Working electrode for an electrochemical sensor

Abstract

An aptamer-based working electrode for an electrochemical sensor, the electrode having surface structures which protect the aptamers from damage arising from (i) passage of the electrode through the skin, or (ii) contact with a tissue or a bodily fluid. The working electrode may be a microneedle of a wearable biosensor.

Description

[0001] WORKING ELECTRODE FOR AN ELECTROCHEMICAL SENSOR

[0002] FIELD

[0003]

[0001] , The present disclosure relates generally to electrochemical sensors useful in determining the amount of an analyte in a sample. More particularly, the disclosure provides a working electrode used in an electrochemical sensor.

[0004] BACKGROUND

[0005]

[0002] , Electrochemical sensors have found utility in a broad range of applications including in the detection of target analytes in the environment, manufacturing process streams, and biological fluids for example. Advantageously, these sensors are able to provide continuous quantitative output allowing for the ongoing monitoring of the material under analysis.

[0006]

[0003] , A number of electrochemical sensor types function on the basis of an interaction between a recognition element with the target analyte. The recognition element may be an aptamer (DNA, RNA or XNA), a peptide, or a peptide nucleic acid, for example. In the presence of target analyte, the recognition element undergoes a reversible, partially reversible, or irreversible change in conformation.

[0007]

[0004] , The recognition element may be linked at one end to the surface of a working electrode, and at another end to a solution-phase redox reporter such as methylene blue or ferrocene. In some sensors the redox reporter can be proximal or distal to the working electrode surface dependant on the conformation of the recognition element. When the redox species is proximal electron transfer kinetics between the redox reporter and electrode increase, and vice- versa. Variation in electron transfer kinetics are determined by measuring the working electrode current. The amount of analyte about the working electrode may be determined by reference to the measured current. Such sensors are often referred to as electrochemical sensors.

[0008]

[0005] , In other types of sensor conformational changes in the recognition element are detected by optical means. Quencher / fluorophore pairs can be used as dual recognition element labels to generate fluorogenic recognition elements. Such recognition element exhibit an increase in fluorescence upon binding. A quencher linked to the recognition element absorbs energy from a fluorophore that is directly adjacent when the recognition elements assumes a certain conformation preventing fluorescence. In the presence of target analyte, the recognition element undergoes a conformational change leading to separation of the fluorophore and quencher thereby generating a fluorescent signal.

[0009]

[0006] . Electrochemical sensors in particular are developing at a rapid rate and are showing significant promise as a tool for real time in vivo continuous monitoring of analytes such as drugs, hormones, markers, metabolites, and the like. In such applications, the sensor electrodes (typically working, counter, and reference electrodes) are contacted to a bodily fluid which is expected to contain the target analyte, such as blood or interstitial fluid. For example, the electrodes may be introduced into a vein via a catheter or a canula so as to contact blood passing therethrough. Less invasive methods involve the use of electrodes in the form or needles, microneedles or fine wires which are inserted through the skin and into underlying dermal tissues so as to contact the interstitial fluid.

[0010]

[0007] , A problem arises in that a sensor that functions acceptably ex vivo shows fails to maintain that function after the electrodes are brought into contact with dermal tissue. A reduction in sensitivity, specificity, limit of detection, output current, signal-to-background ratio, dynamic range, signal stability or reproducibility may be noted.

[0011]

[0008] , It is an aspect of the present disclosure to provide an improvement to prior art working electrodes in any one or more of the aforementioned functions, or another function. It is a further aspect of the present disclosure to provide a useful alternative to prior art working electrodes.

[0012]

[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.

[0013] SUMMARY

[0014]

[0010] , In a first aspect, but not necessarily the broadest aspect, there is provided a working electrode for an electrochemical sensor, the working electrode having a plurality of analyte recognition elements associated therewith, the working electrode comprising surface structures configured to protect the plurality of analyte recognition elements from an adverse influence arising from (i) passage of the working electrode through a tissue of an animal subject, or (ii) disposition of the working electrode in a tissue or a fluid of an animal subject.

[0015] [Oi l], In one embodiment of the first aspect, the passage or the disposition of the working electrode through a tissue of an animal subject results in a contact-mediated or pressure-mediated disruption of the plurality of analyte recognition elements.

[0016]

[0012] , In one embodiment of the first aspect, the disruption is selected from any one or more of: removal from a surface of the electrode, reduction in the ability to change conformation in the presence or target analyte, and reduction in the ability to extend outwardly from a surface of the electrode.

[0017]

[0013] , In one embodiment of the first aspect, the recognition elements comprise a nucleic acid, a peptide or a protein.

[0018]

[0014] , In one embodiment of the first aspect, the surface structures provide or contribute to confined spaces adjacent to a surface of the working electrode, the surface having the plurality of analyte recognition elements associated therewith, the plurality of analyte recognition elements extending into the confined spaces.

[0019]

[0015] , In one embodiment of the first aspect, the surface structures form overhangs facing generally toward the interior or the electrode, with the plurality of analyte recognition elements extending from the overhangs.

[0016] , In one embodiment of the first aspect, the surface structures form an enclosure having an opening, with the plurality of analyte recognition elements extending into the enclosure.

[0020]

[0017] , In one embodiment of the first aspect, the surface structures are generally granular structures.

[0021]

[0018] , In one embodiment of the first aspect, the surface structures are discrete structures.

[0022]

[0019] , In one embodiment of the first aspect, a majority of the surface structures have a measurable dimension of at least about 0.01, 0.1, 0.2, 0.3, 0. 4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 microns.

[0023]

[0020] , In one embodiment of the first aspect, the surface structures are formed fully, predominantly, or in part by an additive process.

[0024]

[0021] , In one embodiment of the first aspect, the additive process is a metal deposition process.

[0025]

[0022] , In one embodiment of the first aspect, the metal deposition process is an electroplating process.

[0026]

[0023] , In one embodiment of the first aspect, the surface structures are not formed by a subtractive process.

[0027]

[0024] , In one embodiment of the first aspect, the subtractive process is a metal removal process.

[0028]

[0025] . In one embodiment of the first aspect, the metal removal process comprises etching, abrading, scratching, ablating, cutting, or shearing.

[0029]

[0026] , In one embodiment of the first aspect, the metal deposition process is an electroplating process comprising electrodeposition of a metal coating onto a substrate metal.

[0030]

[0027] , In one embodiment of the first aspect, the metal coating comprises gold, or a gold alloy.

[0031]

[0028] , In one embodiment of the first aspect, the metal coating is deposited under conditions facilitating the formation of a rough surface or a non-smooth surface.

[0032]

[0029] , In one embodiment of the first aspect, the roughness or the non-smoothness of the surface is discernible as such by the unaided human eye.

[0033]

[0030] , In one embodiment of the first aspect, the roughness or the non-smoothness of the surface is discernible by an analytical method.

[0034]

[0031] , In one embodiment of the first aspect, the analytical method is an optical method.

[0035]

[0032] , In one embodiment of the first aspect, the optical method is selected from: light scattering, laser speckle, while light speckle, holographic interferometry, interferometric fringe contrast, multiple beam (Tolanski), FECO interference scan, and Nomarksi polarised scan interferometry.

[0036]

[0033] , In one embodiment of the first aspect, the analytical method output is in the units of microns (pm) or micro-inches (p-in, p”).

[0037]

[0034] , In one embodiment of the first aspect, the roughness or non-smoothness is at least about 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% that of a gold ingot, a minted gold bar, a Good Delivery gold bar or native gold.

[0035] , In one embodiment of the first aspect, the deposition is controlled so as to provide a surface having a required roughness of non-smoothness by control of any one or more of the following electroplating parameters: rate of metal deposition, temperature, magnitude of applied electrical potential, duty cycle of applied electrical current, electrical current, electrical current density, deposition time, bath composition including concentration of the metal ion to be deposited, pH, degree of agitation, distance between electrodes, position of electrodes.

[0038]

[0036] , In one embodiment of the first aspect, the applied electrical potential and / or temperature is / are controlled so as to control the roughness or non-smoothness of the working electrode surface.

[0039]

[0037] , In one embodiment of the first aspect, the magnitude of the applied electrical potential is determined by reference to ground, or another reference potential.

[0040]

[0038] , In one embodiment of the first aspect, the magnitude of the applied electrical potential is greater than about 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1.0 V, 1.1 V, 1.2V, 1.3 V, 1.4V,

[0041] 1.5V, 1.6V, 1.7V, 1.8V, 1.9V, 2.0V, 2.5V, 5.0V, 7.5V, or 10V.

[0042]

[0039] , In one embodiment of the first aspect, the temperature is greater than about 20°C,

[0043] 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C,

[0044] 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, or 50°C.

[0045]

[0040] , In one embodiment of the first aspect, the magnitude of the applied potential is about 1.7V or greater, and the temperature is about 45°C or greater.

[0046]

[0041] , In one embodiment of the first aspect, the working electrode is a needle, a microneedle or a wire.

[0047]

[0042] , In a second aspect, there is provided a method of producing a working electrode for an electrochemical sensor, the method comprising forming surface structures on the working electrode surface, and associating a plurality of analyte recognition elements with the electrode surface, wherein the surface structures configured to protect the plurality of analyte recognition elements from an adverse influence arising from passage of the working electrode through a tissue of an animal subject, or disposition of the working electrode in a tissue of an animal subject.

[0048]

[0043] , In one embodiment of the second aspect, passage of the working electrode through a tissue of an animal subject results in a contact-mediated disruption of the plurality of analyte recognition elements.

[0049]

[0044] , In one embodiment of the second aspect, disposition of the working electrode in a tissue of an animal subject results in a pressure-mediated or a contact-mediated disruption of the plurality of analyte recognition elements.

[0050]

[0045] , In one embodiment of the second aspect, the disruption is selected from any one or more of: removal from a surface of the electrode, reduction in the ability to change conformation in the presence or target analyte, and reduction in the ability to extend outwardly from a surface of the electrode.

[0051]

[0046] , In one embodiment of the second aspect, the surface structures provide or contribute to confined spaces adjacent to a surface of the working electrode, the surface having the plurality of analyte recognition elements associated therewith, the plurality of analyte recognition elements extending into the confined spaces.

[0052]

[0047] , In one embodiment of the second aspect, the surface structures form overhangs facing generally toward the interior or the electrode, with the plurality of analyte recognition elements extending from the overhangs.

[0053]

[0048] , In one embodiment of the second aspect, the surface structures form an enclosure having an opening, with the plurality of analyte recognition elements extending into the enclosure.

[0054]

[0049] , In one embodiment of the second aspect, the surface structures are generally granular structures.

[0055]

[0050] , In one embodiment of the second aspect, the surface structures are discrete structures.

[0056]

[0051] , In one embodiment of the second aspect, the surface structures have a measurable dimension in the range of wherein a majority of the surface structures have a measurable dimension of at least about 0.01, 0.1, 0.2, 0.3, 0. 4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 microns.

[0057]

[0052] , In one embodiment of the second aspect, the surface structures are formed fully, predominantly, or in part by an additive process.

[0058]

[0053] , In one embodiment of the second aspect, the additive process is a metal deposition process.

[0059]

[0054] , In one embodiment of the second aspect, the metal deposition process is an electroplating process.

[0060]

[0055] , In one embodiment of the second aspect, the surface structures are not formed by a subtractive process.

[0061]

[0056] , In one embodiment of the second aspect, the subtractive process is a metal removal process.

[0062]

[0057] , In one embodiment of the second aspect, the metal removal process comprises etching, abrading, scratching, ablating, cutting, or shearing.

[0063]

[0058] , In one embodiment of the second aspect, the metal deposition process is an electroplating process comprising electrodeposition of a metal coating onto a substrate metal.

[0064]

[0059] , In one embodiment of the second aspect, the metal coating comprises gold, or a gold alloy.

[0065]

[0060] , In one embodiment of the second aspect, the metal coating is deposited under conditions facilitating the formation of a rough surface or a non-smooth surface.

[0066]

[0061] , In one embodiment of the second aspect, the roughness or the non-smoothness of the surface is discernible as such by the unaided human eye.

[0067]

[0062] , In one embodiment of the second aspect, the roughness or the non-smoothness of the surface is discernible by an analytical method.

[0068]

[0063] . In one embodiment of the second aspect, the analytical method is an optical method.

[0069]

[0064] , In one embodiment of the second aspect, the optical method is selected from: light scattering, laser speckle, while light speckle, holographic interferometry, interferometric fringe contrast, multiple beam (Tolanski), FECO interference scan, and Nomarksi polarised scan interferometry.

[0070]

[0065] . In one embodiment of the second aspect, the analytical method output is in the units of microns (pm) or micro-inches (p-in, p”).

[0071]

[0066] , In one embodiment of the second aspect, the roughness or non-smoothness is at least about 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% that of gold ingot, a minted gold bar, a Good Delivery gold bar or native gold.

[0072]

[0067] , In one embodiment of the second aspect, the deposition is controlled so as to provide a surface having a required roughness of non-smoothness by control of any one or more of the following electroplating parameters: rate of metal deposition, temperature, magnitude of applied electrical potential, duty cycle of applied electrical current, electrical current, electrical current density, deposition time, bath composition including concentration of the metal ion to be deposited, pH, degree of agitation, distance between electrodes, position of electrodes.

[0073]

[0068] , In one embodiment of the second aspect, the applied electrical potential and / or temperature is / are controlled so as to control the roughness or non-smoothness of the working electrode surface.

[0074]

[0069] , In one embodiment of the second aspect, the magnitude of the applied electrical potential is determined by reference to ground, or another reference potential.

[0075]

[0070] , In one embodiment of the second aspect, the magnitude of the applied electrical potential is greater than about 0.5 V, 0.6 V, 0.7 V, 0.8 V, 0.9 V, 1.0 V, 1.1 V, 1.2V, 1.3 V, 1.4V,

[0076] 1.5V, 1.6V, 1.7V, 1.8V, 1.9V, 2.0V, 2.5V, 5.0V, 7.5V, or 10V.

[0077]

[0071] , In one embodiment of the second aspect, the temperature is greater than about 20°C,

[0078] 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31 °C, 32°C, 33°C, 34°C, 35°C,

[0079] 36°C, 37°C, 38°C, 39°C, 40°C, 41 °C, 42°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, or 50°C.

[0080]

[0072] , In one embodiment of the second aspect, the magnitude of the applied potential is about 1.7V or greater, and the temperature is about 45°C or greater.

[0081]

[0073] , In one embodiment of the second aspect, the working electrode is a needle, a microneedle or a wire.

[0082]

[0074] , In a third aspect, there is provided a working electrode for an electrochemical sensor produced by the method of any embodiment of the second aspect.

[0083]

[0075] , In a fourth aspect, there is provided an electrochemical sensor comprising the working electrode of any embodiment of the first or third aspects.

[0084]

[0076] , In one embodiment of the fourth aspect, the electrochemical sensor comprises a counter electrode and a reference electrode.

[0085]

[0077] , In one embodiment of the fourth aspect, the analyte recognition element comprises an aptamer, a peptide or a protein.

[0086]

[0078] , In a fifth aspect, there is provided a method for determining an amount of a target analyte in a tissue or a fluid of a subject animal, the method comprising contacting the working electrode to the tissue or the fluid of the subject animal.

[0079] , In one embodiment of the fifth aspect, the step of contacting comprises passing the working electrode through a tissue of an animal subject, or disposing the working electrode in a tissue of the animal subject.

[0087]

[0080] . In one embodiment of the fifth aspect, the step of contacting comprises the working electrode piercing the tissue or another tissue of the subject animal.

[0088]

[0081] , In one embodiment of the fifth aspect, the tissue or another tissue is a dermal tissue or an adjacent tissue.

[0089]

[0082] , In one embodiment of the fifth aspect, the fluid is interstitial fluid or blood.

[0090]

[0083] , In one embodiment of the fifth aspect, the method comprises applying an interrogating potential to the working electrode and analysing a current arising in the working electrode from the interrogating potential so as to determine an amount of the target analyte.

[0091] BRIEF DESCRIPTION OF THE FIGURES

[0092]

[0084] , FIG. 1A, FIG. IB and FIG. 1C illustrate an apparatus for gold electroplating of needle electrodes.

[0093]

[0085] , FIG. 2A, FIG 2B and FIG. 2C each illustrate a needle electrode formed from a steel base and coated with, respectively, smooth gold, moderately rough gold, and highly rough gold. The varying levels of roughness were produced by altering the voltage potential applied during gold electroplating.

[0094]

[0086] , FIG. 3 A, FIG 3B each illustrate a needle electrode formed from a steel base and coated with, respectively, smooth gold, and rough gold. The levels of roughness were produced by altering the temperature of the bath during gold electroplating.

[0095]

[0087] , FIG. 4A is a scanning electron micrograph of a smooth gold surface produced by electroplating. Multiplication x4, 500. Bar = 1 pm.

[0096]

[0088] , FIG. 4B is a scanning electron micrograph of a rough gold surface produced by electroplating. Multiplication x4, 500. Bar = 1 pm.

[0097]

[0089] , FIG. 4C is an enlarged version of FIG. 4B showing more clearly the detail of the surface of a rough gold electrode formed by an additive process. Multiplication x4,600. Bar = 1 pm.

[0098]

[0090] , FIG. 4D is a highly diagrammatic illustration of a cross-section of a working electrode having a smooth gold layer formed from regular and closely packed grains, and an outer rough layer formed from irregular and more loosely packed grains to provide protective structures.

[0099]

[0091] , FIG. 4E is a highly diagrammatic illustration of a cross-section of a working electrode, being an enlargement of FIG. 4D. The arrows indicate spaces within the rough surface layer where recognition elements are protected from insertional damage by surrounding structures such as overhanging portion of irregular grains.

[0100]

[0092] , FIG. 5 is a histogram showing the size distribution of grains in the micrograph of FIG. 4B.

[0101]

[0093] , FIG. 6A and FIG. 6B are each a voltammogram resulting from interrogation of a working electrode in the form of a gold electroplated stainless steel needle functionalised with a vancomycin sensitive aptamer. The surface of the electrode of FIG. 6A is smooth gold. The surface of the electrode of FIG. 6B is rough gold formed by an additive process.

[0102]

[0094] , FIG. 7A and FIG. 7B are each a voltammogram of replicate experiments resulting from interrogation of a working electrode in the form of a gold wire functionalised with a vancomycin sensitive aptamer. The surface of the gold wire was roughened by a subtractive process before functionalization.

[0103]

[0095] , 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.

[0104]

[0096] , The drawings are not necessarily prepared to any particular scale or dimension and are not necessarily presented as being a completely accurate presentation of the various embodiments.

[0105] DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

[0106]

[0097] , After considering this description it will be apparent to one skilled in the art how the invention is implemented in various alternative embodiments and alternative applications. However, although various embodiments of the present invention 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 invention. 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.

[0107]

[0098] . 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.

[0108]

[0099] , 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 invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may.

[0109]

[0100] . The term “subject” includes an animal subject, and particularly a vertebrate subject, and more particularly a mammalian subject, for whom monitoring and / or diagnosis of a target analyte, is desired. Suitable subjects include, but are not limited to, primates such as humans, monkeys, and apes; avians (birds); livestock animals such as sheep, goats, cows, horses, deer, donkeys, and pigs; laboratory test animals such as rabbits, mice, rats, guinea pigs, and hamsters; companion animals such as hamsters, cats, and dogs; bats; and captive wild animals such as tigers, lions, foxes, deer, and dingoes. In preferred embodiments, the subject is a human.

[0110]

[0101] , The present invention is predicated at least in part on the inventors ’ finding that the output signal of an electrochemical sensor is diminished after the working electrode is urged through the surface of the skin and into the dermal tissues of an animal subject. Without wishing to be limited by theory, it is proposed that signal loss arises from the loss, or loss of function of analyte recognition elements such as aptamers and peptides extending from the surface of the working electrode. For example, where the working electrode is a microneedle functionalized with aptamers, the aptamers may be exposed to contact-mediated forces (such as frictional forces) and pressures as the microneedle pierces the skin to seat the functionalised terminal region of the microneedle in the dermal tissues.

[0111]

[0102] . According to the present disclosure, analyte recognition elements may be preserved to at least some extent where the surface to which they are attached comprises protective surface structures. Each surface structure, or a combination of surface structures, may provide a confined space such that recognition elements that extend into the confined space are exposed to lesser adverse influences as compared with an electrode having no such surface structures. The confined spaces remain in fluid communication with the test fluid such that the recognition elements are exposed to target analyte, while at the same time preventing or inhibiting exposure of the recognition elements to dermal materials such as dead cells, live cells, cellular fragments, and components of the extracellular matrix including proteins (such as collagen and elastin).

[0112]

[0103] , The surface structures of the working electrode may provide overhangs beneath which a recognition elements may be disposed, the overhang providing hindrance to the contact of dermal materials with the recognition elements. An overhang may be provided by one structure or a combination of surface structures.

[0113]

[0104] , The surface structures of the working electrode may provide enclosures within which aptamer recognition elements may be disposed. The enclosure may comprise one or more pores that allow the entry of target analyte but excludes dermal materials that could potentially damage the recognition elements.

[0114]

[0105] , In some embodiments, the analyte recognition elements are attached to a surface structure, while in other embodiments the recognition elements are attached to a surface of the working electrode proximal to one or more surface structures.

[0115]

[0106] , Protective surface structures may not form where an electrode surface is subjected to a subtractive process involving the removal of electrode material such as etching. While a subtractive process may result in surface features such as channels, depressions, valleys, scratches, dimples and the like, confined spaces may not be formed. Protective structures such as overhangs and enclosures cannot, or are not readily formed, by subtractive processes.

[0116]

[0107] . Accordingly, in one embodiment of the present disclosure the surface structures are formed by an additive process whereby a conductive material is built up in a manner that provides protective surface structures. The skilled person is aware of a number of techniques by which metals may be deposited onto a surface, as follows.

[0117]

[0108] , Vapour deposition methods are reliant on the coating material being presented to the surface to be coated in a vapour state via condensation, chemical reaction, or conversion. Examples of vapour deposition methods include physical vapour deposition (PVD) and chemical vapour deposition (CVD). In PVD, the surface to be coated is subjected to plasma bombardment. In CVD, thermal energy heats gases in a coating chamber, driving the deposition reaction. Vapour deposition methods are usually performed within a vacuum chamber.

[0118]

[0109] , The vapour deposition method may be a physical vapour deposition method, including an ion plating method, a plasma-based method, an ion implantation method, a sputtering method, a sputter deposition method, a laser surface alloying method and a laser cladding method.

[0119]

[0110] , Physical vapour deposition methods are typically reliant on dry vacuum deposition in which a coating material is deposited over the surface to be coated. Reactive PVD hard coating methods generally require a method for depositing the metal, an active gas (such as nitrogen, oxygen, or methane), and plasma bombardment of the substrate.

[0120]

[0111] , PVD methods include ion plating, ion implantation, sputtering, and laser surface alloying.

[0121]

[0112] , Plasma-based plating is one form of ion plating, whereby the surface to be coated is positioned proximal to a plasma. Ions and neutrons from the plasma are accelerated by a negative bias onto the surface to be coated with a range of energies.

[0122]

[0113] , This technique produces coatings that typically range from 0.008 mm to 0.025 mm, although conditions can be altered to achieve thicker and thinner coatings. Ion plating can provide excellent surface covering ability, good adhesion, flexibility in tailoring film properties (e.g., morphology, density, and residual film stress), and in-situ cleaning of the substrate prior to film deposition. Ion plating methods are capable of depositing a wide variety of metals including copper and gold.

[0123]

[0114] , The electrode surface may be coated using sputtering or sputter deposition methods.

[0124] Sputtering alters the physical properties of a surface. In this process, a gas plasma discharge is provided between a cathode coating material and an anode substrate. Positively charged gas ions are accelerated into the cathode. The impact displaces atoms from the cathode, which then impact the anode and coat the substrate. A film forms on the upwardly facing reflective surface as atoms adhere to the substrate. The deposits are typically thin, ranging from 0.00005 mm to 0.01 mm. This method is often used to coat with silver, aluminium, chromium, titanium, copper, molybdenum, tungsten, and gold. Three techniques for sputtering are available to the skilled person for potential use in the present disclosure: diode plasmas, RF diodes, and magnetron- enhanced sputtering.

[0125]

[0115] , Sputter deposition is capable of depositing coatings of metals, alloys, compounds, and dielectrics on surfaces. Compared to other deposition processes, sputter deposition is relatively inexpensive, and may be preferred in some applications for reasons of economy only.

[0126]

[0116] , Laser cladding is one type of laser surface alloying which may be used to selectively coat a defined area. Typically, a thin layer of metal (which may be a powder metal) is bonded with a base metal via the application of heat and pressure. A metal powder may be fed into a carbon dioxide laser beam above the upwardly facing reflective surface, melted in the beam, and then deposited on the surface. Powder feeding may be performed using a carrier gas in a manner analogous to thermal spray systems. Large areas may be coated by moving the substrate under the beam and overlapping deposition tracks.

[0117] , Laser cladding may generally be used to apply the same or similar materials to those operable with thermal spraying methods. Deposition rates may be altered by modulating any one or more of laser power, feed rates, and traverse speed. Coating thicknesses can range from several hundred microns to several millimetres, although process conditions may be varied to provide for thickness outside of this range.

[0127]

[0118] , In one embodiment of the first aspect, the vapour deposition method is a chemical vapour deposition method, including a sputtering method, an ion plating method, a plasma- enhanced method, a low-pressure method, a laser-enhanced method, an active reactive evaporation, an ion beam method, and a laser evaporation method. The various methods are distinguished by the manner in which the precursor gases are converted into reactive gas mixtures.

[0128]

[0119] , The steps in a typical CVD process are as follows: generation of the reactive gas mixture, transport of reactant gas to the surface to be coated, adsorption of the reactants on the surface to be coated, and reaction of the adsorbents to form the coating.

[0129]

[0120] , To explain further, the reactant gas mixture is contacted with the substrate of the reflector array. The coating material is delivered by a precursor material (termed a reactive vapour) which may be dispensed as a gas, liquid, or in solid phase. The gases are fed into a chamber under ambient pressures and temperatures while solids and liquids are provided at high temperature and / or low pressure. Once resident in the chamber, energy is applied to the substrate surface to facilitate the coating reaction with the carrier gas.

[0130]

[0121] , Pre-treatment of the substrate surface is generally required in vapour deposition methods, and particularly in CVD. Mechanical and / or chemical means may be used before the substrate enters the deposition reactor. Cleaning is typically effected by ultrasonic cleaning and / or vapour degreasing. To facilitate adhesion of the coating, vapour honing may be used. During the coating process, surface cleanliness is maintained to prevent particulates from entering in the coating. Mild acids or bases may be used to slough oxide layers which may have formed during the heat-up step. Post-treatment of the coating may include exposure to heat to cause diffusion of the coating material across the surface.

[0131]

[0122] , In one embodiment of the first aspect, the coating material which forms the protective structures is deposited on the substrate surface by a thermal spray method, including a combustion torch method, a flame spraying method, a high velocity oxy fuel method, a detonation gun method, an electric arc spraying method and a plasma spraying method.

[0132]

[0123] , The coating material may be sprayed from rod or wire stock or from powder material. An operator feeds material to a flame so as to melt it. The molten stock is then stripped from the end of the wire and atomized by a high-velocity stream of compressed air (or other gas), thereby coating the material onto the substrate surface. Depending on the surface, bonding may occur due to mechanical engagement with the roughened surface and / or because of electrostatic forces.

[0133]

[0124] . Parameters that affect the deposition of metals in thermal spray applications include the particle's temperature, velocity, angle of impact, and the extent of any reaction with gases during the deposition process.

[0125] , There currently exists three basic categories of thermal spray technologies: combustion torch methods (including flamespray, high-velocity oxy fuel, and detonation gun methods), electric (wire) arc methods, and plasma arc methods.

[0134]

[0126] , Flame spraying methods involve feeding gas and oxygen through a combustion flame spray torch. The coating material (in powder or wire form) is fed into the flame. The coating material is heated to about or higher than its melting point, and then accelerated by combustion of the coating material. The so-formed molten droplets flow on the surface to form a continuous and even coating.

[0135]

[0127] , High-velocity oxy fuel (HVOF) methods require the coating material to be heated to a temperature of about or greater than its melting point, and then deposited on the upwardly facing reflective surface by a high-velocity combustion gas stream. The method is typically carried out in a combustion chamber to enable higher gas velocities. Fuels used in this method include hydrogen, propane, or propylene.

[0136]

[0128] , Combustion torch and detonation gun methods combine oxygen and acetylene with pulsed powder containing carbides, metal binders, and oxides. The mix is introduced into a water- cooled barrel, and detonated to generate expanding gas that heats and accelerates the powder materials while converting same into a plastic-like state (typically at temperatures of 1,100 degrees Celsius to 19,000 degrees Celsius). A coating may be built up by way of repeated, controlled detonations. Typical coating thicknesses range from 0.05 mm to 0.5 mm, although thinner and thicker coatings can be achieved.

[0137]

[0129] , In electric arc spraying, an electric arc is formed between the termini of two wires composed of the coating material. The arc continuously melts the wire while a gas jet blows the molten droplets toward the surface. Coating material may be applied thinly or thickly as required.

[0138]

[0130] , Plasma spraying relies on introduction of a flow of gas (typically argon) between a water-cooled anode and a cathode. A direct current arc passes through the gas stream causing ionization and the formation of a plasma. The plasma heats the coating material (in powder form) to a molten state. Compressed gas directs the material onto the surface to be coated.

[0139]

[0131] , A preferred method of forming the protective surface structures is electroplating

[0140] (also known as electrochemical deposition or electrodeposition), is a process for metal coating a solid substrate through the reduction of cations of that metal by means of a direct electric current. The working electrode acts as the cathode (negative electrode) of an electrolytic cell; the electrolyte is a solution of a salt of the metal to be coated, and the anode (positive electrode) is usually either a block of that metal, or of some inert conductive material. The current is provided by an external power supply.

[0141]

[0132] , The term "electroplating" may also refer to an electro-oxidation process (i.e positive or anodic current on the working electrode), although such processes are more commonly referred to as anodizing rather than electroplating. One such example is the formation of silver chloride on silver wire in chloride solutions to make silver / silver-chloride (AgCl) electrodes.

[0142]

[0133] , Gold is a preferred metal for forming the protective structures given the ready ability to thiol-link aptamers thereto. Pure gold (i.e. not alloyed with another metal, also termed “soft gold”) is preferred . Soft gold plating may produce a more coarse grain structure than does “hard” (i.e. alloyed) gold plating. It is proposed that the protective structures are more likely produced with the coarser grains arising in soft gold coatings.

[0143]

[0134] , With regard to the substrate used in the coating process (i.e. the electrode surface), any suitable material may be used, including a metal such as steel, stainless steel (including 304, 316, 316L), tungsten, copper, platinum, nickel, cobalt, titanium, and silver. In one embodiment, the substrate is preformed into the final form of the electrode suitable for the intended use, such form including a needle, a microneedle or a wire. For example, a stainless steel acupuncture needle provides a suitable electrode substrate in some applications.

[0144]

[0135] , In some embodiments, a strike layer is coated onto the bare electrode surface as a preliminary step. The strike layer may function to provide a seed layer upon which a bulk layer of gold may be coated. In a sense, the strike layer may be considered to provide an “adhesive” surface allowing for more effective bonding of further gold coatings. A strong bond between the electrode substrate and the furthergold coating (as facilitated by the strike layer) may prevent cracking and delamination of the functionalised electrode surface, especially during insertion of the elected through skin. The strike layer may be formed from any suitable material such as nickel or gold. Preferably, the strike layer is formed from gold.

[0145]

[0136] , A smooth gold layer may be applied after the strike layer. The function of this layer may be to ensure an even base coating over the electrode substrate surface so as to limit or completely obviate any regions of exposed electrode surface. Exposed regions may persist even after functionalisation of the electrode by any aptamer or other analyte recognition element. Exposed regions of the electrode (not being gold) may not allow, for example, thiol-mediated association of an analyte recognition element with an electrode. The total number of analyte recognition elements on a given electrode may therefore be compromised, leading to reduced analyte sensitivity.

[0146]

[0137] , A further function of the smooth gold layer may be to limit the amount of electrochemical background against the analyte-dependent current signal flowing through the electrode. It is contemplated that exposed regions of electrode substrate surface may allow for current flow that is unrelated to the proximity of a redox reporter to the electrode, and therefore not informative as to analyte concentration. Avoidance of exposed electrode substrate may also provide for a more stable and / or a more reproducible signal.

[0147]

[0138] , The smooth gold layer may be deposited at a slower rate than that of the outermost

[0148] (rough) gold layer with which the analyte recognition elements are associated.

[0149]

[0139] , It is preferred that the smooth gold layer is formed by a metal deposition method that favours a smooth morphology, and avoiding inhomogeneities that that leads to roughness. The method may further favour compactness in the crystal structure so as to avoid pinholes that can leave the electrode substrate exposed. Methods promoting a smooth gold layer of uniform thickness are further preferred.

[0150]

[0140] , Conditions for achieving a certain smooth gold layer thickness may rely on modulation of deposition time, with a thick coating being provided by longer time periods.

[0141] , The outermost layer of the working electrode is applied to the smooth gold surface.

[0151] As discussed elsewhere herein, that layer is generally rough so as to provide the required protective structures. Preferably the outermost layer is gold that is made rough by more rapid deposition times. Again, the thickness of the outmost layer may be achieved by modulation of deposition time.

[0152]

[0142] , A working electrode comprising protective surface structures may present visually as a rough surface, a dull surface, or a surface that is less than smooth or shiny. Roughness may be determined alternatively by more quantitative means such as scattering, laser speckle, while light speckle, holographic interferometry, interferometric fringe contrast, multiple beam (Tolanski), FECO interference scan, and Nomarksi polarised scan interferometry, for example.

[0153]

[0143] , A working electrode may be assessed by routine means for the ability to resist an adverse influence arising from passage of the working electrode through a tissue of an animal subject, or disposition of the working electrode in a tissue of an animal subject. For example, a candidate working electrode may be wired into an appropriate circuit to form a sensor for an analyte. The candidate working electrode may be contacted to a test liquid having an amount of the target analyte contained therein. The electrode is then used to pierce the skin (living or cadaver) of a subject (human or non-human) and then contacted for a second time to the test liquid. A candidate electrode may be considered useful if the signal retained after piercing the skin is greater than about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0154]

[0144] , The working electrode has an analyte recognition element associated therewith. 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.

[0155]

[0145] , 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.

[0156]

[0146] , 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.

[0147] , 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.

[0157]

[0148] , 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.

[0158]

[0149] , A non-natural nucleic acid molecule useful in the context of the present invention 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).

[0159]

[0150] , 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.

[0160]

[0151] , One method of identifying aptamers useful in the context of the present invention 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.

[0161]

[0152] , 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.

[0162]

[0153] , 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 methylamino 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.

[0163]

[0154] , Side chain modifications may be 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, benzyloxy-tyrosine, and P- phenylalanine are commonly commercially available, and can be conveniently used to chemically modify the peptide side chain during peptide synthesis.

[0155] , 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 side-chains for stabilization of secondary structures may be pursued.

[0164]

[0156] , 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.

[0165]

[0157] , The analyte recognition element in some embodiments is a plurality of polynucleotide molecules. Each of the plurality has an identical nucleotide sequence, the sequence selected so as to specifically bind or otherwise associate with a target analyte in a fluid comprising a non-target species. Where an analyte recognition element is described to bind “specifically” or “selectively” to a target analyte, it is not intended that either term be considered as absolute. Typically, non-target species bind also to the binding element, but with a significantly lower affinity. In that regard, the target analyte is nevertheless detectable against the background signal of low affinity non-target binding. A target analyte may have a binding affinity of greater than about 2-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, or greater than about 500-fold, with respect to binding of a non-target species.

[0166]

[0158] , A particularly useful class of polynucleotides, known as “aptamers”, have been shown herein to be useful in the context of in vivo detection of a target analyte present in interstitial fluid, and particularly in the epidermal, dermal, and subdermal tissue layers.

[0167]

[0159] , Binding of target analyte to an aptamer or other type of analyte recognition element may be detected by a reporter element associated therewith and configured to generate an output, typically in the form of an electric current. The output electric current is typically used as microprocessor input and interpreted according to a predetermined criterion embodied in software instructions. Where the criterion is satisfied, the signal is interpreted as specific binding of a target analyte.

[0168]

[0160] , A particularly useful combination is found in an aptamer having an associated redox reporter. For example, the aptamer may be modified at one terminus with methylene blue, with the other terminus being immobilised on the first conductive element, or working electrode. Upon binding of a target analyte, the immobilised aptamer changes in conformation, the conformational change in turn altering the electron transfer efficiency between the redox reporter and the working electrode. In one particular example, a methylene blue-modified, binding- responsive aptamer is immobilised onto a gold surface of a working electrode via a thiol linkage. Another species of redox reporter is ferrocene.

[0169]

[0161] , The increase in Faradaic current produced by the working electrode (resulting from interaction between the redox reporter and the working electrode surface), is detected by a potentiostat, with target analyte capable of being specifically detected at picomolar levels. As will be appreciated, a counter electrode is required to function as a cathode or an anode to the working electrode.

[0170]

[0162] , A third conductive element may be included in the apparatus, functioning as a reference electrode. The reference electrode is configured to act as reference with regard to a known electrochemical reaction having a definable redox potential. For example, the reference electrode may be based on the reaction of silver-silver chloride (Ag / AgCl), with the fixed redox potential of that reaction providing a base value for measuring the redox potential of the working electrode (the counter electrode acting as a cathode or anode to the working electrode, as mentioned supra). The counter electrode may be combined with the reference electrode, such that the combined electrode acts as both a counter electrode and a reference electrode. In this configuration it is preferred that the current flowing between the working electrode and the combined counter / reference electrode in use does not perturb the redox potential of the counter / reference electrode is a way that is detrimental to the functioning of the sensor device.

[0171]

[0163] , When a voltage is applied, a very minor current flows between a separate reference electrode, such that a potential can be measured. Because the electrical circuit measuring the voltage difference is typically of a high impedance, the resistance is high, and the voltage is measured with the flow of only very small currents. Current is measured as the potential of the working electrode against the fixed potential of the reference electrode. The difference in these potentials produces an alteration in the current which constitutes the change in electrical signal. Typically, the changes in output signal are stoichiometrically proportional to target analyte binding.

[0172]

[0164] , An aptamer having a redox reporter that is coated onto a working electrode produces a change in electrical signal that is proportional to the target analyte in interstitial fluid of the epidermal, dermal, or subdermal layer of animal skin, and substantially in real time. This finding allows for the routine use of aptamer-based biosensors in clinical and non-clinical settings for the real time analysis of relevant analytes. Advantageously, there is no need for any intermittent sampling of biological fluid (for example by venepuncture, finger prick, biopsy, or urine collection). As a further advantage, the results are available immediately thereby allowing for timely intervention as required.

[0173]

[0165] , A sensor comprising the present working electrode may incorporated into an electrochemical sensor operable by way of voltammetry, including square wave voltammetry, cyclic voltammetry, chronoamperometry, chronopotentiometry, impedance spectroscopy, electrochemically implemented surface plasmon resonance, optical waveguide lightmode spectroscopy, ellipsometry, and quartz crystal microbalance, or a field-effect transistor based method. Generally, the sensor will be configured to be operable by way of voltammetry (and particularly square wave voltammetry), and in that regard will be configured to connect to a voltage source and particularly a controllable voltage source. Voltage is applied across at least the working electrode and the counter electrode when both are in contact with a biological fluid.

[0174]

[0166] , Some embodiments of the sensor may be configured to be “wearable”, in that the apparatus is easily fitted to an animal subject (and particularly to a human subject), and without

[0175] Y1 causing any significant impact on the activities of the subject. In that regard, the working electrode and any further electrodes may be microneedles configured to breach the skin, and dwell within interstitial fluid of the dermis or the sub-dermis. The microneedles are sufficiently fine in construction so as to cause little, if any, discomfort upon insertion into the skin.

[0176]

[0167] . As a wearable apparatus, there will typically be configuration to allow for retention on the skin surface of the subject. For example, the apparatus may be retained by way of an adhesive or a stretchable band.

[0177]

[0168] , In some embodiments, the electrode(s) are mounted on a mounting element. The mounting element will be typically electrically non-conducting to prevent shorting between the conductive elements. Where a conducting material is used, insulation may be used to prevent shorting. The mounting element will typically have a generally planar surface. In some embodiments, the mounting element is rigid, semi-rigid, or at least partially rigid, for facilitating penetration of the microneedles on application to the skin. The mounting element may also be partially flexible or semi-flexible, so that, in use, the microneedle-based electrochemical biosensor can conform to an outer surface or an outer shape of at least part of a subject’s body.

[0178]

[0169] , The mounting element may be fabricated from or contain woven and non-woven fabrics including electronic fabrics; natural or synthetic fibres; natural or synthetic textiles; silk; organic materials; natural or artificial composite materials, including polymeric materials; glass; ceramics, including polymer ceramics; porous materials; polymers such as rigid or semi-rigid plastics and machinable polymers such as such as acrylic, polycarbonate, polyether ether ketone, or PEEK; synthetic polymers such as polymethyl methacrylate or acrylic glass, and other plastics made from methacrylate; thermoplastics and thermosetting plastics such as acrylic resin, polycarbonate, and polyether ether ketone; thermoplastic polymers such as polyethylene terephthalate; doped polymers such as polyacetylene, polypyrrole, polyindole, and polyaniline; intrinsically conducting polymers; metals, including aluminium, copper, gold including colloidal gold, silver including colloidal silver, chromium, platinum, titanium; metal alloys including stainless steel; carbon including colloidal carbon, carbon-nano materials, and carbon composites such as graphene and graphite; semiconductors such as silicon, germanium, and gallium arsenide; doped semiconductors; and, organosilicates. In some embodiments, the mounting element is made from a synthetic polymer such as polymethyl methacrylate.

[0179]

[0170] , The electrode(s) may be provided in any form, including a microneedle, a wire or even a simple planar electrode or a rod electrode or as a coating on an insulating substrate. For use where no piercing of the skin is required (for example ex vivo use, or implantation into a blood vessel via catheter guided means) the conductive element may not be rigid and indeed may be very flexible.

[0180]

[0171] , Microneedles can be fabricated in a range of various shapes and geometries, although the specific microneedle geometry will for transdermal applications be optimised to breach the stratum corneum for reliable skin penetration. A microneedle-based electrochemical sensor may however further comprise an applicator configured to apply a force to the microneedles to facilitate the microneedles to breach the stratum corneum and to penetrate through the skin layers. For non-human applications, the stratum corneum may be replaced by an analogous, or even a non-analogous layer on the surface of the animal. In some applications the microneedles may be required to breach other structures overlying the stratum corneum or other surface layer of an animal, such structures including scales and feathers. In some applications, the microneedles are configured to breach the external surface or an organ (such as a capsular structure), a blood vessel, or any other structure of an animal body.

[0181]

[0172] , Generally, each microneedle will have the shape of a protruding needle structure extending from the mounting element. Typically, the microneedles will extend generally perpendicular from the mounting element.

[0182]

[0173] , The protruding needle structure of the microneedle can be of any needle-type shape.

[0183] For example, the microneedle may taper smoothly from a base to form a pointed tip (e.g., cone shape), may have multiple lateral sides extending from a base that converge to form a pointed tip (e.g., pyramid shape or triangular prism), be tapered in just one dimension, or have a base with curved sides of relatively constant diameter, which is segmented to form a pointed tip (e.g., a segment of a cylindrical shape). Typically, the pointed tip will be sharp. The microneedle may or may not include shape changes along its length. Further, any edge or side of the microneedle shape may be bevelled, curved, or rounded.

[0184]

[0174] , In some embodiments, the shape of the microneedle is a cone, or a pyramid such as a triangular pyramid, square pyramid, or hexagonal pyramid. In other embodiments, the microneedle has the shape of a tetrahedron or a triangular prism. In further embodiments, the microneedle has the shape of a rocket, turret, arrowhead, spike, or spear.

[0185]

[0175] , It will be appreciated that a range of other shapes could be used. For example, the shape of the microneedle may a circular or an elliptical cylinder, which is truncated. Any of the other microneedle shapes described herein may or may not be truncated. The term “truncated”, as used in this context, may refer to a shape cut on a plane parallel to the base, which may be referred to as a parallel-truncated shape or more specifically, a frustum, or a shape cut at an angle relative to an axis of the microneedle, which may be referred to as an angular-truncated shape. For angular- truncated shapes, the angle of truncation relative to an axis of the microneedle shape will be at least about 50° and no more than about 75°. In some embodiments, the truncation angle is between about 55° and about 70°, about 55° and about 65°, and about 50° and about 60°. In other embodiments, the truncation angle is about 50°, about 60°, or about 65°, or about 70°. In a particular embodiment, the shape of the microneedle is a truncated circular cylinder, with a truncation angle of about 60° relative to its axis.

[0186]

[0176] , It will be appreciated that the same or different microneedle shapes could be provided on the mounting element. For example, the microneedle could be shaped as a plate or blade with a sharp edge.

[0187]

[0177] , Microneedles are typically divided into 4 types: solid microneedles, coated microneedles, dissolving microneedles, and hollow microneedles. In some embodiments the microneedles are hollow. However, it will be appreciated that the microneedle platform could comprise a combination of the 4 types of microneedles. For example, the microneedle platform may comprise a combination of solid and hollow microneedles. For example, there may be no requirement for a microneedle functioning as a counter electrode or a reference electrode to be hollow.

[0188]

[0178] , A hollow microneedle will generally have a hollow interior defined by an interior wall with an opening at the terminus intended to contact the biological fluid. The hollow interior may or may not conform to the outer shape of the microneedle. In some embodiments, the hollow microneedle has a generally circular hollow interior, like a bore hole. The circular hollow interior may have a diameter of between at least about 0.1 mm and no more than about 5 mm. In some embodiments, the diameter of the hollow interior is between about 0.5 mm and about 1 mm. The opening to the hollow interior is preferably in proximity to the terminus, but could also be at the top face of the hollow microneedle.

[0189]

[0179] , The exterior wall of a microneedle may be configured to abut against the stratum corneum of a subject to control depth of penetration into the skin layers of the subject. The exterior wall of a microneedle may also be provided with a shoulder or ledge for this purpose.

[0190]

[0180] , The exterior wall of a microneedle may have a smooth or rough surface, and can include surface features, such raised portions, etchings, serrations, anchors, barbs, or the like, which may assist engaging a biological tissue once the microneedles have breached the stratum corneum to secure the microneedles within the subject. It will be appreciated that the ability of the microneedle-based electrochemical biosensor to remain in situ is particularly beneficial, as this ensures that continuous measurements over a prolonged period of time are made at the same site within the subject. Furthermore, constraining the location in which measurements are performed ensures more accurate longitudinal monitoring. In some embodiments, the microneedle-based electrochemical biosensor is configured to remain in situ for at least one minute, at least one hour, at least about 8 hours, at least about 18 hours, at least one day (about 24 hours), at least about 3 days, at least about 4 days, or at least one week. In some applications it may be necessary or desirable to remain in situ for one month.

[0191]

[0181] , The exterior wall of the microneedle may or may not have void spaces. In some embodiments, the exterior wall of the hollow microneedle is porous, or has a porous layer, which may increase the effective surface area of the microneedle, or may allow a target of interest to enter the pores, but exclude one or more other targets or substances, depending on the size of the target of interest. The pores may be less than about 10 pm in diameter, preferably less than about 1 pm in diameter.

[0192]

[0182] , It will be appreciated that the size of the microneedles, and their arrangement on the mounting element, may vary depending upon the intended application of the microneedlebased electrochemical biosensor.

[0193]

[0183] , The microneedles will be of a length at least greater than the thickness of the stratum corneum and to penetrate the skin layers to a depth of at least 100 pm, to be positioned in a biological tissue to contact a biological fluid of a subject. In some embodiments, the length will be at least about 10% greater than the thickness of the stratum corneum, at least about 20% greater than the thickness of the stratum corneum, at least about 50% greater than the thickness of the stratum corneum, at least about 75% greater than the thickness of the stratum corneum, or at least about 100% greater than the thickness of the stratum corneum. In some embodiments, the length is less than about 1500 gm, less than about 1000 gm, less than about 750 gm, less than about 600 gm, less than about 500 gm, less than about 400 gm, less than about 300 gm, less than about 250 gm, greater than about 100 gm, greater than about 50 gm, greater than about 20 gm, or greater than about 10 gm. In other embodiments, the length is between about 100 gm and about 1000 gm, about 200 gm and about 1000 gm, about 500 gm and about 1000 gm, about 750 gm and about 1000 gm, about 800 gm and about 1000 gm, about 900 gm and about 1000 gm, about 100 gm and about 900 gm, about 200 gm and about 900 gm, about 500 gm and about 900 gm, about 750 gm and about 900 gm, about 800 gm and about 900 gm, about 100 gm and about 800 gm, about 200 gm and about 800 gm, about 500 gm and about 800 gm, or about 750 gm and about 800 gm. In other embodiments, the length is about 600 gm, about 750 gm, about 800 gm, about 900 gm, or about 1000 gm.

[0194]

[0184] . In some embodiments, the microneedles have a tiered arrangement and thus would not all be of the same length. In such embodiments, the length of the microneedles may range between about 400 gm and about 800 gm.

[0195]

[0185] , The base width of the microneedle may be at least less than about 50% of the length, less than about 25% of the length, less than about 20% of the length, less than about 15% of the length, less than about 10% of the length, or less than about 5% of the length. In some embodiments, the base width is at least about 100 gm but no more than about 400 gm. In other embodiments, the diameter is about 200 gm, or about 300 gm.

[0196]

[0186] , The diameter of the microneedle may be at least less than about 50% of the length, less than about 25% of the length, less than about 20% of the length, less than about 15% of the length, less than about 10% of the length, or less than about 5% of the length. In some embodiments, the diameter is between at least about 0.1 mm and no more than about 5 mm. In some embodiments, the diameter is between about 0.5 mm and about 1 mm.

[0197]

[0187] , The microneedles may be provided in various arrangements, and the number of microneedles provided on the mounting element will depend on available surface area. The mounting element may comprise up to about 100 microneedles. In some embodiments, the mounting element comprises between at least 3 microneedles and less than about 50 microneedles. In other embodiments, the mounting element comprises between at least 3 microneedles and less than about 30 microneedles. In yet other embodiments, the mounting element comprises between at least 3 microneedles and less than about 20 microneedles. In yet other embodiments, the mounting element comprises between at least 3 microneedles and less than about 10 microneedles.

[0198]

[0188] , Typically, the microneedle arrangement will be of a relatively low density, as this is likely to facilitate breach of the stratum corneum by the microneedles and may avoid potential problems with skin penetration by high density arrangements. In some embodiments, the mounting element comprises at least about 4 microneedles / cm2, at least about 8 microneedles / cm2, or at least about 16 microneedles / cm2.

[0189] , The microneedles may be arranged in pairs, in groups, or as a matrix. A pair arrangement would comprise an even number of microneedles. A group arrangement may comprise between 1 and about 5 groups, with each group comprising between about 4 to about 8 microneedles. A matrix arrangement may comprise either an even or odd number of microneedles as such an arrangement may or may not have the same number of rows and / or columns. In some embodiments, the microneedles are arranged in matrix selected from the group consisting of 2x2, 2x3, 2x4, 2x5, 2x6, 3x23x3, 3x4, 3x5, 3x6, 4x2, 4x3, 4x4, 4x5, 4x6, 5x2, 5x3, 5x4, 5x5, 5x6, 6x2, 6x3, 6x4, 6x5, and 6x6. In any arrangement, the microneedles may be spaced less than about 5 mm, about 4 mm, about 3 mm, about 2 mm, about 1 mm, or about 0.5 mm, and more than about 0.1 mm, from each other. The space may be measured from the centre-to-centre point of each respective microneedle.

[0199]

[0190] , Like the mounting element, the microneedles can be made from any suitable material. For example, the microneedles could be made from or contain woven and non-woven fabrics including electronic fabrics; natural or synthetic fibres; natural or synthetic textiles; silk; organic materials; natural or artificial composite materials, including polymeric materials; glass; ceramics, including polymer ceramics; porous material; polymers such as rigid or semi-rigid plastics and machinable polymers such as such as acrylic, polycarbonate, polyether ether ketone, or PEEK; synthetic polymers such as polymethyl methacrylate or acrylic glass, and other plastics made from methacrylate; thermoplastics and thermosetting plastics such as acrylic resin, polycarbonate, and polyether ether ketone; thermoplastic polymers such as polyethylene terephthalate; doped polymers such as polyacetylene, polypyrrole, polyindole, and polyaniline; intrinsically conducting polymers; metals, including aluminium, copper, gold including colloidal gold, silver including colloidal silver, chromium, platinum, titanium; metal alloys including stainless steel; carbon including colloidal carbon, carbon-nano materials, and carbon composites such as graphene and graphite; semiconductors such as silicon, germanium, and gallium arsenide; doped semiconductors; and, organosilicates. In some embodiments, the microneedles are made from a synthetic polymer such as polymethyl methacrylate.

[0200]

[0191] , The mounting element and microneedles could be fabricated from the same, similar and / or dissimilar materials, and could be integrally formed, or fabricated separately and attached or bonded together. In particular embodiments, the mounting element and microneedles are fabricated from the same material and integrally formed.

[0201]

[0192] . The structure of the mounting element and / or microneedles can be fabricated using any suitable technique. For example, in the case of silicon-based microneedle structures, this could be performed using etching techniques. Polymer or plastic microneedle structures, for example, could be fabricated using additive manufacturing such as 3D printing, or moulding including injection moulding. Solid polymeric microneedles are commonly fabricated from moulding methods such as injection moulding and micro-moulding. Generally, the injection moulding process involves heating a material to its melting temperature Tm and subsequently adjusting the processing temperature in order to inject the material into a mould at a given speed. Although injection moulding equipment can be expensive, it is compatible with large scale manufacturing. UV rapid prototyping is a technology which may also be used to fabricate hollow polymeric microneedle structures. This technique uses computer models to 3D print polymeric builds by guiding light over a photocurable material for selective polymerisation. Once electro-polymerised, the microneedle structure is subject to a cleaning step prior to curing. Microneedle structures may also be fabricated using micro-Computer Numerical Control (CNC) micromachining methods. This fabrication method provides a flexibility in fabrication of the mounting element and shape, spacing, tip geometry, and material(s) selection (e.g., soft metals such as aluminium, 316L stainless steel and copper, machinable polymers such as polymethyl methacrylate, and ceramics) for the microneedles. CNC-based fabrication methods are also highly cost-effective, precisely reproducible (e.g., 1 pm precision), likely to have less residual material, and suitable for automated fabrication.

[0202]

[0193] , Where a support element is provided to support the analyte recognition element (for example, to support a plurality of aptamers), it is considered that the support element is functioning as a conductive element (such as an electrode) per se, or part of conductive element (such as an electrode). In other embodiments, the microneedle is directly coated with analyte recognition element (such as aptamers) and is therefore considered a conductive element (such as an electrode)

[0203]

[0194] , However implemented, an electrode may be a solid or semi-solid electrode, or a surface coated electrode.

[0204]

[0195] , The electrode may be on at least part of an exterior wall of a microneedle. For example, a solid electrode may be a coil around, or a sleeve or a panel on, the exterior wall of a microneedle. In another example, a solid electrode could form the tip of the microneedle. In yet another example, a surface coated electrode may be coated or deposited onto at least part of an exterior wall of the microneedle. A surface coated electrode could also be coated or deposited onto at least part of an interior wall of a hollow microneedle.

[0205]

[0196] , A hollow microneedle could also have an electrode disposed within the hollow interior of a hollow microneedle. With such a structure, the microneedle may further protect the electrode as it breaches the stratum corneum and penetrates the skin layers and / or when the microneedles are in situ (e.g., minimise degradation of the functionality of the recognition element or the effect of contaminants on the ability for the recognition material to detect the produced response signal).

[0206]

[0197] , When disposed within the hollow interior of a hollow microneedle, the electrode may be configured to have various forms. For example, the electrode may partly or completely conform to the shape of the hollow interior of the hollow microneedle. In another example, the electrode may completely fill the hollow interior of the hollow microneedle, and be substantially flush from the opening to the hollow interior. Alternatively, the electrode may partially or substantially fill the hollow interior. In such examples, the electrode may form a recess within the hollow interior of the hollow microneedle. In yet another example, the electrode may partially fill the hollow interior of the hollow microneedle such that the electrode is spaced-apart from the interior wall of the hollow microneedle. In yet another example, the electrode may substantially fill the hollow interior of the hollow microneedle such that there is minimal to no space between the electrode and the interior wall of the hollow microneedle. In preferred embodiments, the electrode substantially fills the hollow interior of the hollow microneedle such that there is minimal to no space between the electrode and the interior wall of the hollow microneedle.

[0207]

[0198] , The electrode will typically comprise a conductive or semi-conductive material capable of exchanging electrons with a redox reporter. The electrode material can be metallic or non-metallic, or a biocompatible conductive material. In some embodiments, the electrode material is selected from the group consisting of intrinsically conducting polymers; metals, including aluminium, copper, gold including colloidal gold, silver including colloidal silver and silver chloride, chromium, platinum, titanium, iridium, rhodium; metal alloys including stainless steel; carbon including colloidal carbon, carbon-nano materials, and carbon composites such as graphene and graphite; semiconductors such as silicon, germanium, gallium arsenide, indium oxide, tin oxide, and mixtures of indium and tin oxides; and, doped semiconductors. The electrode material may also be a porous metal; porous metals are potentially excellent electrode materials because of their high specific surface area. The pores may be of any suitable size as described above. Further, the electrode material may be in the form of a paste with embedded metal wire (e.g., a carbon paste embedded with metal wires), a solid, a semi-solid, or as one or more coating layers.

[0208]

[0199] , While the exposed surface area of an electrode may vary depending upon the intended application of the microneedle-based electrochemical biosensor, the shape of the microneedle, and the placement of the electrode, it should be optimised to maximise the signal-to- noise ratio to improve detection of the produced response signal. An electrode disposed within the hollow interior of a hollow microneedle may, for example, have an exposed surface area of at least 1 mm2, at least 1 pm2, at least 2 pm2, or at least 10 pm2.

[0209]

[0200] . A conductive element, however formed or for whatever purpose may be configured to minimise the rate and / or extent of biological fouling to increase sensor lifetime and minimising sensor drift in vivo. Sensor lifetime and sensor drift may be relevant considerations in the design of a biosensor device for continuous monitoring.

[0210]

[0201] , In vivo stability of electrochemical target detection in a biological fluid may be achieved by applying an effective antifouling layer on one or more electrodes. For example, a polymer layer comprising or consisting of polyvinyl chloride (PVC), p-phenylenediamine (PPD), polyurethane (PU), chitosan and / or a zwitterionic polymer or a combination of any thereof, can be applied to one or more electrodes to minimise the rate and extent of fouling. Polyethylene glycol and oligoethylene glycol molecules are commonly also used to achieve antifouling properties.

[0211]

[0202] , While the electrodes can be fabricated using any suitable technique, it will be appreciated that the electrode fabrication technique will depend on the nature of the microneedlebased electrochemical biosensor and the microneedle arrangement on the mounting element. For example, e-beam evaporation or sputtering can be used to coat an electrode to a surface of a microneedle.

[0212]

[0203] , As another example, an electrode may be disposed within the hollow interior of a hollow microneedle by packing the hollow interior with a conductive material. In yet another example, electrode structures (which may be referred to as an electrode micro pillar or the like) may be fabricated on a substrate, which may be referred to as the electrode substrate, by high precision stereolithography printing, 3D printing, or using a micro-Computer Numerical Control (CNC) micromachining method, which would eliminate the need for packing micron-sized hollow interiors of hollow microneedles. In addition, a CNC-fabrication technique would be a simple, highly cost-effective, precisely reproducible (1 pm precision), electrode fabrication method. The electrode structures may be integrally formed on an electrode substrate, although it is possible that the electrode structures could be fabricated separately and attached or bonded to the electrode substrate. The CNC-fabricated electrode substrate and electrode structures may be initially fabricated from one or more of the same materials as described for the mounting element and / or microneedles. For example, a synthetic polymer such as polymethyl methacrylate.

[0213]

[0204] , Using high-precision techniques enables the electrode structures to be fabricated to substantially conform to the dimensions of the circular hollow section of the hollow microneedles, as well as their positioning on the base, so that the relevant interfaces of the mounting element and the electrode substrate can be accurately coupled together. For example, the electrode has the shape of a circular cylinder, which may or may not have a chamfered edge to provide mechanical strength and stability.

[0214]

[0205] , Connection of a conductive element to an electronics processing unit is preferably stable and robust. This may be achieved by creating, during CNC-fabrication of the electrode substrate, interconnection tracks or channels on a face of the mounting element and apertures to connect each electrically isolated functionalised conductive element, for example by electronic pogo-pins, to an electronics processing unit.

[0215]

[0206] . Techniques such as e-beam evaporation or sputtering can be used to coat the various elements as required, tracks or channels using masking methods. If necessary, post-treatment to confer electrical isolation may be performed to allow for each conductive element to become individually addressable.

[0216]

[0207] , The various elements, interconnection tracks or channels may be coated with one or more conductive materials. The thickness of each coating layer may be between about 10 nm and about 400 nm. Each coating layer may or may not be of the same thickness.

[0217]

[0208] , The material(s) used for the working electrode and the counter electrodes may be different to the electrode material(s) used for the reference electrode. For example, the counter electrode may comprise chromium or titanium (Ti), and the reference electrode may comprise silver at a thickness of about 200 nm. One or more of the electrodes may or may not be coated with AgCl, for example by being externally reacted to Ag / AgCl (e.g., by FeCh 0.1M for one minute). Some non-limiting examples of electrode compositions, are, 20nm Ti, 200nm Au, 20nm Ti, 200nm Au, 20nm Ti, 200nm Au (after rotating the devices - to ensure complete deposition), 40nm Ti, 400nm Au and 20nm Ti, lOOnm Au. When using sputtering to form the electrodes the Ti layer may be optional. An Au layer in the range lOnm to 500nm, more preferably 15nm to 200nm and most preferred 20nm to 50nm is suitable. The respective components of the present apparatus may be coupled together by any suitable means. For example, the components may be attached, joined, or bonded together. In one embodiment, the components are bonded together using a sealant such as a micro-heat shrink sealant, or a photocurable / biocompatible resin. The sealant or resin could also be drop-casted to the base of the electrode and sucked into any gap between the interior wall of the hollow microneedle and electrode by capillary forces following by UV / heat treatment curing to remove any gap. The electrode would then be sealed within the hollow interior of the hollow microneedle. Other methods include the use of solid adhesives laser-cut to size, solid adhesive layers supported on a flexible substrate; laser-cut or cut using machine tools to size, interference fits, O-rings, and gaskets. Examples of suitable machine tools include blades, shear cut tools, male / female punch tools, and crush cut tools.

[0218]

[0209] , It is advantageous in the current disclosure to provide a method by which the area of the at least working electrode exposed to the liquid to be analysed is controlled. There are many methods known in the art, including having a cut-out region in an adhesive layer to define an electrode area, wherein the adhesive layer incorporating the cut-out region is applied over the electrode layer; forming the electrode layer in the presence of a mask, which is subsequently removed to leave a defined area of electrode; forming an electrode layer, coating a portion of the electrode layer to be defined as the electrode area with a releasable masking agent, overcoating with an insulating layer, including coating portions of the electrode layer that it is desired not to be exposed in the final device and then selectively removing the masking layer and any insulating layer overlying it to expose a defined electrode area.

[0219]

[0210] , With regard to the analyte recognition element of the present apparatus, a redoxreporter modified aptamer may be used. A redox-reporter modified aptamer is capable of selectively binding a specific target in a biological fluid of the subject, and upon target binding, producing a target-induced change in current between the redox reporter and the electrode. The target-induced change in current produced between the redox reporter and the electrode may be referred to herein as the response signal.

[0220]

[0211] , One or more aptamer types may be used, allowing for detection of the same or different target analytes at the same time. Further, it is adaptable to be differentially responsive to targets, responsive to different targets, responsive to different combinations of targets, or responsive to different concentrations of targets.

[0221]

[0212] , The aptamer may be of any size that binds to a target analyte, including between about 10 and about 200 nucleotides in length, about 30 and about 200 nucleotides in length, about 50 and about 200 nucleotides in length, about 70 and about 200 nucleotides in length, about 90 and about 200 nucleotides in length, about 100 and about 200 nucleotides in length, about 130 and about 200 nucleotides in length, about 150 and about 200 nucleotides in length, about 170 and about 200 nucleotides in length, about 10 and about 100 nucleotides in length, about 20 and about 100 nucleotides in length, about 30 and about 100 nucleotides in length, about 50 and about 100 nucleotides in length, about 70 and about 100 nucleotides in length, and about 90 and about 100 nucleotides in length. In some embodiments, the aptamer is between about 30 and about 100 nucleotides in length. In other embodiments, the aptamer is between about 10 and about 200 nucleotides in length, or about 30 and about 100 nucleotides in length.

[0213] , The aptamer may be attached or immobilised to the surface of a conductive element. In some embodiments, the aptamer comprises an attachment moiety for attaching or immobilising the aptamer to an electrode surface such as a functional group or compound, preferably via a covalent bond. Suitable attachment moieties for attaching or immobilising the aptamer to the electrode surface include, but are not limited to, a sulphur analogue of an alcohol such as a thiol, an amine, a carboxylic acid, an alcohol, a carbodiimide, a perfluorosulfonic acid polymer such as Nafion, a tetrameric biotin-binding protein such as avidin, a water-soluble vitamin such as vitamin H, an azide, and the like. The skilled person is familiar with means to attach or immobilise the aptamer to the electrode surface, for example by the use of a thiol linkage.

[0222]

[0214] , While the attachment moiety may be directly attached to the aptamer, in some embodiments, the attachment moiety is attached to the aptamer via a linker such as an alkyl chain, including a C1-C20 alkyl, especially a C6 or Cl l alkyl, most especially a C6 alkyl linker (i.e., (CH2)e linker), a polymer such as polyethylene glycol (PEG), or a nucleic acid sequence, including DNA and RNA sequences. In some embodiments, the linker is an alkyl chain such as a C1-C20 alkyl, especially a C6 or Cl l alkyl, most especially a C6 alkyl linker (i.e., (CH2)e linker). In such embodiments, the aptamer may be attached or immobilised to the electrode surface at the 5' terminus. Suitable linkers and synthetic routes for producing linkers are known in the art such as Lai et al (2006) Langmuir 22: 10796-10800, the entire contents of which is incorporated herein by reference.

[0223]

[0215] , In some embodiments, the attached or immobilised aptamer forms a self-assembled monolayer on the electrode surface. The self-assembled monolayer may consist of an aptamer and a diluent molecule that fills spaces between the aptamers on gold surfaces. One useful diluent molecule is 6-mer capto-1 -hexanol (MCH). MCH serves two main functions: (1) it allows a certain amount of aptamers to assemble in a functional orientation for sensing, and (2) it minimizes the contribution of electrochemical currents that do not originate from methylene blue.

[0224]

[0216] , In other embodiments, the attached or immobilised aptamer forms a multilayer on the electrode surface.

[0225]

[0217] , It may be desirable to improve the stability of the aptamer. Several approaches are known in the art, including capping the terminal ends of the aptamer, substituting naturally occurring nucleotides with unnatural nucleotides (e.g., 2'-F, 2'-OCH3, 2'-H, 2'-OH or 2'-NH2 modified nucleotides such as 2'-fluorine-substituted pyrimidines, 2'-amino pyrimidines, and 2'-O- methyl ribose purines and pyrimidines), using unnatural internucleotide linkages such as phosphorothioate, methylphosphonate or triazole linkages, using altered sugar moieties, conjugating a molecule such as biotin to the 3' terminus, 3' terminus capping with inverted thymidine (dT), conjugating protein-like side chains, for example, to the nucleotides such as the 5-position of deoxyuridine (dU) (e.g., 5-(N-benzylcarboxyamide)-2-deoxyuridine), to develop “Spiegelmers”, which are composed entirely of unnatural L-ribonucleic acid backbone, and the like. Eurther approaches are discussed in, for example, Shuaijian et al (2017) Int J Mol Sci 18(8): 1683, the content of which is incorporated herein by reference in its entirety.

[0218] , The aptamer may be modified to increase the sensitivity and binding kinetics of the aptamer for the target of interest. It is noted that one or more of the approaches for improving the stability of an aptamer may have this result, particularly conjugating protein-like side chains e.g., to the nucleotides such as the 5-position of deoxyuridine (dU) (e.g., 5-(N-benzylcarboxyamide)- 2-deoxyuridine). Additional modifications to increase the sensitivity and binding kinetics of the aptamer for the target of interest may be achieved using methods described in Ricci et al (2016) Acc Chem Res, 49(9): 1884-1892, including population shift, allostery, matched receptor sets, sequestration, and cooperativity. Further approaches contemplated by the disclosure may include attaching retaining structures which retain the aptamer in the second configuration to increase the aptamer recovery time, such as complementary primers attached to the ends of the aptamer, which bind together upon target binding to retain the aptamer in the second configuration beyond a recovery interval and at least one blocker bound to the aptamer which prevents the primers from binding together prior to target binding, or functional groups which interact with each other upon target binding to retain the aptamer in the second configuration beyond a recovery interval. Such approaches are discussed in WO2018 / 031559A1, the entire content of which is incorporated herein by reference.

[0226]

[0219] , With reference to the reporter element, a redox moiety may be used. The redox moiety may be a purely organic redox label, an organo-metallic redox label, or a biological redox label. Examples of suitable redox moieties include, but are not limited to, viologen, methylene blue, Nile blue, ferrocene, vinylferrocene, anthraquinone, thionine, ethidium bromide, daunomycin, anthraquinone-C5, dabcyl, 2,6-dichlorophenal-indophenol, gallocyanine, ROX, pentamethylferrocene, ferrocene-C5, neutral red, and horseradish peroxidase, ruthenium, bispyridine, tris-pyridine, bis-imidizole, cytochrome c, plastocyanin, and cytochrome c', and suitable derivatives thereof. In some embodiments, the polynucleotide is modified with a redox moiety selected from the group consisting of methylene blue, Nile blue, ferrocene, and anthraquinone.

[0227]

[0220] , The redox moiety may be attached at any suitable point on the polynucleotide provided that the conformational change which occurs upon target analyte binding to the aptamer alters the accessibility of the redox reporter relative to the conductive element, producing a target- induced change in current between the redox reporter and the electrode on which the aptamer is immobilised to produce a detectable signal response. In some embodiments, the redox moiety is attached at the 3' terminus or 5' terminus of the aptamer. In particular embodiments, the redox moiety is attached at the 3' terminus, and the aptamer is attached to the electrode through the opposite terminus.

[0228]

[0221] , The redox reporter modified aptamer may be applied on a surface or part thereof of the conductive element. For example, a redox reporter modified aptamer could be applied as a coating on a surface of an electrode or incorporated into an electrode material.

[0229]

[0222] , In some embodiments, the redox reporter modified polynucleotide is a coating on a surface of an electrode. The coating may be effected by any suitable technique such as chemisorption, or chemical cross-linking. For example, the technique may include contacting the surface of the electrode with the redox-reporter modified aptamer for a time period sufficient for a moiety for attaching or immobilising the redox-reporter modified aptamer on the surface of the electrode to attach to the surface of the electrode such as via a covalent bond. Suitable, non-limiting methods may include chemisorption of thiolated aptamers on a gold electrode, attachment of biotinylated aptamer to avidin-modified electrode, immobilisation of an azide-ended aptamer to alkyne-modified electrode, covalent immobilisation of amine-ended aptamers by amide coupling to carboxyl groups on the functionalised electrode, covalent immobilisation of amine-ended aptamer to functionalised electrode containing amine groups using glutaraldehyde, and the like. Exemplary methods are described in Xiao et al (2007) Nat Protocols 2(11): 2875-2880; Negahdary et al (2018) J Biomed Phys Eng 8(2): 167-178; and Mishra et al (2018) Biosensors 8(2): 28.

[0230]

[0223] , The number and / or density of polynucleotides may depend on the target of interest

[0231] (including target size and expected levels or concentration to be detected), application of the present apparatus, and the detection method. The density should result in a detectable response signal upon target binding. In some embodiments, the density is between about IxlO10and about IxlO14molecules / cm2, about 5xl010and about 5xlO13molecules / cm2, about IxlO11and about IxlO13molecules / cm2, about 5xl0nand about 5xl012molecules / cm2(and all integers therebetween).

[0232]

[0224] , When the redox-reporter modified polynucleotide interacts or binds with a target of interest, the aptamer undergoes a conformational change. For example, the polynucleotide has a first conformation in the absence of target binding and a second conformation upon target binding. In the second conformation, a portion of the polynucleotide (e.g., a first terminus of the aptamer such as the 3' or 5' terminus) may be closer to the electrode than in the first confirmation (i.e., the spacing between the portion of the aptamer and the electrode is decreased in the second conformation). Alternatively, in the second confirmation, a portion of the polynucleotide may be further from the electrode than in the first conformation (i.e., the spacing between the portion of the polynucleotide and the electrode is increased in the second confirmation). In another example, the conformational change from the first to the second may enable the redox moiety to become more flexible or accessible, allowing it to diffuse, or otherwise move towards the electrode. The change in proximity of the redox moiety to the electrode produces a distinguishable redox signal.

[0233]

[0225] , The analyte recognition element of the present apparatus need not be polynucleotide, and may be a protein, such as a peptide, and enzyme or an antibody.

[0234]

[0226] , Moreover, the apparatus may have multiple different types of analyte recognition element to allow for the detection of multiple different types of target analyte concomitantly, in a multiplexing manner.

[0235]

[0227] , The conformational change of the analyte recognition element upon target binding may be detected by different methods. In preferred embodiments, the method of detection is an electrochemical detection technique such as potentiometry, cyclic voltammetry, fast scan cyclic voltammetry, square wave voltammetry, or chronoamperometry, which are suitable detection mechanisms for detecting changes in electrochemical properties. In particular embodiments, the method of detection is potentiometry, square wave voltammetry, or chronoamperometry.

[0228] , A potentiometric measurement technique is one where the open circuit potential of the electrochemical cell is directly measured. This potential is measured between the reference electrode and the working electrode. This is contrasted with amperometric measurements that measure current while controlling the cell potential, for example.

[0236]

[0229] , Chronoamperometry is a powerful tool for measuring diffusion-controlled reactions. In chronoamperometry the potential is stepped at the beginning of the experiment and then remains constant throughout the duration of the measurement. The current that results from this stimulus is plotted as a function of time.

[0237]

[0230] , Voltammetry techniques vary the potential as a function of time. The resulting current is plotted as a function of potential. For example, cyclic voltammetry sweeps the potential of the cell linearly across a potential range, while fast scan cyclic voltammetry does this at a faster rate. Square wave voltammetry uses a square wave superimposed over a staircase function to provide a sweeping measurement that provides 2 sampling instances per potential. As a result of this sampling technique, the contribution to the total current that results from Faradaic currents is minimised. Like cyclic voltammetry, the current is plotted as a function of potential.

[0238]

[0231] , A sensor of the present disclosure is useful for real-time, continuous in vivo target detection in a biological fluid.

[0239]

[0232] , Measurements of target analyte may be captured by voltammetric interrogation of the working electrode to assess sensing. Interrogation can be by any voltammetric method, including cyclic voltammetry, differential pulse voltammetry, alternating current voltammetry, square wave voltammetry, potentiometry or amperometry, as known in the art. Waveforms may be selected as known in the art. For voltammetry, each scan is performed across a range of potentials which results in distinct signals being generated from the sensing redox reporter and reference redox reporter. Scans can be performed at discreet time intervals or substantially continuously over the measurement period. In some embodiments, the measurement period may be every second, every minute, every 5 to 10 minutes, or hourly.

[0240]

[0233] , The target analyte for the present apparatus is configured to detect may be a low molecular weight organic compound such as a metabolite. In some embodiments, the metabolite is a primary metabolite. Non-limiting examples include vitamins, amino acids (e.g., phenylalanine), nucleosides, organic acids, carbohydrates, and proteins. In other embodiments, the metabolite is a secondary metabolite, e.g., alkaloids and creatinine.

[0241]

[0234] , In some embodiments, the target is a protein. The protein may be selected from the group consisting of enzymes (e.g., amylase, creatinine kinase, lactate dehydrogenase, angiotensin II converting enzyme), hormones (e.g., insulin and thyroxine), cystatin C, albumin, globulin, C- reactive protein, TNFa, IL-6, ICAM1, TLR2, TLR4, presepsin, D-dimer, viral proteins, bacterial proteins, parasitic proteins, antibodies (including antibodies produced in response to an infection, such as a bacterial or viral infection including an influenza infection), botulinum toxin.

[0242]

[0235] , The target may also be a glycoprotein. The glycoprotein may be an N-linked glycoprotein, O-linked glycoprotein, or non-enzymatic glycosylated glycoprotein.

[0236] . In some embodiments, the target is a peptide or polypeptide. Non-limiting examples of which include peptide hormones, neuropeptides (e.g., oxytocin) and cytokines (e.g., IL-6, IL-1, TNF-a, and interferon). In some embodiments, the target may be a cytokine storm.

[0243]

[0237] , In some embodiments, the target is a hormone, including steroid hormones.

[0244] Examples of non-limiting hormones include dopamine, testosterone, estrogen, progesterone, cortisol, and cortisone.

[0245]

[0238] , The target may also be a growth factor, including vitamins, hormones, and platelet- derived growth factors.

[0246]

[0239] , In other embodiments, the target is a complex organic substance, including nucleic acids (DNA and RNA), nucleosides, nucleotides, polynucleotides, and oligonucleotides.

[0247]

[0240] , The target may also be selected from monosaccharides, polysaccharides, and oligosaccharides.

[0248]

[0241] , The target may be a virus or virus-like particle, bacteria or bacterium, parasite, pesticide.

[0249]

[0242] , In some embodiments, the target may be a nutrient. Nutrients include glucose, amino acids, vitamins, minerals, and fatty acids.

[0250]

[0243] , The target may be an ion or electrolytes. Non-limiting examples include sodium, potassium, bicarbonate, chloride, and calcium.

[0251]

[0244] , In some embodiments, the target is a large molecule drug or biologic such as insulin. In other embodiments, the target is a therapeutic drug, especially a small molecule therapeutic drug, which may be an administered or ingested pharmaceutical or medicament. Examples of therapeutic drugs included, but are not limited to, antibiotics such as piperacillin, penicillin, cefaclor, cefodizime, cefpirome, cefpodoxime, ceftobiprole, ciprofloxacin, fleroxacin, gemifloxacin, moxifloxacin, doripenem, ertapenem, imipenem, linezolid, terezolid, azithromycin, clarithromycin, dirithromycin, erythromycin, telithromycin, fosfomycin, tigecycline, daptomycin, and metranidazole, glycopeptide antibiotics such as bleomycin, complestatin, corbomycin, dalbavancin, decaplanin, oritavancin, ramoplanin, teicoplanin, telavancin, and vancomycin, aminoglycoside antibiotics such as amikacin, tobramycin, kanamycin, streptomycin, and gentamicin, antiepileptics or anticonvulsants such as carbamazepine, valproic acid, phenobarbital, and phenytoin, mood stabilisers such as pimozide and clozapine, small molecule tyrosine kinase inhibitors, anticancer drugs such as doxorubicin, 6-thioguanine, paclitaxel, docetaxel, camptothecin, megestrol acetate, navelbine, cytarabine, fludarabine, 6-mercaptopurine, 5- fluorouracil, teniposide, vinblastine, vincristine, colchicine, carboplatin, procarbazine, and etopside, TNF inhibitors, antifungal agents, antiretroviral agents, psychiatric drugs, antidepressants, antipsychotics and antianxiety drugs such as including alprazolam, amoxapine, bentazepam, bromazepam, clorazipine, clobazam, clotiazepam, diazepam, lorazepam, flunitrazepam, flurazepam, lormetazepam, medazepam, nitrazepam, oxazepam, temazepam, maprotiline, mianserin, nortriptyline, risperidone, sertraline, trazodone, haloperidol, trimipramine maleate fluoxetine, ondansetron, midazolam, chlorpromazine, haloperidol, triazolam, clozapine, fluopromazine, fluphenazine decanoate, fluanisone, perphenazine, pimozide, prochlorperazine, sulpiride, thioridazine, paroxitine, citalopram, bupropion, phenelzine, olanzapine, divalproex sodium and venlafaxine, immunosuppressants such as tacrolimus, cyclosporine, and mycophenolate mofetil, chemotherapy drugs such as carboplatin, cisplatin, methotrexate, doxorubicin, and irinotecan, phosphodiesterase inhibitors such as theophylline, cardiac glycosides such as digoxin, digitoxin, adenosine triphosphate (ATP), opioids such as opioid receptor agonists and antagonists, compounds which exhibit mixed agonist / antagonist activity and compounds which exhibit partial agonist activity, including morphine, depomorphine, etorphine, 6- acetylmorphine, diacetylmorphine, hydromorphone, oxymorphone, levorphanol, methadone, levomethadyl, meperidine, fentanyl, sufentanyl, alfentanil, codeine, hydrocodone, oxycodone, thebaine, desomorphine, nicomorphine, dipropanoylmorphine, benzylmorphine, ethylmorphine, pethidine, tramadol, dextropropoxyphene; naloxone and naltrexone; buprenorphine, nalbuphine, butorphanol, pentazocine, and ethylketocyclazocine.

[0252]

[0245] , In yet other embodiments, the target is an illicit drug (e.g., amphetamine, meth amphetamine, cocaine or procaine, heroin, and cannabinoids) or non-illicit drug such as a prescription drug taken for non-medical reasons. It may also be a non-illicit substance of abuse such as alcohol.

[0253]

[0246] , In further embodiments, the target is a poison, carcinogen, or toxin including environmental contaminants.

[0254]

[0247] , The target could also be selected from allergens, infection agents, and biohazards.

[0255]

[0248] , In some embodiments, the target is a cytotoxic agent. Non-limiting examples include amsacrine, bexarotene, bortezomib, carboplatin, cetuximab, cisplatin, dacarbazine, docetaxel, hydroxycarbamide (hydroxyurea), irinotecan, oxaliplatin, paclitaxel, pentostatin, procarbazine, temozolomide, topotecan, trastuzumab, and tretinoin.

[0256]

[0249] , The target may be a biomarker or other species indicative of a condition (e.g., drug abuse), disease (e.g., infectious diseases), disorder (e.g., neurological disorders), a physiological or pathologic process that occurs in a subject (e.g., drug metabolism), or a normal or an abnormal process taking place in a subject, which may be a sign of an underlying condition or disease. Nonlimiting examples of biomarkers which may be indicative of disease risk and / or disease severity include azathioprine, benzoylecgonine, branched chain amino acids, bulsulfan, caffeine, carboplatin, cathinones, chloramphenicol, desipramine, doxepin, ecgonine, eslicarbazepine acetate, ethanol, ethosuximide, ethyl glucuronide, felbamate, fentanyl, flecainide, homocysteine, imipramine, irinotecan, ketones, lacosamide, lactic acid, lamotrigine, levetiracetam, levodopa, lidocane, lignocaine, lipids, mda, mdma, methotrexate, metranidazole, mexilitine, mycophenolate acid, mycophenolate mofetil, n-acetyl-d- glucosamine, n-acetyl-procainamide, neomycin, netilmicin, nortriptyline, oxcarbazepine, paromomycin, pdgf, perhexiline, phenytoin, plazomycin, pregabalin, procainamide, propofol, quinidine, rufinamide, salicylate, sirolimus, sodium valproate, stiripentol, telithromycin, theophylline, tiagabine, topiramate, tygercycline, urocanic acid, valproic acid, vigabatrin, zonisamide, A9-THC, amiodarone, amitriptyline, cholesterol (e.g., LDL cholesterol), creatinine, glycogen, lithium, phosphate, procalcitonin, triglycerides, troponin, warfarin, urea, and uric acid.

[0250] , The present sensor is particularly suitable for use as a wearable device, allowing measurements to be performed whilst the subject is undergoing normal activities and / or over a prolonged period of time. The wearable device may be a collar, a bracelet or other suitable jewellery piece, a watch, a garment, a strap, an adhesive, or a patch. A person skilled in the art would appreciate that means may be provided to assist adhering and / or securing the wearable device, when in use, to a subject, e.g., micro-anchors, or the like.

[0257]

[0251] , The wearable device may comprise a housing structure comprising one or more other components, such as electronics processing unit. The electronics processing unit is configured to be in direct or indirect electrical communication with at least one conductive element, and generally will include any one or more of a power source, a data processing unit, an analog front-end, and a wireless transmitter.

[0258]

[0252] , The housing structure may be configured to encase, at least partially, the apparatus, where the conductive elements (such as microneedles) are exposed from a plane of the housing structure. The microneedles may be protected by a protective cover, which may be removed to expose the protruding microneedles before use.

[0259]

[0253] , The device may further comprise means for monitoring temperature or pH of the biological fluid where validity of an output is dependent thereon, or where adjustment to operation or output is possible.

[0260]

[0254] , The housing structure may be configured to encase and be coupled to the apparatus by any appropriate mechanism. For example, electromagnetic coupling, mechanical coupling, adhesive coupling, magnetic coupling, or the like. In some embodiments, the coupling mechanism enables the apparatus and the housing structure to be attached and detached, which would enable the housing structure and its other components to be re-usable, while the apparatus can be discarded and replaced with another apparatus as necessary.

[0261]

[0255] . The wearable device may further comprise a computer program product executable as a software application, resident on a mobile communication device in communication with the electronics processing unit, wherein the computer program product is able to control one or more of (i) detection of electrochemical measurements conducted at the microneedle-based platform, (ii) data analysis, (iii) data transmission, (iv) device configuration, and (v) device power management. Examples of suitable mobile communication devices include, but are not limited to, smartphones, smartwatches, tablets, smartglasses, laptops or other personal computers.

[0262]

[0256] , In some embodiments, the apparatus itself comprises a processor with program instructions configured to drive onboard functions such as voltammetry, and transmitting output to a remote device via a wireless module, such as a Bluetooth™ module.

[0263]

[0257] , The apparatus of the present disclosure may be particularly suitable for therapeutic drug monitoring (TDM). TDM is typically performed regularly at single or fixed time points to maintain drug concentrations within a narrow therapeutic window in a subject’s bloodstream. As the microneedle-based electrochemical biosensor is capable of real-time, continuous detection of a target such as a therapeutic drug, the measurements captured by the microneedle-based electrochemical biosensor can allow clinicians to adjust and maintain optimised drug dosing within the therapeutic window in response to real-time concentration changes. Further, as the microneedle-based electrochemical biosensor can be configured to perform measurements over a prolonged period, it enables variations in a subject’s condition over time to be taken into account, providing a clear advantage over single-point measurements, which may not be indicative of the actual condition of a subject.

[0264]

[0258] , To further describe the present disclosure in its various aspects, reference is made to the following non-limiting Examples.

[0265]

[0259] , The present disclosure will now be more fully described by reference to the following non-limiting examples.

[0266] EXAMPLE 1: Production of a working electrode by an additive electroplating method (soft gold with old strike layer), and testing thereof.

[0267] Materials

[0268]

[0260] . Electro-cleaner solution bath and brush application, TriVai 24k acid gold strike pretreatment and 24K pure gold bath solution (99%) acquired from gold plating services (Layton , UT, USA). Sulfuric acid 316 stainless steel needles. Sulfuric acid, ACS reagents (95-98%). Commercial Pt / Ti mesh acquired from Hele Titanium Technology Co., Ltd (China). Printed circuit boards. Shenzen JBD Technology Co., Ltd (China). Gold plate was coated onto stainless steel acupuncture needles.

[0269] Electrochemical set-up

[0270]

[0261] , For all experiments, the set-up contained five wells each one having two dedicated

[0271] Pt / Ti anodes. 16 needles were placed on each PCB board and a total of five PCBs were connected to a 3D-printed lid, providing 80 needles plated per batch. Needles were submerged in the gold plating solution. Alligator clips were used to connect anode and cathode. A power supply model Rigol DP932E was used in all electroplating steps. Aluminium hot plates were used to adjust solution temperature. The power supply was controlled by custom developed code running on Spyder. Reference is made to FIG. 1A, FIG. IB and FIG. 1C.

[0272] Gold electroplating at different applied potentials

[0273]

[0262] , All solutions were pre-heated at 30°C for about 15 minutes before to start the experiment. After temperature stabilization, the needles were first positioned at the activation solution and a potential of 5V was applied for 30 seconds with a current cut-off of 1.875 mA / needle. After activation, needles were submerged in deionized water for about 2 minutes and moved to activation step when a potential of -1.8V was applied for 50 seconds. Subsequently, the same washing step was applied before gold plating. For gold plating at different potentials, the potential was adjusted in the code to interface with power supply using three different conditions: condition 1 at -1.2V; condition 2 at -1.7V and condition 3: at -2.0V. For all three conditions plating time was constant and fixed at 900 seconds. In the experimental design, a 2-electrode Jig was used and the potential applied on the electrochemical cell is the relative potential against Pt / Ti anode. Vancomycin-sensitive aptamers with methylene blue redox reporter were thiol-linked to the gold surface by an art-accepted method. Gold electroplating at different temperatures

[0274]

[0263] , Activation and gold strike solution were pre-heated at 30°C for about 15 minutes before to start the experiment. The same procedure was used for gold plating solution varying the temperature at two different conditions: 1) at 30°C and 2) 45°C. After temperature stabilization, the needles were first positioned at the activation solution and a potential of 5V was applied for 30 seconds with a current cut-off of 1.875 mA / needle. After activation, needles were submerged in deionized water for about 2 minutes and moved to activation step when a potential of -1.8V was applied for 50 seconds. Subsequently, the same washing step was applied before gold plating. Gold plating was performed at different temperatures fixing the potential and plating time at -1.3V and 600 seconds, respectively.

[0275] Results

[0276]

[0264] , FIG. 2 shows the effects of increasing electroplating voltage (temperature being maintained at a constant 30°C -). At -1.2V (FIG, 2A) the soft gold plate appears shiny to the eye. At -1.7V (FIG. 2B) some surface structures have formed, which manifest as a dulled surface. At -2.0V (FIG. 2C), significant roughening of the gold surface is seen indicating further formation of surface structures.

[0277]

[0265] , FIG. 3 shows the effect of increasing temperature of the electroplating solution (voltage being maintained at a constant -1.3 V over 600 s). At 45°C (FIG. 3B) roughening is more pronounced than for 30°C (FIG. 3A).

[0278]

[0266] , Without wishing to be limited by theory in any way, it is proposed that increasing voltage and temperature each independently increase the rate of gold deposition. An increased rate of gold deposition may favour the formation of protective surface structures on the electrode surface.

[0279]

[0267] , FIG. 4B shows surface structures on the surface of an electrode plated with soft gold under conditions favouring roughening of the surface. These structures are not present on a smooth gold surface (FIG. 4A). The structures shown in FIG. 4B are generally granular and are proposed to provide protection to aptamers that are linked to the working electrode surface. As will be appreciated from FIG. 4B and FIG. 4C, the structures appear to be discretely formed. The structures form confined spaces, with aptamer disposed in the confined spaces being protected from contact by tissues and fluids contacted by the electrode surface as it passes through dermal tissues in the course of being introduced into a subject.

[0280]

[0268] , The peripheral regions of a structure provide an overhang, with a confined space being formed beneath the overhang. Aptamer extending from the underside of an overhang, or extending from any other surface an into the confined space formed by the overhang may be less prone to damage given that subject tissues are excluded from entering the confined spaces.

[0281]

[0269] , Moreover, a confined space may be formed laterally to a structure where the structure is laterally proximal to another structure.

[0282]

[0270] . A further type of confined space is formed by the stacking arrangement of structures as is evident from FIG. 4B and FIG. 4C. The stacking arrangement forms tunnels running generally toward the inner regions of the electrode.

[0271] , The diameter of over 700 of the structures shown in FIG. 4C have been determined and plotted as a histogram (FIG. 5). A majority of the structures have a diameter in the range of 0.478 microns to 1.828 microns.

[0283]

[0272] , A majority of the structures have a diameter in any range defined having a lower value of any one of about 0.328, 0.478, 0.628, 0.778, 0.928, 1.078, 1.228, 1.378, or 1.528, 1.678 or 1.828, each of the lower values being independently selected with an upper value of any one of about 1.978, 1.828, 1,678, 1,528, 1.378, 1.228, 1.078, 0.928, 0.778 Or 0.628.

[0284]

[0273] , Expressed alternatively, the data reveals that the majority of structures have a diameter of 1.06 + 0.42 microns.

[0285]

[0274] , As used herein, the term “majority” may mean a proportion of greater than about

[0286] 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0287]

[0275] , Reference is made to FIG. 4D a possible cross-sectional structure of a working electrode formed from a substrate, a smooth gold layer formed from closely packed regular shaped grains (10) with an outer rough layer formed from irregularly shaped grains (20). Aptamers (not drawn) extend from the surfaces of the grains (20) near the surface.

[0288]

[0276] , The arrows in FIG. 4E indicates spaces formed by the irregularly shaped and loosely packed grains near the surface. The spaces are surrounded by the grains (20) with overhangs protecting any aptamer within a space from damage arising from insertion of the electrode through a tissue (such as skin) of an animal subject. The tissue will contact the outwardly facing surfaces of the uppermost grains (20), although will be unable to enter the arrowed spaces. Aptamers in the spaces will therefore avoid contact with the animal subject tissue and remain functional. A more regularly packed grain layer will not provide any spaces similar to those arrowed, and therefore provide less protection for aptamers.

[0289]

[0277] , The more uniform smooth layer formed from grains (10) function to substantially completely cover the underlying electrode substrate thereby avoiding exposure of the substrate. Background signal is therefore reduced, as discussed elsewhere herein. The strike layer also provides a surface encouraging close bonding of the overlying rough layer formed from grains (20).

[0290]

[0278] , The differences in the level of aptamer loss (and therefore aptamer protection) between smooth and rough electrode surfaces have been demonstrated experimentally. Working electrodes having either a smooth soft gold electrode surface, or a rough soft gold electrode surface were functionalised with a vancomycin sensitive aptamer. Each type of working electrode was inserted into pig skin and then extracted therefrom- .The sensors were then interrogated before and after pig skin insertion with square wave voltammetry at 100 Hz, with an amplitude of 35 mV and a 1 mV potential step in PBS lx containing 0.7 mM MgCh at 37 °C. .

[0291]

[0279] . Reference is made to FIG 5 A showing a voltage sweep of the smooth gold electrode before (blue line) and after (red line) insertion into pig skin. Only 11% of the vancomycin signal is retained after passage through pig skin.

[0280] , Reference is made to FIG 5B showing a voltage sweep of the rough gold electrode before (blue line) and after (red line) insertion into deceased pig skin. 82% of the vancomycin signal is retained after passage through pig skin.

[0292]

[0281] , These results are interpreted thus. A smooth gold electrode surface provides no or few protective structures, and accordingly aptamer is subjected to adverse influences as the electrode passes through the pig skin. Aptamer may be sheared off the electrode surface or otherwise damaged, leading to a significant reduction in vancomycin-specific signal when the electrode is place into the standard vancomycin solution.

[0293]

[0282] , By contrast, the rough gold surface provides protection to the aptamers, and therefore after passage of the electrode through the pig skin the vast majority of aptamers remain on the electrode surface and are able to function to detect vancomycin.

[0294] EXAMPLE 2: Production of a working electrode by an additive method (gold strike, smooth gold intervening layer, outer rough gold layer), and testing thereof.

[0295]

[0283] , A working electrode was produced generally in accordance with the method of

[0296] Example 1, with the exception of a different solution composition for activation, gold strike and gold solutions. The gold strike layer was produced by electroplating, similar to Example 1.

[0297]

[0284] , Materials: self-made electro-cleaner (also called activation solution bath), acid gold strike pre-treatment and gold bath solution (99%). Activation solution composition: 0.8 w.t.% NaOH + 2 w.t.% Na2CCh in MilliQ water. Gold strike composition: 0.1 w.t.% KAu(CN)4 in 0.25 M H2SO4. Internal soft gold solution composition: 0.4 w.t.% KAu(CN)2 in oxalate buffer. Sulfuric acid 316 stainless steel needles. Sulfuric acid, ACS reagents (95-98%). Commercial Pt / Ti mesh acquired from Hele Titanium Technology Co., Ltd (China). Printed circuit boards. Shenzen JBD Technology Co., Ltd (China). Gold plate was coated onto stainless steel acupuncture needles.

[0298]

[0285] , All solutions were pre-heated at 30°C for about 15 minutes before to start the experiment. After temperature stabilization, the needles were first positioned at the activation solution and a potential of 5V was applied for 30 seconds. After activation, needles were submerged in deionized water for about 2 minutes and moved to activation step when a potential of -2.1V was applied for 200 seconds. Subsequently, the same washing step was applied before gold plating. For smooth gold plating, a potential of -1.4V for 1200 seconds was applied. For the formation of rough gold, a potential of -1.9V was applied for 900 seconds. Similarly to example 1, a 2-el ectrode Jig was used and the potential applied on the electrochemical cell is the relative potential against Pt / Ti anode. Vancomycin-sensitive aptamers with methylene blue redox reporter were thiol-linked to the gold surface by an art-accepted method.

[0299] EXAMPLE 3: Comparative example for production of a working electrode by a subtractive method (roughening of smooth gold surface by acid etching), and testing thereof.

[0300]

[0286] , The surface of a gold wire was roughened electrochemically in 0.5 M sulfuric acid by alternating the potential of the electrode between Emitiai = 0.0 V to Ehigh = 2.0 V (versus Ag / AgCl), back and forth, for 16 000 pulses. Each pulse was 20 ms long. The roughed gold wire was functionalised with a vancomycin-sensitive aptamers having a methylene blue redox reporter by thiol bonding to the roughened surface and passivation.

[0301]

[0287] . Deceased pig skin was pierced with a hypodermic needle to form an aperture. After removal of the needle, the aperture naturally closed over due to the elastic resilience of the skin. The functionalised gold wire was inserted into and then withdrawn from the closed over aperture. Sensitivity to vancomycin was assessed before insertion of the wire, after insertion or the wire, and after removal of the wire. The vancomycin was provided as a solution in artificial interstitial fluid with 2 mg / mL BSA and 2mg / mL globulin. Interrogation was by square wave voltammetry (300 Hz, 25 mV amplitude).

[0302]

[0288] , Reference is made to the voltammograms of FIG. 6A and FIG. 6B representing replicate experiments. In both cases, loss of sensitivity to vancomycin is noted as a result of passage of the wire through the pig skin. Given that the skin had already been pierced by a hypodermic needle, greater losses would be expected if the working electrode was rigid and forced through uncompromised skin.

[0303]

[0289] , In any event, the loss is sensitivity for the working electrode roughened by a subtractive process (FIG. 6A and FIG. 6B) is significantly higher than where the electrode is formed rough de novo by way of an additive process (FIG. 5A and FIG. 5B). This comparative result may be interpreted thus. The additive process formed protective structures on the working electrode surface which function to shield the aptamers from damage as the electrode is passed through the pig skin. The subtractive process failed to form protective structures (or formed significantly lower numbers of protective structures, or formed structures that were protective, but less protective) as compared with the additive process.

[0304]

[0290] , 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.

[0305]

[0291] , 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 working electrode for an electrochemical sensor, the working electrode having a plurality of analyte recognition elements associated therewith, the working electrode comprising surface structures configured to protect the plurality of analyte recognition elements from an adverse influence arising from (i) passage of the working electrode through a tissue of an animal subject, or (ii) disposition of the working electrode in a tissue or a fluid of an animal subject.

2. The working electrode of claim 1, wherein the passage or the disposition of the working electrode through a tissue of an animal subject results in a contact-mediated or pressure-mediated disruption of the plurality of analyte recognition elements.

3. The working electrode of claim 1 or claim 2, wherein the surface structures provide or contribute to confined spaces adjacent to a surface of the working electrode, the surface having the plurality of analyte recognition elements associated therewith, the plurality of analyte recognition elements extending into the confined spaces.

4. The working electrode of any one of claims 1 to 3, wherein the surface structures form overhangs facing generally toward the interior or the electrode, with the plurality of analyte recognition elements extending from the overhangs.

5. The working electrode of any one of claims 1 to 4, wherein the surface structures form an enclosure having an opening, with the plurality of analyte recognition elements extending into the enclosure.

6. The working electrode of any one of claims 1 to 5, wherein the surface structures are generally granular structures.

7. The working electrode of any one of claims 1 to 6, wherein the surface structures are discrete structures.

8. The working electrode of any one of claims 1 to 7, wherein the surface structures are formed fully, predominantly, or in part by an additive process.

9. The working electrode of claim 8, wherein the additive process is a metal deposition process.

10. The working electrode of claim 9, wherein the metal deposition process is an electroplating process.

11. The working electrode of claim 10, wherein the metal deposition process is an electroplating process comprising electrodeposition of a metal coating onto a substrate metal.

12. The working electrode of claim 11, wherein the metal coating comprises gold, or a gold alloy.

13. The working electrode of claim 11 or claim 12, wherein the metal coating is deposited under conditions facilitating the formation of a rough surface or a non-smooth surface.

14. The working electrode of claim 13, wherein the roughness or the non-smoothness of the surface is discernible as such by the unaided human eye or by an analytical method.

15. The working electrode of claim 13 or claim 14, wherein the roughness or non-smoothness is at least about 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% that of a gold ingot, a minted gold bar, a Good Delivery gold bar, or native gold.

18. The working electrode of any one of claims 1 to 17 comprising a substantially continuous intervening layer disposed between a metal substrate of the working electrode and an outer layer forming the surface structures.

19. The working electrode of any one of claims 1 to 18, that is a needle, a microneedle or a wire.

20. A method of producing a working electrode for an electrochemical sensor, the method comprising forming surface structures on the working electrode surface, and associating a plurality of analyte recognition elements with the electrode surface, wherein the surface structures configured to protect the plurality of analyte recognition elements from an adverse influence arising from passage of the working electrode through a tissue of an animal subject, or disposition of the working electrode in a tissue of an animal subject.

21. The method of claim 20, wherein the surface structures are formed fully, predominantly, or in part by an additive process.

22. The method of claim 21, wherein the additive process is a metal deposition process.

23. The method of claim 22, wherein the metal deposition process is an electroplating process.

24. The method of any one of claims 20 to 23, wherein the surface structures are not formed by a subtractive process.

25. The method of any one of claims 20 to 24, wherein the subtractive process is a metal removal process.

26. The method of claim 25, wherein the metal removal process comprises etching, abrading, scratching, ablating, cutting, or shearing.

27. The method of claim 23, wherein the metal deposition process is an electroplating process comprising electrodeposition of a metal coating onto a substrate metal.

28. The method of claim 27, wherein the metal coating comprises gold, or a gold alloy.

29. The method of claim 27 or claim 28, wherein the metal coating is deposited under conditions facilitating the formation of a rough surface or a non-smooth surface.

30. The method of claim 29, wherein the deposition is controlled so as to provide a surface having a required roughness of non-smoothness by control of any one or more of the following electroplating parameters: rate of metal deposition, temperature, magnitude of applied electrical potential, duty cycle of applied electrical current, electrical current, electrical current density, deposition time, bath composition including concentration of the metal ion to be deposited, pH, degree of agitation, distance between electrodes, position of electrodes.

31. The method of claim 30, wherein the applied electrical potential and / or temperature is / are controlled so as to control the roughness or non-smoothness of the working electrode surface.

32. The method of claim 31, wherein the magnitude of the applied potential is about 1.7V or greater, and the temperature is about 45°C or greater.

33. The working electrode of any one of claims 20 to 32 comprising forming a substantially continuous intervening layer between a metal substrate of the working electrode and an outer layer forming the surface structures.

34. A working electrode for an electrochemical sensor produced by the method of any one of claims 20 to 33.

35. An electrochemical sensor comprising the working electrode of any one of claims 1 to 19 or claim 34.

36. A method for determining an amount of a target analyte in a tissue or a fluid of a subject animal, the method comprising contacting the working electrode of any one of claims 1 to 19 or claim 34 to the tissue or the fluid of the subject animal by passing the working electrode through a tissue of the subject animal, or disposing the working electrode in a tissue of the subject animal subject.

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