Improvements in electrodes for electrochemical sensors

Abstract

Provided is a reference electrode for an electrochemical sensor, the reference electrode comprising: a solid electrically conductive element; one or more chemical species involved in the generation of a reference electrical potential in the conductive element; wherein the reference electrode comprises, or is associated with, a migration inhibitor configured to inhibit, prevent or retard migration of the one or more chemical species into a fluid distal to the reference electrode while allowing electrical communication between the conductive element and the fluid.

Description

IMPROVEMENTS IN ELECTRODES FOR ELECTROCHEMICAL SENSORSFIELD

[0001] . The present disclosure relates generally to electrochemical sensors, and particularly those having a working electrode and a reference electrode. The disclosure provides an improvement in working electrode performance by limiting the extent to which a deleterious chemical species associated with a reference electrode is able to contact the working electrode.BACKGROUND

[0002] . Electrochemical sensors have been proven to be a reliable means for the real time detection of target analytes in diverse media including air, water, process intermediates, and biological fluids. In such sensors, a biological or a chemical analyte recognition element is applied to an electrically conductive element to form a working electrode. Upon interrogation of the sensor, a current is produced. A feature of the current may be used determine the amount of analyte in the test fluid.

[0003] . Electrochemical aptamer-based (EAB) sensors are showing increasing promise in industry and medicine. In an EAB sensor, the working electrode is coated with a plurality of aptamers that specifically recognise a target analyte. A redox reporter (such as methylene blue) is typically covalently linked to the aptamer at its free end. The recognition manifests as a conformational change in the aptamer upon analyte binding. The conformational change in turn alters the accessibility of the redox reporter to the working electrode surface, thereby producing an analyte-induced change in the level of electron transport between the redox reporter and the electrode.

[0004] . In some circumstances, binding of the analyte brings the redox reporter proximal to the electrode surface, thereby increasing the level of electron transport and in turn increasing current through the electrode. In other circumstances, binding moves the redox reporter distal to the electrode surface resulting in the opposite effects. Regardless, binding of the analyte results in a detectable change in electrode current.

[0005] . Electrochemical sensors are typically interrogated by the application of an electrical potential across a working electrode and a counter electrode, often by way of square wave voltammetry or cyclic voltammetry. Faradaic current is measured (again across theworking and counter electrodes) with a current feature such as signal decay kinetics being exploited to determine the amount of analyte about the working electrode.

[0006] . While electrochemical sensors are undoubtedly useful, problems in their practical implementation arise. One problem is that of performance degradation, which may manifest as signal loss leading to a decrease in responsiveness of the working electrode to the target analyte. Prior artisans have proposed that fouling of the working electrode is causative, especially where the test solution is a biological fluid. It is thought that any of cells, proteins, lipids, and enzymes, may interfere with the conformational change of a recognition element, or may degrade the element itself. Where the sensor relies on a DNA or RNA aptamer, it is proposed that nucleases present in biological fluids actively cleave the aptamer from the working electrode surface. Prior artisans have therefore sought to implement modified aptamers that are resistant to nuclease activity. In one approach, XNA aptamers are used because of their inherent resistance to nucleases. Where the analyte recognition element is a protein, prior artisans have sought to make modifications to confer protease resistance.

[0007] . Problems of signal loss are particularly evident where the sensor is an aptamerbased sensor or a protein-based sensor that is disposed in situ within the body a subject, and the sensor remains in place for real time analyte detection over a period of hours or days.

[0008] . It is an aspect of the present disclosure to provide an improvement to prior art electrochemical sensors. It is a further aspect of the present disclosure to provide a useful alternative to prior art electrochemical sensors.

[0009] . The discussion of documents, acts, materials, devices, articles, and the like is included in this specification solely for the purpose of providing a context for the present disclosure. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.SUMMARY

[0010] . In a first aspect, but not necessarily the broadest aspect, the present disclosure provides a reference electrode for an electrochemical sensor, the reference electrode comprising: a solid electrically conductive element; one or more chemical species involved in the generation of a reference electrical potential in the conductive element; wherein the reference electrode comprises, or is associated with, a migration inhibitor configured to inhibit, prevent or retard migration of the one or more chemical species into a fluid distal to the reference electrode while allowing electrical communication between the conductive element and the fluid.[Oi l]. In one embodiment of the first aspect, the migration inhibitor is configured to inhibit, prevent or retard migration of the one or more chemical species to a working electrode in functional association with the reference electrode via the fluid.

[0012] . In one embodiment of the first aspect, the one or more chemical species is soluble in the fluid.

[0013] . In one embodiment of the first aspect, the one or more chemical species is an ion.

[0014] . In one embodiment of the first aspect, the ion is not a chloride ion or a hydrogen ion.

[0015] . In one embodiment of the first aspect, the ion is a metal ion.

[0016] . In one embodiment of the first aspect, the metal ion is a silver ion, or another cation.

[0017] . In one embodiment of the first aspect, the migration inhibitor is a selective anion exchange material.

[0018] . In one embodiment of the first aspect, the migration inhibitor is configured to selectively inhibit, prevent or retard the passage of a cation while allowing passage of an anion.

[0019] . In one embodiment of the first aspect, the cation is a silver ion and the anion is a chloride ion.

[0020] . In one embodiment of the first aspect, the migration inhibitor forms a complete or partial physical barrier to inhibit, prevent or retard the migration of the one or more chemical species into the fluid.

[0021] . In one embodiment of the first aspect, the physical barrier inhibits or prevents bulk mixing of fluid on opposing sides thereof.

[0022] . In one embodiment of the first aspect, the physical barrier does not substantially inhibit, prevent or retard the migration of chloride ions or hydrogen ions between the fluid and the conductive element.

[0023] . In one embodiment of the first aspect, the migration inhibitor captures the one or more chemical species.

[0024] . In one embodiment of the first aspect, the migration inhibitor presents an energy barrier to the migration of the one or more chemical species.

[0025] . In one embodiment of the first aspect, the migration inhibitor is formed by a layer or a membrane applied to, or disposed about, the conductive element.

[0026] . In one embodiment of the first aspect, the layer or the membrane is formed in situ on a surface of the conductive element.

[0027] . In one embodiment of the first aspect, the layer or the membrane is formed by contacting the conductive element surface with (i) a suspension or solution comprising the migration inhibitor (ii) a suspension or solution of a monomer or an oligomer of a polymer migration inhibitor; removing a liquid component of the suspension or solution to cause respectively deposition of the migration inhibitor on the surface, or formation of the polymer migration inhibitor on the surface.

[0028] . In one embodiment of the first aspect, removal of the liquid component is effected by way of evaporation.

[0029] . In one embodiment of the first aspect, the layer or the membrane is formed by causing or allowing the layer or the membrane to transition to a substantially insoluble state in a test fluid.

[0030] . In one embodiment of the first aspect, the transition to a substantially insoluble state material of the layer or the membrane results from chemical crosslinking of the material in situ.

[0031] . In one embodiment of the first aspect, the migration inhibitor is biocompatible.

[0032] . In one embodiment of the first aspect, the migration inhibitor is a polymer.

[0033] . In one embodiment of the first aspect, the polymer is an ionomer.

[0034] . In one embodiment of the first aspect, the polymer comprises a backbone structure having extending therefrom ionically active functional groups.

[0035] . In one embodiment of the first aspect, the ionically active functional groups are charged groups.

[0036] . In one embodiment of the first aspect, the charged groups are anion groups.

[0037] . In one embodiment of the first aspect, the charged groups are cation groups.

[0038] . In one embodiment of the first aspect, the charged groups are quaternary ammonium groups.

[0039] . In one embodiment of the first aspect, the polymer is a crosslinked quaternary ammonium functionalized poly(2,6-dimethyl-l,4-phenylene oxide).

[0040] . In one embodiment of the first aspect, the polymer is formed by contacting a surface of the conductive element with a solution comprising oligomers of quaternary ammonium functionalized poly(2,6-dimethyl-l,4-phenylene oxide), the oligomers being crosslinked to form a crosslinked quaternary ammonium functionalized poly(2,6-dimethyl-l,4-phenylene oxide) layer.

[0041] . In embodiments where the charged groups in the polymer surrounding the conductive elements are cationic, the material inhibits, prevents or retards the passage of cations by creating an energy barrier that the cations have to overcome to be able to pass through the material, whereas anions experience a smaller or no energy barrier in passing through the material.

[0042] . In one embodiment of the first aspect, the backbone structure comprises carbon and fluorine atoms.

[0043] . In one embodiment of the first aspect, the polymer is a fluoropolymer or a fluorocopolymer.

[0044] . In one embodiment of the first aspect, the ionically active functional groups are charged at about neutral pH, or at about a physiological pH.

[0045] . In one embodiment of the first aspect, the ionically active functional groups are acidic functional groups.

[0046] . In one embodiment of the first aspect, the acidic functional groups are sulfonic acid functional groups.

[0047] . In one embodiment of the first aspect, the polymer is ethanesulfonyl fluoride, 2-[ 1 - [difluoro- [(trifluoroethenyl)oxy] methyl]- l,2,2,2-tetrafhroroethoxy]-l,l,2,2,-tetrafluoro-, with tetrafluoroethylene tetrafluoroethylene -perfluoro-3,6-dioxa-4-methyl -7- octenesulfonic acid copolymer.

[0048] . In embodiments where the charged groups in the polymer surrounding the conductive elements are anionic, the material inhibits, prevents or retards the passage of contaminant cations by the anionic groups in the polymer capturing the cations, thus inhibiting, preventing or retarding their passage through the material, whereas hydrogen ions experience a smaller or no tendency to be captured by the anionic groups in passing through the material.

[0049] . In one embodiment of the first aspect, the conductive element is configured to pierce the skin of a human or another mammal.

[0050] . In one embodiment of the first aspect, the conductive element is configured to penetrate the skin of a human or another mammal.

[0051] . In one embodiment of the first aspect, the conductive element is a wire, a needle or a microneedle.

[0052] . In one embodiment of the first aspect, the migration inhibitor selectively inhibits, prevents or retards migration of the one or more chemical species.

[0053] . In a second aspect, the present disclosure provides a working electrode for an electrochemical sensor, the working electrode comprising: a solid electrically conductive element; a plurality of analyte recognition elements associated with the conductive element; wherein the working electrode comprises, or is associated with, a migration inhibitor configured to inhibit, prevent or retard migration of one or more chemical species involved in the generation of a reference electrical potential in the conductive element from a reference electrode of the electrochemical sensor from contacting the conductive element or the analyte recognition elements while allowing electrical communication between the conductive element and the fluid.

[0054] . In one embodiment of the second aspect, the migration inhibitor is configured to inhibit, prevent or retard migration of the one or more chemical species to the working electrode from the reference electrode in functional association therewith via the fluid.

[0055] . In one embodiment of the second aspect, the one or more chemical species is soluble in the fluid.

[0056] . In one embodiment of the second aspect, the one or more chemical species is an ion.

[0057] . In one embodiment of the second aspect, the ion is not a chloride ion or a hydrogen ion.

[0058] . In one embodiment of the second aspect, the ion is a metal ion.

[0059] . In one embodiment of the second aspect, the metal ion is a silver ion, or another cation.

[0060] . In one embodiment of the second aspect, the migration inhibitor is a selective anion exchange material.

[0061] . In one embodiment of the second aspect, the migration inhibitor is configured to selectively inhibit, prevent or retard the passage of a cation while allowing passage of an anion.

[0062] . In one embodiment of the second aspect, the cation is a silver ion and the anion is a chloride ion.

[0063] . In one embodiment of the second aspect, the migration inhibitor forms a complete or partial physical barrier to inhibit, prevent or retard the migration of the one or more chemical species into the fluid.

[0064] . In one embodiment of the second aspect, the physical barrier inhibits or prevents bulk mixing of fluid on opposing sides thereof.

[0065] . In one embodiment of the second aspect, the physical barrier does not substantially inhibit, prevent or retard the migration of chloride ions or hydrogen ions between the fluid and the conductive element.

[0066] . In one embodiment of the second aspect, the migration inhibitor captures the one or more chemical species.

[0067] . In one embodiment of the second aspect, the migration inhibitor presents an energy barrier to the migration of the one or more chemical species.

[0068] . In one embodiment of the second aspect, the migration inhibitor is formed by a layer or a membrane applied to, or disposed about, the conductive element.

[0069] . In one embodiment of the second aspect, the layer or the membrane is formed in situ on a surface of the conductive element.

[0070] . In one embodiment of the second aspect, the layer or the membrane is formed by contacting the conductive element surface with (i) a suspension or solution comprising the migration inhibitor (ii) a suspension or solution of a monomer or an oligomer of a polymer migration inhibitor; removing a liquid component of the suspension or solution to cause respectively deposition of the migration inhibitor on the surface, or formation of the polymer migration inhibitor on the surface.

[0071] . In one embodiment of the second aspect, removal of the liquid component is effected by way of evaporation.

[0072] . In one embodiment of the second aspect, the layer or the membrane is formed by causing or allowing the layer or the membrane to transition to a substantially insoluble state in a test fluid.

[0073] . In one embodiment of the second aspect, the transition to a substantially insoluble state material of the layer or membrane results from chemical crosslinking of the material in situ.

[0074] . In one embodiment of the second aspect, the migration inhibitor is biocompatible.

[0075] . In one embodiment of the second aspect, the migration inhibitor is a polymer.

[0076] . In one embodiment of the second aspect, the polymer is an ionomer.

[0077] . In one embodiment of the second aspect, the polymer comprises a backbone structure having extending therefrom ionically active functional groups.

[0078] . In one embodiment of the second aspect, the ionically active functional groups are charged groups.

[0079] . In one embodiment of the second aspect, the charged groups are anion groups.

[0080] . In one embodiment of the second aspect, the charged groups are cation groups.

[0081] . In one embodiment of the second aspect, the charged groups are quaternary ammonium groups.

[0082] . In one embodiment of the second aspect, the polymer is a crosslinked quaternary ammonium functionalized poly(2,6-dimethyl-l,4-phenylene oxide).

[0083] . In one embodiment of the second aspect, the polymer is formed by contacting a surface of the conductive element with a solution comprising oligomers of quaternaryammonium functionalized poly(2,6-dimethyl-l,4-phenylene oxide), the oligomers being crosslinked to form a crosslinked quaternary ammonium functionalized poly(2,6-dimethyl- 1,4-phenylene oxide) layer.

[0084] . In embodiments where the charged groups in the polymer surrounding the conductive elements are cationic, the material inhibits, prevents or retards the passage of cations by creating an energy barrier that the cations have to overcome to be able to pass through the material, whereas anions experience a smaller or no energy barrier in passing through the material.

[0085] . In one embodiment of the second aspect, the backbone structure comprises carbon and fluorine atoms.

[0086] . In one embodiment of the second aspect, the polymer is a fluoropolymer or a fluorocopolymer.

[0087] . In one embodiment of the second aspect, the ionically active functional groups are charged at about neutral pH, or at about a physiological pH.

[0088] . In one embodiment of the second aspect, the ionically active functional groups are acidic functional groups.

[0089] . In one embodiment of the second aspect, the acidic functional groups are sulfonic acid functional groups.

[0090] . In one embodiment of the second aspect, the polymer is ethanesulfonyl fluoride, 2-[l-[difhroro-[(trifluoroethenyl)oxy]methyl]- l,2,2,2-tetrafluoroethoxy]-l,l,2,2,- tetrafluoro-, with tetrafluoroethylene tetrafluoroethylene -perfluoro-3,6-dioxa-4-methyl-7- octenesulfonic acid copolymer.

[0091] . In embodiments where the charged groups in the polymer surrounding the conductive elements are anionic, the material inhibits, prevents or retards the passage of contaminant cations by the anionic groups in the polymer capturing the cations, thus inhibiting, preventing or retarding their passage through the material, whereas hydrogen ions experience a smaller or no tendency to be captured by the anionic groups in passing through the material.

[0092] . In one embodiment of the second aspect, the conductive element is configured to pierce the skin of a human or another mammal.

[0093] . In one embodiment of the second aspect, the conductive element is configured to penetrate the skin of a human or another mammal.

[0094] . In one embodiment of the second aspect, the conductive element is a wire, a needle or a microneedle.

[0095] . In one embodiment of the second aspect, the migration inhibitor selectively inhibits, prevents or retards migration of the one or more chemical species.

[0096] . In a third aspect, the present disclosure provides an electrochemical sensor comprising: a reference electrode comprising or being associated with one or more chemical species involved in the generation of a reference electrical potential; a working electrode; and a migration inhibitor configured to inhibit, prevent or retard migration of the one or more chemical species from the reference electrode to the working electrode while allowing electrical communication between the working electrode and a fluid about the working electrode.

[0097] . In one embodiment of the third aspect, the migration inhibitor is configured to inhibit, prevent or retard migration of the one or more chemical species from contacting a working electrode or an analyte recognition element of the working electrode.

[0098] . In one embodiment of the third aspect, the migration inhibitor is disposed between the reference electrode and the working electrode.

[0099] . In one embodiment of the third aspect, the one or more chemical species is soluble in the fluid.

[0100] . In one embodiment of the third aspect, the one or more chemical species is an ion.

[0101] . In one embodiment of the third aspect, the ion is not a chloride ion or a hydrogen ion.

[0102] . In one embodiment of the third aspect, the ion is a metal ion.

[0103] . In one embodiment of the third aspect, the metal ion is a silver ion, or another cation.

[0104] . In one embodiment of the third aspect, the migration inhibitor is a selective anion exchange material.

[0105] . In one embodiment of the third aspect, the migration inhibitor is configured to selectively inhibit, prevent or retard the passage of a cation while allowing passage of an anion.

[0106] . In one embodiment of the third aspect, the cation is a silver ion and the anion is a chloride ion.

[0107] . In one embodiment of the third aspect, the migration inhibitor forms a complete or partial physical barrier to inhibit, prevent or retard the migration of the one or more chemical species into the fluid.

[0108] . In one embodiment of the third aspect, the physical barrier inhibits or prevents bulk mixing of fluid on opposing sides thereof.

[0109] . In one embodiment of the third aspect, the physical barrier does not substantially inhibit, prevent or retard the migration of chloride ions or hydrogen ions between the fluid and the working electrode.

[0110] . In one embodiment of the third aspect, the migration inhibitor captures the one or more chemical species.

[0111] . In one embodiment of the third aspect, the migration inhibitor presents an energy barrier to the migration of the one or more chemical species.

[0112] . In one embodiment of the third aspect, the migration inhibitor is formed by a layer or a membrane applied to, or disposed about, the conductive element.

[0113] . In one embodiment of the third aspect, the layer or the membrane is formed in situ on a surface of the conductive element.

[0114] . In one embodiment of the third aspect, the layer or the membrane is formed by contacting the conductive element surface with (i) a suspension or solution comprising the migration inhibitor (ii) a suspension or solution of a monomer or an oligomer of a polymer migration inhibitor; removing a liquid component of the suspension or solution to cause respectively deposition of the migration inhibitor on the surface, or formation of the polymer migration inhibitor on the surface.

[0115] . In one embodiment of the third aspect, removal of the liquid component is effected by way of evaporation.

[0116] . In one embodiment of the third aspect, the layer or the membrane is formed by causing or allowing the layer or the membrane to transition to a substantially insoluble state in a test fluid.

[0117] . In one embodiment of the third aspect, the transition to a substantially insoluble state material of the layer or the membrane results from chemical crosslinking of the material in situ.

[0118] . In one embodiment of the third aspect, the migration inhibitor is biocompatible.

[0119] . In one embodiment of the third aspect, the migration inhibitor is a polymer.

[0120] . In one embodiment of the third aspect, the polymer is an ionomer.

[0121] . In one embodiment of the third aspect, the polymer comprises a backbone structure having extending therefrom ionically active functional groups.

[0122] . In one embodiment of the third aspect, the ionically active functional groups are charged groups.

[0123] . In one embodiment of the third aspect, the charged groups are anion groups.

[0124] . In one embodiment of the third aspect, the charged groups are cation groups.

[0125] . In one embodiment of the third aspect, the charged groups are quaternary ammonium groups.

[0126] . In one embodiment of the third aspect, the polymer is a crosslinked quaternary ammonium functionalized poly(2,6-dimethyl-l,4-phenylene oxide).

[0127] . In one embodiment of the third aspect, the polymer is formed by contacting a surface of the conductive element with a solution comprising oligomers of quaternary ammonium functionalized poly(2,6-dimethyl-l,4-phenylene oxide), the oligomers being crosslinked to form a crosslinked quaternary ammonium functionalized poly(2,6-dimethyl- 1,4-phenylene oxide) layer.

[0128] . In embodiments where the charged groups in the polymer surrounding the conductive elements are cationic, the material inhibits, prevents or retards the passage of cations by creating an energy barrier that the cations have to overcome to be able to pass through the material, whereas anions experience a smaller or no energy barrier in passing through the material.

[0129] . In one embodiment of the third aspect, the backbone structure comprises carbon and fluorine atoms.

[0130] . In one embodiment of the third aspect, the polymer is a fluoropolymer or a fluorocopolymer.

[0131] . In one embodiment of the third aspect, the ionically active functional groups are charged at about neutral pH, or at about a physiological pH.

[0132] . In one embodiment of the third aspect, the ionically active functional groups are acidic functional groups.

[0133] . In one embodiment of the third aspect, the acidic functional groups are sulfonic acid functional groups.

[0134] . In one embodiment of the third aspect, the polymer is ethanesulfonyl fluoride, 2-[ l-[difhroro-[(trifluoroethenyl)oxy]methyl]- 1,2,2,2-tetrafluoroethoxy]-! , 1 ,2,2,- tetrafluoro-, with tetrafluoroethylene tetrafluoroethylene -perfhroro-3,6-dioxa-4-methyl-7- octenesulfonic acid copolymer.

[0135] . In embodiments where the charged groups in the polymer surrounding the conductive elements are anionic, the material inhibits, prevents or retards the passage of contaminant cations by the anionic groups in the polymer capturing the cations, thus inhibiting, preventing or retarding their passage through the material, whereas hydrogen ions experience a smaller or no tendency to be captured by the anionic groups in passing through the material.

[0136] . In one embodiment of the third aspect, the conductive element is configured to pierce the skin of a human or another mammal.

[0137] . In one embodiment of the third aspect, the conductive element is configured to penetrate the skin of a human or another mammal.

[0138] . In one embodiment of the third aspect, the conductive element is a wire, a needle or a microneedle.

[0139] . In one embodiment of the third aspect, the migration inhibitor selectively inhibits, prevents or retards migration of the one or more chemical species.

[0140] . In one embodiment of the third aspect, the reference electrode is according to any embodiment of the first aspect, and / or the working electrode is according to any embodiment of the second aspect.

[0141] . In one embodiment of the third aspect, the electrochemical sensor is configured such that the reference electrode functions also as a counter electrode, or the electrochemical sensor comprises a dedicated counter electrode.

[0142] . In one embodiment of the third aspect, the reference electrode and working electrodes are disposed in a fixed mutual spatial relationship.

[0143] . In one embodiment of the third aspect, the reference electrode and working electrodes are separated by a distance of less than about 20 mm, 19 mm, 18 mm, 17 mm, 16 mm, 15 mm, 14 mm, 13 mm, 12 mm, 11 mm, 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, or 1 mm.

[0144] . In one embodiment of the third aspect, the reference electrode and / or working electrode is configured to pierce the skin of a human or another mammal.

[0145] . In one embodiment of the third aspect, the reference electrode and / or the working electrode is a wire, a needle or a microneedle.

[0146] . In one embodiment of the third aspect, the electrochemical sensor is configured such that, when applied to a subject, the reference electrode and the working electrode contact an interstitial fluid below the surface of the skin.BRIEF DESCRIPTION OF THE FIGURES

[0147] . FIG. 1 illustrates in basic form a three-electrode electrochemical sensor of the prior art, demonstrating the migration of silver ions from the reference electrode to the working electrode.

[0148] . FIG. 2 illustrates in basic form a three-electrode electrochemical sensor whereby the reference electrode is coated with an ionomer that selectively allows for the bidirectional passage of hydrogen ions therethrough, while containing silver ions leached from the electrode.

[0149] . FIG. 3 illustrates in basic form a three-electrode electrochemical sensor whereby the working electrode is coated with an ionomer that selectively allows for the bidirectional passage of hydrogen ions therethrough, while preventing the inward passage of silver ions thereby preventing contact with the conductive portion of the electrode and the associated recognition elements.

[0150] . FIG. 4 is cross-sectional diagram of a reference electrode in the form of a microneedle, the microneedle being coated with a selectively permeable membrane except for at the sharpened terminus region.

[0151] . FIG. 5 is a graph of normalized current as a function of time for an electrochemical sensor having an Ag|AgCl reference electrode and an aptamer-functionalized working electrode, the latter being either devoid of a Nafion™ coating, or with increasing Nafion™ coating thicknesses. The BASi electrode referred to in the legend refers to a current state of the art Ag|AgCl reference electrode with no coating.

[0152] . Unless otherwise indicated herein, features of the drawings labelled with the same numeral are taken to be the same features, or at least functionally similar features, when used across different drawings.

[0153] . The drawings are not prepared to any particular scale or dimension and are not presented as being a completely accurate presentation of the various embodiments.DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS THEREOF

[0154] . After considering this description it will be apparent to one skilled in the art how the disclosure is implemented in various alternative embodiments and alternative applications. However, although various embodiments of the present disclosure will be described herein, it is understood that these embodiments are presented by way of example only, and not limitation. As such, this description of various alternative embodiments should not be construed to limit the scope or breadth of the present disclosure. Furthermore, statements of advantages or other aspects apply to specific exemplary embodiments, and not necessarily to all embodiments, or indeed any embodiment covered by the claims.

[0155] . Throughout the description and the claims of this specification the word “comprise” and variations of the word, such as “comprising” and “comprises” is not intended to exclude other additives, components, integers, or steps.

[0156] . Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to the same embodiment, but may.

[0157] . The present disclosure is predicated at least in part on the inventors’ discovery that certain contaminants can cause signal loss or other degradation in the performance of an electrochemical sensor. The contaminant may be intrinsic to the sensing matrix, for example biomolecules in the case of a sensor in contact with a biological fluid, or the contaminant may have some extrinsic source. A contaminant may originate from a component of the sensor itself, such as one of the electrodes. In that regard it is proposed that a chemical species originating from a reference electrode of an electrochemical sensor may be a source of malfunction, such as signal loss. Such malfunction may arise due to the chemical species having a deleterious effect on the working electrode of the sensor.

[0158] . It will be appreciated from the Background section that a working electrode must have a given potential applied, or be operated within a given range of potentials in the course of interrogation. Those potentials must be defined by reference to another known potential that is substantially invariable. A reference electrode is typically used to provide that known potential. In a two-electrode sensor the reference electrode functions also as a counter electrode, while in a three -electrode sensor the reference electrode is dedicated to that function.

[0159] . The potential of the reference electrode may be generated by way of a redox reaction having an equilibrium, such as that provided by the redox pair silver-silver chloride (Ag|AgCl). It has been found that in some circumstances, a chemical species originating from the reference electrode (such as silver ions) can interfere with the functioning of the working electrode of the sensor. Without wishing to be limited by theory in any way, it is proposed that when using an Ag|AgCl reference electrode and an aptamer coated working electrode, silver ions can leach from the reference electrode and deposit as silver ions or as elemental silver on the working electrode. This deposition of silver ions leads to an increased rate of degradation on the aptamer layer on the working electrode.

[0160] . Given this finding, it has been discovered that such degradation can be at least limited by the use of a migration inhibitor configured to inhibit, prevent, or retard migration of a deleterious chemical species migrating to, and contacting, the working electrode.

[0161] . The migration inhibitor may function to completely exclude the chemical species, and prevent entry of the chemical species into a bulk region of the migration inhibitor.

[0162] . Alternatively, the migration inhibitor may allow for entry of the chemical species into a bulk region, but once within the chemical species is captured. The chemical species may exit via the same route it entered, but it is not permitted to exit by any other route. In some embodiments, the migration inhibitor functions so as to sequester the chemical species within, not allowing for any exit whatsoever.

[0163] . In another embodiment, the migration inhibitor allows passage of chemical species therethrough, however the passage is not a free passage. In that circumstance the movement of the chemical species is retarded to some extent, such that the amount of chemical species permitted across the migration inhibitor per unit time in decreased.

[0164] . The migration inhibitor may take the form of a selectively permeable membrane that is disposed on or about the reference electrode, where the membrane is capable of slowing or preventing the passage of the chemical species through it, thus retarding it or preventing it from reaching the bulk fluid of the electrochemical cell forming the basis of the electrochemical sensor.

[0165] . The migration inhibitor may function as a sink for the undesirable species, thus preventing the species from reaching the bulk fluid until the sink is full.

[0166] . In a further alternative embodiment, the migration inhibitor may function to impose an energy barrier, for example via electrostatic repulsion, to the deleterious chemical species passing through the membrane, so as to retard or prevent its passage into the bulk fluid.

[0167] . Alternatively, the migration inhibitor may be a physical barrier that functions to inhibit or prevent the bulk mixing of fluid on opposing sides thereof. The term “bulk mixing” in some contexts may be taken to require that the components of the respective fluids are equally free to move across the barrier. Thus, in the context of the present disclosure the physical barrier may allow for movement of solvent across the barrier, but does not equally allow for the movement of all dissolved solutes across the barrier.

[0168] . The migration inhibitor may be applied to, or disposed in relation to, either the reference electrode or the working electrode. When applied to, or disposed in relation to, the reference electrode, the migration inhibitor functions to inhibit migration of thedeleterious chemical species to the working electrode. Conversely, when applied to, or disposed in relation to, the working electrode, the migration inhibitor functions to inhibit migration of the deleterious chemical species to the surface of the working electrode or a recognition element associated therewith. Accordingly, the term “electrode” herein is intended to include in the alternative the reference electrode or the working electrode. In some instances, such interpretation will clearly not apply, an example being where the intention is to refer to a counter electrode.

[0169] . The migration inhibitor may be a pre -formed membrane that is applied to, or disposed in relation to, at least the portion of the electrode that is intended to be exposed to the fluid under test. The pre-formed membrane may be sealed to at least substantially prevent the access of the deleterious species to the bulk fluid except by way of it having to pass through the membrane.

[0170] . The membrane may be formed in situ on the electrode by any suitable means known in the art, for example dipping the electrode into a solution of the membrane material dissolved in a solvent, spraying such a solution onto the electrode, spin-coating such a solution onto the electrode or any other means for depositing a material. After the solution layer is coated onto the electrode the solvent is removed via evaporation to leave behind a layer of solid membrane material on the electrode. In an optional step the layer of solid membrane material can be further treated to render it sufficiently insoluble in the fluid in which the reference electrode is to be used, where it is sufficiently insoluble if the membrane remains intact for the intended use life of the electrode.

[0171] . In one example of the practical application of the disclosure to a reference electrode, a silver wire is exposed to bleach to form a silver chloride layer on its surface. The exposed portion of the silver chloride coated silver wire is dipped into a 5 wt% solution of Nafion™ polymer in a mixture of water and higher alcohols (DuPont) and the resulting layer of solution is air-dried, using ambient or heated air, to remove the solvent.

[0172] . As discussed further infra, Nafion™ is a cation exchange polymer with a fluorinated carbon backbone and negatively charged sulfonic acid groups on side chains attached to the polymer backbone. Optionally, the wire can be dipped into the solution one or more additional times and dried, to increase the thickness of the resulting polymer layer.The coated wire is then heated to 120°C for one hour in an oven to phase invert the Nafion™, leaving it sufficiently insoluble in the fluid in which it is designed to be used.

[0173] . When the reference electrode is in use, the silver chloride on the surface of the silver wire is sparingly soluble in the fluid in which it is immersed. The small degree of solubility generates free silver ions. In prior art reference electrodes these silver ions enter the bulk fluid and possibly migrate to the working electrode. According to the present disclosure any free silver ions generated at the surface of the electrode enter the Nafion™ layer surrounding the electrode, associating with the anionic groups in the Nafion™ via electrostatic interaction and are retarded from exiting the Nafion™ into the bulk fluid. Importantly, the Nafion™ freely allows the passage of hydrogen ions through it, that can carry electric charge and allow the reference electrode to still function in its intended use as a source of a reference potential in an electrochemical cell. Other polymers comprising anionic groups are also suitable in this embodiment of the disclosure, for example Flemion™, a fluorinated polymer with carboxylic acid groups incorporated in its structure, polymers with a styrene backbone and incorporated anionic groups or other cation exchange polymers or cation exchange resins and other cation exchange materials as are known in the art.

[0174] . In one embodiment of the disclosure a silver wire is exposed to bleach to form a silver chloride layer on its surface. The exposed portion of the silver chloride coated silver wire is dipped into a solution of a custom synthesised PPO- (poly(2,6-dimethyl-l,4- phenylene oxide)) based anion exchange polymer comprising quaternary ammonium groups, dissolved in DMSO. The resulting layer of solution is air-dried, using ambient or heated air, to remove the solvent. Optionally, the wire can be dipped into the solution one or more additional times and dried to increase the thickness of the resulting polymer layer. The polymer is designed to be crosslinked using UV light. The dried polymer layer is exposed to UV light to crosslink the polymer chains, rendering it sufficiently insoluble to be useful in the disclosure. Importantly, the anion exchange polymer freely allows the passage of chloride or other anions ions through it, that can carry electric charge and allow the reference electrode to still function In its intended use as a source of a reference potential in an electrochemical cell. In this embodiment of the disclosure, the fixed positively charged groups in the polymer layer present an electrostatic barrier to the freeflow on cations through the membrane, thus rendering it permselective for anion flow. This characteristic retards or prevents the passage of the undesirable silver ions from the silver chloride layer to the bulk fluid. Any other suitable anion exchange material can also be used in this embodiment of the disclosure. To be suitable a membrane formed of the material may retard or prevent the passage of undesirable cations, for example silver ions and be sufficiently insoluble in the presence of the fluid where it is intended the electrode is to be used.

[0175] . As well as polymers, other materials with anion or cation exchange functionality may be operable in the context of this disclosure, for example inorganic materials such as clays or zeolites. Binders may be added to such materials to improve the integrity, robustness, and longevity of the membrane layer when in use.

[0176] . The disclosure may be more fully understood by reference to the non -limiting drawings and examples.

[0177] . FIG. 1 shows a simplified diagram of a prior art three-electrode electrochemical sensor. The working electrode (10) is coated in methylene blue modified DNA aptamers (not drawn) that specifically interact with a target analyte. The reference electrode (20) is of the Ag|AgCl type which is known and used in the art. A counter electrode (30) is also provided. The electrodes (10, 20, 30) are each a microneedle, and are drawn after having been urged through the skin surface (40) so as to contact the underlying interstitial fluid (ISF) (50). Silver ions (60) continuously leach from the reference electrode and migrate in all x, y, z directions along concentration gradients. As shown by the arrows, some silver ions (60) migrate toward the working electrode (10). The working electrode (10) (i.e., the conductive portion and the coating of redox -modified aptamers) is directly exposed to the surrounding ISF (50) and accordingly silver ions (60) are able to contact and deposit on the electrode. It is proposed that such deposition leads to signal loss possibly due to loss of aptamer from the working electrode (10).

[0178] . Reference is made to FIG. 2 showing one embodiment of the disclosure whereby the reference electrode (10) has a Nafion™ coating (60) across the entire surface that contacts the ISF (50). Nafion™ is trade mark of DuPont for the polymer ethanesulfonyl fluoride, 2-[l-[difluoro-[(trifluoroethenyl)oxy]methyl]-l,2,2,2-tetrafluoroethoxy]-1,1, 2, 2, -tetrafluoro-, with tetrafluoroethylene tetrafluoroethylene -perfluoro-3,6-dioxa-4- methyl-7-octenesulfonic acid copolymer.

[0179] . The coating (60) forms a selective barrier, allowing for the bidirectional passage of hydrogen ions (protons), but inhibiting or preventing the migration of silver ions from the reference electrode (20) into the surrounding ISF (50). Exposure of the working electrode (10) to silver ions is therefore inhibited or prevented.

[0180] . The free exchange of hydrogen ions across the coating (60) provides for electrical connection between the reference electrode (20) and the surrounding ISF (50). The working electrode (10) is also in contact with the ISF (50), and accordingly the reference electrode (20) is able to function as a source of reference potential. Inhibition of the migration of silver ions into the ISF prevents or decreases exposure of the adjacent working electrode to damaging silver ions. The responsiveness of the working electrode to the target analyte is therefore maintained, or is at least subject to less degradation than would be the case where there is no Nafion™ coating on the reference electrode.

[0181] . Reference is made to FIG. 3 showing an alternative embodiment of the disclosure to that shown in FIG. 2, whereby the working electrode is coated with Nafion™ across the entire surface that contacts the ISF. In this embodiment, silver ions are freely released from the reference electrode into the ISF however contact of the ions with the working electrode is prevented or at least limited. The Nafion™ coating allows for the bidirectional passage of hydrogen ions, and as for the embodiment of FIG. 2 allows for electrical connection between the working electrode and the ISF. The so-formed electrical connection allows for an interrogating potential to be applied across the working electrode and the counter electrode. Furthermore, current can be measured across the two electrodes to determine the amount of an analyte about the working electrode. Additionally, the working electrode (10) is also in electrical contact with the ISF (50), and accordingly the reference electrode (20) is able to function as a source of reference potential.

[0182] . Given the benefit of the present specification, the skilled person is able to conceive of other coatings that function so as to allow passage of hydrogen ions or electrons to provide for electrical connection, yet inhibit or prevent migration of a chemical species from the reference electrode to contact the working electrode. Nafion™ is a type of ionomer. Ionomers are generally polymers having repeated units of electrically neutralunits and ionized units. These units are covalently bonded to the polymer backbone as pendant group moieties. Typically, less than about 15 mole percent are ionized. The ionized units may be acidic groups, such as carboxylic acid groups and sulfonic acid groups. Further examples of ionomers include polystyrene sulfonate, and Hycar™.

[0183] . Ionomers are copolymers, and therefore distinguished from other poly electrolytes.A further point of difference arises in the relatively low level of ion containing repeating units. That defining level may be considered to be about 15%.

[0184] . In some embodiments the migration of the deleterious chemical species to the working electrode is inhibited by using means to limit mixing of fluid immediately about an electrode (working or reference) with the bulk fluid surrounds. For example, an electrode may be contained within a chamber having perforations which are of sufficiently small diameter to inhibit movement of the fluid from one side of the chamber wall to the other. Such perforations will nevertheless allow for electrical connection, given the connection does not require the actual bulk movement of charged species (such as protons and electrons).

[0185] . An electrode may be formed as a needle, microneedle or a wire configured to pierce the skin. Such arrangements will be useful where an electrochemical sensor is applied to the skin of a subject (such as a human or other mammal). In that case, the subject urges the electrodes through the skin, such that they contact the ISF and allow for the detection of clinically important analytes such as glucose in that fluid. The application of a coating (such as Nafion™) to a sharpened electrode may dull the point leading to difficulties in penetrating the skin during application. In that regard, some modification may be required.

[0186] . An example of such modification is demonstrated in FIG. 4, which shows in crosssection the solid conductive portion (100) of a reference electrode, the electrode having a coating (60). The thickness of the coating (60) is exaggerated for clarity. The coating (60) forms a sleeve which terminates before the pointed region (100b) of the conductive portion (100), leaving to pointed region bare, and therefore able to easily penetrate the skin. The pointed region (100b) is exposed to the surrounding fluid and is a potential source of contaminant species. To prevent leaching of the contaminant species in the surrounding fluid, the pointed region may be treated or not treated so as to prevent such leaching. For example, the pointed region (100b) may be electroplated or otherwise coated with a non-leaching material. Alternatively, a sharpened metal cap fabricated from a hard but inert material may be fitted over the pointed region (100b). As a further alternative, the conductive portion may be fabricated in two parts (100a) (100b), the former being fabricated from a leaching material and the latter from a non-leaching material. For example, the pointed region may consist of an uncoated stainless steel material, whereas in the sleeved region the stainless steel has been coated with silver metal and silver chloride. EXAMPLE 1: Preparation and testing of Nafion™-coated reference electrode

[0187] . Reference electrodes were prepared by dipping a silver chloride coated silver wire into a 5 wt% Nafion™ solution various numbers of times, with air drying between each dip. The coated electrodes were then placed in an oven at 120°C for one hour to phase invert the Nafion™ and render it insoluble in aqueous solutions. The thickness of the coating was increased according to the number of times each electrode was dipped.

[0188] . The different electrodes (each having a different thickness of coating) were used as the reference electrode in an electrochemical cell in combination with a platinum wire counter electrode and a gold wire working electrode functionalized with aptamers capable of binding to vancomycin that were tagged with methylene blue as an indicator redox group (MB -aptamers).

[0189] . All three electrodes of the cell were placed in a vial containing phosphate buffered saline plus 0.7 mM MgCh, maintained a 37°C via partial immersion of the vial in a water bath. The electrochemical sensors thus formed were interrogated over approximately 20 hours to study the rate of degradation of the working electrode.

[0190] . Reference is made to FIG. 5. Normalized SWV peak current at 300 Hz for MB- aptamer functionalized working electrodes in electrochemical cells with reference electrodes coated with different thicknesses of Nafion™. The x number in the legend refers to the number of times the reference electrode wire was dipped in the Nafion™ solution. BASi is an uncoated Ag|AgCl reference electrode. FIG. 5 shows the change over time of the square wave voltammetry (SWV) peak current, using a 300 Hz square wave frequency, that has been normalized by dividing the current at time t by the current from the initial interrogation.

[0191] . It will be noted that over the experimental period of 20 hours, increasing the coating thickness from 0 (BASi) to 8 Nafion™ layers resulted in improved maintenance of theworking electrode signal, as demonstrated by the slower the rate of decrease in the normalized current.

[0192] . Those skilled in the art will appreciate that the disclosure described herein is susceptible to further variations and modifications other than those specifically described. It is understood that the disclosure comprises all such variations and modifications which fall within the spirit and scope of the present disclosure.

[0193] . While the disclosure is described mainly by reference to a microneedle electrode, the disclosure is equally applicable to an electrode of any form and dimension.

[0194] . Similarly, the disclosure is described mainly by reference to the use of an electrochemical sensor to sense an analyte in the ISF of a mammal. The disclosure may be operable with other test fluids such as blood, saliva, urine, potable water, waste water, industrial process intermediates, industrial products, foods, and many others.

[0195] . An aptamer is only one type of analyte recognition element that is workable in the context of the present disclosure. As used herein, the term “analyte recognition element” includes any molecule(s) that specifically interact with a target analyte of interest, the interaction causing a discernible change in the molecule(s). An analyte recognition element may be a polymer, and may comprise from about 5 to about 100 monomers, or from about 15 to about 50 monomers.

[0196] . An aptamer is an exemplary form of analyte recognition element. Aptamers are small (usually from 20 to 60 nucleotides) RNA or DNA oligonucleotides formed from a single strand and able to bind a target analyte with high affinity and specificity. Aptamers may be considered as nucleotide analogues of antibodies, but aptamer production is an in vitro cell-free process that is significantly easier and cheaper than the production of antibodies by cell culture or in vivo methods. Aptamers typically comprise a polynucleotide sequence that promotes the assumption of 3-dimensional shapes in the form of helices and single-stranded loops. Indeed, the specificity of aptamer binding is dictated not by the primary polynucleotide sequence, but instead by its 3-dimensional structure, at least is part. In some circumstances, binding will be influenced by hydrophobic interactions, hydrogen bonding, Van der Waals forces, base-stacking, and intercalation.

[0197] . An analyte recognition element may be a biological molecule or an analogue thereof. An exemplary analyte recognition element may be comprised of DNA, RNA, PNA, XNA. Single-stranded and double-stranded arrangements are contemplated.

[0198] . An analyte recognition element may comprise a non-natural nucleic acid. As used herein, the term “non-natural nucleic acid” is intended to include a polymer that is biosimilar to a natural nucleic acid polymer such as DNA or RNA, but having a chemical structure that is altered and not found in nature. As a result of the altered structure, the non- natural nucleic acid may be more resistant than a natural nucleic acid against degradation (such as cleavage of a chemical bond) occasioned by nucleases found in biological fluids such as blood and the ISF.

[0199] . A non-natural nucleic acid may derive from a naturally occurring nucleic acid, but having had an alteration to its chemical structure such that the chemical structure is considered non-natural. More typically, the non-natural nucleic acid will be synthesised de novo in an altered form.

[0200] . A non-natural nucleic acid molecule useful in the context of the present disclosure may be an altered form of an aptamer. The non-natural nucleic acid may be an oligomer having a non-natural backbone, being a molecular analogue to DNA or RNA. Examples of non-natural backbone oligomers include, but are not limited, to 2' -fluoroarabinoside nucleic acid (FANA), 2'-O-methyl RNA, locked nucleic acid (LNA), and threose nucleic acid (TNA). Collectively, these non-natural backbone oligomers are referred to as xeno nucleic acids (XNAs).

[0201] . Apart from the altered chemical structure which confirms stability in biological fluids, a non-natural nucleic acids may share one or more general features of aptamers such as length, base sequence (primary structure), secondary structure and tertiary structure.

[0202] . One method of identifying aptamers useful in the context of the present disclosure is to use a method of the prior art (such as SELEX) to identify a natural DNA or RNA aptamer, and optionally to then modify the identified aptamer so as to have a non-natural chemical structure. Alternatively, methods such as SELEX may be adapted by the use enzymes configured to synthesise and amplify non-natural nucleic acids in the first instance.

[0203] . An analyte recognition element may be a protein. The protein may in the form of a peptide, optionally having a length of between 10 and 100 amino acids or longer. The protein may be in the form of a monomer, dimer, trimer, tetramer or higher. Antibodies, antibody fragments (such as Fab fragments) and antibody-like molecules may be useful, whether polyclonal or monoclonal.

[0204] . As for polynucleotides, proteins may be subject to modification. For example, backbone modification may be used to improve proteolytic stability of the peptide. Backbone modification includes the substitution of L-amino acids by D-amino acids, insertion of methyl-amino acids, and the incorporation of P-amino acids and peptoids. Introducing these non-natural amino acids into the peptide sequence, particularly at a proteolysis site, is an effective strategy for improving resistance to proteases or other deleterious factors.

[0205] . Side chain modifications may be is achieved by replacing the natural amino acids with their analogues during peptide synthesis, to improve their binding affinity and target selectivity. Variants of natural amino acid analogues such as homoarginine, benzyloxytyrosine, and P-phenylalanine are commonly commercially available, and can be conveniently used to chemically modify the peptide side chain during peptide synthesis.

[0206] . The weak forces in proteins, such as hydrogen bonds, van der Waals forces, and intramolecular hydrophobic interactions may not be adequate for a stable secondary structure conformation. Additional modifications of the backbone, N- or C-termini, or sidechains for stabilization of secondary structures may be pursued.

[0207] . Cyclization is another potentially useful protein modification technique that can include various strategies, such as head-to-tail, backbone -to-side chain, and side chain-to- side chain cyclization. Cyclization can increase proteolytic stability, and allows mimicking and stabilization of the secondary structure.

[0208] . Accordingly, the spirit and scope of the present disclosure is not to be limited by the foregoing examples, but is to be understood in the broadest sense allowable by law.

Claims

CLAIMS:

1. A reference electrode for an electrochemical sensor, the reference electrode comprising: a solid electrically conductive element; one or more chemical species involved in the generation of a reference electrical potential in the conductive element; wherein the reference electrode comprises, or is associated with, a migration inhibitor configured to inhibit, prevent or retard migration of the one or more chemical species into a fluid distal to the reference electrode while allowing electrical communication between the conductive element and the fluid.

2. The reference electrode of claim 1 , wherein the migration inhibitor is configured to inhibit, prevent or retard migration of the one or more chemical species to a working electrode in functional association with the reference electrode via the fluid.

3. The reference electrode of claim 1 or claim 2, wherein the one or more chemical species is soluble in the fluid.

4. The reference electrode of any one of claims 1 to 3, wherein the one or more chemical species is an ion.

5. The reference electrode of claim 4, wherein the ion is not a chloride ion or a hydrogen ion6. The reference electrode of claim 4 or claim 5, wherein the ion is a metal ion.

7. The reference electrode of claim 6, wherein the metal ion is a silver ion, or another cation.

8. The reference electrode of any one of claims 1 to 7, wherein the migration inhibitor is a selective anion exchange material.

9. The reference electrode of any one of claims 1 to 8, wherein the migration inhibitor is configured to selectively inhibit, prevent or retard the passage of a cation while allowing passage of an anion.

10. The reference electrode of claim 9, wherein the cation is a silver ion and the anion is a chloride ion.

11. The reference electrode of any one of claims 1 to 10, wherein the migration inhibitor forms a complete or partial physical barrier to inhibit, prevent or retard the migration of the one or more chemical species into the fluid.

12. The reference electrode of claim 11, wherein the physical barrier inhibits or prevents bulk mixing of fluid on opposing sides thereof.

13. The reference electrode of claim 11 or claim 12, wherein the physical barrier does not substantially inhibit, prevent or retard the migration of chloride ions or hydrogen ions between the fluid and the conductive element.

14. The reference electrode of any one of claims 1 to 13, wherein the migration inhibitor captures the one or more chemical species.

15. The reference electrode of any one of claims 1 to 13, wherein the migration inhibitor presents an energy barrier to the migration of the one or more chemical species.

16. The reference electrode of any one of claims 1 to 15, wherein the migration inhibitor is formed by a layer or a membrane applied to, or disposed about, the conductive element.

17. The reference electrode of claim 16, wherein the layer or the membrane is formed in situ on a surface of the conductive element.

18. The reference electrode of claim 16 or claim 17, wherein the layer or the membrane is formed by contacting the conductive element surface with (i) a suspension or solution comprising the migration inhibitor (ii) a suspension or solution of a monomer or an oligomer of a polymer migration inhibitor; removing a liquid component of the suspension or solution to cause respectively deposition of the migration inhibitor on the surface, or formation of the polymer migration inhibitor on the surface.

19. The reference electrode of claim 18, wherein removal of the liquid component is effected by way of evaporation.

20. The reference electrode of any one of claims 16 to 19, wherein the layer or the membrane is formed by causing or allowing the layer or the membrane to transition to a substantially insoluble state in a test fluid.

21. The reference electrode of claim 20, wherein the transition to a substantially insoluble state material of the layer or the membrane results from chemical crosslinking of the material in situ.

22. The reference electrode of any one of claims 1 to 21, wherein the migration inhibitor is biocompatible.

23. The reference electrode of any one of claims 1 to 22, wherein the migration inhibitor is a polymer.

24. The reference electrode of claim 23, wherein the polymer is an ionomer.

25. The reference electrode of claim 23 or claim 24, wherein the polymer comprises a backbone structure having extending therefrom ionically active functional groups.

26. The reference electrode of claim 25, wherein the ionically active functional groups are charged groups.

27. The reference electrode of claim 26, wherein the charged groups are anion groups.

28. The reference electrode of claim 26, wherein the charged groups are quaternary ammonium groups.

29. The reference electrode of any one of claims 23 to 28, wherein the polymer is a crosslinked quaternary ammonium functionalized poly(2,6-dimethyl-l,4-phenylene oxide).

30. The reference electrode of claim 29, wherein the polymer is formed by contacting a surface of the conductive element with a solution comprising oligomers of quaternary ammonium functionalized poly(2,6-dimethyl-l,4-phenylene oxide), the oligomers being crosslinked to form a crosslinked quaternary ammonium functionalized poly(2,6-dimethyl-l,4- phenylene oxide) layer.

31. The reference electrode of any one of claims 21 to 30, wherein, where the charged groups in the polymer surrounding the conductive elements are cationic, the material inhibits, prevents or retards the passage of cations by creating an energy barrier that the cations have to overcome to be able to pass through the material, whereas anions experience a smaller or no energy barrier in passing through the material.

32. The reference electrode of any one of claims 25 to 31, wherein the backbone structure comprises carbon and fluorine atoms.

33. The reference electrode of any one of claims 23 to 32, wherein the polymer is a fluoropolymer or a fluorocopolymer.

34. The reference electrode of any one of claims 25 to 33, wherein the ionically active functional groups are charged at about neutral pH, or at about a physiological pH.

35. The reference electrode of any one of claims 25 to 34, wherein the ionically active functional groups are acidic functional groups.

36. The reference electrode of claim 35, wherein the acidic functional groups are sulfonic acid functional groups.

37. The reference electrode of any one of claims 23 to 36, wherein the polymer is ethanesulfonyl fluoride, 2-[l-[difhioro-[(trifhroroethenyl)oxy]methyl]-l,2,2,2-tetrafluoroethoxy]- 1,1, 2, 2, -tetrafluoro-, with tetrafluoroethylene tetrafhroroethylene-perfluoro-3,6-dioxa-4-methyl- 7-octenesulfonic acid copolymer.

38. The reference electrode of any one of claims 21 to 37, wherein, where the charged groups in the polymer surrounding the conductive elements are anionic, the material inhibits, prevents or retards the passage of contaminant cations by the anionic groups in the polymer capturing the cations, thus inhibiting, preventing or retarding their passage through the material, whereas hydrogen ions experience a smaller or no tendency to be captured by the anionic groups in passing through the material.

39. The reference electrode of any one of claims 1 to 38, wherein the conductive element is configured to pierce the skin of a human or another mammal.

40. The reference electrode of any one of claims 1 to 39, wherein the conductive element is configured to penetrate the skin of a human or another mammal.

41. The reference electrode of any one of claims 1 to 40, wherein the conductive element is a wire, a needle or a microneedle.

42. The reference electrode of any one of claims 1 to 41, wherein the migration inhibitor selectively inhibits, prevents or retards migration of the one or more chemical species.

43. A working electrode for an electrochemical sensor, the working electrode comprising: a solid electrically conductive element; a plurality of analyte recognition elements associated with the conductive element; wherein the working electrode comprises, or is associated with, a migration inhibitor configured to inhibit, prevent or retard migration of one or more chemical species involved in the generation of a reference electrical potential in the conductive element from a reference electrode of the electrochemical sensor from contacting the conductive element or the analyte recognition elements while allowing electrical communication between the conductive element and the fluid.

44. The working electrode of claim 43, wherein the migration inhibitor is configured to inhibit, prevent or retard migration of the one or more chemical species to the working electrode from the reference electrode in functional association therewith via the fluid.

45. The working electrode of claim 43 or claim 44, wherein the one or more chemical species is soluble in the fluid.

46. The working electrode of any one of claims 43 to 45, wherein the one or more chemical species is an ion.

47. The working electrode of claim 46, wherein the ion is not a chloride ion or a hydrogen ion.

48. The working electrode of claim 46 or claim 47, wherein the ion is a metal ion.

49. The working electrode of claim 48, wherein the metal ion is a silver ion, or another cation.

50. The working electrode of any one of claims 43 to 49, wherein the migration inhibitor is a selective anion exchange material.

51. The working electrode of any one of claims 43 to 50, wherein the migration inhibitor is configured to selectively inhibit, prevent or retard the passage of a cation while allowing passage of an anion.

52. The working electrode of claim 51, wherein the cation is a silver ion and the anion is a chloride ion.

53. The working electrode of any one of claims 43 to 52, wherein the migration inhibitor forms a complete or partial physical barrier to inhibit, prevent or retard the migration of the one or more chemical species into the fluid.

54. The working electrode of claim 53, wherein the physical barrier inhibits or prevents bulk mixing of fluid on opposing sides thereof.

55. The working electrode of claim 53 or claim 54, wherein the physical barrier does not substantially inhibit, prevent or retard the migration of chloride ions or hydrogen ions between the fluid and the conductive element.

56. The working electrode of any one of claims 43 to 55, wherein the migration inhibitor captures the one or more chemical species.

57. The working electrode of any one of claims 43 to 55, wherein the migration inhibitor presents an energy barrier to the migration of the one or more chemical species.

58. The working electrode of any one of claims 43 to 57, wherein the migration inhibitor is formed by a layer or a membrane applied to, or disposed about, the conductive element.

59. The working electrode of claim 58, wherein the layer or the membrane is formed in situ on a surface of the conductive element.

60. The working electrode of claim 59 or claim 59, wherein the layer or the membrane is formed by contacting the conductive element surface with (i) a suspension or solution comprising the migration inhibitor (ii) a suspension or solution of a monomer or an oligomer of a polymer migration inhibitor; removing a liquid component of the suspension or solution to cause respectively deposition of the migration inhibitor on the surface, or formation of the polymer migration inhibitor on the surface.

61. The working electrode of claim 60, wherein removal of the liquid component is effected by way of evaporation.

62. The working electrode of any one of claims 58 to 61, wherein the layer or the membrane is formed by causing or allowing the layer or the membrane to transition to a substantially insoluble state in a test fluid.

63. The working electrode of claim 62, wherein the transition to a substantially insoluble state material of the layer or the membrane results from chemical crosslinking of the material in situ.

64. The working electrode of any one of claims 43 to 63, wherein the migration inhibitor is biocompatible.

65. The working electrode of any one of claims 43 to 64, wherein the migration inhibitor is a polymer.

66. The working electrode of claim 65, wherein the polymer is an ionomer.

67. The working electrode of claim 65 or claim 66, wherein the polymer comprises a backbone structure having extending therefrom ionically active functional groups.

68. The working electrode of claim 67, wherein the ionically active functional groups are charged groups.

69. The working electrode of claim 68, wherein the charged groups are anion groups.

70. The working electrode of claim 68, wherein the charged groups are cation groups.

71. The working electrode of claim 68, wherein the charged groups are quaternary ammonium groups.

72. The working electrode of any one of claims 65 to 71, wherein the polymer is a crosslinked quaternary ammonium functionalized poly(2,6-dimethyl-l,4-phenylene oxide).

73. The working electrode of claim 72, wherein the polymer is formed by contacting a surface of the conductive element with a solution comprising oligomers of quaternary ammonium functionalized poly(2,6-dimethyl-l,4-phenylene oxide), the oligomers being crosslinked to form a crosslinked quaternary ammonium functionalized poly(2,6-dimethyl-l,4- phenylene oxide) layer.

74. The working electrode of any one of claims 63 to 73, wherein, where the charged groups in the polymer surrounding the conductive elements are cationic, the material inhibits, prevents or retards the passage of cations by creating an energy barrier that the cations have to overcome to be able to pass through the material, whereas anions experience a smaller or no energy barrier in passing through the material.

75. The working electrode of any one of claims 67 to 74, wherein the backbone structure comprises carbon and fluorine atoms.

76. The working electrode of any one of claims 65 to 75, wherein the polymer is a fluoropolymer or a fluorocopolymer.

77. The working electrode of any one of claims 67 to 76, wherein the ionically active functional groups are charged at about neutral pH, or at about a physiological pH.

78. The working electrode of any one of claims 67 to 77, wherein the ionically active functional groups are acidic functional groups.

79. The working electrode of claim 78, wherein the acidic functional groups are sulfonic acid functional groups.

80. The working electrode of any one of claims 65 to 79, wherein the polymer is ethanesulfonyl fluoride, 2-[l-[difhioro-[(trifhroroethenyl)oxy]methyl]-l,2,2,2-tetrafluoroethoxy]- 1,1, 2, 2, -tetrafluoro-, with tetrafluoroethylene tetrafhroroethylene-perfluoro-3,6-dioxa-4-methyl- 7-octenesulfonic acid copolymer.

81. The working electrode of any one of claims 63 to 80, wherein, where the charged groups in the polymer surrounding the conductive elements are anionic, the material inhibits, prevents or retards the passage of contaminant cations by the anionic groups in the polymer capturing the cations, thus inhibiting, preventing or retarding their passage through the material, whereashydrogen ions experience a smaller or no tendency to be captured by the anionic groups in passing through the material.

82. The working electrode of any one of claims 43 to 81, wherein the conductive element is configured to pierce the skin of a human or another mammal.

83. The working electrode of any one of claims 43 to 82, wherein the conductive element is configured to penetrate the skin of a human or another mammal.

84. The working electrode of any one of claims 43 to 83, wherein the conductive element is a wire, a needle or a microneedle.

85. The working electrode of any one of claims 43 to 84, wherein the migration inhibitor selectively inhibits, prevents or retards migration of the one or more chemical species.

86. An electrochemical sensor comprising: a reference electrode comprising or being associated with one or more chemical species involved in the generation of a reference electrical potential; a working electrode; and a migration inhibitor configured to inhibit, prevent or retard migration of the one or more chemical species from the reference electrode to the working electrode while allowing electrical communication between the working electrode and a fluid about the working electrode.

87. The electrochemical sensor of claim 86, wherein the migration inhibitor is configured to inhibit, prevent or retard migration of the one or more chemical species from contacting the working electrode or an analyte recognition element of the working electrode.

88. The electrochemical sensor of claim 86 or claim 87, wherein the migration inhibitor is disposed between the reference electrode and the working electrode.

89. The electrochemical sensor of any one of claims 86 to 88, wherein the one or more chemical species is soluble in the fluid.

90. The electrochemical sensor of any one of claims 86 to 89, wherein the one or more chemical species is an ion.

91. The electrochemical sensor of claim 90, wherein the ion is not a chloride ion or a hydrogen ion.

92. The electrochemical sensor of claim 90 or claim 91, wherein the ion is a metal ion.

93. The electrochemical sensor of claim 92, wherein the metal ion is a silver ion, or another cation.

94. The electrochemical sensor of any one of claims 86 to 93, wherein the migration inhibitor is a selective anion exchange material.

95. The electrochemical sensor of any one of claims 86 to 94, wherein the migration inhibitor is configured to selectively inhibit, prevent or retard the passage of a cation while allowing passage of an anion.

96. The electrochemical sensor of claim 85, wherein the cation is a silver ion and the anion is a chloride ion.

97. The electrochemical sensor of any one of claims 86 to 96, wherein the migration inhibitor forms a complete or partial physical barrier to inhibit, prevent or retard the migration of the one or more chemical species into the fluid.

98. The electrochemical sensor of claim 97, wherein the physical barrier inhibits or prevents bulk mixing of fluid on opposing sides thereof.

99. The electrochemical sensor of claim 97 or claim 98, wherein the physical barrier does not substantially inhibit, prevent or retard the migration of chloride ions or hydrogen ions between the fluid and the working electrode.

100. The electrochemical sensor of any one of claims 86 to 99, wherein the migration inhibitor captures the one or more chemical species.

101. The electrochemical sensor of any one of claims 86 to 99, wherein the migration inhibitor presents an energy barrier to the migration of the one or more chemical species.

102. The electrochemical sensor of any one of claims 86 to 101, wherein the migration inhibitor is formed by a layer or a membrane applied to, or disposed about, the working electrode.

103. The electrochemical sensor of claim 102, wherein the layer or the membrane is formed in situ on a surface of the working electrode.

104. The electrochemical sensor of claim 102 or claim 103, wherein the layer or the membrane is formed by contacting the working electrode surface with (i) a suspension or solution comprising the migration inhibitor (ii) a suspension or solution of a monomer or an oligomer of a polymer migration inhibitor; removing a liquid component of the suspension or solution to cause respectively deposition of the migration inhibitor on the surface, or formation of the polymer migration inhibitor on the surface.

105. The electrochemical sensor of claim 104, wherein removal of the liquid component is effected by way of evaporation.

106. The electrochemical sensor of any one of claims 102 to 105, wherein the layer or the membrane is formed by causing or allowing the layer or the membrane to transition to a substantially insoluble state in a test fluid.

107. The electrochemical sensor of claim 106, wherein the transition to a substantially insoluble state material of the layer or the membrane results from chemical crosslinking of the material in situ.

108. The electrochemical sensor of any one of claims 86 to 107, wherein the migration inhibitor is biocompatible.

109. The electrochemical sensor of any one of claims 86 to 108, wherein the migration inhibitor is a polymer.

110. The electrochemical sensor of claim 109, wherein the polymer is an ionomer.

111. The electrochemical sensor of claim 109 or claim 110, wherein the polymer comprises a backbone structure having extending therefrom ionically active functional groups.

112. The electrochemical sensor of claim 111, wherein the ionically active functional groups are charged groups.

113. The electrochemical sensor of claim 112, wherein the charged groups are anion groups.

114. The electrochemical sensor of claim 112, wherein the charged groups are quaternary ammonium groups.

115. The electrochemical sensor of any one of claims 109 to 114, wherein the polymer is a crosslinked quaternary ammonium functionalized poly(2,6-dimethyl-l,4-phenylene oxide).

116. The electrochemical sensor of claim 115, wherein the polymer is formed by contacting a surface of the working electrode with a solution comprising oligomers of quaternary ammonium functionalized poly(2,6-dimethyl-l,4-phenylene oxide), the oligomers being crosslinked to form a crosslinked quaternary ammonium functionalized poly(2,6-dimethyl-l,4-phenylene oxide) layer.

117. The electrochemical sensor of any one of claims 107 to 116, wherein, where the charged groups in the polymer surrounding the working electrode are cationic, the material inhibits, prevents or retards the passage of cations by creating an energy barrier that the cations have toovercome to be able to pass through the material, whereas anions experience a smaller or no energy barrier in passing through the material.

118. The electrochemical sensor of any one of claims 111 to 118, wherein the backbone structure comprises carbon and fluorine atoms.

119. The electrochemical sensor of any one of claims 109 to 118, wherein the polymer is a fluoropolymer or a fluorocopolymer.

120. The electrochemical sensor of any one of claims 111 to 119, wherein the ionically active functional groups are charged at about neutral pH, or at about a physiological pH.

121. The electrochemical sensor of any one of claims 111 to 120, wherein the ionically active functional groups are acidic functional groups.

122. The electrochemical sensor of claim 121, wherein the acidic functional groups are sulfonic acid functional groups.

123. The electrochemical sensor of any one of claims 109 to 122, wherein the polymer is ethanesulfonyl fluoride, 2-[l-[difhioro-[(trifhroroethenyl)oxy]methyl]-l,2,2,2-tetrafluoroethoxy]- 1,1, 2, 2, -tetrafluoro-, with tetrafluoroethylene tetrafhroroethylene-perfluoro-3,6-dioxa-4-methyl- 7-octenesulfonic acid copolymer.

124. The electrochemical sensor of any one of claims 107 to 123, wherein, where the charged groups in the polymer surrounding the working electrode are anionic, the material inhibits, prevents or retards the passage of contaminant cations by the anionic groups in the polymer capturing the cations, thus inhibiting, preventing or retarding their passage through the material, whereas hydrogen ions experience a smaller or no tendency to be captured by the anionic groups in passing through the material.

125. The electrochemical sensor of any one of claims 86 to 124, wherein the working electrode is configured to pierce the skin of a human or another mammal.

126. The electrochemical sensor of any one of claims 86 to 125, wherein the working electrode is configured to penetrate the skin of a human or another mammal.

127. The electrochemical sensor of any one of claims 86 to 126, wherein the working electrode is a wire, a needle or a microneedle.

128. The electrochemical sensor of any one of claims 86 to 127, wherein the migration inhibitor selectively inhibits, prevents or retards migration of the one or more chemical species.

129. The electrochemical sensor of any one of claims 86 to 128, wherein the reference electrode according to any one of claims 1 to 42, and / or the working electrode is according to any one of claims 43 to 85.

130. The electrochemical sensor of any one of claims 86 to 129, configured such that the reference electrode functions also as a counter electrode, or the electrochemical sensor comprises a dedicated counter electrode.

131. The electrochemical sensor of any one of claims 86 to 130, wherein the reference electrode and working electrodes are disposed in a fixed mutual spatial relationship.

132. The electrochemical sensor of any one of claims 86 to 131, wherein the reference electrode and working electrode are separated by a distance of less than about 20 mm, 19 mm, 18 mm, 17 mm, 16 mm, 15 mm, 14 mm, 13 mm, 12 mm, 11 mm, 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, or 1 mm.

133. The electrochemical sensor of any one of claims 86 to 132, wherein the reference electrode and / or working electrode is configured to pierce the skin of a human or another mammal.

134. The electrochemical sensor of any one of claims 86 to 133, wherein the reference electrode and / or the working electrode is a wire, a needle or a microneedle.

135. The electrochemical sensor of any one of claims 86 to 134, configured such that, when applied to a subject, the reference electrode and the working electrode contact an interstitial fluid below the surface of the skin.

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