Improved electrochemical biosensors

Abstract

Electrochemical biosensors are described, such as inverted molecular pendulums (iMP) comprising a charged polymer bound to a receptor and to a redox reporter, wherein the iMP is operationally affixed to an electrode. The electrochemical biosensors are configured to detect analytes, such as analytes present in biological or other fluids. Also described are biosensor devices comprising such biosensors, and methods of using the biosensors and devices to detect the analytes.

Description

[0001] IMPROVED ELECTROCHEMICAL BIOSENSORS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Nos.

[0004] 63 / 712,342, filed October 25, 2024, and 63 / 759,729, filed February 18, 2025, which are incorporated by reference herein in their entireties.

[0005] TECHNICAL FIELD

[0006] The present disclosure relates to electrochemical biosensors configured to detect analytes, such as analytes present in biological or other fluids, biosensor devices comprising such biosensors, and methods of using the biosensors and devices to detect the analytes.

[0007] BACKGROUND

[0008] There is a need for improved biosensor devices, including improved electrochemical biosensors that are reusable, durable, and tunable such that the biosensors can be configured to operate using various receptor configurations and electrochemical monitoring modalities. However, the development of such improved biosensor devices is challenging.

[0009] SUMMARY

[0010] The present disclosure relates in several embodiments to improved electrochemical biosensors, devices, and methods of using the biosensors.

[0011] According to a first aspect, the present disclosure relates in several embodiments to an electrochemical biosensor. The electrochemical biosensor includes:

[0012] a plurality of charged polymers, each charged polymer having first and second ends; an electrode, wherein the first end of each charged polymer is operationally affixed to the electrode, and wherein the second end of each charged polymer is structured to bind to a receptor for a target analyte; and

[0013] a first redox reporter operationally bound to the charged polymer, wherein the first redox reporter is reactive at positive potential when the charged polymer presents a net negative charge and reactive at negative potential when the charged polymer presents a net positive charge; and

[0014] wherein upon application of an electric field and binding of the charged polymer to a receptor, the biosensor is characterized by: an unbound state of the charged polymers, where no target analyte is bound to the receptor, at which the charged polymers are displaced towards the biosensor electrode surface and electron transfer from the charged polymers towards the biosensor electrode occurs at an unbound electron transfer rate; and a bound state of the charged polymers, where the target analyte is bound to the receptor, at which the charged polymers are displaced towards the biosensor electrode surface and electron transfer from the charged polymers towards the biosensor electrode occurs at a bound electron transfer rate.

[0015] The electrochemical biosensor may include the following details, which can be combined with one another in any combinations unless clearly mutually exclusive:

[0016] (i) an inverted molecular pendulum (iMP) may include the charged polymer bound to the receptor and to the first redox reporter, and the iMP may be operationally affixed to the electrode.

[0017] (ii) the electrode may be multilayered.

[0018] (iii) the electrode may include a gold layer and a parylene coating layer.

[0019] (iv) the charged polymers may be operationally affixed to the electrode by covalent bonding via an intermediate linking moiety.

[0020] (v) each charged polymer may be structured to bind to the receptor via an adapter, wherein the adapter has a first portion configured to bind to the second end of the polymer and the adapter has a second portion configured to bind to the receptor.

[0021] (vi) upon binding of the target analyte to the receptor at the applied electric field, an electrochemical signal may be produced translating a difference between the unbound electron transfer rate and the bound electron transfer rate.

[0022] (vii) upon application of the electric field, the first redox reporter may cause an electron transfer as the iMPs approach the biosensor electrode surface.

[0023] (viii) the electron transfer rate may be dependent on a time rate at which the iMPs are displaced.

[0024] (ix) the unbound electron transfer rate may be dependent on a time rate at which the unbound iMPs are displaced.

[0025] (x) the bound electron transfer rate may be dependent on a time rate at which the bound iMPs are displaced.

[0026] (xi) the iMPs displacement towards the biosensor electrode surface may substantially correspond to a tilting movement or a collapsing movement of the iMPs. (xii) upon application of the electric field, the first redox reporter may touch the biosensor electrode surface, and the electron transfer may be based on a redox reaction or electron tunneling current.

[0027] (xiii) the first redox reporter may be bound to the charged polymer proximal to the second end thereof.

[0028] (xiv) the first redox reporter may be covalently bound to the charged polymer.

[0029] (xv) the charged polymer may include a double-stranded DNA (dsDNA), singlestranded DNA (ssDNA), one or more charged polymers, one or more uncharged polymers, or any combination thereof.

[0030] (xvi) the charged polymer may be negatively charged and optionally comprises a DNA / DNA duplex, a PNA / DNA duplex, a PNA / PNA duplex, optionally wherein one or both of the PNA are modified with negatively charged amino acids, a rigid anionic polyelectrolyte, a rigid negatively charged peptide, or any combination thereof.

[0031] (xvii) the first redox reporter may include ferrocene, [Co(GA)2(phen)] (GA=glycolic acid, phen=l, 10-phenathroline), metal nanoparticles (e.g., Au, Pt, Pd, Ag, Cu), pyrroloquinoline quinone (PQQ), benzoquine, Osmium(III) complexes such as Os(bpy)Ci23+, diphenylamine, or any combination thereof.

[0032] (xviii) the first redox reporter may have a redox state change above 0 mV.

[0033] (xix) the charged polymer may be positively charged and optionally comprises a PNA / PNA duplex with lysines, a rigid cationic polyelectrolyte, a rigid positively charged peptide, or any combination thereof.

[0034] (xx) the first redox reporter may include methylene blue, ruthenium(III) complexes such as Ru(NH3)e3+, neutral red, toluidine blue, phenosafranine, or any combination thereof.

[0035] (xxi) the first redox reporter may have a redox state change below 0 mV.

[0036] (xxii) the charged polymer may have a length ranging from about 5 nm to about 20 nm. (xxiii) the charged polymer may include a ssDNA having a length ranging from about lOmer to about lOOmer.

[0037] (xxiv) the charged polymer may include a dsDNA having a length ranging from about 15mer to about 60mer.

[0038] (xxv) the charged polymer may be rigid along a length thereof, flexible or collapsible along the length thereof, or has a combination of one or more rigid and flexible or collapsible portions along the length thereof.

[0039] (xxvi) the iMPs may form a molecular monolayer at the surface of the biosensor electrode. (xxvii) the receptor may include an antibody, an antibody receptor, a nanobody, an antigen, an aptamer, an aptamer fragment, a molecular imprint, a protein receptor, DNA, a microorganism, a protein / enzyme substrate, or any combination thereof.

[0040] (xxviii) the antibody receptor may include an Fc receptor.

[0041] (xxix) the biosensor electrode may include a glassy carbon electrode, a carbon nanotube-modified electrode, an indium tin oxide (ITO) electrode, a platinum electrode, a silver electrode, a gold electrode, or a palladium electrode.

[0042] (xxx) the biosensor electrode may include a gold nanostructured microelectrode or a gold wire electrode.

[0043] (xxxi) the target analyte may include a small molecule, a macromolecule, a prokaryotic cell, a eukaryotic cell, a prokaryotic cell-derived component, a eukaryotic cell-derived component (e.g., nucleic acid material), a virus, a bacterium, an antibody, a protein, a cellular extract, or any combination thereof.

[0044] (xxxii) the biosensor may include a second redox reporter bound to the charged polymer proximal to the first end thereof.

[0045] (xxxiii) the second redox reporter may be covalently bound to the charged polymer. (xxxiv) the second redox reporter may be reactive at a different potential than the first redox reporter.

[0046] (xxxv) upon binding of the first end of the charged polymer to the surface of the biosensor electrode, the second redox reporter may cause an electron transfer to the biosensor electrode surface.

[0047] (xxxvi) the plurality of iMPs may be bound to the surface of the biosensor electrode at a density sufficient to sterically hinder the iMPs bound state from being displaced towards the biosensor electrode.

[0048] (xxxvii) the application of the electric field may be in chronoamperometric configuration, by square wave voltammetry, or selectably in either chronoamperometric configuration or by square wave voltammetry.

[0049] According to a second aspect, the present disclosure relates in several embodiments to a method of detecting a target analyte in a sample. The method includes:

[0050] providing an electrochemical biosensor described herein;

[0051] contacting said biosensor with the sample; and

[0052] detecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte; and optionally wherein the receptor is an antibody or a fragment thereof, wherein the binding of the target analyte to the antibody or fragment thereof occurs when the sample is at a first temperature and / or a first pH, and dissociation of the target analyte occurs at a second temperature, the method further comprises:

[0053] (i) optionally applying heat and / or ionic molecules to the sample, thereby causing the sample to have the second temperature and / or a second pH sufficient to result in the dissociation;

[0054] (ii) optionally applying a wash solution to remove the sample from the biosensor; and

[0055] optionally contacting the biosensor with a further sample and detecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte bound to antibody or a fragment thereof.

[0056] According to a third aspect, the present disclosure relates in several embodiments to method for in situ detection of a target analyte in a biological fluid. The method includes: contacting an electrochemical biosensor described herein with the biological fluid; and detecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte.

[0057] According to a fourth aspect, the present disclosure relates in several embodiments to the use of an electrochemical biosensor described herein to detect the presence of a target analyte in a sample. The sample may be a biological fluid. The biological fluid may be saliva, blood, urine, tears, sweat or feces.

[0058] According to a fifth aspect, the present disclosure relates in several embodiments to a kit including a biosensor described herein, wherein the receptor is an Fc receptor. The kit further includes one or more antibodies or fragments thereof, comprising: an antigen-binding site adapted to specifically bind to a target analyte; and an Fc region or Fc-receptor binding fragment thereof.

[0059] According to a sixth aspect, the present disclosure relates in several embodiments to a method of detecting one or more target analytes in a sample. The method includes providing a kit described herein; contacting the biosensor with the sample and the one or more antibodies or fragments thereof; and

[0060] detecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte bound to the one or more antibodies or fragments thereof.

[0061] The method may include the following details, which can be combined with one another in any combinations unless clearly mutually exclusive:

[0062] (i) the detecting the electrochemical signal may include the one or more antibodies or fragments thereof binding to the one or more target analytes and to the Fc receptor.

[0063] (ii) the application of the electric field may be in chronoamperometric configuration, by square wave voltammetry, or selectably in either chronoamperometric configuration or by square wave voltammetry.

[0064] (iii) the binding of the one or more antibodies or fragments thereof to the one or more target analytes and to the Fc receptor may occur when the sample is at a first temperature and / or a first pH, and dissociation of one or more antibodies or fragments thereof to the one or more target analytes and to the Fc receptor occurs at a second temperature.

[0065] (iv) The method may include applying heat and / or ionic molecules to the sample, thereby causing the sample to have the second temperature and / or a second pH sufficient to result in the dissociation.

[0066] (v) The method may further include applying a wash solution to remove the sample from the biosensor.

[0067] (vi) The method may further include contacting the biosensor with a further sample and the one or more antibodies or fragments thereof; and detecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte bound to the one or more antibodies or fragments thereof.

[0068] According to a seventh aspect, the present disclosure relates in several embodiments to a device for detecting a target analyte in a sample. The device includes:

[0069] a collector for collecting the sample;

[0070] a sensing component comprising a biosensor as described herein; and

[0071] a connector for connecting the sensing component to an electrochemical measurement device;

[0072] wherein the collector and the sensing component are configured for allowing contact between the iMPs of the biosensor and the collected sample. The device may include the following details, which can be combined with one another in any combinations unless clearly mutually exclusive:

[0073] (i) the sensing component may include two working electrodes, a counter electrode and a reference electrode.

[0074] (ii) each electrode may be in the form of a wire.

[0075] (iii) the electrodes may be disposed in a matrix of a non-conductive material.

[0076] (iv) the iMPs may be bound to a first end of the biosensor electrode wire and the iMPs may be exposed to the sample when the first end is in contact with the collected sample.

[0077] (v) a second end of the wires may be in contact with the connector.

[0078] (vi) the device may further include a voltage controller electrically connected to at least one of the electrodes, wherein the voltage controller is configured to deliver voltage to at least one of the electrodes in chronoamperometric configuration, by square wave voltammetry, or selectably in either chronoamperometric configuration or by square wave voltammetry.

[0079] According to an eighth aspect, the present disclosure relates in several embodiments to a biosensor device for detecting an analyte in a sample. The biosensor device includes:

[0080] a substrate;

[0081] at least one electrode provided on the substrate;

[0082] a first dielectric layer provided on the substrate, the first dielectric layer comprising a well or aperture associated with the electrode such that the electrode is responsive to the presence of a sample received within the well or aperture; and a second dielectric layer provided on the first dielectric layer comprising an aperture extending through the second dielectric layer fluidly connected to the well or aperture in the first dielectric layer.

[0083] The biosensor device may include the following details, which can be combined with one another in any combinations unless clearly mutually exclusive:

[0084] (i) at least one electrode may be functionalized so as to interact with a sample received within the well or aperture of the first dielectric layer.

[0085] (ii) the at least one electrode may include a surface and a plurality of iMPs as described herein configured to bind an analyte within the sample.

[0086] (iii) the substrate may be a rigid substrate.

[0087] (iv) the electrode may include a plurality of metal layers, the metal layers comprising or being formed from copper, nickel, platinum, palladium, silver, silver chloride, gold or other noble metals. (v) the plurality of metal layers may include the palladium layer in between the gold layer and the nickel layer.

[0088] (vi) the biosensor device may include a hydrophobic coating disposed around the well or aperture.

[0089] (vii) the hydrophobic layer may include parylene.

[0090] According to a ninth aspect, the present disclosure relates in several embodiments to an electrochemical biosensor. The electrochemical biosensor includes:

[0091] a plurality of charged polymers, each charged polymer having first and second ends; an electrode, wherein the first end of each charged polymer is operationally affixed to the electrode, and wherein the second end of each charged polymer is structured to bind to a first receptor for a target analyte;

[0092] a second receptor for the target analyte;

[0093] one or more of a first redox reporter operationally bound to the second receptor, wherein the first redox reporter is reactive at positive potential when the charged polymer presents a net negative charge and reactive at negative potential when the charged polymer presents a net positive charge; and

[0094] wherein upon application of an electric field and binding of the charged polymer to the first receptor and binding of the second receptor to the target analyte to provide a target analyte-second receptor complex, the biosensor is characterized by:

[0095] an unbound state of the charged polymers, where no target analyte-second receptor complex is bound to the first receptor, at which the charged polymers are displaced towards the biosensor electrode surface and electron transfer from the one or more first redox reporters towards the biosensor electrode occurs at an unbound electron transfer rate; and a bound state of the charged polymers, where the target analyte-second receptor complex is bound to the first receptor, at which the charged polymers are displaced towards the biosensor electrode surface and electron transfer from the one or more first redox reporters towards the biosensor electrode occurs at a bound electron transfer rate.

[0096] The electrochemical biosensor may include the following details, which can be combined with one another in any combinations unless clearly mutually exclusive:

[0097] (i) an inverted molecular pendulum (iMP) may include the charged polymer bound to the first receptor, and wherein the iMP is operationally affixed to the electrode.

[0098] (ii) the electrode may be multilayered. (iii) the electrode may include a gold layer and may have a parylene coating layer. (iv) the charged polymers may be operationally affixed to the electrode by covalent bonding via an intermediate linking moiety.

[0099] (v) each charged polymer may be structured to bind to the first receptor via an adapter, wherein the adapter has a first portion configured to bind to the second end of the polymer and the adapter has a second portion configured to bind to the first receptor.

[0100] (vi) upon binding of the target analyte-second receptor complex to the first receptor at the applied electric field, an electrochemical signal may be produced translating a difference between the unbound electron transfer rate and the bound electron transfer rate.

[0101] (vii) upon binding of the target analyte- second receptor complex to the first receptor and application of the electric field, the first redox reporter may cause an electron transfer as the iMPs approach the biosensor electrode surface.

[0102] (viii) the electron transfer rate may be dependent on a time rate at which the iMPs are displaced.

[0103] (ix) the unbound electron transfer rate may be dependent on a time rate at which the unbound iMPs are displaced.

[0104] (x) the bound electron transfer rate may be dependent on a time rate at which the bound iMPs are displaced.

[0105] (xi) the iMPs displacement towards the biosensor electrode surface may substantially correspond to a tilting movement or a collapsing movement of the iMPs.

[0106] (xii) upon binding of the target analyte- second receptor complex to the first receptor and application of the electric field, the first redox reporter may touch the biosensor electrode surface, and the electron transfer may be based on a redox reaction or electron tunneling current.

[0107] (xiii) the first receptor may be adapted to bind to the target analyte at a first target analyte binding site, the second receptor may be adapted to bind to the target analyte at a second target analyte binding site, and the first receptor may be adapted to bind the target analyte-second receptor complex.

[0108] (xiv) the first redox reporter may be covalently bound to the second receptor.

[0109] (xv) the charged polymer may include a double-stranded DNA (dsDNA), singlestranded DNA (ssDNA), one or more charged polymers, one or more uncharged polymers, or any combination thereof.

[0110] (xvi) the charged polymer may be negatively charged and optionally comprises a DNA / DNA duplex, a PNA / DNA duplex, a PNA / PNA duplex, optionally wherein one or both of the PNA are modified with negatively charged amino acids, a rigid anionic polyelectrolyte, a rigid negatively charged peptide, or any combination thereof.

[0111] (xvii) the first redox reporter may include ferrocene, [Co(GA)2(phen)] (GA=glycolic acid, phen=l, 10-phenathroline), metal nanoparticles (e.g., Au, Pt, Pd, Ag, Cu), pyrroloquinoline quinone (PQQ), benzoquine, Osmium(III) complexes such as Os(bpy)Ci23+, diphenylamine, or any combination thereof.

[0112] (xviii) the first redox reporter may have a redox state change above 0 mV.

[0113] (xix) the charged polymer may be positively charged and optionally comprises a PNA / PNA duplex comprising one or more lysines, one or more arginines, a rigid cationic polyelectrolyte, a rigid positively charged peptide, or any combination thereof.

[0114] (xx) the first redox reporter may include methylene blue, ruthenium(III) complexes such as Ru(NH3)e3+, neutral red, toluidine blue, phenosafranine, or any combination thereof.

[0115] (xxi) the first redox reporter may have a redox state change below 0 mV.

[0116] (xxii) the charged polymer may have a length ranging from about 5 nm to about 20 nm. (xxiii) the charged polymer may include a ssDNA having a length ranging from about lOmer to about lOOmer.

[0117] (xxiv) the charged polymer may include a dsDNA having a length ranging from about 15mer to about 60mer.

[0118] (xxv) the charged polymer may be rigid along a length thereof, flexible or collapsible along the length thereof, or has a combination of one or more rigid and flexible or collapsible portions along the length thereof.

[0119] (xxvi) the iMPs may form a molecular monolayer at the surface of the biosensor electrode.

[0120] (xxvii) the first receptor, the second receptor, or both the first receptor and the second receptor, may include an antibody, an antibody receptor, a nanobody, an antigen, an aptamer, an aptamer fragment, a molecular imprint, a protein receptor, DNA, a microorganism, a protein / enzyme substrate, or any combination thereof.

[0121] (xxviii) the antibody receptor may include an Fc receptor.

[0122] (xxix) the biosensor electrode may include a glassy carbon electrode, a carbon nanotube-modified electrode, an indium tin oxide (ITO) electrode, a platinum electrode, a silver electrode, a gold electrode, or a palladium electrode.

[0123] (xxx) the biosensor electrode comprises a gold nanostructured microelectrode or a gold wire electrode. (xxxi) the biosensor may further include a second redox reporter bound to the charged polymer.

[0124] (xxxii) the second redox reporter may be covalently bound to the charged polymer. (xxxiii) the second redox reporter may be non-covalently bound to the charged polymer. (xxxiv) the second redox reporter may be reactive at a different potential than the first redox reporter.

[0125] (xxxv) upon binding of the first end of the charged polymer to the surface of the biosensor electrode, the second redox reporter may cause an electron transfer to the biosensor electrode surface.

[0126] (xxxvi) the plurality of iMPs may be bound to the surface of the biosensor electrode at a density sufficient to sterically hinder the iMPs bound state from being displaced towards the biosensor electrode.

[0127] (xxxvii) the application of the electric field may be in chronoamperometric configuration, by square wave voltammetry, or selectably in either chronoamperometric configuration or by square wave voltammetry.

[0128] (xxxviii) the positively charged polymer may include a PNA / PNA duplex and the first redox reporter may include one or more methylene blue redox reporters.

[0129] (xxxix) one strand or both strands of the PNA / PNA duplex may have a net positive charge.

[0130] (xxxx) one or more methylene blue redox reporters may be operationally bound to one strand or both strands of the PNA / PNA duplex.

[0131] (xxxxi) the biosensor may be adapted for use in consecutive measurements of the analyte without substantial loss of the electrochemical signal.

[0132] (xxxxii) the biosensor may be adapted for detecting the presence of the analyte in a sample comprising whole blood or whole blood diluted in a sample buffer fluid.

[0133] According to a tenth aspect, the present disclosure relates in several embodiments to a method of detecting a target analyte in a sample. The method includes:

[0134] providing the electrochemical biosensor as described herein;

[0135] contacting said biosensor with the sample; and

[0136] detecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte; and

[0137] optionally wherein the first receptor, the second receptor, or both the first receptor and the second receptor, is an antibody or a fragment thereof, wherein the binding of the target analyte to the antibody or fragment thereof occurs when the sample is at a first temperature and / or a first pH, and dissociation of the target analyte occurs at a second temperature, the method further comprises:

[0138] (i) optionally applying heat and / or ionic molecules to the sample, thereby causing the sample to have the second temperature and / or a second pH sufficient to result in the dissociation;

[0139] (ii) optionally applying a wash solution to remove the sample from the biosensor; and

[0140] optionally contacting the biosensor with a further sample and detecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte bound to antibody or a fragment thereof.

[0141] According to an eleventh aspect, the present disclosure relates in several embodiments to a method for in situ detection of a target analyte in a sample comprising a biological fluid. The method includes:

[0142] contacting the electrochemical biosensor as described herein with the biological fluid;

[0143] and

[0144] detecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte.

[0145] According to a twelfth aspect, the present disclosure relates in several embodiments to a use of an electrochemical biosensor as described herein to detect the presence of a target analyte in a sample, or use of an electrochemical biosensor as described herein to detect the presence of a target analyte in a sample comprising a biological fluid.

[0146] The methods or the use may include the following details, which can be combined with one another in any combinations unless clearly mutually exclusive:

[0147] (i) the biological fluid may include saliva, blood, urine, tears, sweat or feces.

[0148] (ii) the target analyte may include a small molecule, a macromolecule, a prokaryotic cell, a eukaryotic cell, a prokaryotic cell-derived component, a eukaryotic cell-derived component (e.g., nucleic acid material), a virus, a bacterium, an antibody, a protein, a cellular extract, or any combination thereof.

[0149] According to a thirteenth aspect, the present disclosure relates in several embodiments to a kit comprising a biosensor described herein. The kit includes: the second receptor of the biosensor provided suspended in a carrier fluid for addition to the sample to allow binding of the second receptor to the target analyte to provide the target analyte-second receptor complex.

[0150] According to a fourteenth aspect, the present disclosure relates in several embodiments to a method of detecting one or more target analytes in a sample. The method includes:

[0151] providing a kit as described herein;

[0152] contacting the biosensor with the sample, comprising:

[0153] adding the second receptor of the biosensor to the sample to allow binding of the second receptor to the target analyte to provide the target analyte- second receptor complex;

[0154] contacting the sample comprising the target analyte-second receptor complex with the first receptor; and

[0155] detecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte-second receptor complex.

[0156] The method may include the following details, which can be combined with one another in any combinations unless clearly mutually exclusive:

[0157] (i) the detecting the electrochemical signal may include the target analyte-second receptor complex binding to the first receptor.

[0158] (ii) the application of the electric field may be in chronoamperometric configuration, by square wave voltammetry, or selectably in either chronoamperometric configuration or by square wave voltammetry.

[0159] (iii) the binding of the first receptor and / or the second receptor to the target analyte may occur when the sample is at a first temperature and / or a first pH, and dissociation of the first receptor and / or the second receptor to the target analyte may occur at a second temperature, and further includes applying heat and / or ionic molecules to the sample, thereby causing the sample to have the second temperature and / or a second pH sufficient to result in the dissociation.

[0160] (iv) the method may further include applying a wash solution to remove the sample from the biosensor.

[0161] (v) the method may further include contacting the biosensor with a further sample; and detecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte-second receptor complex bound to the to the first receptor. (vi) the method may further include a second redox reporter bound to the charged polymer.

[0162] According to a fifteenth aspect, the present disclosure relates in several embodiments to a device for detecting a target analyte in a sample. The device includes:

[0163] a collector for collecting the sample;

[0164] a sensing component comprising the biosensor as described herein; and

[0165] a connector for connecting the sensing component to an electrochemical measurement device;

[0166] wherein the collector and the sensing component are configured for allowing contact between the iMPs of the biosensor and the collected sample.

[0167] The device may include the following details, which can be combined with one another in any combinations unless clearly mutually exclusive:

[0168] (i) the sensing component may further include two working electrodes, a counter electrode and a reference electrode.

[0169] (ii) each electrode may be in the form of a wire.

[0170] (iii) the electrodes are disposed in a matrix of a non-conductive material.

[0171] (iv) the iMPs may be bound to a first end of the biosensor electrode wire and the iMPs are exposed to the sample when the first end is in contact with the collected sample.

[0172] (v) a second end of the wires may be in contact with the connector.

[0173] (vi) the device may further include a voltage controller electrically connected to at least one of the electrodes, wherein the voltage controller is configured to deliver voltage to at least one of the electrodes in chronoamperometric configuration, by square wave voltammetry, or selectably in either chronoamperometric configuration or by square wave voltammetry.

[0174] According to a sixteenth aspect, the present disclosure relates in several embodiments to a biosensor device for detecting an analyte in a sample. The biosensor device includes: a substrate;

[0175] at least one electrode provided on the substrate;

[0176] a first dielectric layer provided on the substrate, the first dielectric layer comprising a well or aperture associated with the electrode such that the electrode is responsive to the presence of a sample received within the well or aperture; and a second dielectric layer provided on the first dielectric layer comprising an aperture extending through the second dielectric layer fluidly connected to the well or aperture in the first dielectric layer.

[0177] The biosensor device may include the following details, which can be combined with one another in any combinations unless clearly mutually exclusive:

[0178] (i) the at least one electrode may be functionalized so as to interact with a sample received within the well or aperture of the first dielectric layer.

[0179] (ii) the at least one electrode may include a surface and a plurality of iMPs as described herein and configured to bind an analyte within the sample.

[0180] (iii) the substrate may be a rigid substrate.

[0181] (iv) the electrode may include a plurality of metal layers, the metal layers comprising or being formed from copper, nickel, platinum, palladium, silver, silver chloride, gold or other noble metals.

[0182] (vii) the plurality of metal layers may include the palladium layer in between the gold layer and the nickel layer.

[0183] (viii) the biosensor device may further include a hydrophobic coating disposed around the well or aperture.

[0184] (ix) the hydrophobic layer may include parylene.

[0185] (x) the positively charged polymer may include a PNA / PNA duplex and the first redox reporter may include one or more methylene blue redox reporters.

[0186] (xi) one strand or both strands of the PNA / PNA duplex may have a net positive charge. (xii) the one or more methylene blue redox reporters may be operationally bound to one strand or both strands of the PNA / PNA duplex.

[0187] BRIEF DESCRIPTION OF THE DRAWINGS

[0188] The present disclosure may be further understood through reference to the attached figures in combination with the detailed description that follows.

[0189] FIG. 1 A, FIG. IB and FIG. 1C are schematics of an example biosensor.

[0190] FIG. 2A and FIG. 2B are schematics of another example biosensor.

[0191] FIG. 3 is a schematic of yet another example biosensor.

[0192] FIG. 4 is a graph reporting example modeled plots of current vs. time for an unbound inverted molecular pendulum (iMP) and an analyte-bound iMP. FIG. 5A is a schematic of an example biosensor having two redox markers.

[0193] FIG. 5B is a graph reporting example modeled plot of current vs. voltage in response to the example biosensor of FIG. 5 A having two redox markers.

[0194] FIG. 6A, FIG. 6B and FIG. 6C are schematics of an example steady-state pendulum configuration.

[0195] FIG. 7A is a schematic cross-sectional view of an example improved biosensor substrate and electrode.

[0196] FIG. 7B is a schematic plan view of an example improved biosensor substrate and electrode.

[0197] FIG. 8 is a schematic of an example biosensor. Inset of FIG. 8 is a graph reporting example modeled current vs. ratio of distance (d) between iMPs on the electrode surface and the length (1) of the iMP linkers.

[0198] FIG. 9 is a set of graphs reporting example data on current vs. time when using electrodes having various surface areas.

[0199] FIG. 10A and FIG. 10B are schematics of example biosensors.

[0200] FIG. 11 is a schematic of an example biosensor that includes a DNA pendulum linker and a ferrocene reporter (left) and an example biosensor that includes a PNA pendulum linker and a methylene blue reporter (left).

[0201] FIG. 12 is a schematic of an example biosensor that includes a PNA pendulum linker and a methylene blue reporter, being displaced toward the electrode in an unbound state (left) and in an analyte-bound state (right).

[0202] FIG. 13 is a graph reporting example data from four consecutive measurements from a biosensor that includes PNA pendulum linkers and methylene blue reporters.

[0203] FIG. 14 is a graph reporting example peak current from an example biosensor having either DNA pendulum linkers and ferrocene reporters (“DNA-FC pendulum”, on the left side of the graph) or positively charged PNA pendulum linkers and MB reporters (“PNA-MB pendulum”, on the right side of the graph).

[0204] FIG. 15 is a schematic of an example temporal pendulum assay configuration.

[0205] FIG. 16 is a schematic of an example spatial pendulum assay configuration.

[0206] FIG. 17 is a schematic of an example recruitment pendulum assay configuration. FIG. 18A and FIG. 18B are schematics of another example recruitment pendulum assay configuration.

[0207] FIG. 19 is a schematic of yet another example recruitment pendulum assay configuration. FIG. 20 is a graph reporting example data from a double-stranded recruitment pendulum assay.

[0208] FIG. 21 is a schematic of an example single-stranded recruitment pendulum assay configuration.

[0209] FIG. 22A is a graph reporting example data from a single-stranded recruitment pendulum assay.

[0210] FIG. 22B is another graph reporting example data from a single-stranded recruitment pendulum assay.

[0211] DETAILED DESCRIPTION

[0212] The present disclosure relates in various embodiments to improved electrochemical biosensors configured to detect analytes, such as analytes present in biological or other fluids, biosensor devices comprising such biosensors, and methods of using the improved biosensors and devices to detect the analytes.

[0213] Electrochemical biosensors, biosensor devices, and methods of using the biosensors and devices to detect analytes have been described, for example, in International Patent Application No.’s PCT / CA2021 / 050270 and PCT / EP2022 / 084871, the disclosures of which are incorporated by reference herein in their entireties. In some embodiments, the biosensors, biosensor devices, and methods described herein are improvements upon the biosensors, biosensor devices, and methods known in the art.

[0214] In general, the disclosed biosensors include a plurality of charged polymers (for example as described herein in example inverted molecular pendulum probes, or “iMPs”) operationally affixed to an electrode. The charged polymers (e.g., as described herein in example iMPs) are structured so as to bind to an analyte and receive an electrical current via a charge applied to the electrode.

[0215] In implementations, that charged polymer has first and second ends, with the first end of each charged polymer operationally affixed to the electrode, and the second end of each charged polymer is structured to bind to a receptor for a target analyte. The second end of the charged polymer may be bound directly to the receptor, or may be prepared in a modular manner by binding to a multi-purpose adapter. For example, in some non-limiting embodiments, the multi-purpose adapter may bean Fc receptor, and the receptor may be an IgG antibody. In some non-limiting example embodiments, the multi-purpose adapter may be streptavidin, and the receptor may be a biotinylated receptor. The adapter can be structured to bind to a receptor that can then bind to an analyte. The adapter can be structured to bind to any receptor that can specifically bind to a particular analyte. Accordingly, in some embodiments, the biosensors may be provided to multi-analyte detection functionality. The biosensors have one or more redox reporters operationally bound to the charged polymer. A first redox reporter is reactive at positive potential when the charged polymer presents a net negative charge and reactive at negative potential when the charged polymer presents a net positive charge. The charged polymers are made operational by binding them to a receptor (either directly or via an adapter).

[0216] In that operational condition, when applied in the presence of a sample, an electric field is applied to the electrode. Upon application of the electric field, the biosensor takes on an unbound state, where no target analyte is bound to the receptors, at which the charged polymers are displaced towards the electrode surface and electron transfer from the charged polymers towards the electrode occurs at an unbound electron transfer rate, or the sensor takes on a bound state, where the target analyte is bound to the receptors, at which the charged polymers are displaced towards the electrode surface and electron transfer from the charged polymers towards the electrode occurs at a bound electron transfer rate.

[0217] In some embodiments, a redox mediator reagent can be provided to the assay sample solution, such that the mediator is catalyzed or catalyzes the pendulum’s redox reporter signal to increase electron transfer (for example, without limitation, using ferricyanide as a redox mediator reagent in combination with using a methylene blue redox marker).

[0218] The following non-limiting examples illustrate representative biosensors, devices and methods that may be prepared in accordance with the present disclosure.

[0219] EXAMPLES

[0220] The present examples are provided for illustrative purposes only. They are not intended to and should not be interpreted to encompass the full breadth of the invention.

[0221] Example 1. - Rigid iMP structures

[0222] This Example relates to an example biosensor that includes charged polymers comprising double stranded nucleic acid charged polymers, also referred to as double stranded nucleic acid linkers. As shown for example in FIG.1A, FIG IB and FIG. 1C, electrochemical biosensors may be provided that include nucleic acid iMP linkers (also referred to herein as “pendulum linker”) such as double stranded nucleic acid linkers (e.g. by hybridizing two single-stranded nucleic acid strands), to generate a rigid pendulum linker, that may be attached to a transducing metal surface with chemistries (e.g., “attachment chemistry”) such as thiol chemistry. A “blocking molecule” (e.g. 6-Mercapto-l -hexanol, MCH) may be co-deposited onto the transducing metal surface (e.g., in a pre-determined ratio of attachment chemistry to blocking molecule) e.g., to control iMP density of deposition onto the transducing metal surface. Other blocking molecules can be used, including, without limitation, MCO (8-mercapto-l -octanol), MHA (6-mercaptohexanoic acid), PEG (polyethylene glycol), short / charged oligonucleotide sequences (DNA or PNA), thiolated short carbon chains, and the like. The blocking molecule may be backfilled onto the transducing metal surface e.g., to insulate the metal surface surrounding the iMP attachment sites. The insulating of the metal surface by the blocking molecules may assist in reducing or preventing non-specific iMP electrostatically attraction to the surface of the metal substrate. In FIG.1A, FIG IB and FIG. 1C, the distal end of the linker comprises a receptor (also referred to herein as a “biorecognition element”) configured to bind analytes e.g., molecular analytes, and a first redox reporter (also referred to herein as a “redox marker”) which reports on the binding event of the molecular analyte. The biosensor operates on molecular resistance-enabled electronic transduction when applying a potential to the transduction surface (e.g., “metal substrate”) which causes the iMP of the biosensor to electrostatically attract to the surface of the metal substrate at a rate dependent on the drag coefficient of the sensor through a fluid, and with selection of a redox reporter, which reacts at or below the applied potential. The drag coefficient increases upon analyte binding to the biorecognition element (FIG. 1C), and this change is electrochemically reported by a lag in the redox marker reaching the metal substrate surface for electron transfer (“slower time to fall” as shown in FIG. 1C vs. “faster time to fall” as shown in FIG. IB). The biosensor iMP pivots about the flexible thiol linker, allowing access of the redox marker to the transduction surface. Flexible regions of the linker themselves can also modulate redox marker access to the transduction surface. With an plurality of biosensor iMPs, the peak measured current will be delayed in time for an iMPs bound state vs an iMP unbound state, alternatively represented by an increase in the sensor’s time to fall, tau.

[0223] 2. Flexible / iMP structure This Example relates to an example biosensor that includes iMPs comprising singlestranded nucleic acid linkers. As shown for example in FIG. 2A and FIG 2B, a single-stranded iMP linker may include a single stranded, flexible or collapsible nucleic acid linker which enables a “collapsing” displacement as the linker is attracted to an opposing charged electrode surface (e.g., “metal substrate”). The linker can be negatively or positively charged, and the attached redox marker may be positively or negatively charged, respectively, to enable molecular drag electrical transduction.

[0224] The drag coefficient increases upon analyte binding to the biorecognition element (FIG.

[0225] 2B), and this change is electrochemically reported by a lag in the redox marker reaching the metal substrate surface for electron transfer (“slower time to fall” as shown in FIG. 2B vs. “faster time to fall” as shown in FIG. 2A).

[0226]

[0227] 3. iMP having an Fc

[0228] This Example relates to an example biosensor that includes iMPs comprising singlestranded nucleic acid linkers. FIG. 3 shows a schematic of an example biosensor iMP in which the biorecognition element is an Fc receptor. The example biosensor iMP can then bind any antibody or fragment thereof having an FC region (e.g., “IgG” antibody) For example, an IgG may be added to a sample fluid to form a biosensor iMP without the need for complex conjugation chemistry, enabling a simple platform for biosensor iMP construction. For example, such a biosensor can be used for rapid screening of antibodies to determine antibodies having the best antigen binding characteristics, allowing screening and development of biosensors targeted to detect specific analytes, and / or to enable rapid detection on demand for novel analyte targets.

[0229]

[0230] 4. Biosensor

[0231] FIG. 4 is a graph reporting example modeled plots of current vs. time for an unbound inverted molecular pendulum (iMP) and an analyte-bound iMP.

[0232] Biosensor transduction is enabled by any potential application to simultaneously electrostatically attract the iMP to the transduction surface and to promote redox reporter electron transfer with sufficient excitation energy. Chronoamperometry is one such electrochemical technique which applies a fixed potential step, producing a current output which reports a transient capacitive double layer charging current with a following faradaic current decay from the redox reporter. The faradaic current decay will be delayed in time for analyte-bound, slower iMP sensors due to fluidic resistance, causing a characteristically lagged current trace. This lag correlates with target analyte concentration, where shallower trace slope denotes higher concentration of analyte due to slower biosensor iMP displacements (as shown for example by the “dotted” trace below the solid trace in FIG. 4). Alternative transduction techniques (e.g., square wave voltammetry) may be used that enable electrostatic attraction of the iMP sensor to the transduction surface. In square wave voltammetry (SWV), peak current is the current measured at the end of a forward or reverse pulse. The peak current is used to calculate the standard rate constant and to evaluate the kinetics of an electrode process.

[0233] Example 5. Biosensor iMPs having a second redox marker proximal to electrode attachment

[0234] This Example relates to example biosensor iMPs having a second redox marker proximal to electrode attachment. FIG. 5A shows an example biosensor having an additional redox reporter (e.g., “redox marker 2”) at the proximal end of the iMP, e.g., on the opposite nucleic acid strand to the distal redox reporter (e.g., “redox marker 1”), and reactive at a different potential.

[0235] As shown for example in FIG. 5B, this example iMP enables determination of attachment chemistry efficiency and probe density calculation (e.g., measuring signal “2” in the example graph plot). The distal redox reporter on the non-surface-attached strand enables hybridization reporting (e.g. reporting on hybridization of the two linker strands) and biosensor mechanism transduction (e.g., responsiveness of the iMP to an applied current), e.g., measuring signal “1” in the example graph plot.

[0236] Example 6. Steady-state biosensor

[0237] This Example relates to an example steady-state biosensor.

[0238] Biosensors having iMPs such as shown for example in Example 1, also referred to herein as “kinetic pendulums” or “kinetic iMPs” monitor iMP motion as a function of current measured when iMPs fall and interact with the transduction surface. In practice, a plurality of kinetic iMPs falling may generate current through a Gaussian distribution of electron transfer events about a certain time, tau, which will be larger if receptors are bound with a target analyte.

[0239] To generate this tau-dependent current, a voltage pulse is applied, such as chronoamperometry, where a single step potential can simultaneously pull down the iMPs and cause the redox marker to change electronic states. These two phenomena occur within a few hundred microseconds so the entire measurement is taken within a short pulse.

[0240] In contrast, “steady-state pendulums” or “steady state iMPs” monitor electron transfer through the transduction surface as a function of the number of iMPs that have fully fallen to the transduction surface without steric hindrance. In practice, a plurality of iMPs would fall to the transduction surface before the redox marker changes electronic states, therefore typically requiring a voltage function which does not simultaneously pull down pendulums and cause electron transfer. This can occur with scanning potential techniques such as square wave voltammetry which applies small voltage pulses in a ramp from a low to high potential. With steady-state iMPs, analyte-bound iMPs are larger which cause greater steric hindrance effects which reduce their ability to fully rest on the transduction surface when pulled down, leading to a different electron transfer compared to unbound iMPs.

[0241] For example as shown in FIG. 6 A, FIG. 6B and FIG. 6C, a steady-state iMP biosensor was generated using square wave voltammetry as a transduction technique. The measured signal in a steady state iMP biosensor is due to unbound iMPs that have reached the transduction surface upon application of a scanning potential. Analyte-bound pendulums will be sterically hindered to the surface due to the increased size of the iMP distal end, leading to lower electron transfer. This mechanism is a function of iMP receptor-analyte occupancy so can be modulated by the size of target analytes. This mechanism has been tested with biotinpendulums binding a streptavidin target (FIG. 6A, FIG. 6B, FIG. 6C). In FIG. 6A, a high electron transfer signal is associated with the unbound iMP displacement toward the transduction surface. In FIG. 6B, binding of streptavidin analyte to the biotin receptor is associate with a lower analyte-bound signal. In FIG. 6C, the electron transfer signal may be higher for larger target analytes compared to smaller target analytes, due to reduced occupancy of iMP receptors. For example, the larger target analyte in FIG. 6C is streptavidin bound by an anti-streptavidin antibody. In contrast, in FIG. 6B, the smaller target analyte is streptavidin.

[0242] A plurality of iMPs may be associated with the transduction surface at a density sufficient to result in increased steric hindrance between the iMPs in the analyte-bound state.

[0243]

[0244] 7. Biosensor device stack-.

[0245] This example relates to an improved biosensor device stack-up, wherein the biosensor has an electrode with multiple layers.

[0246] Previous biosensors are provided on flexible printed circuit boards (“PCB”) substrates. Improved biosensors may be functionalized onto rigid, multi-layer PCB substrates (e.g., ENEPIG PCB substrates), e.g., as shown in FIG. 7A and FIG. 7B.

[0247] ENEPIG PCB has advantages such as prevention of percolating problems without having to use a thick layer of gold (thereby providing a cheaper process). Nickel will migrate into gold over time, but a palladium layer above the nickel (e.g., as shown in FIG. 7A) will block the percolation. For example, a rigid PCB substrate, such as FR4, can be used. In addition, parylene coating (e.g., as shown in FIG. 7A) may be used to passivate the surface of the PCB. Advantageously, parylene does not adhere to the gold surface, such that a photolithography / masking step is not required to remove the material from the gold surface after passivation. A parylene coating may cover edges of gold electrodes to mask edges which may expose underlying metals or leaching volatiles, prevent shorting between metals, and benefits easier liquid spotting during functionalization due to the hydrophobic barrier around gold electrodes. This arrangement may prevent moisture from damaging the PCB while in use and aid in discrete functionalization of the working gold electrodes. The PCB surface may be hydrophobic (e.g., parylene coating) and the gold working electrodes may be hydrophilic allowing for more discrete functionalization of individual working electrodes. Parylene may also cap areas of the PCB to prevent further leaching of contaminants onto gold surfaces. Use of rigid PCB instead of a flexible substrate reduces risk of contaminants due to higher baking temperatures and lower flexing of substrate associated with changes in temperature and use over time. Rigid PCBs are easier and cheaper to manufacture at high volumes and at lower cost.

[0248] In some embodiments, ENIG and other methods (e.g., less expensive methods known in the art) of manufacturing PCBs can be used, for example for use in PNA-MB pendulums, for example because MB has reactivity in a potential window outside of the gold electroactivity window.

[0249] In some embodiments, ENIG and other methods (e.g., less expensive methods) of manufacturing PCBs can be used, for example for use in pendulum configurations that may use slow pulse potentials (e.g., spatial pendulum configurations or recruitment pendulum configurations), which are less affected by background capacitance or noise that is associated with fast sampling.

[0250] In some embodiments, biosensor devices may be developed using reel-to-reel gold print on plastic due to low volume cost, better reproducibility, and miniaturization. In some embodiments, multiple working electrodes allow multiplexed sensors and control sensors to be patterned onto single strips.

[0251]

[0252] 8. of attachment of iMPs and of iMP linkers

[0253] As shown for example in FIG. 8, biosensors may be tuned by varying the iMP linker lengths (“ / ”) and spacing distance (“t ”) of iMP attachment to maximize faradaic current. Maximum current may be generated when the spacing is such that the biosensor iMPs (e.g., kinetic iMPs, steady state iMPs, or both) are not sterically hindered by each other. iMP spacing, e.g., probe density, can be tuned by adjusting the ratio of a co-deposition of iMPs and a blocking agent (e.g. 6-Mercapto-l -hexanol, MCH), also referred to herein as a “blocking molecule”. The blocking agent forms an inert monolayer around iMPs to control probe density as well as insulate the metal surface of the sensor from extraneous redox activity. An additional backfill of blocking agent may also be performed to fill gaps and replace non-specifically bound probes. The charge (“ ”) of the iMP linker can also be modulated using negatively charged DNA, neutral PNA, and positively charged PNA modifications. Net positive, neutral, and negative charge allows modulation of iMP falling time due to changes in electrostatic attraction / repulsion of the probe to the transduction surface. Modifying the viscosity or ionic strength of the sample medium can also tune the sensitivity of detection, such as the addition of protein, glycerol, or other viscosity-increasing compounds or salt and other charged moieties. Added viscosity delays the faradaic electron transfer for iMPs, and increased ionic strength decreases Debye length where the iMP interacts with the electric field. Additionally, the size of the biorecognition element and target analyte will also modulate the given fall time of biosensor iMPs and can be modelled to appropriately select e, 1, and d to maximize the sensitivity of the system.

[0254] 9. Electrode size

[0255] Chronoamperometric signal from an analyte-bound iMP is made up both a background current (capacitive) and faradaic current (from the probe itself), and the background can exceed the faradaic current if the electrode size is too large. As shown for example in FIG. 9, decreasing electrode size to improve faradaic : capacitive current ratio, with a current optimized size (e.g., of 0.035 mm2) allows greater assay sensitivity. Electrode size can be modulated by increasing the accessible gold surface through the passivation layer on test strips, or by introducing surface roughening or nano- structuring of gold.

[0256] 10. Continuous monitoring This example relates to an example biosensor and the use of the biosensor for continuous monitoring, or repeated biosensing, of analytes.

[0257] Biosensors can be used in continuous monitoring or repeated use applications, for example when using antibody biorecognition / capture elements. Within the sample solution after target analyte binding and detection, the pH of solution can be altered either to a more basic or acidic pH, causing the antibody to change conformation and release target. After a wash step to remove target, the pH can be returned to a more neutral / physiological pH allowing the antibody to return to its proper binding conformation and is now able to be reused to bind target and detect again.

[0258] Varying the potential from positive to negative or vice-versa may allow the biosensor to be reset for reusing the biosensor for another scanning and iMP falling event. For example, applied positive potential pulls a negatively charged iMP to the electrode surface, which brings a redox-probe closer to the electrode surface. Applied negative potential, on the other hand, pushes the probe away from the surface.

[0259] Example 11. Denaturing a double-stranded nucleic acid linker to allow replacement of one strand of the double-stranded linker

[0260] Another embodiment of a biosensor includes the ability to heat the functionalized probe to denature the probe to remove the redox-labelled strand if the redox label has degraded in use. Then a new redox-labelled strand can be introduced to hybridize the tethered strand, replenishing the probe with a new redox-label, making this a reusable sensor. Alternatively, if the other strand is attached to the electrode, the other strand (e.g., having the receptor attached) may be replaced using a similar denaturing and hybridizing process.

[0261] Example 12. iMPs having peptide nucleic acid (PNA) pendulum linkers and methylene blue (MB) redox reporters

[0262] This example relates to example biosensors having PNA pendulum linkers instead of DNA pendulum linkers. Compared to DNA, PNA has several advantages. For example:

[0263] PNA is more stable and is DNAse-resistant and protease-resistant.

[0264] PNA has a very strong hybridization strength and duplex strands have better stability over DNA. PNA probe hybridization is very efficient, and probe functionalization retains <5% CV. Unmodified PNA has 0 net charge, but charge can be added at any base pair, allowing to manipulate overall charge of pendulum linker molecule (+ / -). In comparison, DNA is negatively charged.

[0265] For example, PNA strands can be synthesized with positively charged side chains, such as lysine or arginine residues to provide PNA strands having a net positive charge.

[0266] Positively charging PNA can be used with methylene blue as a redox reporter.

[0267] The overall charge of PNA can be manipulated (+ / -). Also, the charge (+ / -) location within a PNA strand can be selected (e.g., charge can be changed at PNA base-pairs at a proximal, medial or distal location from the electrode, on a single strand or on both strands of a PNA). This allows either speeding up (more charge) or slowing down (less charge) the rate of pendulum movement. Selecting and controlling the charge (+ / -) of one or more PNA strands, at one or more base-pairs, also allows for better control of probe density and easier more controllable deposition conditions.

[0268] For example, applying positive charge to one or more base-pairs of one PNA strand (e.g., the electrode-tethered PNA strand) allows to keep the pendulum motion relatively slow (which may allow easier to measure kinetics).

[0269] This example also relates to example biosensors having MB redox markers. Compared to ferrocene, MB has several advantages. For example:

[0270] MB is much more stable than ferrocene. MB can be scanned repeatedly, demonstrating reproducible signal integrity with the less decay than is observed with ferrocene (e.g., decay caused by aging and repeated potential applied). Accordingly, MB signal is very stable over multiple measurements compared to ferrocene, and MB shows no signal reduction over consecutive measurements.

[0271] Using positively charged PNA with MB uses a scanning potential window that is not affected by an incomplete monolayer on a gold electrode surface. MB’s scanning potential window does not overlap with the gold oxidation peak at +0.2V. In contrast, ferrocene’s scanning potential window is in a region where an incomplete monolayer can cause high background current peaks which can lead to false positive detection.

[0272] MB has a two-electron reduction providing a much more robust signal compared to single electron oxidation of ferrocene.

[0273] MB signal can be measured in whole blood while ferrocene may have signal integrity issues. iMPs having PNA pendulum linkers and MB reporters can include any bio recognition element as described herein (for example, without limitation, antibody, aptamer, DNA sequences, among others).

[0274] iMPs having PNA pendulum linkers and MB reporters can use positively charged PNA linkers.

[0275] iMPs having PNA pendulum linkers and MB reporters can use MB redox reporter which reduces at negative potential.

[0276] In use, a negative potential is applied to the electrode to cause displacement of the iMPs having PNA pendulum linkers towards the biosensor electrode surface.

[0277] In some embodiments, iMPs having PNA pendulum linkers and MB reporters can include kinetic pendulums, stead-state pendulums, or recruitment pendulums.

[0278] FIG. 11 shows, on the left side, a schematic of an example iMP that includes a DNA pendulum linker and a ferrocene reporter. A positive potential (e.g., +0.5V) may be applied to the electrode to cause displacement of the iMP having the DNA pendulum linker towards the biosensor electrode surface. FIG. 11 also shows, on the right side, a schematic of an example iMP that includes a PNA pendulum linker and a methylene blue MB reporter. The example PNA pendulum linker has positive charge on the tethered strand, indicated by

[0279]

[0280] whereas the untethered strand that is hybridized to the tethered strand is neutral, indicated by “0”. A negative potential (e.g., -0.5V) may be applied to the electrode to cause displacement of the iMP having the PNA pendulum linker towards the biosensor electrode surface.

[0281] FIG. 12 shows another a schematic of an example iMP that includes a PNA pendulum linker and a MB reporter. In use, a negatively charged potential is applied to the electrode which pulls down the positively charged PNA linker towards the electrode. The potential is selected to simultaneously pull down the pendulum and to reduce the methylene blue once it reaches the electrode surface. For example, when incorporating a PNA pendulum linker in a kinetic iMP, an unbound iMP that includes a PNA pendulum linker and a methylene blue MB reporter (as shown on the left side) has faster displacement (“faster time to fall”) towards the electrode surface than an analyte-bound iMP that includes a PNA pendulum linker and a methylene blue MB reporter (as shown on the right side) which has a slower displacement (“slower time to fall”) towards the electrode surface.

[0282] Example 13. iMPs having peptide nucleic acid (PNA) pendulum linkers and methylene blue (MB) reporters show stable signal over multiple measurements. FIG. 13 is a graph reporting example data from four consecutive measurements from a iMPs having PNA pendulum linkers and MB reporters, demonstrating good signal integrity of the methylene blue reporter.

[0283] Signal integrity during multiple measurements is important to enable consecutive measurement of unbound and bound iMPs. Good signal integrity of iMPs having PNA pendulum linkers and MB reporters provides minimizes signal loss during sample incubation, and improved signal calibration.

[0284] Example 14. iMPs having peptide nucleic acid (PNA) pendulum linkers and methylene blue (MB) reporters show high signal integrity in whole blood.

[0285] This Example shows that iMPs having PNA pendulum linkers and MB reporters show high signal integrity in whole blood, which is useful for a quantitative assay in blood. In whole blood and blood dilutions, signal detection from iMPs having a MB reporter is more stable over multiple scans as compared to iMPs having a ferrocene reporter.

[0286] FIG. 14 is a graph reporting example peak current from an example biosensor having either iMPs including DNA pendulum linkers and ferrocene reporters (“DNA-FC pendulum”, on the left side of the graph) or iMPs including positively charged PNA pendulum linkers and MB reporters (“PNA-MB pendulum”, on the right side of the graph). The graph shows that, in response to a positive potential applied to the electrode to cause displacement of the DNA-FC pendulum towards the biosensor electrode surface, a small peak current signal is observed in venous blood diluted 1:1 to PBS buffer, and a diminished peak is observed in whole venous blood (left side of graph). In contrast, in response to a negative potential applied to the electrode to cause displacement of the PNA-MB pendulum towards the biosensor electrode surface, a strong peak current signal is observed in venous blood diluted 1 : 1 to PBS buffer, and a similar, overlapping strong peak is observed in whole venous blood (right side of graph). The signal of PNA-MB pendulums in whole blood overlaps that of 1 : 1 blood diluted with PBS, compared to the signal decay observed for DNA-FC. The clear MB signal in whole blood and diluted blood is a useful improvement compared to the significant reduction of ferrocene signal, which shows highly diminished signal in whole blood.

[0287] 15. Comparisons of example characteristics of pendulum assay formats. This Example provides example summaries of some of the characteristics of three iMP assay formats: (1) temporal pendulum, (2) spatial pendulum, and (3) recruitment pendulum.

[0288] (1) Temporal pendulum (also referred to herein as “kinetic pendulum” or “kinetic iMP”):

[0289] FIG. 15 is a schematic of an example temporal pendulum configuration.

[0290] As shown for example in FIG. 15, in use, when a target analyte is bound to the biorecognition element (as shown on the right side of FIG. 15) it increases the drag / resistance of the pendulum as it falls to the transduction surface, delaying the electron transfer event and output current as compared to the unbound state (as shown on the left side of FIG. 15). Output current has a broadened slope for bound pendulums as compared to unbound pendulums due to the extended time to fall. DNA or PNA can be used with associated redox reporters (e.g. ferrocene or methylene blue, respectively), can be single stranded or duplex, and the redox reporter can be on the pendulum or on a secondary capture molecule. The larger the target analyte (e.g. proteins, aggregates, bacteria, viruses) the bigger the signal change. The temporal pendulum is a “turn-on” system”, such that bound signal is higher than unbound signal. The temporal pendulum system typically uses fast pulse potential.

[0291] (2) Spatial pendulum (also referred to herein as “steady-state pendulum” or “steady-state iMP”):

[0292] FIG. 16 is a schematic of an example spatial pendulum configuration.

[0293] As shown for example in FIG. 16, in use, when a target analyte is bound to the biorecognition element (as shown in the bottom of FIG. 16; as compared to an unbound state as shown in the top of FIG. 16) it is sterically hindered from fully reaching the transduction surface. As a result, output current is reduced for bound pendulums as the electron transfer is only efficient for unbound pendulums. DNA or PNA can be used with associated redox reporters (e.g., ferrocene and methylene blue, respectively), can be single stranded or duplex, and the redox reporter can be on the pendulum or on a secondary capture molecule. The larger the target (e.g. proteins, aggregates, bacteria, viruses) the bigger the signal change. The spatial pendulum is a “turn-off’ system, such that bound signal is lower than unbound signal. The temporal pendulum system typically uses slow pulse potential.

[0294] (3) Recruitment pendulum (also referred to herein as “recruitment iMP”):

[0295] FIG. 17 is a schematic of an example recruitment pendulum configuration. As shown for example in FIG. 17, in use, the target analyte first binds with a secondary molecule which has a conjugated redox reporter. Next, the target analyte-secondary molecule complex binds to the biorecognition element of the pendulum (as shown in the right of FIG.

[0296] 17; as compared to an unbound state as shown in the left of FIG. 17), and in response to an applied potential, the pendulum pulls down the target analyte-secondary molecule complex to the transduction surface for efficient electron transfer. Output current will be much higher for analyte-bound pendulums (as shown in the right of FIG. 17), with minimal background current from unbound pendulums (as shown in the left of FIG. 17), as unbound secondary molecules will not efficiently transfer electrons through the pendulum monolayer. In use, typically no washing step is required.

[0297] Alternatively, in use, the target analyte first binds to the biorecognition element of the pendulum, and next a secondary molecule which has a conjugated redox reporter binds to the target analyte that is bound to the biorecognition element, thereby providing target analytesecondary molecule complex bound to the biorecognition element of the pendulum.

[0298] DNA or PNA can be used with associated redox reporters (e.g. ferrocene and methylene blue, respectively), can be single stranded or duplex, and the redox reporter is on a secondary capture molecule. For example, and without limitation, the secondary capture molecule can be a redox-conjugated antibody, redox-conjugated aptamer, redox-conjugated nano / microparticle, and the like. Secondary capture molecules can be engineered using standard methods known in the art to provide secondary capture molecules having increased size, conjugation of a plurality of redox markers, and so on. For example, in some non-limiting embodiments, secondary capture molecules can be fusion proteins comprising an antibody fused to another protein, or can be an antibody fused to one or more synthetic particles. One or more conjugations of the redox marker onto the secondary capture molecule can be done to increase the redox signal.

[0299] The recruitment pendulum configuration can be used for detection of various target analytes having various sizes, from small to large (e.g., from small molecules to cells). The recruitment pendulum is a “turn-on” system, such that analyte-bound signal is higher than unbound signal. The recruitment pendulum system may use a fast pulse potential or a slow pulse potential.

[0300]

[0301] 16. This Example relates to an example recruitment pendulum assay configuration is which the redox reporter is attached to a secondary capture molecule (e.g., an antibody) that binds to a target analyte. In use, the recruitment pendulum assay configuration has no signal for unbound pendulums, which may improve signal resolution. The secondary capture molecule (e.g., an antibody) may have a larger size than the target analyte (e.g., small proteins), thereby providing increased drag of target analyte-secondary molecule complex bound to the biorecognition element of the pendulum as the bound pendulum is displaced toward the electrode, as compared to the drag produced by the analyte bound to the biorecognition element alone. A configuration wherein the reporter is attached to the secondary capture molecule provides a configuration having no background signal for an unbound state. In the bound state, the pendulum linker enables movement for the secondary capture molecule (e.g. antibody) to reach the transduction surface to transfer electrons from the redox reporter, in response to an applied potential.

[0302] FIG. 18A is a schematic of an example recruitment pendulum assay configuration, which has positive charge (“+ charge”) on the tethered strand (attached to an electrode, not shown, by e.g. thiol chemistry) of a PNA pendulum (“PNA pendulum linker P2”). As shown in FIG. 18 A, the example recruitment pendulum assay configuration has a biorecognition element (“biotin”) which binds to the target analyte (“monomeric streptavidin with flag tag”). The example secondary capture molecule (“anti-flag antibody”) binds to flag tag on streptavidin.

[0303] For example, MB reporters can be conjugated to antibody using N-hydroxysuccinimide (NHS) ester chemistry. A plurality of MB redox markers can be conjugated to a secondary antibody to enhance the signal.

[0304] As shown for example in FIG. 18 A, a recruitment pendulum assay configuration may also include another redox reporter (e.g. ferrocene) for quality control assays to confirm deposition of pendulums to the electrode.

[0305] Measurements of assays using negative control pendulums (e.g., pendulums that do not bind target analyte, e.g. those that lack a biorecognition element configured to bind the target analyte) may be used to subtract unbound secondary antibody signal from bound pendulums. In practice, the signal from unbound secondary antibody is minimal as it is not concentrated to the transduction surface by the pendulum pull-down mechanism; in other words, unbound secondary antibody is free floating in sample, having diffuse motion. Electron transfer efficiency of these diffuse, free floating, molecules is low through the pendulum monolayer, making this system usable without requiring a washing step and can be performed in an unprocessed patient sample, eliminating need for microfluidics and additional processing steps. For example, FIG. 18B shows a schematic of an example recruitment pendulum assay configuration. In the right side of FIG. 18B, in response to an applied potential, the recruitment pendulum pulls down the biorecognition element-bound target that is also bound to the secondary antibody bearing methylene blue redox markers, which increases the electron transfer efficiency from the redox marker to the electrode surface. In contrast, in the left side of FIG. 18B, an unbound, secondary antibody bearing methylene blue redox marker that is diffuse in solution has low electron transfer efficiency to the electrode surface. A high density of blocking molecules forming a high-density blocking layer on the electrode surface may further impede electron transfer efficiency to the electrode surface from unbound secondary antibodies. In some embodiments, a high density of blocking molecules refers to a blocking molecule layer substantially having full coverage of the electrode surface (e.g., gold surface) such that substantially no electrochemical activity of gold is measured within the potential windows, and the blocking molecules having length and charge sufficient to reduce nonspecific binding, biofouling, and / or diffuse electron transfer. A high-density blocking layer may also reduce non-specific binding and pendulum density which sterically hinders unbound secondary from reaching the surface. The example recruitment pendulum assay configuration may be implemented without a wash step between assays.

[0306]

[0307] 17. recruitment for a

[0308] This Example relates to an example recruitment pendulum - protein assay configuration in which the biorecognition element is an antibody that binds to a target analyte protein, and a redox reporter is attached to a secondary capture molecule (e.g., an antibody) that also binds to a target analyte.

[0309] FIG. 19 is a schematic of an example recruitment pendulum assay configuration that includes a PNA antibody pendulum with a MB redox reporter attached to a secondary antibody.

[0310] As shown for example in the left of FIG. 19, the unbound state is a pendulum configuration that includes an antibody configured to bind a protein analyte, the antibody being conjugated to duplex PNA, and includes no redox reporter for an analyte binding assay. However, a different redox reporter may be attached to the pendulum linker (e.g., a ferrocene redox reporter, as shown in FIG. 19) which can be used for probe deposition quality control.

[0311] As shown for example in the right of FIG. 19, the target is a protein of interest bound to a secondary antibody which may bind to a different epitope to the epitope that is bound by the pendulum’s antibody. As shown for example in the right of FIG. 19, a MB redox reporter is conjugated to the secondary antibody. As shown for example in the right of FIG. 19, the bound state is a “sandwich” configuration, in which the target protein-secondary antibody complex binds the pendulum, and binding assay signal is provided by the MB redox reporter.

[0312] Negative control pendulums (e.g., those that do not bind target protein) may be used for subtracting background unbound secondary antibody signal from bound pendulums.

[0313] Advantages of the recruitment pendulum configuration include the following: The pendulum linker improves access of the MB redox reporter to the transduction surface, due to the pendulum pivot mechanism. The pendulum linker extends the binding location into the assay solution, such that less diffusion of protein target is needed (e.g., the target protein is not required to diffuse to an antibody bound directly to a surface, as in some existing protein assay techniques (e.g., sandwich ELISA), so more efficient binding of the target protein analyte may be achieved. Also, the recruitment pendulum configuration does not need a step for washing unbound secondary as only bound secondary antibodies are brought down to the transduction surface upon potential application during measurement. The PNA and blocking molecule monolayer is optimized to minimize unbound signal and enable bound signal through the monolayer to the transduction surface by the pendulum’s pull-down.

[0314] 18. Example double-stranded PNA-MB recruitment pendulum configuration

[0315] FIG. 20 is a graph reporting example data from a recruitment pendulum assay, in which PNA-MB pendulums were used to detect flag-tagged monomeric streptavidin in solution. A recruitment pendulum configuration as shown in FIG. 18A and FIG. 18B was used for this assay.

[0316] As shown in FIG. 20, no MB redox signal was observed under conditions where no flag-tagged monomeric streptavidin or anti-flag antibody was present in solution (“blank”). When the sample solution contained only an anti-flag antibody conjugated to an MB reporter, a low level of MB redox signal was observed (“Neg Ctrl”). However, when the sample solution contained (1) flag-tagged monomeric streptavidin and (2) an anti-flag antibody conjugated to an MB reporter, and (3) a pendulum was used that binds to flag-tagged monomeric streptavidin (e.g., a biotin biorecognition element was present on the pendulum), a high level of MB redox signal was observed.

[0317]

[0318] 19. i-stranded recruitment

[0319] FIG. 21 is a schematic of an example single-stranded recruitment pendulum assay configuration. The example single-stranded recruitment pendulum assay configuration is similar to the example double stranded recruitment pendulums such as those shown in FIG.

[0320] 18A and FIG. 18B, except the pendulum linker (e.g., PNA pendulum linker) is single-stranded.

[0321] For example, FIG. 21 shows a schematic of an example single-stranded recruitment pendulum assay configuration. In the right side of FIG. 21, in response to an applied potential, the recruitment pendulum pulls down the biorecognition element-bound target that is also bound to the secondary antibody bearing methylene blue redox markers, which increases the electron transfer efficiency from the redox marker to the electrode surface. In contrast, a targetbound secondary antibody bearing methylene blue redox marker that is free-floating in solution has low electron transfer efficiency to the electrode surface. In addition, in the left side of FIG.

[0322] 21, an unbound secondary antibody that is free-floating in solution and is not bound to it’s target also has low electron transfer efficiency to the electrode surface. A high density of blocking molecules forming a high-density blocking layer on the electrode surface may further impede electron transfer efficiency to the electrode surface from unbound secondary antibodies. A high-density blocking layer may also reduce non-specific binding and pendulum density which sterically hinders unbound secondary from reaching the surface. The example recruitment pendulum assay configuration may be implemented without a wash step between assays.

[0323] A single-stranded linker typically has more structural flexibility than a double-stranded linker. Accordingly, a single-stranded recruitment pendulum may advantageously fall faster due to linker flexibility, which may decrease the time between target binding and redox signal. In a single-stranded recruitment pendulum assay configuration, a methylene blue redox marker-bearing secondary antibody can reorient more flexibly and more quickly to transduce more efficiently to the electrode surface, produce higher redox signal. A single-stranded recruitment pendulum assay configuration may have less steric hindrance and repulsion from neighboring pendulums attached to the electrode surface, as it can reorient as it falls towards the electrode surface. A single-stranded recruitment pendulum assay configuration does not require hybridization of a complementary strand during functionalization, which simplifies manufacturing process and eliminates a heating step during hybridization and possible damage to biorecognition element. 20. ^-stranded PNA-MB recruitment

[0324]

[0325] FIG. 22A is a graph reporting example data from a single-stranded recruitment pendulum assay, in which single-stranded PNA-MB pendulums were used to detect flag-tagged monomeric streptavidin in solution. A recruitment pendulum configuration as shown in FIG.

[0326] 21 was used for this assay. This example assay used single-strand recruitment pendulums having biotin as the biorecognition element. The target was flag-tagged monomeric streptavidin. The secondary antibody was an anti-flag antibody conjugated with MB (“MB-secondary”).

[0327] The assay procedure was as follows. As a negative control (“Neg Ctrl”), a solution containing MB-secondary alone was applied to a biosensor device having assay wells comprising the single-stranded recruitment pendulum configuration. A negative potential was applied and current (nA) was measured for 60 minutes. The assay well of the biosensor device was then washed, and a negative potential was applied and current (nA) was measured. Next, a sample solution containing 500ng / mL of the monomeric streptavidin having the flag tag was mixed with the MB-secondary to allow the MB-secondary to bind to the flag-tagged streptavidin. The sample solution containing the MB-secondary bound to the flag-tagged streptavidin was then applied to the assay well, a negative potential was applied and current (nA) was measured for 20 minutes without washing. Current measurements were taken at 0 minutes, 5 minutes, 10 minutes, and 20 minutes after applying the sample solution containing the MB-secondary bound to the flag-tagged streptavidin. As shown in FIG. 22A, very low background signal was observed for the negative control. Current signal is observed within 5 minutes of applying the sample solution containing the MB-secondary bound to the flag-tagged streptavidin to the assay well. FIG. 22B is another graph reporting the same example assay data as in FIG. 22 A, showing current measured at 0 minutes, 5 minutes, 10 minutes, and 20 minutes after applying the sample solution containing the MB-secondary bound to the flag-tagged streptavidin. As shown in FIG. 22B, the current signal appears to begin to plateau by 20 minutes after applying the sample solution.

[0328]

[0329] 21. NT-

[0330] In some non-limiting embodiments, a technique such as outlined in Examples 18 or 19 may be used to assay for N-terminal Pro-Brain Natriuretic Peptide (NT-proBNP) in blood samples. NT-proBNP is a hormone released into the blood by the heart in response to stress, such as heart failure. It is a biomarker used to diagnose and monitor heart failure, as well as to assess the risk of future cardiovascular events. For example, in some embodiments, a pendulum, e.g., a PNA-MB pendulum (e.g. having positive charge), for example in recruitment pendulum configuration, may be used. For example, an anti -NT-proBNP antibody may be conjugated to the pendulum, and a secondary antibody that binds to NT-proBNP (e.g., at a different epitope to the anti -NT -proBNP antibody conjugated to the pendulum) has a redox reporter (e.g., one or more MB). The secondary antibody may bind to NT-proBNP in the sample solution, and then the secondary antibody -bound NT-proBNP may bind to the pendulum-conjugated anti -NT -proBNP antibody. Applying a potential (e.g., a negative potential) to the electrode may then cause displacement of the pendulums to the electrode surface, allowing efficient electron transfer from MB in bound pendulums. Interference compounds such as non-specific proteins and unbound secondary antibody will have minimal reactivity with the transduction surface without the pendulum pull-down, meaning a wash is not required.

[0331] Example 22. Using potential during deposition and assay to improve kinetics

[0332] For a positively charged PNA pendulum monolayer, a weak positive potential pulse (e.g., between 0 to +0.5V) may be applied to remove non-specifically adsorbed PNA from the surface of a gold electrode, promoting stronger PNA thiol bonds to form with the gold electrode.

[0333] For a positively charged PNA pendulum monolayer, during MCH backfilling steps and washing steps, weak positive potential pulses (e.g., between 0 to +0.5V) can also be applied to improve MCH backfilling, to remove non-specifically adsorbed MCH from the surface and stand up bound PNA pendulums to improve MCH access to the surface to bind.

[0334] For target analyte binding in an assay, a weak positive potential pulse or sustained potential (e.g., between 0 to +0.5V) can be used to stand up the positively charged PNA probes from their equilibrium position, allowing for better presentation of biorecognition element to target analyte in solution.

[0335] For a negatively charged DNA pendulum monolayer, a weak negative potential pulse (e.g., between 0 to -0.5V) may be applied to remove non-specifically adsorbed DNA from the surface of a gold electrode, promoting stronger DNA thiol bonds to form with the gold electrode.

[0336] For a negatively charged DNA pendulum monolayer, during MCH backfilling steps and washing steps, weak negative potential pulses (e.g., between 0 to -0.5 V) can also be applied to improve MCH backfilling, to remove non-specifically adsorbed MCH from the surface and stand up bound PNA pendulums to improve MCH access to the surface to bind.

[0337] For target analyte binding in an assay, a weak negative potential pulse or sustained potential (e.g., between 0 to -0.5 V) can be used to stand up the negatively charged DNA probes from their equilibrium position, allowing for better presentation of biorecognition element to target analyte in solution.

[0338] ***

[0339] The above disclosure contains various examples of electrochemical biosensors and methods of use thereof. Aspects of these various examples may all be combined with one another, even if not expressly combined in the present disclosure, unless they are clearly mutually exclusive.

[0340] In addition, various example materials are discussed herein and are identified as examples, as suitable materials, and as materials included within a more generally described type of material, for example by use of the term “including” or “such-as.” All such terms are used without limitation, such that other materials falling within the same general type exemplified but not expressly identified may be used in the present disclosure as well.

[0341] Furthermore, unless it is otherwise clear that a single entity is intended, terms such as “a,” “an,” and “the” are not intended to refer to only a singular entity and include the general class of which a specific example is described for illustration. In addition, unless it is clear that a precise value is intended, numbers recited herein should be interpreted to include variations above and below that number that may achieve substantially the same results as that number, or variations that are “about” the same number. Finally, a derivative as disclosed herein may include a chemically modified molecule that has an addition, removal, or substitution of a chemical moiety of the parent molecule.

[0342] It is understood the use of the alternative (e.g., “or”) herein is taken to mean either one or both or any combination thereof of the alternatives. The term “and / or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other. For example, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0343] As used herein, terms “comprising”, “including”, “having” and “containing”, and their grammatical variants, as used herein are intended to be non-limiting so that one item or multiple items in a list do not exclude other items that can be substituted or added to the listed items. It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0344] Various components of the biosensors described herein may be identified by trade name in this application. All such trade names refer to the relevant composition or instrument as it existed as of the earliest filing date of this application, or the last date a product was sold commercially under such trade name, whichever is later. One of ordinary skill in the art will appreciate that variant compositions and instruments sold under the trade name at different times will typically also be suitable for the same uses.

[0345] The above disclosed subject matter is to be considered illustrative, and not restrictive, and the appended claims are intended to cover all such modifications, enhancements, and other implementations which fall within the true spirit and scope of the present disclosure. Thus, to the maximum extent allowed by law, the scope of the present disclosure is to be determined by the broadest permissible interpretation of the following claims and their equivalents and shall not be restricted or limited by the foregoing detailed description.

Claims

CLAIMS1. An electrochemical biosensor comprising:a plurality of charged polymers, each charged polymer having first and second ends; an electrode, wherein the first end of each charged polymer is operationally affixed to the electrode, and wherein the second end of each charged polymer is structured to bind to a receptor for a target analyte; anda first redox reporter operationally bound to the charged polymer, wherein the first redox reporter is reactive at positive potential when the charged polymer presents a net negative charge and reactive at negative potential when the charged polymer presents a net positive charge; andwherein upon application of an electric field and binding of the charged polymer to a receptor, the biosensor is characterized by:an unbound state of the charged polymers, where no target analyte is bound to the receptor, at which the charged polymers are displaced towards the biosensor electrode surface and electron transfer from the charged polymers towards the biosensor electrode occurs at an unbound electron transfer rate; and a bound state of the charged polymers, where the target analyte is bound to the receptor, at which the charged polymers are displaced towards the biosensor electrode surface and electron transfer from the charged polymers towards the biosensor electrode occurs at a bound electron transfer rate.

2. The biosensor of claim 1, wherein an inverted molecular pendulum (iMP) comprises the charged polymer bound to the receptor and to the first redox reporter, and wherein the iMP is operationally affixed to the electrode.

3. The biosensor of claim 1 or 2, wherein the electrode is multilayered.

4. The biosensor of any one of claims 1-3, wherein the electrode comprises a gold layer and has a parylene coating layer.

5. The biosensor of any one of claims 1-4, wherein the charged polymers are operationally affixed to the electrode by covalent bonding via an intermediate linking moiety.

6. The biosensor of any one of claims 1-5, wherein each charged polymer is structured to bind to the receptor via an adapter, wherein the adapter has a first portion configured to bind to the second end of the polymer and the adapter has a second portion configured to bind to the receptor.

7. The biosensor of any one of claim 1-6, wherein upon binding of the target analyte to the receptor at the applied electric field, an electrochemical signal is produced translating a difference between the unbound electron transfer rate and the bound electron transfer rate.

8. The biosensor of any one of claims 2-7, wherein upon application of the electric field, the first redox reporter causes an electron transfer as the iMPs approach the biosensor electrode surface.

9. The biosensor of any one of claims 2 to 8, wherein the electron transfer rate is dependent on a time rate at which the iMPs are displaced.

10. The biosensor of any one of claims 2 to 9, wherein the unbound electron transfer rate is dependent on a time rate at which the unbound iMPs are displaced.

11. The biosensor of any one of claims 2 to 10, wherein the bound electron transfer rate is dependent on a time rate at which the bound iMPs are displaced.

12. The biosensor of any one of claims 2 to 11, wherein the iMPs displacement towards the biosensor electrode surface substantially corresponds to a tilting movement or a collapsing movement of the iMPs.

13. The biosensor of any one of claims 1 to 12, wherein upon application of the electric field, the first redox reporter touches the biosensor electrode surface, and the electron transfer is based on a redox reaction or electron tunneling current.

14. The biosensor of any one of claims 1 to 13, wherein the first redox reporter is bound to the charged polymer proximal to the second end thereof.

15. The biosensor of any one of claims 1 to 14, wherein the first redox reporter is covalently bound to the charged polymer.

16. The biosensor of any one of claims 1 to 15, wherein the charged polymer comprises a double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), one or more charged polymers, one or more uncharged polymers, or any combination thereof.

17. The biosensor of any one of claims 1 to 15, wherein the charged polymer is negatively charged and optionally comprises a DNA / DNA duplex, a PNA / DNA duplex, a PNA / PNA duplex, optionally wherein one or both of the PNA are modified with negatively charged amino acids, a rigid anionic polyelectrolyte, a rigid negatively charged peptide, or any combination thereof.

18. The biosensor of any one of claims 1 to 17, wherein the first redox reporter comprises ferrocene, [Co(GA)2(phen)] (GA=gly colic acid, phen=l, 10-phenathroline), metal nanoparticles (e.g., Au, Pt, Pd, Ag, Cu), pyrroloquinoline quinone (PQQ), benzoquine, Osmium(III) complexes such as Os(bpy)Ci23+, diphenylamine, or any combination thereof.

19. The biosensor of any one of claims 1 to 18, wherein the first redox reporter has a redox state change above 0 mV.

20. The biosensor of any one of claims 1 to 15, wherein the charged polymer is positively charged and optionally comprises a PNA / PNA duplex comprising one or more lysines, one or more arginines, a rigid cationic polyelectrolyte, a rigid positively charged peptide, or any combination thereof.

21. The biosensor of any one of claims 1 to 15 and 20, wherein the first redox reporter comprises methylene blue, ruthenium(III) complexes such as Ru(NH3)e3+, neutral red, toluidine blue, phenosafranine, or any combination thereof.

22. The biosensor of any one of claims 1 to 15, 20 and 21, wherein the first redox reporter has a redox state change below 0 mV.

23. The biosensor of any one of claims 1 to 22, wherein the charged polymer has a length ranging from about 5 nm to about 20 nm.

24. The biosensor of any one of claims 1 to 19, wherein the charged polymer comprises a ssDNA having a length ranging from about lOmer to about lOOmer.

25. The biosensor of any one of claims 1 to 19, wherein the charged polymer comprises a dsDNA having a length ranging from about 15mer to about 60mer.

26. The biosensor of any one of claims 1 to 25, wherein the charged polymer is rigid along a length thereof, flexible or collapsible along the length thereof, or has a combination of one or more rigid and flexible or collapsible portions along the length thereof.

27. The biosensor of any one of claims 2 to 26, wherein the iMPs form a molecular monolayer at the surface of the biosensor electrode.

28. The biosensor of any one of claims 1 to 27, wherein the receptor comprises an antibody, an antibody receptor, a nanobody, an antigen, an aptamer, an aptamer fragment, a molecular imprint, a protein receptor, DNA, a microorganism, a protein / enzyme substrate, or any combination thereof.

29. The biosensor of claim 28, wherein the antibody receptor comprises an Fc receptor.

30. The biosensor of any one of claims 1 to 29, wherein the biosensor electrode comprises a glassy carbon electrode, a carbon nanotube-modified electrode, an indium tin oxide (ITO) electrode, a platinum electrode, a silver electrode, a gold electrode, or a palladium electrode.

31. The biosensor of any one of claims 1 to 30, wherein the biosensor electrode comprises a gold nanostructured microelectrode or a gold wire electrode.

32. A method of detecting a target analyte in a sample comprising:providing the electrochemical biosensor of any one of claims 1 to 31; contacting said biosensor with the sample; anddetecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte; andoptionally wherein the receptor is an antibody or a fragment thereof, wherein the binding of the target analyte to the antibody or fragment thereof occurs when the sample is at a first temperature and / or a first pH, and dissociation of the target analyte occurs at a second temperature, the method further comprises:(iii) optionally applying heat and / or ionic molecules to the sample, thereby causing the sample to have the second temperature and / or a second pH sufficient to result in the dissociation;(iv) optionally applying a wash solution to remove the sample from the biosensor; and(v) optionally contacting the biosensor with a further sample and detecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte bound to antibody or a fragment thereof.

33. A method for in situ detection of a target analyte in a sample comprising a biological fluid, comprising:contacting the electrochemical biosensor of any one of claims 1 to 31 with the biological fluid; anddetecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte.

34. Use of the electrochemical biosensor of any one of claims 1 to 31 to detect the presence of a target analyte in a sample.

35. Use of the electrochemical biosensor of any one of claims 1 to 31 to detect the presence of a target analyte in a sample comprising a biological fluid.

36. The method of claim 33 or the use of claim 35, wherein the biological fluid comprises saliva, blood, urine, tears, sweat or feces.

37. The biosensor of any one of claims 1 to 31, the method of any one of claims 32, 33 or 36, or the use of any one of claims 34 to 35, wherein the target analyte comprises a smallmolecule, a macromolecule, a prokaryotic cell, a eukaryotic cell, a prokaryotic cell- derived component, a eukaryotic cell-derived component (e.g., nucleic acid material), a virus, a bacterium, an antibody, a protein, a cellular extract, or any combination thereof.

38. A kit comprising the biosensor of any one of claims 1 to 31 or claim 37, wherein the receptor is an Fc receptor, and further comprising:one or more antibodies or fragments thereof, comprising:an antigen-binding site adapted to specifically bind to a target analyte; and an Fc region or Fc-receptor binding fragment thereof.

39. A method of detecting one or more target analytes in a sample comprising:providing the kit of claim 38;contacting the biosensor with the sample and the one or more antibodies or fragments thereof; anddetecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte bound to the one or more antibodies or fragments thereof.

40. The method of claim 39, wherein:the detecting the electrochemical signal comprises the one or more antibodies or fragments thereof binding to the one or more target analytes and to the Fc receptor.

41. The method of claim 40, wherein the application of the electric field is in chronoamperometric configuration, by square wave voltammetry, or selectably in either chronoamperometric configuration or by square wave voltammetry.

42. The method of claim 40, wherein the binding of the one or more antibodies or fragments thereof to the one or more target analytes and to the Fc receptor occurs when the sample is at a first temperature and / or a first pH, and dissociation of one or more antibodies or fragments thereof to the one or more target analytes and to the Fc receptor occurs at a second temperature, and further comprising:applying heat and / or ionic molecules to the sample, thereby causing the sample to have the second temperature and / or a second pH sufficient to result in the dissociation.

43. The method of claim 42, further comprising:applying a wash solution to remove the sample from the biosensor.

44. The method of claim 43, further comprising:contacting the biosensor with a further sample and the one or more antibodies or fragments thereof; anddetecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte bound to the one or more antibodies or fragments thereof.

45. The biosensor of any one of claims 1-31 or claim 37, further comprising a second redox reporter bound to the charged polymer proximal to the first end thereof.

46. The biosensor of claim 45, wherein the second redox reporter is covalently bound to the charged polymer.

47. The biosensor of claim 46, wherein the second redox reporter is covalently bound to the charged polymer.

48. The biosensor of claim 45, wherein the second redox reporter is reactive at a different potential than the first redox reporter.

49. The biosensor of claim 45, wherein upon binding of the first end of the charged polymer to the surface of the biosensor electrode, the second redox reporter causes an electron transfer to the biosensor electrode surface.

50. The biosensor of any one of claims 2-31, 37, or 45-49, wherein the plurality of iMPs are bound to the surface of the biosensor electrode at a density sufficient to sterically hinder the iMPs bound state from being displaced towards the biosensor electrode.

51. The biosensor of claim 50, wherein the application of the electric field is in chronoamperometric configuration, by square wave voltammetry, or selectably in either chronoamperometric configuration or by square wave voltammetry.

52. A device for detecting a target analyte in a sample comprising:a collector for collecting the sample;a sensing component comprising the biosensor as defined in any one of claims 2 to 31 or 37-51; anda connector for connecting the sensing component to an electrochemical measurement device;wherein the collector and the sensing component are configured for allowing contact between the IMPs of the biosensor and the collected sample.

53. The device of claim 52, wherein the sensing component further comprises two working electrodes, a counter electrode and a reference electrode.

54. The device of claim 53, wherein each electrode is in the form of a wire.

55. The device of any one of claims 53 to 54, wherein the electrodes are disposed in a matrix of a non-conductive material.

56. The device of any one of claims 54 to 55, wherein the iMPs are bound to a first end of the biosensor electrode wire and the iMPs are exposed to the sample when the first end is in contact with the collected sample.

57. The device of any one of claims 54 to 56, wherein a second end of the wires are in contact with the connector.

58. The device of any one of claims 52 to 57, further comprising a voltage controller electrically connected to at least one of the electrodes, wherein the voltage controller is configured to deliver voltage to at least one of the electrodes in chronoamperometric configuration, by square wave voltammetry, or selectably in either chronoamperometric configuration or by square wave voltammetry.

59. A biosensor device for detecting an analyte in a sample, the biosensor device comprising:a substrate;at least one electrode provided on the substrate;a first dielectric layer provided on the substrate, the first dielectric layer comprising a well or aperture associated with the electrode such that the electrode is responsive to the presence of a sample received within the well or aperture; and a second dielectric layer provided on the first dielectric layer comprising an aperture extending through the second dielectric layer fluidly connected to the well or aperture in the first dielectric layer.

60. The biosensor device of claim 59, wherein the at least one electrode is functionalized so as to interact with a sample received within the well or aperture of the first dielectric layer.

61. The biosensor device of claim 60, wherein the at least one electrode comprises a surface and a plurality of iMPs as defined in any one of claims 2 to 31 or 37-51 configured to bind an analyte within the sample.

62. The biosensor device of any one of claims 59 to 61, wherein the substrate is a rigid substrate.

63. The biosensor device of any one of claims 59 to 62, wherein the electrode comprises a plurality of metal layers, the metal layers comprising or being formed from copper, nickel, platinum, palladium, silver, silver chloride, gold or other noble metals.

64. The biosensor device of claim 63, wherein the plurality of metal layers comprises the palladium layer in between the gold layer and the nickel layer.

65. The biosensor device of cany one of claims 59 to 64, further comprising a hydrophobic coating disposed around the well or aperture.

66. The biosensor device of claim 65, wherein the hydrophobic layer comprises parylene.

67. The biosensor of claim 21, wherein the positively charged polymer comprises a PNA / PNA duplex and wherein the first redox reporter comprises one or more methylene blue redox reporters.

68. The biosensor of claim 67, wherein one strand or both strands of the PNA / PNA duplex have a net positive charge.

69. The biosensor of claim 67, wherein the one or more methylene blue redox reporters is operationally bound to one strand or both strands of the PNA / PNA duplex.

70. The biosensor of claim 67, adapted for use in consecutive measurements of the analyte without substantial loss of the electrochemical signal.

71. The biosensor of claim 67, adapted for detecting the presence of the analyte in a sample comprising whole blood or whole blood diluted in a sample buffer fluid.

72. An electrochemical biosensor comprising:a plurality of charged polymers, each charged polymer having first and second ends; an electrode, wherein the first end of each charged polymer is operationally affixed to the electrode, and wherein the second end of each charged polymer is structured to bind to a first receptor for a target analyte;a second receptor for the target analyte;one or more of a first redox reporter operationally bound to the second receptor, wherein the first redox reporter is reactive at positive potential when the charged polymer presents a net negative charge and reactive at negative potential when the charged polymer presents a net positive charge; andwherein upon application of an electric field and binding of the charged polymer to the first receptor and binding of the second receptor to the target analyte to provide a target analyte-second receptor complex, the biosensor is characterized by:an unbound state of the charged polymers, where no target analyte-second receptor complex is bound to the first receptor, at which the charged polymers are displaced towards the biosensor electrode surface and electron transfer from the one or more first redox reporters towards the biosensor electrode occurs at an unbound electron transfer rate; and a bound state of the charged polymers, where the target analyte-second receptor complex is bound to the first receptor, at which the charged polymers are displaced towards the biosensor electrode surface and electron transfer from the one or more first redox reporters towards the biosensor electrode occurs at a bound electron transfer rate.

73. The biosensor of claim 72, wherein an inverted molecular pendulum (iMP) comprises the charged polymer bound to the first receptor, and wherein the iMP is operationally affixed to the electrode.

74. The biosensor of claim 72 or 73, wherein the electrode is multilayered.

75. The biosensor of any one of claims 72-74, wherein the electrode comprises a gold layer and has a parylene coating layer.

76. The biosensor of any one of claims 72-75, wherein the charged polymers are operationally affixed to the electrode by covalent bonding via an intermediate linking moiety.

77. The biosensor of any one of claims 72-76, wherein each charged polymer is structured to bind to the first receptor via an adapter, wherein the adapter has a first portion configured to bind to the second end of the polymer and the adapter has a second portion configured to bind to the first receptor.

78. The biosensor of any one of claim 72-77, wherein upon binding of the target analyte-second receptor complex to the first receptor at the applied electric field, an electrochemical signal is produced translating a difference between the unbound electron transfer rate and the bound electron transfer rate.

79. The biosensor of any one of claims 73-78, wherein upon binding of the target analyte-second receptor complex to the first receptor and application of the electric field, the first redox reporter causes an electron transfer as the iMPs approach the biosensor electrode surface.

80. The biosensor of any one of claims 73-79, wherein the electron transfer rate is dependent on a time rate at which the iMPs are displaced.

81. The biosensor of any one of claims 73-80, wherein the unbound electron transfer rate is dependent on a time rate at which the unbound iMPs are displaced.

82. The biosensor of any one of claims 73-81, wherein the bound electron transfer rate is dependent on a time rate at which the bound iMPs are displaced.

83. The biosensor of any one of claims 73-82, wherein the iMPs displacement towards the biosensor electrode surface substantially corresponds to a tilting movement or a collapsing movement of the iMPs.

84. The biosensor of any one of claims 72-83, wherein upon binding of the target analyte- second receptor complex to the first receptor and application of the electric field, the first redox reporter touches the biosensor electrode surface, and the electron transfer is based on a redox reaction or electron tunneling current.

85. The biosensor of any one of claims 72-84, wherein the first receptor is adapted to bind to the target analyte at a first target analyte binding site, the second receptor is adapted to bind to the target analyte at a second target analyte binding site, and the first receptor is adapted to bind the target analyte-second receptor complex.

86. The biosensor of any one of claims 73-85, wherein the first redox reporter is covalently bound to the second receptor.

87. The biosensor of any one of claims 72-86, wherein the charged polymer comprises a double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), one or more charged polymers, one or more uncharged polymers, or any combination thereof.

88. The biosensor of any one of claims 72-86, wherein the charged polymer is negatively charged and optionally comprises a DNA / DNA duplex, a PNA / DNA duplex, a PNA / PNA duplex, optionally wherein one or both of the PNA are modified with negatively charged amino acids, a rigid anionic polyelectrolyte, a rigid negatively charged peptide, or any combination thereof.

89. The biosensor of any one of claims 72-88, wherein the first redox reporter comprises ferrocene, [Co(GA)2(phen)] (GA=gly colic acid, phen=l, 10-phenathroline), metal nanoparticles (e.g., Au, Pt, Pd, Ag, Cu), pyrroloquinoline quinone (PQQ), benzoquine,Osmium(III) complexes such as Os(bpy)Ci23+, diphenylamine, or any combination thereof.

90. The biosensor of any one of claims 72-89, wherein the first redox reporter has a redox state change above 0 mV.

91. The biosensor of any one of claims 72-86, wherein the charged polymer is positively charged and optionally comprises a PNA / PNA duplex comprising one or more lysines, one or more arginines, a rigid cationic polyelectrolyte, a rigid positively charged peptide, or any combination thereof.

92. The biosensor of any one of claims 72-86 and 91, wherein the first redox reporter comprises methylene blue, ruthenium(III) complexes such as Ru(NH3)e3+, neutral red, toluidine blue, phenosafranine, or any combination thereof.

93. The biosensor of any one of claims 72-86, 91 and 92, wherein the first redox reporter has a redox state change below 0 mV.

94. The biosensor of any one of claims 72-93, wherein the charged polymer has a length ranging from about 5 nm to about 20 nm.

95. The biosensor of any one of claims 72-90, wherein the charged polymer comprises a ssDNA having a length ranging from about lOmer to about lOOmer.

96. The biosensor of any one of claims 72-90, wherein the charged polymer comprises a dsDNA having a length ranging from about 15mer to about 60mer.

97. The biosensor of any one of claims 72-96, wherein the charged polymer is rigid along a length thereof, flexible or collapsible along the length thereof, or has a combination of one or more rigid and flexible or collapsible portions along the length thereof.

98. The biosensor of any one of claims 73-97, wherein the iMPs form a molecular monolayer at the surface of the biosensor electrode.

99. The biosensor of any one of claims 72-98, wherein the first receptor, the second receptor, or both the first receptor and the second receptor, comprises an antibody, an antibody receptor, a nanobody, an antigen, an aptamer, an aptamer fragment, a molecular imprint, a protein receptor, DNA, a microorganism, a protein / enzyme substrate, or any combination thereof.

100. The biosensor of claim 99, wherein the antibody receptor comprises an Fc receptor.

101. The biosensor of any one of claims 72-100, wherein the biosensor electrode comprises a glassy carbon electrode, a carbon nanotube-modified electrode, an indium tin oxide (ITO) electrode, a platinum electrode, a silver electrode, a gold electrode, or a palladium electrode.

102. The biosensor of any one of claims 72-101, wherein the biosensor electrode comprises a gold nanostructured microelectrode or a gold wire electrode.

103. A method of detecting a target analyte in a sample comprising:providing the electrochemical biosensor of any one of claims 72-102; contacting said biosensor with the sample; anddetecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte; andoptionally wherein the first receptor, the second receptor, or both the first receptor and the second receptor, is an antibody or a fragment thereof, wherein the binding of the target analyte to the antibody or fragment thereof occurs when the sample is at a first temperature and / or a first pH, and dissociation of the target analyte occurs at a second temperature, the method further comprises:(iii) optionally applying heat and / or ionic molecules to the sample, thereby causing the sample to have the second temperature and / or a second pH sufficient to result in the dissociation;(iv) optionally applying a wash solution to remove the sample from the biosensor; and(v) optionally contacting the biosensor with a further sample and detecting the electrochemical signal, wherein detection of saidsignal indicates the presence of the target analyte bound to antibody or a fragment thereof.

104. A method for in situ detection of a target analyte in a sample comprising a biological fluid, comprising:contacting the electrochemical biosensor of any one of claims 72-102 with the biological fluid; anddetecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte.

105. Use of the electrochemical biosensor of any one of claims 72-102 to detect the presence of a target analyte in a sample.

106. Use of the electrochemical biosensor of any one of claims 72-102 to detect the presence of a target analyte in a sample comprising a biological fluid.

107. The method of claim 104 or the use of claim 106, wherein the biological fluid comprises saliva, blood, urine, tears, sweat or feces.

108. The biosensor of any one of claims 72-102, the method of any one of claims 103, 104 or 107, or the use of any one of claims 105-106, wherein the target analyte comprises a small molecule, a macromolecule, a prokaryotic cell, a eukaryotic cell, a prokaryotic cell- derived component, a eukaryotic cell-derived component (e.g., nucleic acid material), a virus, a bacterium, an antibody, a protein, a cellular extract, or any combination thereof.

109. A kit comprising the biosensor of any one of claims 72-102 or claim 108, wherein:the second receptor of the biosensor is provided suspended in a carrier fluid for addition to the sample to allow binding of the second receptor to the target analyte to provide the target analyte-second receptor complex.

110. A method of detecting one or more target analytes in a sample comprising:providing the kit of claim 109;contacting the biosensor with the sample, comprising:adding the second receptor of the biosensor to the sample to allow binding of the second receptor to the target analyte to provide the target analyte- second receptor complex;contacting the sample comprising the target analyte-second receptor complex with the first receptor; anddetecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte-second receptor complex.

111. The method of claim 110, wherein:the detecting the electrochemical signal comprises the target analyte-second receptor complex binding to the first receptor.

112. The method of claim 111, wherein the application of the electric field is in chronoamperometric configuration, by square wave voltammetry, or selectably in either chronoamperometric configuration or by square wave voltammetry.

113. The method of claim 111, wherein the binding of the first receptor and / or the second receptor to the target analyte occurs when the sample is at a first temperature and / or a first pH, and dissociation of the first receptor and / or the second receptor to the target analyte occurs at a second temperature, and further comprising:applying heat and / or ionic molecules to the sample, thereby causing the sample to have the second temperature and / or a second pH sufficient to result in the dissociation.

114. The method of claim 113, further comprising:applying a wash solution to remove the sample from the biosensor.

115. The method of claim 114, further comprising:contacting the biosensor with a further sample; anddetecting the electrochemical signal, wherein detection of said signal indicates the presence of the target analyte-second receptor complex bound to the to the first receptor.

116. The biosensor of any one of claims 72-102 or claim 108, further comprising a second redox reporter bound to the charged polymer.

117. The biosensor of claim 116, wherein the second redox reporter is covalently bound to the charged polymer.

118. The biosensor of claim 117, wherein the second redox reporter is non-covalently bound to the charged polymer.

119. The biosensor of claim 116, wherein the second redox reporter is reactive at a different potential than the first redox reporter.

120. The biosensor of claim 116, wherein upon binding of the first end of the charged polymer to the surface of the biosensor electrode, the second redox reporter causes an electron transfer to the biosensor electrode surface.

121. The biosensor of any one of claims 73-102, 108, or 116-120, wherein the plurality of iMPs are bound to the surface of the biosensor electrode at a density sufficient to sterically hinder the iMPs bound state from being displaced towards the biosensor electrode.

122. The biosensor of claim 121, wherein the application of the electric field is in chronoamperometric configuration, by square wave voltammetry, or selectably in either chronoamperometric configuration or by square wave voltammetry.

123. A device for detecting a target analyte in a sample comprising:a collector for collecting the sample;a sensing component comprising the biosensor as defined in any one of claims 73-102 or 108-122; anda connector for connecting the sensing component to an electrochemical measurement device;wherein the collector and the sensing component are configured for allowing contact between the iMPs of the biosensor and the collected sample.

124. The device of claim 123, wherein the sensing component further comprises two working electrodes, a counter electrode and a reference electrode.

125. The device of claim 124, wherein each electrode is in the form of a wire.

126. The device of any one of claims 124-125, wherein the electrodes are disposed in a matrix of a non-conductive material.

127. The device of any one of claims 125-126, wherein the iMPs are bound to a first end of the biosensor electrode wire and the iMPs are exposed to the sample when the first end is in contact with the collected sample.

128. The device of any one of claims 125-127, wherein a second end of the wires are in contact with the connector.

129. The device of any one of claims 123-128, further comprising a voltage controller electrically connected to at least one of the electrodes, wherein the voltage controller is configured to deliver voltage to at least one of the electrodes in chronoamperometric configuration, by square wave voltammetry, or selectably in either chronoamperometric configuration or by square wave voltammetry.

130. A biosensor device for detecting an analyte in a sample, the biosensor device comprising:a substrate;at least one electrode provided on the substrate;a first dielectric layer provided on the substrate, the first dielectric layer comprising a well or aperture associated with the electrode such that the electrode is responsive to the presence of a sample received within the well or aperture; and a second dielectric layer provided on the first dielectric layer comprising an aperture extending through the second dielectric layer fluidly connected to the well or aperture in the first dielectric layer.

131. The biosensor device of claim 130, wherein the at least one electrode is functionalized so as to interact with a sample received within the well or aperture of the first dielectric layer.

132. The biosensor device of claim 131, wherein the at least one electrode comprises a surface and a plurality of iMPs as defined in any one of claims 73-102 or 108-122 configured to bind an analyte within the sample.

133. The biosensor device of any one of claims 130-132, wherein the substrate is a rigid substrate.

134. The biosensor device of any one of claims 130-133, wherein the electrode comprises a plurality of metal layers, the metal layers comprising or being formed from copper, nickel, platinum, palladium, silver, silver chloride, gold or other noble metals.

135. The biosensor device of claim 134, wherein the plurality of metal layers comprises the palladium layer in between the gold layer and the nickel layer.

136. The biosensor device of cany one of claims 130-135, further comprising a hydrophobic coating disposed around the well or aperture.

137. The biosensor device of claim 136, wherein the hydrophobic layer comprises parylene.

138. The biosensor of claim 92, wherein the positively charged polymer comprises a PNA / PNA duplex and wherein the first redox reporter comprises one or more methylene blue redox reporters.

139. The biosensor of claim 138, wherein one strand or both strands of the PNA / PNA duplex have a net positive charge.

140. The biosensor of claim 138, wherein the one or more methylene blue redox reporters is operationally bound to one strand or both strands of the PNA / PNA duplex.

141. The biosensor of claim 138, adapted for use in consecutive measurements of the analyte without substantial loss of the electrochemical signal.

142. The biosensor of claim 138, adapted for detecting the presence of the analyte in a sample comprising whole blood or whole blood diluted in a sample buffer fluid.

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