molecules, phenazines, organosulfur compounds, radical acceptors, or a combination thereof. In some embodiments, the redox active material is poly luminol, methylene blue or ferrocene.

[0043] It is noted that the degradable materials can be included in the polymeric coating to create porous 3D matrix, such as dissolvable polymers. Integration of dissolving polymers can also be utilized to increase the porosity of the polymeric coating layer (e.g., polymers that dissolve in water (salt crystals); other can be removed by degradation (e.g., proteins or protein aggregates); temperature dependent removal (e.g., poly(N-isopropyl acrylamide) (PNIPAAm)). Some exemplary degradable polymers include, but are not limited to, poly(N-104936-2288-6248 1Attorney Docket No.: 002806-000143WQPT isopropyl acrylamide) (PNIPAAm), polyethylene glycol (PEG), alginate, polytetrafluoroethylene (PTFE), polyacetylene (PA), polyaniline (PANI), polypyrrole (PPy), polythiophene (PTH), poly(para-phenylene) (PPP), poly(phenylenevinylene) (PPV), and polyfuran (PF).

[0044] In some embodiments of any one of the aspects described herein, the polymeric coating layer has a porosity of about 5% to about 95%. For example, the polymeric coating layer has a porosity of about 20% to about 75%. In some embodiments, the polymeric coating layer has a porosity of about 25% to about 60%, or about 30% to about 50%. For example, the polymeric coating layer has a porosity of about 35% to 45%.

[0045] In some embodiments of any one of the aspects described herein, the polymeric coating layer comprises macropores. In some embodiments of any one of the aspects described herein, the polymeric coating layer comprises mesopores. In some embodiments of any one of the aspects described herein, the polymeric coating layer comprises nanopores. In some embodiments of any one of the aspects described herein, the polymeric coating layer comprises multiscale pores, i.e., both macropores and mesopores.

[0046] In still another aspect, provided herein is a sensor comprising a surface or electrode described herein. Generally, the sensor comprises a fluid-contact surface and the electrode is immobilized on at least a portion of the fluid-contact surface. In some embodiments of any one of the aspects described herein, the fluid-contact surface further comprises a positive control electrode and / or a negative control electrode immobilized thereon. In some embodiments of any one of the aspects described herein, the sensor comprises one or more microfluidic flow cells.

[0047] In some embodiments of any one of the aspects described herein, the target analyte is a peptide, a polypeptide, a peptidomimetic, a nucleic acid, an oligosaccharide, a polysaccharide, an amino acid, nucleoside, a nucleotide, a carbohydrate, a lipid, a peptidoglycan, a cell, microbial matter, an antigen, a lipid, a steroid, a hormone, a lipopolysaccharide, an endotoxin, a therapeutic agent, a lipid-binding molecule, a cofactor, a small molecule, a toxin, a biological threat agent (e.g., spore, viral, cellular and protein toxin), or any combination thereof.

[0048] The sample suspected of comprising the target analyte can be a biological sample (e.g., blood, saliva, amniotic fluid, sputum, urine, semen, cerebrospinal fluid, bronchial aspirate, perspiration, mucus, liquefied stool sample, synovial fluid, lymphatic fluid, tears, tracheal aspirate, lactation product, breath condensate and any combination thereof); or a food, an ingredient for preparing a food, poultry, meat, fish, beverage, grains, crops, or dairy product;114936-2288-6248 1Attorney Docket No. : 002806-000143WQPT or a non -biological sample (e.g., water, salt water, pond water, river water, reservoir water, brine, drinking water, industrial water, brown water, waste water, sewerage, soil, and mixtures thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIG. 1 are schematics illustrating sensitivity enhancement by applying AC voltage. Potential decay in equilibrium can be disrupted, making increased potential overlap between electrode’s double layer and target’s double layer in a non-equilibrium state.

[0050] FIGS. 2A-2E shows that impedance changes were observed across MIP-ip concentrations ranging from 0 to 1 ng / mL. Impedance was characterized after BSA blocking and MIP binding with and without AC voltage. The resulting bar plot illustrates that the detection limit was enhanced from 0.1 ng / mL to 0.01 ng / mL through the application of AC voltage.

[0051] FIGS. 3A and 3B shows changes in signal response for MIP-ip detection based on AC frequency and potential. Frequencies ranging from 10 to 100,000 Hz and AC potentials ranging from 100 to 500 mV were tested, revealing higher signal response for positive samples (100 pg / mL MIP-ip) compared to negative samples (no MIP-ip) under all conditions.DETAILED DESCRIPTION

[0052] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0053] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose.Enhancing Sensitivity

[0054] In one aspect provided herein is a method of increasing sensitivity of an electrical biosensor by generating non-equilibrium states to disrupt potential decay in the electrical double layer (EDL) during signal measurement. Breaking up potential decay can increase the overlap and lead to more interaction between a sensor or electrode’s double layer and a target124936-2288-6248 1Attorney Docket No. : 002806-000143WQPT molecule’s double layer. As used herein, non-equilibrium state refers to a state where the forces and EDL have changed from equilibrium or static to dynamic. In some embodiments, the nonequilibrium state is induced in an EDL of a sensor or electrode. In some embodiments, the nonequilibrium state is induced in an EDL of a target analyte. In some embodiments, the nonequilibrium state is induced in an EDL of a sensor or electrode and in an EDL of a target analyte. In some embodiments, the non-equilibrium state disrupts exponential decay and increases the potential overlap between the electrode and target plane. In some embodiments, the non-equilibrium state in an EDL of a sensor or electrode inhibits or reduces potential decay in the EDL of the conducting surface. In some embodiments, the non-equilibrium state in an EDL of a target analyte inhibits or reduces potential decay in the target.

[0055] In some embodiments of any one of the aspects described herein, a non-equilibrium state in an EDL is induced by applying a voltage or a potential sweep. In some embodiments, a non-equilibrium state in an EDL is induced by applying AC voltage, DC voltage, or potential sweep. In some embodiments, a non-equilibrium state in an EDL is induced by applying AC voltage. In some embodiments, a non-equilibrium state in an EDL is induced by applying DC voltage. In some embodiments, a non-equilibrium state in an EDL is induced by applying potential sweep. In some embodiments, a non-equilibrium state is induced by not applying voltage or potential sweep. In some embodiments, a non-equilibrium state is induced by adjusting the concentration of complementarity of nucleic acids.

[0056] In some embodiments of any of the aspects described herein, the biosensor comprises a primary, working, electrode comprising a conductive surface and a target binding ligand. In some embodiments, the voltage or potential sweep is applied to a primary, working, electrode. In some embodiments, the biosensor comprises a primary, working, electrode and one or more secondary, non-working, electrodes. As used herein, a secondary, non-working, electrode is an electrode that does not have a target binding ligand and does not detect target analytes. In some embodiments, the voltage or potential sweep is applied to a secondary, nonworking, electrode.

[0057] In some embodiments of the various aspects described herein, the primary, working, electrode and one or more secondary, non-working, electrodes are arranged such that they are coplanar. In some embodiments, the primary, working, electrode and secondary, non-working, electrodes are arranged such that they are non-planar. In some embodiments, the secondary, non-working, electrode is on either side of the primary, working, electrode. In some embodiments, the secondary, non-working, electrode is in a top-down configuration to the primary, working, electrode. In some embodiments, the secondary, non-working, electrode is4936-2288-6248 1 13Attorney Docket No. : 002806-000143WOPT above or below the primary, working, electrode. In some embodiments, the primary, working, electrode and secondary, non-working, electrode are arranged in a radial configuration.

[0058] The distance between the primary, working, electrode and the secondary, nonworking, electrode can be from 0.1cm to about 50cm. In some embodiments of any of the aspects described herein, the distance between the primary, working, electrode and the secondary, non-working, electrode is from about 0.1cm to about 40cm. For example, the distance between the primary, working, electrode and the secondary, non-working, electrode can be from 0.5cm to about 30cm, from 1cm to about 20cm, from 2cm to about 10cm, from 3cm to about 5cm. In some embodiments, the distance between the primary, working, electrode and the secondary, non-working, electrode is about 0.1cm, or about 0.2cm, or about 0.3cm, or about 0.4cm, or about 0.5cm, or about 0.6cm, or about 0.7cm, or about 0.8cm, or about 0.9cm, or about 1cm, or about 2cm, or about 3cm, or about 4cm, or about 5cm, or about 6cm, or about 7cm, or about 8cm, or about 9cm, or about 10cm, or about 20cm, or about 30cm, or about 40cm, or about 50cm, or about 60cm.

[0059] The frequency of the voltage applied to the biosensor can be from about 1 Hz to about 200,000 Hz. In some embodiments of any of the aspects described herein, the frequency of the voltage is from about 1 Hz to about 100,000 Hz. For example, the frequency of the voltage can be from about 10 Hz to about 50,000 Hz, about 20 Hz to 10,000 Hz, about 40 Hz to about 5,000 Hz, about 50 Hz to about 1,000 Hz, about 75 Hz to about 500 Hz, or about 100 Hz to about 250 Hz. In some embodiments, the frequency of the voltage is about 200,000 Hz, or about 150,000 Hz, or about 100,000 Hz, or about, 90,000 Hz, or about 80,000 Hz, or about 70,000 Hz, or about 60,000 Hz, or about 50,000 Hz, or about 40,000 Hz, or about 30,000 Hz, or about 20,000 Hz, or about 10,000 Hz, or about 9,000 Hz, or about 8,000 Hz, or about 7,000 Hz, or about 6,000 Hz, or about 5,000 Hz, or about 4,000 Hz, or about 3,000 Hz, or about 2,000 Hz, or about 1,000 Hz or about 900 Hz, or about 800 Hz, or about 700 Hz, or about 600 Hz, or about 500 Hz, or about 400 Hz, or about 300 Hz, or about 200 Hz, or about 100 Hz, or about 90 Hz, or about 80 Hz, or about 70 Hz, or about 60 Hz, or about 50 Hz, or about 40 Hz, or about 30 Hz, or about 20 Hz, or about 10 Hz or about 9 Hz, or about 8 Hz, or about 7 Hz, or about 6 Hz, or about 5 Hz, or about 4 Hz, or about 3 Hz, or about 2 Hz, or about 1 Hz. In preferred embodiments the AC voltage has a frequency of 100 Hz.

[0060] The potential of the voltage applied to the biosensor can be from about 1 mV to about 1,500 mV. In some embodiments of any of the aspects described herein, the potential of the voltage is from about 1 mV to about 1,000 mV. For example, the potential of the voltage can be from about 10 mV to about 750 mV, about 50 mV to 500 mV, about 100 mV to about144936-2288-6248 1Attorney Docket No. : 002806-000143WQPT400 mV, about 150 mV to about 350 mV, or about 200 mV to about 300 mV. In some embodiments, the potential of the voltage is about 2,000 mV, or about 1,000 mV, or about 900 mV, or about 800 mV, or about 700, mV, or about 600 mV, or about 500 mV, or about 400 mV, or about 300 mV, or about 200 mV, or about 100 mV, or about 90 mV, or about 80 mV, or about 70, mV, or about 60 mV, or about 50 mV, or about 40 mV, or about 30 mV, or about 20 mV, or about 10 mV, or about 9 mV, or about 8 mV, or about 7, mV, or about 6 mV, or about 5 mV, or about 4 mV, or about 3 mV, or about 2 mV, or about 1 mV. In some embodiments the voltage has a potential of 100 mV.

[0061] In some embodiments of any of the aspects described herein, a sample incubates with the sensor for a period of time prior to inducing a non-equilibrium state. In some embodiments, the incubation period of time is sufficient for a target analyte in a sample to bind to the target binding ligand of the sensor. In some cases, the sample can be incubated with the sensor for a period of few second to few minutes prior to inducing a non-equilibrium state. For example, the sample can be incubated with the sensor for a period of from about 5 seconds to about 60 minutes prior to inducing the non-equilibrium state. In some embodiments, the sample can be incubated with the sensor for a period of from about 10 seconds to about 10 minutes, e.g., from about 15 seconds to about 5 minutes, from about 20 seconds to about 2 minutes or from about 30 seconds to about 1 minute prior to inducing the non-equilibrium state In some embodiments, the incubation period is from a minute to a few hours prior to inducing a non-equilibrium state . For example, the incubation period of time is from about 5 minutes to about 5 hours, from about 10 minutes to about 4 hours, from about 15 minutes to about 3 hours, from about 20 minutes to about 2.5 hours, from about 25 minutes to about 2 hours, from about 30 minutes to about 1.5 hours. In some embodiments, the incubation of the sample with the sensor is for about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 55 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hours, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, or about 5 hours. In some embodiments, the inducing a non-equilibrium154936-2288-6248 1Attorney Docket No. : 002806-000143WOPT state is performed after the target analyte binds to the target binding ligand. In some embodiments, the voltage is applied to the primary, working, or secondary, non-working, electrode after the target analyte binds to the target binding ligand.

[0062] In some embodiments of any of the aspects described herein, the measuring of an electrochemical signal from the conductive surface is performed simultaneously with the inducing a non-equilibrium state. In some embodiments, the measuring of an electrochemical signal from the conductive surface is performed after with the inducing a non-equilibrium state.

[0063] Electrochemical methods are methods that rely on a change in the potential, charge or current to characterize the analyte’s chemical reactivity. Some examples include potentiometry, controlled current coulometry, controlled-potential coulometry, amperometry, stripping voltammetry, hydrodynamic voltammetry, polarography, stationary electrode voltammetry, pulsed polarography, electrochemical impedance spectroscopy, cyclic voltammetry, reactance, and resistance. In some embodiments, measuring the electrochemical signal comprises Electrochemical Impedance Spectroscopy (EIS), Potentiodynamic technique, Linear polarization, Voltammetric methods, Amperometric methods, and / or Coulometric methods. In some embodiments, measuring the electrochemical signal comprises EIS. The signals are detected using an electrode or electrochemical sensors coupled to circuits and systems for collection, manipulation and analysis of the signals.Sensors

[0064] In the context of this specification, the term “sensor” refers a device that senses the presence and / or amount of something. For example, the sensor could sense the presence of a chemical such as glucose, a protein such as an antigen, or an antibody in a biological fluid.

[0065] A sensor has two basic components: the sensing surface (or receptor) and the transducer. The sensing surface interacts with the target analyte and the transducer converts this interaction into a readable electronic signal. The sensor performance characteristics depend on both the components. The sensor selectivity and affinity towards the target analyte depends solely on the sensing surface because the analyte interacts only at the sensing surface. Other performance metrics such as sensitivity, resolution, and calibration depend on both components.

[0066] The sensor may have a channel length of between about 5 pm and about 50 pm, or between about 10 pm and about 30 pm, or about 20 pm, and a channel width of between about 1 mm and about 20 mm, or between about 1 mm and about 10 mm, or about 3 mm.164936-2288-6248 1Attorney Docket No. : 002806-000143WOPT

[0067] In some embodiments, the sensor has one or more fluid-contact surfaces, and the electrode is immobilized on at least a portion of the fluid contact surface. In some embodiments, the sensor has one or more wells. In some embodiments, each well of the sensor comprises an inner bottom surface on which one or more analyte specific electrodes are immobilized. In some embodiments, the wells are open cells comprising open tops, enclosed sides and bottom, and one or more analyte-specific electrodes immobilized on the inner fluidcontact surface of the wells. In some embodiments, the sensor comprises 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 32, 48, 64, 96 or more open wells. In one embodiment the sensor is in the form of a 96-well microtiter plate.

[0068] In some embodiments, the wells are microfluidic flow cells comprising an enclosed top, sides and bottom, wherein the top of each flow cell includes a fluid inlet and a fluid outlet, and comprising one or more analyte-specific electrodes immobilized on the inner fluid-contact surface of the wells. In some embodiments, the electrochemical sensor comprises 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 32, 48, 64, 96 or more microfluidic flow cells. Another embodiment is in the form of a 96-well microtiter plate, wherein each well comprises an enclosed top having a fluid inlet and a fluid outlet. In some embodiments, the sensor comprises both one or more open cells and one or more flow cells. Each well contains an array of analyte-specific electrodes (e.g., 32 gold electrodes) that can be individually modified with capture probes to bind the corresponding target analyte (e.g., pathogen, protein, carbohydrate, toxin, drug, etc.) present in the collected sample. In some embodiments, one sample is introduced into each well. In embodiments having two or more wells, portions of the same sample can be introduced into more than one well, or different samples can be introduced into different wells. Thus, in embodiments having multiple wells, multiple samples can be simultaneously assayed.

[0069] In some embodiments, the sensor comprises a conductive surface. The conductive substrate can be in any form having a surface that can be coated. For example, the conductive substrate can be included in the form of a conductive particle, a conductive nano-particle, a conductive micro-particle, a conductive nano-fiber, a conductive micro-fiber, a conductive flake, a conductive chip, a conductive crystal, a conductive porous substrate, a conductive wafer, a conductive wire, a conductive nano-wire, a conductive micro-wire, a conductive channel, a conductive nano-channel, a conductive micro-channel, a conductive rod, a conductive nano-rod, a conductive micro-rod, a conductive foil, a conductive sheet, a conductive web, or combination of these forms. In some implementations, the conductive substrate is part of a microfluidic device, such as a channel or chamber therein.174936-2288-6248 1Attorney Docket No. : 002806-000143WQPT

[0070] Metal patterning techniques, such as standard printed circuit board (PCB) technology, offer a number of versatile fabrication options such as (i) track size and spacing less than 100 pm; (ii) high purity electrolytic gold plating several microns thick suitable for electrochemistry and surface modification chemistries; (iii) ease of small-scale prototyping in standard laboratory settings; and (iv) large scale mass manufacturing capabilities at a fraction of the cost of high-end microarrays. In some embodiments, electrodes as disclosed herein may be fabricated using PCB technology.

[0071] In some embodiments, the conductive substrate includes a metal, a metalloid, a conducting polymer, a conducting glassy material, a conducting amorphous material, a conducting biological membrane, a conducting carbon-based material, or any combination of these.

[0072] In some embodiments, the conductive substrate includes gold. In some embodiments, the conductive substrate includes a silica-based glass (e.g., pure silica or mixtures such as borosilicate glass). In some embodiments, the conductive substrate includes graphite, diamond, glassy carbon, or carbon nano-tubes (CNTs). In some implementations, the conductive substrate is a chip including gold and a silica-based glass.

[0073] In some embodiments of any one of the aspects described herein, the conductive substrate is a flexible substrate. For example, the conductive substrate comprises a flexible material. Exemplary materials for the flexible substrate include, but are not limited to, polyethylene terephthalate, polyethylene naphathalate, polyimides, polymeric hydrocarbons, celluloses, plastics, polycarbonates, polystyrenes, and any combination thereof.

[0074] In some embodiments, the conductive surface comprises a polymeric coating layer. In some embodiments, the polymeric coating layer of the sensor is adapted for contact with an analyte or a sample comprising an analyte. The polymeric coating layer can allow analyte to flow through the pores and be detected, for example, by binding to a target binding ligand such as an antibody, DNA strand, or aptamer. In some implementations, the coatings can be patterned as a conductive wire or a dielectric / insulating surface. Some implementations include coating microfluidic chips, lab-on-a-chip, and organs on a chip. In some implementations, the coatings can be used in nano-gap and micro-gap devices. For example, these devices include nano-gap electrodes, nanostructured-based electrical biosensors, and nano-gap dielectric biosensor for label-free DNA hybridization detection. The coatings can be applied, for example, to the gap between electrodes in the device.Electrode184936-2288-6248 1Attorney Docket No. : 002806-000143WOPT

[0075] In some embodiments, the sensor comprises an electrode. As used herein an “electrode” is a conductor through which current enters or leaves a medium, where the medium is nonmetallic (i.e., it emits or collects electrons or electron “holes”). For example, the medium can be a complex matrix (e.g., blood or serum). The electrode can be inserted into / onto a tissue such as mammalian tissue and be contacted with tissue and / or fluids therein / thereon. The electrode can be large (e.g., with a primary, working, surface area of greater than 1 cm2, greater than 10 cm2, greater than 100 cm2) or the electrode can be small (e.g., with a working surface area of less than 1 cm2, less than 1mm2, less than 100 pm2, less than 10 pm2, less than 1 pm2). The working surface area is the area in contact with the medium and wherein current enters or leaves the medium.

[0076] In some embodiments, the electrodes are mass fabricated onto non-electrically conductive surfaces such as plastic substrates using inexpensive standard technology such as printed circuit board (PCB) technology, roll-to-roll laser ablation or evaporation. Exemplary non-electrically conductive surfaces include plastic, poly(carbonate) (PC), poly(methyl methacrylate) (PMMA), cyclic olefin polymers (COP) or cyclic olefin copolymers (COC), SU- 8, parylene, silicon nitride, kapton, styrene-ethylene-butylene-styrene (SEBS), polydimethylsiloxane (PDMS), polyimide, silicon dioxide, and any combination thereof .

[0077] In some embodiments, the electrode is a planar or a 3 -dimensional electrode. As used herein, a planar electrode electrically interacts with an electroactive species or mediator on a 2-dimensional surface. As used herein, a 3-dimensional electrode is an electrode displaying a very high surface area per unit volume, caused by no planarity. Without being bound by theory, this provides high turbulence at their interface with an electroactive species or mediator, enhancing the mass transfer process of the electroactive species towards the electrode surface. These characteristics strongly improve the electrochemical reaction rate.

[0078] In some embodiments the electrode is “Multiplexed” such that it is configured for a multiplexed assay. As used herein a “multiplexed” assay can be used to simultaneously measure multiple analytes or signals such as two or more (e.g., 3 or more, 5 or more, 10 or more, 50 or more, 100 or more, 1000 or more) during a single run or cycle of the assay. The electrode can therefore be configured as an array of electrodes, microelectrodes or electrochemical sensors each of which can be independently electrically attached to a circuit for monitoring the electrical signals. For example, the array of electrodes can be disposed at the bottom, sides or top of a multiwell plate (e.g., microwell plate) arrayed on a flat surface such as a semiconductor chip (e.g., a sensor array chip) or form part of a multi el ectrode array (e.g., for connection of neurons to electronic circuitry). In some embodiments, the194936-2288-6248 1Attorney Docket No. : 002806-000143WQPT compositions as described herein, can coat more than one sensor since the coating will not conduct between the sensors due to the anisotropy of the conduction, therefore an array of conductors, sensors or electrodes can be coated forming a multiplexed electrode.

[0079] Electrodes can include materials with metallic conduction and semiconductors. For example, electrodes can include metals, metal alloys, semiconductors, doped materials, conducting ceramics and conducting polymers. Without limitation, electrode materials can include carbon (e.g., graphite, glassy carbon, conductive polymers), copper, titanium, brass, mercury, silver, platinum, palladium, gold, rhodium, zinc, lead, tin, iron, Indium Tin Oxide (ITO), aluminum, stainless steel, tungsten, nickel, zirconium, niobium, tantalum, chromium, molybdenum, manganese, rhenium, ruthenium, rhodium, iridium, silicon, doped silicon, II- VI semiconductors (e.g., ZnO, ZnS, CdSe), III-V semiconductors such as (e,g., GaAs, InSb), ceramics (e.g., TiO?, FesO4, MgCr2O4), and conductive polymers (e.g., poly(acetylene)s, poly(p-phenylene vinylene), poly(fluorenes)s, polyphenylenes, polypyrenes, polyazulenes, polynaphthalenes, polyanilines, polyazepines, polyindoles, polycarbazoles, poly (pyrrol e)s, poly(thiophene)s, poly(3,4-ethylenedi oxy thiophene)), polyimide, parylene, benzocyclobutene, and combinations, mixtures and alloys of these.

[0080] Electrodes can also include insulating components such as insulators for electrical and mechanical protection, imparting rigidity and electrical isolation to parts of the electrode.

[0081] In some embodiments, the electrode can be part of an electrochemical cell. For example, the electrode is a primary, working, electrode and the electrochemical cell can include a counter electrode and reference electrode. In some embodiments, a primary, working electrode, comprises counter and reference electrodes. In some embodiments, electrochemical cell includes one or more working electrodes, counter electrodes, and reference electrodes.Polymeric coating layer

[0082] Generally, the polymeric coating layer comprises a polymer. In some embodiments, the polymer coating layer is non-conductive. It is noted that a polymer comprised in the polymeric coating layer can be an electropolymerized polymer, photopolymerized polymer, or auto-polymerized polymer. In some embodiments of any one of the aspects described herein, the polymeric coating layer comprises an electropolymerized polymer. In some embodiments of any one of the aspects described herein, the polymeric coating layer comprises an auto-polymerized polymer. In some embodiments of any one of the aspects described herein, the polymeric coating layer comprises a photopolymerized polymer.204936-2288-6248 1Attorney Docket No.: 002806-000143WQPT

[0083] Exemplary polymers for the polymeric coating layer include, but are not limited to, polysaccharides, polydopamines, poly-p-phenylenes, polypyrroles, coumarin based polymers, polyhydroxyethylmethacrylates, polyindolyenes, polyanilines, polythiophenes, poly(ethylenedioxy) thiophene, poly(heteroaromatic vinylenes), polyvinylphosphates, poly-p- phenylene sulfides, poly(2,5-thienylenes), poly-o-phenylenes, poly(o-phenylene diamines), polysaccharides, polyacetylenes, porphyrins, phthalocyanins, fullerenes, polyparaphenylenes, polyphenylenevinylenes, polyfluorenes, polypyridines, polycarbazoles, polypyridinevinylenes, polyarylvinylenes, poly (p-phenylmethylvinylenes), derivatives and co-polymers thereof, and any combination thereof.

[0084] In some embodiments of any one of the aspects described herein, the polymeric coating layer comprises polydopamine, chitosan, or scopoletin. For example, the polymeric coating layer comprises electropolymerized polydopamine. In another non-limiting example, the polymeric coating layer comprises auto-polymerized chitosan. In yet another example, the polymeric coating layer comprises electropolymerized chitosan. In still another example, the polymeric coating layer comprises electropolymerized scopoletin.

[0085] Generally, the polymer in the polymeric coating layer is chosen to be complementing the target-binding molecule properties. The polymer is capable of being deposited in a controlled method, is compact and is biocompatible. In some embodiments, the polymer is capable of replacing ions in the solution and / or displacing the Debye length by changing the position of Helmholtz plane.

[0086] It is noted that the polymer in the polymeric coating layer is not a proteinaceous material, i.e., the polymer is not a polypeptide.

[0087] It is noted that polymeric layer can be formed on a surface by electropolymerization. Methods for electropolymerizing polymer are well known in the art. Such methods include, but are not limited to, voltammetry, amperometry, etc. In some embodiments, the polymeric layer can be formed on a surface by performing cyclic voltammetry (CV) from -1 to +1 V (e.g., from -0.75 to +0.75 V, from -0.5 to +0.5 V or from -0.2 V to + 0.5 V) at the scan rate of 5-500 mV / s (e.g., 5 mV / s, 10 mV / s, 15 mV / s, 20 mV / s, 25 mV / s, 30 mV / s, 35 mV / s, 40 mV / s, 45 mV / s, 50 mV / s, 55 mV / s, 60 mV / s, 65 mV / s, 70 mV / s, 75 mV / s, 80 mV / s, 85 mV / s, 80 mV / s, 95 mV / s, or 100 mV / s) for at least 2 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) cycles. The thickness of polymer layer can be controlled by altering the number of CV cycles. As CV cycling proceeds, more polymer gets deposited at the surface resulting in the formation of polymeric coating layer on the surface.214936-2288-6248 1Attorney Docket No. : 002806-000143WQPT

[0088] In some embodiments of any one of the aspects described herein, a thickness of the polymeric coating layer is less than or equal to a distance of a target-binding site of the targetbinding molecule from the surface on which the target-binding molecule is present. For example, a thickness of the polymeric coating layer is less than or equal to a distance of a target-binding site of the target-binding molecule from the surface on which the target-binding molecule is present, when the target-binding site is furthest away from the surface.

[0089] In some embodiments of any one of the aspects described herein, a thickness of the polymeric layer is less than or equal to a length of the target-binding molecule in its fully extended conformation.

[0090] In some embodiments of the various aspects described herein, a thickness of the polymeric coating layer is equal to or greater than a Debye length of a target molecule associated with the target-binding molecule.

[0091] As used herein, “Debye length” refers to the distance from the electrode surface at which ions in a solution effectively screen an electrical response induced by the target analyte binding to the capture probe, i.e., the distance at which significant charge separation can occur. Stated in another way, Debye length is a measure of the distance that the counter-ions extend away from the electrode surface. The Debye length is calculated using the Debye-Huckel equation: ~^EkT / Co, where Ao=Debye length, 8=electric constant, k=Boltzman constant, T=temperature, and Co=ionic concentration. Typically, for physiological solutions, the Debye length is about 1 nm or less. This means that for binding of the target molecules to the capture probe that extend more than about 1 nm from the electrode surface into the sample volume, any electrostatic effects outside the Debye length tend to be screened or shielded from detection by the excess free charge. As is known in the art, the salt concentration has an effect on the Debye length according to the Debye-Huckel model. For example, the Debye length is about 1 nanometer (nm) for 100 millimolar (mM) potassium chloride (KC1), about 3.4 nm for 10 mM KC1 and about 10 nm for 1 mM KC1.

[0092] In some embodiments of any one of the aspects, Debye length can range from about 0.1 nm to about lOOnm. For example, the Debye length can be from about 0.2 nm to about 75 nm, from about 0.3 nm to about 50 nm, from about 0.4 nm to about 25 nm, from about 0.5 nm to about 20 nm, or from about 0.75 nm to about 15 nm. In some embodiments of any one of the aspects, Debye length can be from about 1 nm to about 10 nm.

[0093] The polymeric coating layer can have a thickness of from about 0.1 nm to about 200 pm. In some embodiments of any one of the aspects described herein, the polymeric224936-2288-6248 1Attorney Docket No. : 002806-000143WQPT coating layer can have a thickness of from about 0.1 nm to about 500 nm. For example, the polymeric coating layer can have a thickness of from about 0.5 nm to about 250 nm, from about 1 nm to about 200nm or from about 1.5 nm to about 150 nm. In some embodiments, the polymeric coating layer can have a thickness of from about 2 nm to about 100 nm. In some embodiments, the polymeric coating layer can have a thickness of from about 1 nm to about 20 nm. For example, the polymeric coating layer can have a thickness of from about 1 nm to about 10 nm.

[0094] In some embodiments of any one of the aspects described herein, the polymeric coating layer can have a thickness of from about 0.5 pm to about 175 pm. For example, the polymeric coating layer can have a thickness of from about 0.75 pm to about 150 pm, from about 1.5 pm to about 100 pm, from about 2 pm to about 75 pm., or from about 2.5 pm to about 50 pm. In some embodiments, the polymeric coating layer can have a thickness of from about 5 pm to about 25 pm.

[0095] In some embodiments of any one of the aspects described herein, the polymeric coating layer is porous. As used herein, the term “porous” in the context polymeric coating layer means the polymeric coating layer comprises a plurality of spores, holes, openings, bores, apertures, spaces, perforations, or intervals. While the term porous indicates the presence of voids, it does not specify the specific size of the spores, holes, openings, bores, apertures, spaces, perforations, or intervals. The term “porosity” is widely understood as the ratio of void volume to total volume of a three-dimensional porous body, where the total volume is determined by the macroscopic outer dimensions of the body. Porosity can be indicated as a fraction between 0-1 or as a percentage between 0-100%. Porosity can be measure by instruments in the art, such as a porometer. Porosity is inversely proportional to density of the material. Thus, the porosity also can be determined by measuring the density of the coating layer. In some embodiments of any one of the aspects described herein, the porosity can be determined by mercury porosimetry analysis. In some embodiments, mercury porosimetry analysis corresponds to the intrusion of a volume of mercury characteristic of the existence of pores in the polymeric coating layer according to the ASTM D4284-83 standard.

[0096] Generally, the polymeric coating layer has a porosity from about 5% to about 95%. For example, the polymeric coating layer has a porosity from about 10% to about 75%, about 15% to about 70%, about 20% to about 65%, about 25% to about 60%, or about 30% to about 55%. In some embodiments of any one of the aspects described herein, the polymeric coating layer has a porosity from about 35% to about 45%.234936-2288-6248 1Attorney Docket No. : 002806-000143WQPTTarget-binding molecule (capture probe)

[0097] Embodiments of the various aspects described herein include a target-binding molecule. The terms “target-binding ligand”, “target-binding molecule” and “capture probe” are used interchangeably herein and refer to a molecule that binds to or interacts with a target molecule. In other words, a target-binding ligand or molecule is a molecule that is capable of binding with a target molecule. The targeting binding ligand can be a natural or synthetic molecule (e.g., a molecular receptor) that binds to a target molecule. Exemplary target-binding ligands include, but are not limited to, a receptor, a ligand for a receptor, an antibody, an antigen binding fragment of an antibody, an antigen, an enzyme, a nucleic acid, an aptamer or affimers. The target-binding ligand is also referred to as a “capture agent” or “capture molecule” herein.

[0098] In some embodiments of any one of the aspects described herein, the binding of the target-binding ligand to the target molecule is a specific binding such that it is selective to that target above non-targets. For example the dissociation constant between the target-binding ligand and target molecule is at least about 200 nM, alternatively at least about 150 nM, alternatively at least about 100 nM, alternatively at least about 60 nM, alternatively at least about 50 nM, alternatively at least about 40 nM, alternatively at least about 30 nM, alternatively at least about 20 nM, alternatively at least about 10 nM, alternatively at least about 8 nM, alternatively at least about 6 nM, alternatively at least about 4 nM, alternatively at least about 2 nM, alternatively at least about 1 nM, or greater. In certain embodiments, the specific binding refers to binding where the target-binding ligand binds to its target molecule without substantially binding to any other species in the sample / test solution.

[0099] By way of non-limiting examples, a target-binding ligand can be selected from antibodies, adnectins, ankyrins, other antibody mimetics and other protein scaffolds, aptamers, nucleic acid (e.g., an RNA or DNA aptamer), protein, peptide, binding partner, oligosaccharides, polysaccharides, lipopolysaccharides, cellular metabolites, cells, viruses, subcellular particles, haptens, pharmacologically active substances, alkaloids, steroids, vitamins, amino acids, avimers, peptidomimetics, hormone receptors, cytokine receptors, synthetic receptors, sugars or molecularly imprinted polymer. The target-binding ligand can be selective to a specific target or class of targets such as toxins and biomolecules. For example, the target can be ions, molecules, oligomers, polymers, proteins, peptides, nucleic acids, toxins, biological threat agents such as spore, viral, cellular and protein toxins, carbohydrates (e.g., monosaccharides, disaccharides, oligosaccharides, polyols, and polysaccharides) and combinations of these (e.g., copolymers including these).244936-2288-6248 1Attorney Docket No. : 002806-000143WQPT

[0100] In some embodiments of any one of the aspects described herein, the target-binding ligand is an antibody or antigen binding fragment thereof. As used herein, the terms “antibody” and “antibodies” include polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain Fv antibody fragments, Fab fragments, and F(ab)2 fragments. Antibodies having specific binding affinity for a target of interest (e.g., an antigen) can be produced through standard methods. As used herein, the terms “antibody” and “antibodies” refer to intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding and includes chimeric, humanized, fully human, and bispecific antibodies. In some embodiments, binding fragments are produced by recombinant DNA techniques. In additional embodiments, binding fragments are produced by enzymatic or chemical cleavage of intact antibodies. Binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, and single-chain antibodies.

[0101] In some embodiments of any one of the aspects described herein, the target-binding molecule is covalently conjugated with the surface. For example, the target-binding molecule is covalently conjugated with the surface via a linker. In some embodiments of any one of the aspects described herein, the target-binding molecule is covalently conjugated with the surface and the target-binding molecule and the polymeric coating layer are not covalently linked to each other. Some exemplary target-binding molecules include, but are not limited to, receptors, ligand for a receptor, antibodies, antigen binding fragment of an antibody, antigens, enzymes, and nucleic acids. In some embodiments of any one of the aspects described herein, the targetbinding molecule is an antibody or an antigen binding fragment of an antibody.Antifouling material

[0102] Embodiments of the various aspects described herein include an antifouling material. As used herein an antifouling material is a molecule, substance or composition that inhibits, prevents or reduces non-specific adsorption of molecules, e.g., target molecules on a surface, e.g., the polymeric coating. Some exemplary antifouling materials include, but are not limited to, ethanolamine (ETA), hyaluronic acid (HA), and poly vinyl alcohol (PVA).Conductive element

[0103] Embodiments of the various aspects described herein include a conductive element. As used herein a conductive element is a substance or substrate that has the capability to conduct electricity. The conductive element can comprise conducting and / or semi-conducting materials. Further, the conductive element can be in any desired shape or form. For example,254936-2288-6248 1Attorney Docket No. : 002806-000143WOPT the conductive element can be in form of particles (e.g., nanoparticles), rods, flakes (e.g., nanoflakes), tubes (e.g., nanotubes), fibers, sheets, films, and the like. For example, the conductive element can be included in the form of a particle, a nano-particle, a micro-particle, a fiber, a nano-fiber, a micro-fiber, a flake, a nanoflake, a microflake, a tube, a nanotube, a microtube, a crystal, a nanocrystal, a microcrystal, a wire, a nano-wire, a micro-wire, a rod, a nano-rod, a micro-rod, a foil, a sheet, a web, or any combinations of these forms.

[0104] The conductive element can be formed from one or more metals, e.g., copper, gold, silver, platinum, palladium, indium, iridium, rhodium, ruthenium, osmium, nickel, tin, titanium, tantalum, tungsten, chromium, iron, aluminum, zinc, combinations thereof, or alloys of any of the foregoing. In addition, or in the alternative, a nonmetallic conductive material can be used. Exemplary nonmetallic conductive materials include, but are not limited to, graphite or acetylene black, graphene, conductive ceramics such as indium tin oxide (ITO), titanium nitride, tungsten nitride, tantalum nitride, and conductive polymers such as polythiophenes, polyanilines, polypyrroles, and polyetheylenes and their mixtures and derivatives.

[0105] In some embodiments of any one of the aspects, the conductive element comprises a metal or a metalloid. For example, the conductive element comprises gold. In some embodiments of any one of the aspects described herein, the conductive element comprises gold particles (e.g., gold nano-particles), gold wires (e.g., gold nanowires), gold rods (e.g., gold nano-rods), or any combinations thereof.

[0106] In some embodiments of any one of the aspects described herein, the conductive element comprises a conducting carbon-based material. For example, the conductive element comprises an allotrope of carbon atoms arranged in a hexagonal lattice. The allotropes of carbon can include some functionalization, such as oxygen, carboxylates, epoxides, amines, amides and combinations of these, as described below. In some implementations, the functionalization includes poly amine functionalization such as pentaamine functionalization. In some embodiments, the conductive element comprises graphite, graphene, graphene oxide, functionalized graphene oxide, reduced graphene oxide (rGO), functionalized reduced graphene oxide, or carbon nano-tubes (CNTs).

[0107] As used herein “carbon nanotubes” and “graphene” are allotropes of carbon with sp2carbon atoms arranged in a hexagonal, honeycomb lattice. Single layer graphene is a two- dimensional material, and is a single layer of graphite. As used herein, more than one layer of graphene can be referred to as graphene, for example between 1 and 200 layers (e.g., about 1 to 100 layers, about 1 to 50 layers, about 1 to 10 layers). Carbon nanotubes are hollow, cylindrical structures, formed as a sheet of graphene rolled into a cylinder.264936-2288-6248 1Attorney Docket No. : 002806-000143WQPT

[0108] As used herein “graphene oxide” is a material that can be formed from the oxidation of graphene or exfoliation of graphite oxide. In a first step for producing graphene oxide, graphite is oxidized. Several methods for oxidation are known, one common method known as the Hummers and Offeman method, in which graphite is treated with a mixture of sulphuric acid, sodium nitrate and potassium permanganate (a very strong oxidizer). Other methods are known to be more efficient, reaching levels of 70% oxidization, by using increased quantities of potassium permanganate, and adding phosphoric acid combined with the sulphuric acid, instead of adding sodium nitrate. Exfoliation of graphene oxide provides graphite oxide and can be done by several methods. Sonication can be a very time-efficient way of exfoliating graphite oxide, and it is extremely successful at exfoliating graphene (almost to levels of full exfoliation), but it can also heavily damage the graphene flakes, reducing them in surface size from microns to nanometers, and also produces a wide variety of graphene platelet sizes. Mechanically stirring is a much less destructive approach, but can take much longer to accomplish.

[0109] Graphite oxide and graphene oxide are very similar, chemically, but structurally, they are very different. Both are compounds having carbon, oxygen and hydrogen in variable ratios. In the most oxidized state the oxygen amount can be as high as about 60 wt%. the amount of hydrogen varies depending on the functionalization, for example, the number of epoxy bridges, hydroxyl groups and carboxyl groups. The main difference between graphite oxide and graphene oxide is the interplanar spacing between the individual atomic layers of the compounds, caused by water intercalation. This increased spacing, caused by the oxidization process, also disrupts the sp2bonding network, meaning that both graphite oxide and graphene oxide are often described as electrical insulators.

[0110] Reduced graphene oxide (rGO) is prepared from reduction of graphene oxide by thermal, chemical or electrical treatments. For example, treating the graphene oxide with; hydrazine, hydrogen plasma, heating in water, high temperature heating (e.g., under nitrogen / argon) and electrochemical reduction. Whereas graphene can be a single carbon layer ideally comprising only carbon, reduced graphene oxide is similar but contains some degree of oxygen functionalization. The amount of oxygen depends on the degree of reduction and in some materials can vary between about 50 wt% and about 1 wt. % (e.g., between about 30 wt.% and about 5 wt.%).

[0111] Reduced graphene oxide can be functionalized or include functional groups. For example, reduced graphene oxide often includes oxygen in the form of carboxyl groups and hydroxyl groups. In some forms, the carboxyl and hydroxyl groups populate the edges of the274936-2288-6248 1Attorney Docket No. : 002806-000143WOPT rGO sheets, which can be functionalized. Accordingly, in some embodiments, the reduced graphene oxide (rGO) is carboxylated reduced graphene oxide or aminated reduced graphene oxide. As used herein, carbonylated reduced graphene oxide can refer to reduced graphene oxide having carboxyl groups. In some embodiments the amount of oxygen attributable to the carboxyl groups is between about 30 wt.% and about 0.1 wt.% (e.g., between about 10 wt.% and about 1 wt.%). Other forms of functionalization are possible. For example, amine functionalized rGO can be formed by a modified Buchere reaction, wherein ammonia an graphene oxide is reacted using a catalyst such as sodium bisulfite, or epoxide groups on graphene oxide can be opened with p-phenylenediamine. In some embodiments, the amount of nitrogen is between about 30 wt.% and 0.1 wt.% (e.g., between about 10 wt.% and 1 wt.%). In some implementations, a polyamine is used to functionalize rGO. For example, pentaamine functionalized graphene is used in some implementations.

[0112] The tube-shaped carbon nanotubes have diameters in the nanometer scale, such as, for example, between about 0.2 and about 20 nm, preferably between about 0.5 and about 10 nm, and more preferably still between about 1 and about 5 nm. These can be single walled carbon nanotubes (SWCNT), multi walled carbon nanotubes (MWCNT) (e.g., a collection of 2 or more nested tubes of continuously increasing diameters, or mixtures of these). The diameters of MWCNT can be larger than the SWCNT, such as between about 1 and about 100 nm (e.g., between about 1 and about 50 nm, between about 10 and 20 nm, between 5 and 15 nm, between about 30 and 50 nm). Depending on how the precursor graphene sheet is rolled up to make a seamless cylinder that is the carbon nanotube, different isomers of carbon nanotube can be made, for example designated as armchair configuration, chiral configuration, and zigzag configuration. In some embodiments, the carbon nanotubes are carboxylated carbon nanotubes or aminated carbon nanotubes.

[0113] The carbon nanotubes and reduced graphene oxide can include intercalated materials, such as ions and molecules. In some embodiments the carbon nanotubes can be functionalized for example by oxidation to form carboxylic acid groups on the surface, providing CNTs. In addition, in some embodiments, the carbon nanotubes and rGO can be further modified through condensation reactions with the carboxylic acid groups present on the CNTs or rGO (e.g., with alcohols and amines), electrostatic interactions with the carboxylic acid groups (e.g., calcium mediated coupling, or quaternary amines, protonated aminecarboxylate interaction, through cationic polymers or surfactants) or hydrogen bonding through the carboxylic acid groups (e.g., with fatty acids, and other hydrogen bonding molecules). The functionalization can be partial (e.g., wherein less than 90%, less than 80%, less than 60%,284936-2288-6248 1Attorney Docket No. : 002806-000143WOPT less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, of the available carboxylic acid groups are functionalized) or complete, such as functionalizing substantially all the carboxylic acids (e.g., more than 90%, more than 95%, more than 99% of available carboxylic acid groups).

[0114] In some embodiments of any one of the aspects, the conductive element comprises a conductive polymer. Exemplary conductive polymers include, but are not limited to, polyacrylonitrile (PAN), polyanilines, polypyrroles, polyacetylenes, polyphenylene sulfide, polythiophene, polyfluorene, polypyrene, polyazulene, polynaphthalene, polycarbazole, polyindole, polyazepine, poly (3, 4-ethylenedi oxythiophene) (PEDOT), poly (p-phenylene sulfide) (PPS), poly(p-phenylene vinylene), poly(fluorenes)s, polyphenylenes, polypyrenes, polyazulenes, polynaphthalenes, polyanilines, polyazepines, polyindoles, polycarbazoles, poly(pyrrole)s, poly(thiophene)s, poly (p-phenylene vinylene) (PPV), and mixtures thereof.

[0115] In some embodiments of any one of the aspects described herein, the conductive element comprises one or more organic compounds having conducting and / or semiconducting properties. Exemplary organic compounds having conducting and / or semiconducting properties include, but are not limited to polyacetylenes, porphyrins, phthalocyanins, fullerenes, polyparaphenylenes, polyphenylenevinylenes, polyfluorenes, polythiophenes, polypyrroles, polypyridines, polycarbazoles, polypyridinevinylenes, polyarylvinylenes, poly (p-phenylmethylvinylenes), derivatives and co-polymers thereof, and any combination thereof. In some embodiments of any one of the aspects described herein, the organic compounds having conducting and / or semiconducting properties are selected from the group consisting of poly(3 -hexylthiophene) (P3HT), perfluoro-3,6-dioxa-4-methyl-7-octenesulfonic acid copolymer, tetrafluoroethylene-perfluoro-3,6-dioxa-4-methyl-7-octenesulfonic acid copolymer (e.g. nafion), poly(9,9-dioctylfluorene-2,7-diyl-co-bis-N,N-(4-butylphenyl)-bis- N,N-phenyl-l,4-phenylenediamine), poly(9,9-dioctylfluorene-2,7-diyl-co-benzothiadiazole), (6,6)-phenyl-C61 -butyric acid methyl ester, poly(2-methoxy-5-(2'-ethyl-hexyloxy)-l,4- phenylene vinylene), poly(4-vinylphenol) (PVP), and copolymers thereof, and any combination thereof.

[0116] The conductive element can be cross-linked with the surface and / or other components present in the in the polymeric coating layer. In some embodiments, the conductive element is cross-linked with the surface and / or another component in the polymeric coating layer by a cross-linking agent. In some embodiments, the conductive element in the294936-2288-6248 1Attorney Docket No.: 002806-000143WQPT polymeric coating layer is covalently linked to the surface that is coated by the polymeric coating layer.

[0117] In some embodiments of any one of the aspects described herein, the conductive element is cross-linked with another component in the polymeric coating layer by a crosslinking agent selected from Genipin, polyethylene glycol, and glutaraldehyde.The ratio of the conductive element to the cross-linking agent can be from about 100: 1 to about 1 : 1 (w / w). In some embodiments of any one of the aspects described herein, the ratio of the conductive element to the cross-linker is from about 100: 1 to about 10: 1 (w / w). For example, the ratio of the conductive element to the cross-linker can be from about 90: 1 to about 20: 1, about 80: 1 to about 30: 1, about 70: 1 to about 40: 1, or about 60: 1 to about 50: 1 (w / w). In some embodiments, the w / w ratio of the conductive element to cross-linker is about 100: 1, or about 95: 1, or about 90: 1, or about 85: 1, or about 80: 1, about 75: 1, or about 70: 1, or about 65: 1, or about 60: 1, about 55: 1, or about 50: 1, or about 45: 1, or about 40: 1, about 35: 1, or about 30: 1, or about 25: 1, or about 20: 1, or about 15: 1, or about 10: 1, or about 5: 1 or about 1 : 1.Redox active material

[0118] Embodiments of the various aspects described herein include a redox active material. As used herein, term “redox active material” refers to any chemical moiety capable of undergoing a reduction (accepting of an electron(s)) or oxidation (donation of an electron(s)) in the course of a multi-step process transferring electrons to or from a substrate of an oxidoreductase to an electrode. Exemplary redox active materials include, but are not limited to, metallocenes, metallocence derivatives, quinones, polycyclic aromatic hydrocarbons, redox-active organic molecules, phenazines, organosulfur compounds, radical acceptors, or a combination thereof.

[0119] In some embodiments of any one of the aspects described herein, the redox active material is selected from the group consisting of ferrocene, ferrocene derivatives, 3, 3', 5,5'- tetramethylbenzidine (TMB), o-phenylenediamine dihydrochloride (OPD), N,N,N',N'- tetramethyl-p-phenylenediamine, viologens 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), p-nitrophenyl Phosphate (PNPP), 3,3'-diaminobenzidine (DAB), 4-chloro-l- naphthol (4-CN), 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), nitro blue tetrazolium (NBT), methylene blue, tetrathialfulvaene, 2,6-dichloroindophenol (DCIP), 2,6- dichloroindophenyl phosphate, riboflavin 5 '-monophosphate (RMP), ethyl viologen (1,1'- bis(ethyl)-4,4'-bipyridinium or BEBP), Paraquat, hexaamine ruthenium, 1,4-naphthoquione, resorufine, cyanomethylviologen, diquat, triquat, l,r-bis(2-sulfoethyl)-4,4'-bipyridinium,304936-2288-6248 1Attorney Docket No. : 002806-000143WQPT1,1 '-dibenzyl-4,4'-bipyridinium, 4,4'-dicarboxy-2,2'-bipyridyl, 1-hydroxybenzotri azole, veratryl alcohol, violuric acid, 2-methoxy-phenothiazone, 3-hydroxyanthranilic acid, anthraquinone 2,6-disulfonic acid, N-hydroxyacetanilide, phenol red, 3,3',5,5'-tetramethyl benzidine, dichlorophenol red, 2,2',6,6'-tetramethylpiperidine-N-oxyl radical, syringaldehyde and acetosyringone, 2,2,6,6-tetramethylpiperidin-l-yl)oxyl (TEMPO), 2,2'7,7'-tetrakis-(N,N- di-p-methoxyphenyl-amine)-9,9'-spirobifluorene (spiro-MeOTAD), sodium anthraquinone- 2,6-di sulphonate (AQDS), benzoquinones, 2,2'-biimidazole, 2-(2-pyridyl)imidazole, 2,2'- bipyridine, tetracyanoquinodimethane (TCNQ), and combinations thereof.

[0120] In some embodiments of any one of the aspects described herein, the redox active material is ferrocene or a derivative thereof. As used herein, the term “ferrocene derivative” refers to a molecule containing an optionally substituted ferrocene group (e.g., optionally substituted ferrocenyl radical or ferrocene nucleus). Some exemplary ferrocene derivatives include, but are not limited to, aminoferrocene, ferrocene monocarboxylic acid, ferrocyanide, l,l'-ferrocene dicarboxylic acid, l,l'-dimethylferrocene (DMF), polyvinylferrocene, [N- ferrocenoyl]-4-aminophenyl phosphate, ferrocenylmethyl methacrylate, P-ferrocenyl- propenoic acid, and ferrocene monocarboxylic acid (FMCA). In some embodiments of any one of the aspects described herein, the redox active material is aminoferrocene

[0121] In some embodiments of any one of the aspects described herein, the redox active material is covalently linked with other components present in the polymeric coating layer. For example, the redox active material can be covalently linked with the conductive element or the polymer in the in the polymeric coating layer. For example, the redox active material is covalently linked to the polymer in the in the polymeric coating layer. In another non-limiting example, the redox active material is covalently linked to the conductive element in the in the polymeric coating layer.

[0122] In some embodiments of any one of the aspects described herein, the redox active material in the polymeric coating layer is covalently linked to the substrate surface, e.g., conductive substrate surface coated with the polymeric coating layer. It is noted the redox active material can be linked directly to the surface without being linked to the conductive element or the polymer in the in the polymeric coating layer. In some embodiments, the redox active material is not linked directly to the surface. For example, the redox active material is linked to the surface by forming a covalent link to the conductive element or the polymer.

[0123] It is noted that the redox active material can be covalently linked with the conductive element, the polymer or the substrate surface directly (e.g., a bond) or by a crosslinking agent. Exemplary cross-linking agents include, but are not limited to, glutaraldehyde,314936-2288-6248 1Attorney Docket No.: 002806-000143WQPTGenipin, polyethylene glycol, carbodiimide based cross-linkers. Accordingly, in some embodiments, the redox active material is covalently linked with the conductive element by a cross-linking agent. For example, the redox active material covalently linked with the conductive element by a cross-linking agent selected from glutaraldehyde, Genipin, polyethylene glycol, and carbodiimide cross-linker. In some embodiments, the redox active material is covalently linked to the conductive element by formation of a bond between a functional group in the redox active material and a complementary functional group in the conductive element. In some other embodiments, the redox active material is covalently linked to the polymer by formation of a bond between a functional group in the redox active material and a complementary functional group in the polymer.

[0124] The ratio of the redox active material to the cross-linking agent can be from about 100:1 to about 1:1 (w / w). In some embodiments of any one of the aspects described herein, the ratio of the redox active material to the cross-linker is from about 100: 1 to about 10:1 (w / w). For example, the ratio of the redox active material to the cross-linker can be from about 90:1 to about 20:1, about 80:1 to about 30:1, about 70:1 to about 40:1, or about 60:1 to about 50:1 (w / w). In some embodiments, the w / w ratio of the redox active material to cross-linker is about 100:1, or about 95:1, or about 90:1, or about 85:1, or about 80:1, about 75:1, or about 70:1, or about 65:1, or about 60:1, about 55:1, or about 50:1, or about 45:1, or about 40:1, about 35:1, or about 30:1, or about 25:1, or about 20:1, or about 15:1, or about 10:1, or about 5:1 or about 1:1.

[0125] The redox active material can be present in the polymeric coating layer in an amount from about 0.001 to about 10 (w / w).

[0126] The ratio of the redox active material to the polymer in the polymeric coating layer can be from about 1 : 10 to about 1 : 1000 (w / w). In some embodiments of any one of the aspects described herein, the ratio of the redox active material to the polymer in the polymeric coating layer is from about 1:10 to about 1:900 (w / w). For example, the ratio of the redox active material to the polymer in the polymeric coating layer can be from about 1 : 100 to about 1 :800, about 1:200 to about 1:700, about 1:300 to about 1:600, or about 1:400 to about 1:500 (w / w). In some embodiments, the w / w ratio of the redox active material to the polymer in the polymeric coating is about 1 : 10, or about 1 :50, or about 1 : 100, or about 1 : 150, or about 1 :200, about 1:250, or about 1:300, or about 1:350, or about 1:400, about 1:450, or about 1:500, or about 1:550, or about 1:600, about 1:650, or about 1:700, or about 1:750, or about 1:800, or about 1:850, or about 1:900, or about 1:950, or about 1:1000.324936-2288-62481Attorney Docket No. : 002806-000143WOPT

[0127] The ratio of the redox active material to the conductive element in the polymeric coating layer can be from about 1 : 10 to about 1 : 1000 (w / w). In some embodiments of any one of the aspects described herein, the ratio of the redox active material to the conductive element in the polymeric coating layer is from about 1 : 10 to about 1 :900 (w / w). For example, the ratio of the redox active material to the conductive element in the polymeric coating layer can be from about 1 : 100 to about 1 :800, about 1 :200 to about 1 :700, about 1 :300 to about 1 :600, or about 1 :400 to about 1 :500 (w / w). In some embodiments, the w / w ratio of the redox active material to the conductive element in the polymeric coating is about 1 : 10, or about 1 :50, or about 1 : 100, or about 1 : 150, or about 1 :200, about 1 :250, or about 1 :300, or about 1 :350, or about 1 :400, about 1 :450, or about 1 :500, or about 1 :550, or about 1 :600, about 1 :650, or about 1 :700, or about 1 :750, or about 1 :800, or about 1 :850, or about 1 :900, or about 1 :950, or about 1 : 1000.Target analytes

[0128] In some embodiments, the analyte is a biological analyte. In some embodiments, the analyte ion, molecule, oligomer, polymer, protein, peptide, polypeptide, peptidomimetic, nucleic acid, antigen, antibody, nucleic acid, toxin, biological threat agent such as spore, viral, cellular and protein toxin, carbohydrate, monosaccharide, disaccharide, oligosaccharide, polyol, and polysaccharide, lipid, peptidoglycan, cell, microbial matter, steroid, hormone, lipopolysaccharide, endotoxin, therapeutic agent, lipid-binding molecule, co-factor, small molecule, fatty acid, chemical, or combinations of these. The analyte is optionally an antigen or antibody indicative of infection or resistance to infection. The analyte is optionally a clinical chemistry analyte.

[0129] In some embodiments, the analyte is immunological or serological, for example an antigen or antibody.

[0130] In some embodiments the analyte is a hormone, for example a gynaecological hormone such as luteinizing hormone (LH), progesterone, estradiol or follicle-stimulating hormone. In preferred embodiments the probe detects LH. In some embodiments the probe is a LH specific antibody. In some embodiments the probe is an LH monoclonal antibody. Additionally, or alternatively the hormone may be a pregnancy hormone such as human chorionic gonadotropin (hCG).

[0131] In some embodiments the analyte is a clinical chemistry analyte such as an ion, salt, mineral, metabolite, therapeutic drug, toxicology marker, drug of abuse, transport protein,334936-2288-6248 1Attorney Docket No. : 002806-000143WQPT enzyme, specific protein, lipoprotein or marker, for example diabetes or myocardial infarction markers. In some embodiments the analyte is a metabolite selected from the group of glucose, cholesterol, urea, lactic acid, bilirubin, creatinine, triglycerides. In preferred embodiments the probe is selected to detect glucose or cholesterol.

[0132] In some embodiments, the analyte is a tumor marker. Tumor markers can be used in guiding treatment decisions, monitoring treatment, predicting the change of recovery and to predict or monitor for tumor recurrence.Sample

[0133] In accordance with various embodiments described herein, a sample, including any fluid or specimen (processed or unprocessed) that is intended to be evaluated for the presence of an analyte can be subjected to methods, compositions, kits and systems described herein. The sample or fluid can be liquid, supercritical fluid, solutions, suspensions, gases, gels, slurries, and combinations thereof. The sample or fluid can be aqueous or non-aqueous.

[0134] In some embodiments, the sample can be an aqueous fluid. An aqueous fluid includes biological fluids as described below. Optionally, if the sample is water-based but not fluid, an aqueous solution can be added to produce a fluid sample.

[0135] In some embodiments, the sample can include a biological fluid obtained from a subject. Exemplary biological fluids obtained from a subject can include, but are not limited to, blood (including whole blood, plasma, cord blood and serum), lactation products (e.g., milk), amniotic fluids, sputum, saliva, urine, semen, cerebrospinal fluid, bronchial aspirate, perspiration, mucus, liquefied stool sample, synovial fluid, lymphatic fluid, tears, tracheal aspirate, breath condensate and any combination thereof. In some embodiments, a biological fluid can include a homogenate of a tissue specimen (e.g., biopsy) from a subject. In one embodiment, a test sample can comprise a suspension obtained from homogenization of a solid sample or a fragment thereof obtained from a subject.

[0136] In some embodiments, the sample can include a fluid or specimen obtained from an environmental source. For example, the fluid or specimen obtained from the environmental source can be obtained or derived from food products or industrial food products, food produce, poultry, meat, fish, beverages, grains, crops, dairy products, water (including wastewater), surfaces, ponds, rivers, reservoirs, swimming pools, soils, food processing and / or packaging plants, agricultural places, hydrocultures (including hydroponic food farms), pharmaceutical manufacturing plants, animal colony facilities, and any combinations thereof.344936-2288-6248 1Attorney Docket No. : 002806-000143WOPT

[0137] In some embodiments, the sample can be a non-biological fluid. As used herein, the term “non-biological fluid” refers to any fluid that is not a biological fluid as the term is defined herein. Exemplary non-biological fluids include, but are not limited to, water, salt water, brine, drinking water, industrial water, brown water, sewerage, and mixtures thereof. Preferred non- biological fluids are drinking or industrial water or sewerage.

[0138] In some embodiments, the sample is pre-processed prior to contacting with the electrode or the sensor.Linkers

[0139] Embodiments of the various aspects described herein include a linker. For example, the target binding molecule can be linked to the surface of the conductive substrate via a linker. As used herein, the term “linker” means an organic moiety that connects two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR1, C(O), C(O)O, C(O)NR1, SO, SO2, SO2NH or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroaryl alkenyl alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroaryl alkenyl alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl alkenylheteroaryl, alkynylhereroaryl, where one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(RLL)2, C(O), cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R1and RLLindependently are is hydrogen, acyl, aliphatic or substituted aliphatic.

[0140] In some embodiments of any of the aspects, the linker is a flexible linker. As used herein, a “flexible linker” is a linker which does not have a fixed structure (secondary or tertiary structure) in solution and is therefore free to adopt a variety of conformations. Generally, a354936-2288-6248 1Attorney Docket No. : 002806-000143WOPT flexible linker has a plurality of freely rotating bonds along its backbone. In contrast, a rigid linker is a linker which adopts a relatively well-defined conformation when in solution. Rigid linkers are therefore those which have a particular secondary and / or tertiary structure in solution. In some embodiment, the linker is a rigid linker.Some selected definitions

[0141] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected herein. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0142] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the invention, the methods, devices, and materials in this regard are described herein.

[0143] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0144] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.364936-2288-6248 1Attorney Docket No. : 002806-000143WOPT

[0145] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not. In other words, except where the context requires otherwise, the term “comprise” and variations of the term, such as “comprising”, “comprises” and “comprised”, are not intended to exclude further additives, components, integers or steps.

[0146] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0147] As used herein, the term “binding” or “bound” generally refers to a reversible binding of one molecule to molecule via, e.g., van der Waals force, hydrophobic force, hydrogen bonding, and / or electrostatic force. The binding interaction between two molecules can be described by a dissociation constant (Ka) or association constant (K).

[0148] Specific elements of any of the disclosed embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.Exemplary embodiments

[0149] The technology may be described in any one of the following numbered Embodiments:

[0150] Embodiment 1 : A method for detecting a target analyte in a sample, the method comprising: (a) contacting a sample suspected of comprising a target analyte with a sensor, wherein the sensor comprises a conducting surface, and wherein the conductive surface comprises a target binding ligand capable of binding with the target analyte thereon; (b) inducing a non-equilibrium state in an electrical double layer (EDL) of the conducting surface and / or of the target analyte, and / or inhibiting / reducing a potential decay in the EDL of the conducting surface and / or of the target analyte, and / or increasing the overlap between the EDL of the conducting surface and EDL of the target analyte; (c) measuring an electrochemical signal from the conductive surface, and wherein a change in the electrochemical signal374936-2288-6248 1Attorney Docket No. : 002806-000143WQPT indicates binding of the target analyte with the target binding ligand, and wherein step (b) and (c) are carried out simultaneously.

[0151] Embodiment 2: The method of Embodiment 1, wherein the method comprises incubating the sample with sensor for a period of time prior to step (b).

[0152] Embodiment 3: The method of Embodiment 2, wherein said period of time is sufficient for target analyte binding to the target binding ligand.

[0153] Embodiment 4: The method of any one of Embodiments 1-3, wherein step (b) is performed after the target analyte binds to the target binding ligand.

[0154] Embodiment 5: The method of any one of Embodiments 1-4, wherein a means for inducing a non-equilibrium state in an EDL of the conducting surface and / or of the target analyte, and / or inhibiting / reducing the potential decay in the EDL of the conducting surface and / or of the target analyte, or increasing the overlap between the EDL of the conducting surface and EDL of the target analyte, is not applied to the sensor prior to the step of measuring an electrochemical signal.

[0155] Embodiment 6: The method of any one of Embodiments 1-5, wherein said inducing a non-equilibrium state in an EDL of the conducting surface and / or of the target analyte, and / or inhibiting / reducing the potential decay in the EDL of the conducting surface and / or of the target analyte, or increasing the overlap between the EDL of the conducting surface and EDL of the target analyte comprises applying AC voltage, DC voltage or potential sweep to the sensor.

[0156] Embodiment 7: The method of Embodiment 6, wherein said inducing a nonequilibrium state in an EDL of the conducting surface and / or of the target analyte, and / or inhibiting / reducing the potential decay in the EDL of the conducting surface and / or of the target analyte, or increasing the overlap between the EDL of the conducting surface and EDL of the target analyte comprises applying AC voltage to the sensor.

[0157] Embodiment 8: The method of any one of Embodiments 1-7, wherein the conductive surface is a surface of an electrode (i.e., primary, working, electrode).

[0158] Embodiment 9: The method of any one of Embodiments 1-8, wherein the sensor further comprises a secondary, non-working, electrode.

[0159] Embodiment 10: The method of any one of Embodiments 1-9, wherein the working and non-working electrodes are coplanar.

[0160] Embodiment 11 : The method of any one of Embodiments 1-10, wherein the nonworking electrode is positioned on either side of the working electrode.

[0161] Embodiment 12: The method of any one of Embodiments 1-11, wherein the nonworking electrode is positioned in a top-down configuration to the working electrode.384936-2288-6248 1Attorney Docket No. : 002806-000143WOPT

[0162] Embodiment 13: The method of any one of Embodiments 1-12, wherein the nonworking electrode and working electrode are in a radial configuration to each other.

[0163] Embodiment 14: The method of any one of Embodiments 9-13 wherein a distance between the working and non-working electrode is from about 0.1 cm to about 50 cm.

[0164] Embodiment 15: The method of any one of Embodiments 1-14, wherein said inducing a non-equilibrium state in an EDL of the conducting surface and / or of the target analyte, and / or inhibiting / reducing the potential decay in the EDL of the conducting surface and / or of the target analyte, or increasing the overlap between the EDL of the conducting surface and EDL of the target analyte comprises applying AC voltage, DC voltage or potential sweep to the non-working electrode.

[0165] Embodiment 16: The method of Embodiment 15, wherein said inducing a nonequilibrium state in an EDL of the conducting surface and / or of the target analyte, and / or inhibiting / reducing the potential decay in the EDL of the conducting surface and / or of the target analyte, or increasing the overlap between the EDL of the conducting surface and EDL of the target analyte comprises applying AC voltage to the non-working electrode.

[0166] Embodiment 17: The method of any one of Embodiments 1-16, wherein the AC voltage has a frequency between 10 Hz - 100,000 Hz.

[0167] Embodiment 18: The method of any one of Embodiments 1-17, wherein the AC voltage has a frequency between 1 Hz - 200,000 Hz.

[0168] Embodiment 19: The method of any one of Embodiments 1-18, wherein the AC voltage has a frequency between 50 Hz - 10,000 Hz.

[0169] Embodiment 20: The method of any one of Embodiments 1-19, wherein the AC voltage has a frequency between 100 Hz - 1,000 Hz.

[0170] Embodiment 21 : The method of any one of Embodiments 1-20, wherein the AC voltage has a frequency of 100 Hz.

[0171] Embodiment 22: The method of any one of Embodiments 1-21, wherein the AC voltage has a potential between 100 mV to 500 mV.

[0172] Embodiment 23: The method of any one of Embodiments 1-22, wherein the AC voltage has a potential between 10 mV to 1000 mV.

[0173] Embodiment 24: The method of any one of Embodiments 1-23, wherein the AC voltage has a potential between mV 200 to 400 mV.

[0174] Embodiment 25: The method of any one of Embodiments 1-24, wherein the AC voltage has a potential between 250 mV to 350 mV.394936-2288-6248 1Attorney Docket No. : 002806-000143WOPT

[0175] Embodiment 26: The method of any one of Embodiments 1-25, wherein the AC voltage has a potential of 300 mV to 500 mV.

[0176] Embodiment 27: The method of any one of Embodiments 1-26, wherein the change in electrochemical signal is a change in an electrochemical property.

[0177] Embodiment 28: The method of Embodiment 27, wherein the change in an electrochemical property is a change in impedance, a change in capacitance, a change in current, a change in voltammetry, a change in reactance, or a change in resistance.

[0178] Embodiment 29: The method of any one of Embodiments 1-28, wherein measuring the electrochemical signal comprises Electrochemical Impedance Spectroscopy (EIS), Potentiodynamic technique, Linear polarization, Voltammetric methods, Amperometric methods, Coulometric methods.

[0179] Embodiment 30: The method of any one of Embodiments 1-29, wherein measuring the electrochemical signal comprises Electrochemical Impedance Spectroscopy.

[0180] Embodiment 31 : The method of any one of Embodiments 1-30, wherein the conductive surface comprises a coating layer thereon.

[0181] Embodiment 32: The method of any one of Embodiments 1-31, wherein the coating comprises a polymeric or proteinaceous material.

[0182] Embodiment 33: The method of any one of Embodiment 1-32, wherein the coating layer covers at least a non-target binding portion of the target-binding molecule.

[0183] Embodiment 34: The method of Embodiment 33, wherein the coating layer forms an insulating or non-conducting layer between a target-binding portion or site of the targetbinding molecule and the surface of the conductive substrate.

[0184] Embodiment 35: The method of any one of Embodiments 31-34, wherein a targetbinding portion or site of the target-binding molecule is not embedded within the coating layer.

[0185] Embodiment 36: The method of any one of Embodiments 31-35, wherein a targetbinding portion or site of the target-binding molecule is exposed for contact with the target analyte, and wherein said contact with the target analyte is not within the polymeric coating layer.

[0186] Embodiment 37: The method of any one of Embodiments 31-36, wherein a targetbinding portion or site of the target-binding molecule is at a surface of the coating layer.

[0187] Embodiment 38: The method of any one of Embodiments 31-37, wherein the coating layer does not cover a target-binding portion or site of the target-binding molecule.404936-2288-6248 1Attorney Docket No. : 002806-000143WOPT

[0188] Embodiment 39: The method of any one of Embodiments 31-38, wherein a targetbinding portion or site of the target-binding molecule is at a surface of the coating layer, and wherein said surface of the coating layer is not a surface of a pore in the coating layer.

[0189] Embodiment 40: The method of any one of Embodiments 31-39, wherein a targetbinding portion or site of the target-binding molecule is at a surface of the coating layer, and wherein said surface of the coating layer is a surface of a pore in the coating layer.

[0190] Embodiment 41 : The method of any one of Embodiments 31-40, wherein the coating layer reduces or inhibits mobility ions in a liquid solution to the surface of the conductive substrate when the electrode is disposed in said liquid solution.

[0191] Embodiment 42: The method of any one of Embodiments 31-41, wherein the coating layer reduces or inhibits direct contact between ions in a liquid solution and the surface of the conductive substrate when the electrode is disposed in said liquid solution.

[0192] Embodiment 43: The method of any one of Embodiments 31-42, wherein the coating layer has a porosity of about 10% (e.g., about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3% about 2.5%, about 2%, about 1.5%, about 1%, about 0.75%, about 0.5%) or lower.

[0193] Embodiment 44: The method of any one of Embodiments 31-43, wherein the coating layer is substantially non-porous.

[0194] Embodiment 45: The method of any one of Embodiments 31-44, wherein the coating layer completely covers the surface of the conductive substrate.

[0195] Embodiment 46: The method of any one of Embodiments 31-45, wherein a thickness of the coating layer is about 95% or less of a distance between a target-binding portion or site of the target-binding molecule and the surface of the conductive substrate.

[0196] Embodiment 47: The method of any one of Embodiments 31-46, wherein a thickness of the coating layer is at least about 1 nm.

[0197] Embodiment 48: The method of any one of Embodiments 31-47, wherein a thickness of the coating layer is about 20 nm or lower.

[0198] Embodiment 49: The method of any one of Embodiments 31-48, wherein the coating layer comprises an electropolymerized polymer, photopolymerized polymer, or autopolymerized polymer.

[0199] Embodiment 50: The method of any one of Embodiments 31-49, wherein the coating layer comprises an electropolymerized polymer.

[0200] Embodiment 51 : The method of any one of Embodiments 31-50, wherein the coating layer comprises a polymer selected from the group consisting of polysaccharides,414936-2288-6248 1Attorney Docket No. : 002806-000143WOPT polydopamines, poly-p-phenylenes, polypyrroles, coumarin based polymers, polyhydroxyethylmethacrylates, polyindolyenes, polyanilines, polythiophenes, poly(ethylenedioxy) thiophene, poly(heteroaromatic vinylenes), polyvinylphosphates, poly-p- phenylene sulfides, poly(2,5-thienylenes), poly-o-phenylenes, poly(o-phenylene diamines), polysaccharides, polyacetylenes, porphyrins, phthalocyanins, fullerenes, polyparaphenylenes, polyphenylenevinylenes, polyfluorenes, polypyridines, polycarbazoles, polypyridinevinylenes, polyarylvinylenes, poly (p-phenylmethylvinylenes), derivatives and co-polymers thereof, and any combination thereof.

[0201] Embodiment 52: The method of any one of Embodiments 31-51, wherein the coating layer comprises poly dopamine, chitosan, or scopoletin (6-methoxy-7- hydroxy coumarin).

[0202] Embodiment 53: The method of any one of Embodiments 31-52, wherein the coating layer comprises electropolymerized polydopamine, electropolymerized chitosan, electropolymerized scopoletin, or autopolymerized chitosan.

[0203] Embodiment 54: The method of any one of Embodiments 31-53, wherein the coating layer further comprises a dissolvable or degradable material.

[0204] Embodiment 55: The method of any one of Embodiment 54, wherein the dissolvable or degradable material is selected from the group consisting of water dissolvable polymers (salt crystals), materials capable of being removed by degradation (e.g., proteins or protein aggregates), temperature dependent polymers (e.g., poly(N-isopropyl acrylamide) (PNIPAAm)); and any combination thereof.

[0205] Embodiment 56: The method of any one of Embodiments 31-55, wherein the coating layer comprises an antifouling material.

[0206] Embodiment 57: The method of any one of Embodiment 56, wherein the antifouling material is ethanolamine (ETA), hyaluronic acid (HA), or poly vinyl alcohol (PVA).

[0207] Embodiment 58: The method of any one of Embodiments 1-57, wherein the conductive substrate comprises a flexible substrate.

[0208] Embodiment 59: The method of any one of Embodiments 1-58, wherein the conductive substrate comprises a flexible substrate selected from the group consisting of polyethylene terephthalate, polyethylene naphathalate, polyimides, polymeric hydrocarbons, celluloses, plastics, polycarbonates, polystyrenes, silicon and any combination thereof

[0209] Embodiment 60: The method of any one of Embodiments 1-59, wherein the targetbinding molecule is covalently linked to the surface of the conductive substrate.424936-2288-6248 1Attorney Docket No. : 002806-000143WOPT

[0210] Embodiment 61 : The method of any one of Embodiments 1-60, wherein the targetbinding molecule is linked to the surface of the conductive substrate via a linker

[0211] Embodiment 62: The method of any one of Embodiments 1-61, wherein the sensor comprises a fluid-contact surface and at least a portion of the fluid-contact surface is the conductive surface.

[0212] Embodiment 63: The method of any one of Embodiments 1-62, wherein the sensor comprises one or more microfluidic flow cells.

[0213] Embodiment 64: The method of any one of Embodiments 1-63, wherein the sensor comprises one or more open well-cells.

[0214] Embodiment 65: The method of any one of Embodiments 1-64, wherein the fluidcontact surface further comprises a positive control electrode and / or a negative control electrode immobilized thereon

[0215] Embodiment 66: The method of any one of Embodiments 1-65, wherein the targetbinding molecule is a peptide, polypeptide, a nucleic acid (e.g., oligonucleotide or polynucleotide) or a small molecule.

[0216] Embodiment 67: The method of any one of Embodiments 1-66, wherein the target binding molecule is a receptor, a ligand for a receptor, an antibody, antigen binding fragment of an antibody, an antigen, an enzyme, an affimer or an aptamer

[0217] Embodiment 68: The method of any one of Embodiments 1-67, wherein the target binding molecule is an antibody, an antigen binding fragment of an antibody, or an antigen, optionally, the target binding molecule is an antibody or an antigen binding fragment of an antibody

[0218] Embodiment 69: The method of any one of Embodiments 1-68, wherein the target analyte is a peptide, a polypeptide, a peptidomimetic, a nucleic acid, an oligosaccharide, a polysaccharide, an amino acid, nucleoside, a nucleotide, a carbohydrate, a lipid, a peptidoglycan, a cell, microbial matter, an antigen, a lipid, a steroid, a hormone, a lipopolysaccharide, an endotoxin, a therapeutic agent, a lipid-binding molecule, a cofactor, a small molecule, a toxin, a biological threat agent (e.g., spore, viral, cellular and protein toxin), or any combination thereof.

[0219] Embodiment 70: The method of any one of Embodiments 1-69, wherein the target analyte is a protein, an antibody, an antigen binding fragment of an antibody, an antigen, a hormone, a nucleic acid, or a metabolite.

[0220] Embodiment 71 : The method of any one of Embodiments 1-70, wherein the target analyte is a tumor marker or a clinical chemistry target.434936-2288-6248 1Attorney Docket No. : 002806-000143WOPT

[0221] Embodiment 72: The method of any one of Embodiments 1-71, wherein the sample is a biological sample (e.g., blood, saliva, amniotic fluid, sputum, urine, semen, cerebrospinal fluid, bronchial aspirate, perspiration, mucus, liquefied stool sample, synovial fluid, lymphatic fluid, tears, tracheal aspirate, lactation product, breadth condensate any combination thereof).

[0222] Embodiment 73: The method of any one of Embodiments 1-72, wherein the sample is a food, an ingredient for preparing a food, poultry, meat, fish, beverage, food grain, a crop, or dairy product.

[0223] Embodiment 74: The method of any one of Embodiments 1-73, wherein the sample is a non-biological sample.

[0224] Embodiment 75: The method of any one of Embodiments 1-74, wherein the sample is pre-processed prior to contacting with the electrode or the sensor.

[0225] Embodiment 76: The method of any one of Embodiments 1-75, wherein the method comprises washing the sample and sensor prior to step (b).

[0226] Embodiment 77: The method of any one of Embodiments 1-76, wherein the method comprises washing the sample and sensor and adding a buffer prior to step (b).

[0227] Embodiment 78: The method of any one of Embodiments 1-77, wherein the nonworking electrode and working electrode are not coplanar.

[0228] Embodiment 79: The method of any one of Embodiments 1-78, wherein the nonworking electrode and working electrode are in an interdigitated configuration to each other.EXAMPLES

[0229] The following examples illustrate some embodiments and aspects of the invention. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be performed without altering the spirit or scope of the invention, and such modifications and variations are encompassed within the scope of the invention as defined in the claims which follow. The following examples do not in any way limit the invention.Example 1: Sensitivity enhancement strategy via non-equilibrium label-free sensing

[0230] The demand for point-of-care testing is rapidly growing and electrical sensor offers quick diagnostic testing. However, most of them suffers from charge screening effects in electrical double layer (EDL), where all charges are masked, making it challenging to detect target analytes. One potential approach is to disrupting double layer from reaching equilibrium444936-2288-6248 1Attorney Docket No. : 002806-000143WQPT state and to reduce the charge screening environment. Such an approach generates nonequilibrium condition that is desirable for sensitive recognition of targets in label-free sensing.

[0231] Electrochemical sensors play a pivotal role in detecting and quantifying various analytes, contributing to fields such as healthcare monitoring and point-of-care diagnostics. However, the sensitivity of these sensors can be compromised due to the formation of the EDL at the electrode-electrolyte interface. The EDL, a result of the accumulation of charged species near the electrode surface, induces charge screening effects, hindering the accurate recognition of antibody-antigen interaction and subsequently diminishing the sensitivity. This issue becomes particularly pronounced when dealing with low-concentration analytes or in environments with complex matrices, where the interference from ions exacerbates the charge screening effects.

[0232] In conventional sensors, potential decay has occurred in the EDL, which is derived from linearized Poisson-Boltzmann model that illustrates screening as an equilibrium balance between diffusion and drift. The Debye length (XD) is the characteristic distance over which potential decay occurs in electrolyte solutions. It is considerably shorter than typical biomolecular receptors, which is inherent limitations for all electrical sensors. To overcome these limitations, there is an increasing demand for fundamental approaches that can tackle the sensitivity challenges arising from potential decay in the EDL.

[0233] The inventors here report a novel method to increase sensitivity of electrical biosensor by addressing potential decay in EDL and generating non-equilibrium states during signal measurement. This method involves applying AC voltage to the interface between electropolymerzied surface and electrolyte solution for breaking up potential decay and inducing more interaction between electrode’s double layer and target molecule’s double layer.

[0234] A schematic of illustrating the change of electrochemical states by applying AC voltage is demonstrated in Fig. 1. The typical electrode-electrolyte interface possesses the potential decay in both electrode’s double layer (Electrode) and target’s double layer (^target) in equilibrium, which limits potential overlap between electrode and target plane and thus makes it challenging sensitive detection of target analytes. By applying AC voltage with a specific frequency towards the EDL disrupts the typical exponential decay. This leads to the formation of a dynamic double layer under non-equilibrium conditions, allowing for the accurate detection of potential changes induced by the target.

[0235] Preliminary experiments were performed to validate the AC voltage-driven sensitivity enhancement. The fabrication of the sensor has been described in detail in Zupancic, et al.1Briefly, the gold chips were incubated with lOmM DSP (dithiobis (succinimidyl454936-2288-6248 1Attorney Docket No. : 002806-000143WOPT propionate)) (Thermo Fisher Scientific, USA, 22585) solution for 60 min, which functionalizes sensor surface with thiol cross-linker and enables covalent linkage of the antibody. MIP-ip capture antibody (R&D systems, USA) of 100 pg / mL was immobilized for 1 hour. Then, scopoletin was polymerized using cyclic voltammetry (5 cycles, -0.2 - 0.5 V, 10 mV / s) on the sensor surface.

[0236] Sensitivity in the detection of MIP-ip was investigated by applying AC voltage (Figs. 2A-2E). AC voltage was applied to two gold electrodes positioned on either side of the central working electrode. After incubating serially diluted MIP-ip spiked in human plasma for 1 hour, impedance was measured in PBS without AC voltage. Following this, the same sensor was subjected to impedance measurement with AC voltage application (250 mV, 100 Hz). When comparing the changes in impedance before and after MIP-ip incubation in the Bode plot, the impedance increased at a concentration of 0 (Negative control) and 0.01 ng / mL without AC voltage, while decreasing at concentrations of 0.1 and 1 ng / mL. Upon applying AC voltage to the sensor, the impedance significantly decreased under all conditions, and the signal change increased with higher MIP-ip concentration. As a result, calibration curve showed that the detection limit, initially at 0.1 ng / mL when compared to the signal of the negative control, was significantly enhanced by more than one order of magnitude, reaching 0.01 ng / mL upon the application of AC voltage.

[0237] How the signal response varies based on the AC potential and frequency was also examined. By applying AC potential to both positive (0.1 ng / mL MIP-ip) and negative control (no MIP-ip), the inventors aimed to observe specific signal changes under positive conditions. Regarding frequency, the inventors tested the 10-100,000 Hz range at a 300mV applied potential, showing higher signal responses for positive samples compared to negative samples at all tested frequencies (Fig.3A). Particularly, the signal response proportionally increased with increasing frequencies. For AC potential, we explored the 100~500mV range at a constant frequency of 100 Hz. Notably, at 100 mV and 500 mV, we observed higher specific signals compared to the negative control (Fig. 3B). Therefore, we confirmed that the frequency and AC potential play a major role in enhancing the sensitivity of the label-free sensor. Without wishing to be bound by a theory, this enhancement is attributed to the generation of a more pronounced overlap between TLiectrode and Target in a non-equilibrium state.REFERENCES:464936-2288-6248 1Attorney Docket No. : 002806-000143WOPT1. Zupancic, U., Jolly, P., Estrela, P., Moschou, D., & Ingber, D. E. (2021). Graphene Enabled Low-Noise Surface Chemistry for Multiplexed Sepsis Biomarker Detection in Whole Blood. Advanced Functional Materials, 3 / ( 16), 2010638.474936-2288-6248 1

Claims

Attorney Docket No. : 002806-000143WQPTCLAIMSWhat is claimed is:

1. A method for detecting a target analyte in a sample, the method comprising: a. contacting a sample suspected of comprising a target analyte with a sensor, wherein the sensor comprises a conducting surface, and wherein the conductive surface comprises a target binding ligand capable of binding with the target analyte thereon; b. inducing a non-equilibrium state in an electrical double layer (EDL) of the conducting surface and / or of the target analyte, and / or inhibiting / reducing a potential decay in the EDL of the conducting surface and / or of the target analyte, and / or increasing the overlap between the EDL of the conducting surface and EDL of the target analyte; c. measuring an electrochemical signal from the conductive surface, and wherein a change in the electrochemical signal indicates binding of the target analyte with the target binding ligand, and wherein step (b) and (c) are carried out simultaneously.

2. The method of claim 1, wherein the method comprises incubating the sample with sensor for a period of time prior to step (b).

3. The method of claim 2, wherein said period of time is sufficient for target analyte binding to the target binding ligand.

4. The method of any one of claims 1-3, wherein step (b) is performed after the target analyte binds to the target binding ligand.

5. The method of any one of claims 1-4, wherein a means for inducing a non-equilibrium state in an EDL of the conducting surface and / or of the target analyte, and / or inhibiting / reducing the potential decay in the EDL of the conducting surface and / or of the target analyte, or increasing the overlap between the EDL of the conducting surface and EDL of the target analyte, is not applied to the sensor prior to the step of measuring an electrochemical signal.

6. The method of any one of claims 1-5, wherein said inducing a non-equilibrium state in an EDL of the conducting surface and / or of the target analyte, and / or inhibiting / reducing the potential decay in the EDL of the conducting surface and / or of the target analyte, or484936-2288-6248 1Attorney Docket No. : 002806-000143WOPT increasing the overlap between the EDL of the conducting surface and EDL of the target analyte comprises applying AC voltage, DC voltage or potential sweep to the sensor.

7. The method of claim 6, wherein said inducing a non-equilibrium state in an EDL of the conducting surface and / or of the target analyte, and / or inhibiting / reducing the potential decay in the EDL of the conducting surface and / or of the target analyte, or increasing the overlap between the EDL of the conducting surface and EDL of the target analyte comprises applying AC voltage to the sensor.

8. The method of any one of claims 1-7, wherein the conductive surface is a surface of an electrode (i.e., primary, working, electrode).

9. The method of any one of claims 1-8, wherein the sensor further comprises a secondary, non-working, electrode.

10. The method of any one of claims 1-9, wherein the working and non-working electrodes are coplanar.

11. The method of any one of claims 1-10, wherein the non-working electrode is positioned on either side of the working electrode.

12. The method of any one of claims 1-11, wherein the non-working electrode is positioned in a top-down configuration to the working electrode.

13. The method of any one of claims 1-12, wherein the non-working electrode and working electrode are in a radial configuration to each other.

14. The method of any one of claims 9-13 wherein a distance between the working and nonworking electrode is from about 0.1 cm to about 50 cm.

15. The method of any one of claims 1-14, wherein said inducing a non-equilibrium state in an EDL of the conducting surface and / or of the target analyte, and / or inhibiting / reducing the potential decay in the EDL of the conducting surface and / or of the target analyte, or increasing the overlap between the EDL of the conducting surface and EDL of the target analyte comprises applying AC voltage, DC voltage or potential sweep to the non-working electrode.

16. The method of claim 15, wherein said inducing a non-equilibrium state in an EDL of the conducting surface and / or of the target analyte, and / or inhibiting / reducing the potential decay in the EDL of the conducting surface and / or of the target analyte, or increasing the overlap between the EDL of the conducting surface and EDL of the target analyte comprises applying AC voltage to the non-working electrode.494936-2288-6248 1Attorney Docket No. : 002806-000143WOPT17. The method of any one of claims 1-16, wherein the AC voltage has a frequency between 10 Hz - 100,000 Hz.

18. The method of any one of claims 1-17, wherein the AC voltage has a frequency between1 Hz - 200,000 Hz.

19. The method of any one of claims 1-18, wherein the AC voltage has a frequency between 50 Hz - 10,000 Hz.

20. The method of any one of claims 1-19, wherein the AC voltage has a frequency between 100 Hz - 1,000 Hz.

21. The method of any one of claims 1-20, wherein the AC voltage has a frequency of 100 Hz.

22. The method of any one of claims 1-21, wherein the AC voltage has a potential between 100 mV to 500 mV.

23. The method of any one of claims 1-22, wherein the AC voltage has a potential between10 mV to 1000 mV.

24. The method of any one of claims 1-23, wherein the AC voltage has a potential between mV 200 to 400 mV.

25. The method of any one of claims 1-24, wherein the AC voltage has a potential between 250 mV to 350 mV.

26. The method of any one of claims 1-25, wherein the AC voltage has a potential of 300 mV to 500 mV.

27. The method of any one of claims 1-26, wherein the change in electrochemical signal is a change in an electrochemical property.

28. The method of claim 27, wherein the change in an electrochemical property is a change in impedance, a change in capacitance, a change in current, a change in voltammetry, a change in reactance, or a change in resistance.

29. The method of any one of claims 1-28, wherein measuring the electrochemical signal comprises Electrochemical Impedance Spectroscopy (EIS), Potentiodynamic technique, Linear polarization, Voltammetric methods, Amperometric methods, Coulometric methods.

30. The method of any one of claims 1-29, wherein measuring the electrochemical signal comprises Electrochemical Impedance Spectroscopy.

31. The method of any one of claims 1-30, wherein the conductive surface comprises a coating layer thereon.504936-2288-6248 1Attorney Docket No. : 002806-000143WOPT32. The method of any one of claims 1-31, wherein the coating comprises a polymeric or proteinaceous material.

33. The method of any one of claim 1-32, wherein the coating layer covers at least a nontarget binding portion of the target-binding molecule.

34. The method of claim 33, wherein the coating layer forms an insulating or non-conducting layer between a target-binding portion or site of the target-binding molecule and the surface of the conductive substrate.

35. The method of any one of claims 31-34, wherein a target-binding portion or site of the target-binding molecule is not embedded within the coating layer.

36. The method of any one of claims 31-35, wherein a target-binding portion or site of the target-binding molecule is exposed for contact with the target analyte, and wherein said contact with the target analyte is not within the polymeric coating layer.

37. The method of any one of claims 31-36, wherein a target-binding portion or site of the target-binding molecule is at a surface of the coating layer.

38. The method of any one of claims 31-37, wherein the coating layer does not cover a targetbinding portion or site of the target-binding molecule.

39. The method of any one of claims 31-38, wherein a target-binding portion or site of the target-binding molecule is at a surface of the coating layer, and wherein said surface of the coating layer is not a surface of a pore in the coating layer.

40. The method of any one of claims 31-39, wherein a target-binding portion or site of the target-binding molecule is at a surface of the coating layer, and wherein said surface of the coating layer is a surface of a pore in the coating layer.

41. The method of any one of claims 31-40, wherein the coating layer reduces or inhibits mobility ions in a liquid solution to the surface of the conductive substrate when the electrode is disposed in said liquid solution.

42. The method of any one of claims 31-41, wherein the coating layer reduces or inhibits direct contact between ions in a liquid solution and the surface of the conductive substrate when the electrode is disposed in said liquid solution.

43. The method of any one of claims 31-42, wherein the coating layer has a porosity of about 10% (e.g., about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3% about 2.5%, about 2%, about 1.5%, about 1%, about 0.75%, about 0.5%) or lower.

44. The method of any one of claims 31-43, wherein the coating layer is substantially non- porous.514936-2288-6248 1Attorney Docket No. : 002806-000143WOPT45. The method of any one of claims 31-44, wherein the coating layer completely covers the surface of the conductive substrate.

46. The method of any one of claims 31-45, wherein a thickness of the coating layer is about 95% or less of a distance between a target-binding portion or site of the target-binding molecule and the surface of the conductive substrate.

47. The method of any one of claims 31-46, wherein a thickness of the coating layer is at least about 1 nm.

48. The method of any one of claims 31-47, wherein a thickness of the coating layer is about 20 nm or lower.

49. The method of any one of claims 31-48, wherein the coating layer comprises an electropolymerized polymer, photopolymerized polymer, or auto-polymerized polymer.

50. The method of any one of claims 31-49, wherein the coating layer comprises an electropolymerized polymer.

51. The method of any one of claims 31-50, wherein the coating layer comprises a polymer selected from the group consisting of polysaccharides, polydopamines, poly-p-phenylenes, polypyrroles, coumarin based polymers, polyhydroxyethylmethacrylates, polyindolyenes, polyanilines, polythiophenes, poly(ethylenedioxy) thiophene, poly(heteroaromatic vinylenes), polyvinylphosphates, poly-p-phenylene sulfides, poly(2,5-thienylenes), poly-o-phenylenes, poly(o-phenylene diamines), polysaccharides, polyacetylenes, porphyrins, phthalocyanins, fullerenes, polyparaphenylenes, polyphenylenevinylenes, polyfluorenes, polypyridines, polycarbazoles, polypyridinevinylenes, polyarylvinylenes, poly (p-phenylmethylvinylenes), derivatives and co-polymers thereof, and any combination thereof.

52. The method of any one of claims 31-51, wherein the coating layer comprises polydopamine, chitosan, or scopoletin (6-methoxy-7-hydroxycoumarin).

53. The method of any one of claims 31-52, wherein the coating layer comprises electropolymerized polydopamine, electropolymerized chitosan, electropolymerized scopoletin, or autopolymerized chitosan.

54. The method of any one of claims 31-53, wherein the coating layer further comprises a dissolvable or degradable material.

55. The method of any one of claim 54, wherein the dissolvable or degradable material is selected from the group consisting of water dissolvable polymers (salt crystals), materials capable of being removed by degradation (e.g., proteins or protein aggregates), temperature dependent polymers (e.g., poly(N-isopropyl acrylamide) (PNIPAAm)); and any combination thereof.524936-2288-6248 1Attorney Docket No. : 002806-000143WOPT56. The method of any one of claims 31-55, wherein the coating layer comprises an antifouling material.

57. The method of any one of claim 56, wherein the antifouling material is ethanolamine (ETA), hyaluronic acid (HA), or poly vinyl alcohol (PVA).

58. The method of any one of claims 1-57, wherein the conductive substrate comprises a flexible substrate.

59. The method of any one of claims 1-58, wherein the conductive substrate comprises a flexible substrate selected from the group consisting of polyethylene terephthalate, polyethylene naphathalate, polyimides, polymeric hydrocarbons, celluloses, plastics, polycarbonates, polystyrenes, silicon and any combination thereof60. The method of any one of claims 1-59, wherein the target-binding molecule is covalently linked to the surface of the conductive substrate.

61. The method of any one of claims 1-60, wherein the target-binding molecule is linked to the surface of the conductive substrate via a linker62. The method of any one of claims 1-61, wherein the sensor comprises a fluid-contact surface and at least a portion of the fluid-contact surface is the conductive surface.

63. The method of any one of claims 1-62, wherein the sensor comprises one or more microfluidic flow cells.

64. The method of any one of claims 1-63, wherein the sensor comprises one or more open well-cells.

65. The method of any one of claims 1-64, wherein the fluid-contact surface further comprises a positive control electrode and / or a negative control electrode immobilized thereon66. The method of any one of claims 1-65, wherein the target-binding molecule is a peptide, polypeptide, a nucleic acid (e.g., oligonucleotide or polynucleotide) or a small molecule.

67. The method of any one of claims 1-66, wherein the target binding molecule is a receptor, a ligand for a receptor, an antibody, antigen binding fragment of an antibody, an antigen, an enzyme, an affimer or an aptamer534936-2288-6248 1Attorney Docket No. : 002806-000143WOPT68. The method of any one of claims 1-67, wherein the target binding molecule is an antibody, an antigen binding fragment of an antibody, or an antigen, optionally, the target binding molecule is an antibody or an antigen binding fragment of an antibody69. The method of any one of claims 1-68, wherein the target analyte is a peptide, a polypeptide, a peptidomimetic, a nucleic acid, an oligosaccharide, a polysaccharide, an amino acid, nucleoside, a nucleotide, a carbohydrate, a lipid, a peptidoglycan, a cell, microbial matter, an antigen, a lipid, a steroid, a hormone, a lipopolysaccharide, an endotoxin, a therapeutic agent, a lipid-binding molecule, a cofactor, a small molecule, a toxin, a biological threat agent (e.g., spore, viral, cellular and protein toxin), or any combination thereof.

70. The method of any one of claims 1-69, wherein the target analyte is a protein, an antibody, an antigen binding fragment of an antibody, an antigen, a hormone, a nucleic acid, or a metabolite.

71. The method of any one of claims 1-70, wherein the target analyte is a tumor marker or a clinical chemistry target.

72. The method of any one of claims 1-71, wherein the sample is a biological sample (e.g., blood, saliva, amniotic fluid, sputum, urine, semen, cerebrospinal fluid, bronchial aspirate, perspiration, mucus, liquefied stool sample, synovial fluid, lymphatic fluid, tears, tracheal aspirate, lactation product, breadth condensate any combination thereof).

73. The method of any one of claims 1-72, wherein the sample is a food, an ingredient for preparing a food, poultry, meat, fish, beverage, food grain, a crop, or dairy product.

74. The method of any one of claims 1-73, wherein the sample is a non-biological sample.

75. The method of any one of claims 1-74, wherein the sample is pre-processed prior to contacting with the electrode or the sensor.

76. The method of any one of claims 1-75, wherein the method comprises washing the sample and sensor prior to step (b).

77. The method of any one of claims 1-76, wherein the method comprises washing the sample and sensor and adding a buffer prior to step (b).

78. The method of any one of claims 1-77, wherein the non-working electrode and working electrode are not coplanar.

79. The method of any one of claims 1-78, wherein the non-working electrode and working electrode are in an interdigitated configuration to each other.544936-2288-6248 1

Images