WO2025107019A1

WO2025107019A1 - Sensor having an extended dynamic range

Info

Publication number: WO2025107019A1
Application number: PCT/AU2024/051222
Authority: WO
Inventors: Robert Batchelor; Alastair Hodges; Julian GERSON; Kaylyn LEUNG; Tod KIPPIN; Kevin Plaxco
Original assignee: Nutromics Technology Pty Ltd; University of California Berkeley; University of California San Diego UCSD
Current assignee: Nutromics Technology Pty Ltd; University of California Berkeley; University of California San Diego UCSD
Priority date: 2023-11-22
Filing date: 2024-11-18
Publication date: 2025-05-30
Anticipated expiration: 2026-05-22

Abstract

A working electrode for use with an electrochemical analyte recognition element-based sensor. The working electrode has associated analyte recognition elements specific for a target analyte. The recognition elements including a first population allowing for quantitation of the target analyte across a first concentration range, and a second population allowing for quantification of the target analyte across a second concentration range. The first and second concentration ranges are different.

Description

SENSOR HAVING AN EXTENDED DYNAMIC RANGE

STATEMENT OF FEDERALLY SPONSORED RESEARCH

[001]. This invention was made with government support under EB022015 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD

[002]. The present disclosure relates generally to electrochemical aptamer-based (EAB) sensors useful for the detection of a target analyte. The sensor may be used, for example, to detect target analyte in a test specimen, in situ within the body of an animal, or in the environment. In particular, the sensor comprises a working electrode having an extended dynamic range with regard to the target analyte.

BACKGROUND

[003]. EAB sensors show significant promise in human health, animal health, and other fields. These sensors have been demonstrated to be highly selective and capable of the detection of target analytes including pharmaceutical compounds, toxins, metabolites, proteins, hormones, industrial process intermediates, and environment contaminants.

[004]. In health-related applications, an EAB sensor may be embodied in the form of a microneedle-based patch applied to skin. The microneedle is coated with a redox-modified aptamer to a working electrode which is inserted into the skin so as to contact a biological fluid. The tip of the microneedle functions as the sensor electrode, with the binding element being associated with the tip. This arrangement provides a minimally invasive platform for real-time, continuous in vivo target analyte detection. Alternatively, the working electrode may be a wire that is inserted into a subcutaneous tissue, or even into a blood vessel.

[005]. EAB sensors may also be used to detect target analyte in a test specimen comprising a fluid that has been removed from its normal environment. Such applications include an in vitro clinical specimen and a water specimen removed from a waste water stream.

[006]. An electrochemical sensor typically comprises a working electrode being coated in a binding element (such as an aptamer) that undergoes a conformational change upon analyte binding. A redox reporter (such as methylene blue) may be covalently linked to the binding element. The conformational change in the binding element alters the accessibility of the redox reporter to the electrode surface, thereby producing an analyte-induced change in the amount or rate of electron transport between the redox reporter and the electrode. In some circumstances, binding of the analyte enhances the ability of the redox reporter to move proximal to the electrode surface, thereby increasing the level of electron transport and in turn increasing current through the electrode. In other circumstances, binding reduces the ability of the redox reporter to move proximal to the electrode surface resulting in the opposite effects. Regardless, binding of the analyte results in a detectable change in electrochemical signal.

[007]. Electrochemical sensors are typically interrogated by the application of an electrical potential or potential waveform across the working electrode and a counter electrode, and then measuring current flow produced in response.

[008]. For some applications, the sensor is required to detect target analyte across very wide concentration ranges. As one example, clinically relevant levels of some therapeutic drugs span a broad range of concentrations and, in the context of therapeutic drug monitoring, using an EAB sensor, it is necessary for the sensor to accurately report all concentrations within that range. For therapeutic drug monitoring of the antibiotic vancomycin, quantification of trough concentrations below 1 p M, and also peak (i.e., C_max) concentrations of millimolar levels is desired.

[009]. Many EAB sensors are able to quantify high levels of target analyte without saturating; however, at the same time, insufficiently sensitive to very low levels of target analyte. Conversely, EAB sensors able to detect very low levels of analyte become easily saturated, thereby precluding utility in quantitating higher analyte levels.

[010]. Prior artisans have shown that sensitivity of an EAB sensor may be modulated by actively heating and cooling the working electrode of an EAB sensor. The need to introduce temperature -control increases the complexity and cost of an EAB sensor, whilst also providing a potential point of malfunction. Moreover, heating requires the input of large amounts of electrical energy which will significantly drain the battery of a portable EAB sensor, such as that incorporated into a wearable device.

[Oi l]. It is an aspect of the present disclosure to provide an improvement to prior art EAB sensors to extend the dynamic range for a target analyte. It is a further aspect of the present disclosure to provide a useful alternative to prior art EAB sensors. [012]. The discussion of documents, acts, materials, devices, articles, and the like, is included in this specification solely for the purpose of providing a context for the present disclosure. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

SUMMARY

[013]. In a first aspect, but not necessarily the broadest aspect, the present disclosure provides one or more working electrodes for use with an electrochemical analyte recognition element-based sensor, the one or more working electrodes having associated therewith a plurality of analyte recognition elements specific for a target analyte, the plurality of analyte recognition elements comprising a first population of analyte recognition elements allowing for quantitation of the target analyte across a first concentration range, and a second population of analyte recognition elements allowing for quantification of the target analyte across a second concentration range, the first concentration range being different to the second concentration range.

[014]. In one embodiment of the first aspect, the analyte recognition elements are independently selected from: an aptamer, an XNA (incorporating non-naturally occurring nucleotides), such as a PNA (peptide nucleic acid, such as N-(20aminoethyl)-glycine peptides), or hybrid polymer including a hybrid species comprised of two or more monomer types.

[015]. In one embodiment of the first aspect, wherein the first concentration range is defined as the range between a lower chosen fraction of the upper saturation limit and an upper chosen fraction of the upper saturation limit for the first population of analyte recognition elements, the second concentration range is defined as the range between the lower chosen fraction of the upper saturation limit and a chosen upper fraction of the upper saturation limit for the second population of analyte recognition elements.

[016]. In one embodiment of the first aspect, the first and second concentration ranges are discrete, abutting, or overlapping.

[017]. In one embodiment of the first aspect, the midpoint of the first concentration range is lower than the midpoint of the second concentration range. [018]. In one embodiment of the first aspect, the first and second populations of analyte recognition elements respond differently to the same change in concentration of target analyte, with one of the populations having a different relative response to the change than the other population.

[019]. In one embodiment of the first aspect, the different relative response to the change is a greater or lesser change in a measured electrochemical output.

[020]. In one embodiment of the first aspect, the plurality of analyte recognition elements comprise a third, a fourth or more analyte recognition element populations, each of which allows for improved quantification of the target analyte concentration across a third, a fourth, or more concentration ranges.

[021]. In one embodiment of the first aspect, the second, third, fourth or more populations of analyte recognition elements allow for improved quantitation of the target analyte across a wider range of concentrations compared with that allowed for by the first population of analyte recognition elements.

[022]. In one embodiment of the first aspect, each analyte recognition element of the plurality of analyte recognition elements is associated with a single working electrode.

[023]. In one embodiment of the first aspect, the analyte recognition elements of the plurality of analyte recognition elements are distributed across two or more working electrodes, a single recognition element species being associated with one or the other of the two or more working electrodes.

[024]. In one embodiment of the first aspect, the two or more electrodes are mutually connected, or are mutually connectable, or are mounted on a single mounting portion.

[025]. In one embodiment of the first aspect, the analyte recognition elements of each of the analyte recognition element populations are associated exclusively with a dedicated working electrode associated with a dedicated electrical connection. According to this embodiment each working electrode can be separately interrogated by allowing connection between a measuring circuit and the dedicated electrical connection for that working electrode. Which working electrode connection is chosen as being the one that indicates the analyte concentration, can be based upon chosen criteria applied to the electrical signal arising from one or more of the plurality of working electrodes. [026]. In one embodiment of the first aspect, each of the analyte recognition element populations comprises respectively a analyte recognition element species having a different binding affinity for the target analyte.

[027]. In one embodiment of the first aspect, each respective analyte recognition element species has a different base sequence composition and/or length.

[028]. In one embodiment of the first aspect, the plurality of analyte recognition elements were contacted with the one or more working electrodes by co-deposition onto a surface of the working electrode.

[029]. In one embodiment of the first aspect, each of the first, second, third, fourth or more analyte recognition element populations are co-deposited such that each analyte recognition element species is present in substantially equal numbers.

[030]. In one embodiment of the first aspect, a substantially equal number is +/- about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of a mathematically equal number.

[031]. In one embodiment of the first aspect, each of the first, second, third, fourth or more analyte recognition element populations are co-deposited such that at least one analyte recognition element species is present in substantially different numbers to the other analyte recognition elements.

[032]. In one embodiment of the first aspect, each analyte recognition element of the plurality of analyte recognition elements is connected at or about a first end thereof to a surface of the one or more working electrodes.

[033]. In one embodiment of the first aspect, the connection comprises one or more covalent bonds.

[034]. In one embodiment of the first aspect, the connection consists of, or comprises, a thiol linkage.

[035]. In one embodiment of the first aspect, each analyte recognition element of the plurality of analyte recognition elements is associated with a redox reporter species.

[036]. In one embodiment of the first aspect, the redox reporter species is covalently connected to each analyte recognition element.

[037]. In one embodiment of the first aspect, the redox reporter species is methylene blue or a functional equivalent thereof. [038]. In one embodiment of the first aspect, different redox reporter species with different

E° values are each associated with a different analyte recognition element population.

[039]. In one embodiment of the first aspect, the redox reporter species is connected to each analyte recognition element of the plurality of analyte recognition elements at or about a second end thereof.

[040]. In one embodiment of the first aspect, the target analyte is an inorganic species, a small molecule, an organic molecule, a therapeutic drug molecule or a metabolite thereof, or a molecule endogenous to an animal.

[041]. In one embodiment of the first aspect, the therapeutic drug molecule is vancomycin or another glycopeptide antibiotic.

[042]. In one embodiment of the first aspect, each analyte recognition element of the first population of analyte recognition elements is the analyte recognition element 4-Trunc, and each analyte recognition element of the second population of analyte recognition elements is the analyte recognition element 3-Trunc.

[043]. In one embodiment of the first aspect, the molecule endogenous to an animal is creatinine.

[044]. In one embodiment of the first aspect, each analyte recognition element of the first population of analyte recognition elements is the analyte recognition element Cre 1GC, and each analyte recognition element of the second population of analyte recognition elements is the analyte recognition element Cre OG.

[045]. In one embodiment of the first aspect, each analyte recognition element of the plurality of analyte recognition elements is capable of detecting the target analyte selectively amongst one, more than one, or all non-target analytes present in a biological fluid.

[046]. In one embodiment of the first aspect, the biological fluid is selected from interstitial fluid, blood, saliva, a lacrimal secretion, a lactational secretion, a nasal secretion, a tracheal secretion, a bronchial secretion, an alveolar secretion, a gastric secretion, a gastric content, a glandular secretion, a vaginal secretion, a uterine secretion, a prostate secretion, semen, urine, sweat, cerebrospinal fluid, a glomerular filtrate, a hepatic secretion, bile, and an exudate. [047]. In one embodiment of the first aspect, the biological fluid is interstitial fluid or blood present in situ in the body of an animal.

[048]. In one embodiment of the first aspect, each of the one or more working electrodes is a wire, a needle, or a microneedle.

[049]. In a second aspect, the present disclosure provides a method for producing a working electrode for use in an electrochemical analyte recognition element-based sensor, the method comprising the steps of: providing one or more electrodes; providing a first population of analyte recognition elements allowing for quantitation of the target analyte across a first concentration range, and a second population of analyte recognition elements allowing for quantification of the target analyte across a second concentration range, the first concentration range being different to the second concentration range; and contacting the one or more electrodes with the first and second populations of analyte recognition elements under conditions allowing the analyte recognition elements of the first and second populations to associate with a surface of the one of more electrodes. [050]. In one embodiment of the second aspect, the analyte recognition elements are independently selected from: an aptamer, a XNA (incorporating non-naturally occurring nucleotides), such as a PNA (peptide nucleic acid), a hybrid polymer including a hybrid species comprised of two or more monomer types.

[051]. In one embodiment of the second aspect, wherein the first concentration range is defined as the range between a lower chosen fraction of the upper saturation limit and an upper chosen fraction of the upper saturation limit for the first population of analyte recognition elements, the second concentration range is defined as the range between the lower chosen fraction of the upper saturation limit and a chosen upper fraction of the upper saturation limit for the second population of analyte recognition elements.

[052]. In one embodiment of the second aspect, the first and second concentration ranges are discrete, abutting, or overlapping.

[053]. In one embodiment of the second aspect, the midpoint of the first concentration range is lower than the midpoint of the second concentration range. [054]. In one embodiment of the second aspect, upon exposure to a fixed concentration of target analyte and application of an interrogating potential waveform, the first population of analyte recognition elements provides a different relative response to that of the second population of analyte recognition elements.

[055]. In one embodiment of the first aspect, the different relative response is a greater or lesser change in a measured electrochemical output.

[056]. In one embodiment of the second aspect, the plurality of analyte recognition elements comprise a third, a fourth or more analyte recognition element populations, each of which allows for improved quantification of the target analyte across a third, a fourth, or more concentration ranges.

[057]. In one embodiment of the second aspect, the second, third, fourth or more populations of analyte recognition elements allow for improved quantitation of the target analyte across a wider range of concentrations compared with that allowed for by the first population of analyte recognition elements.

[058]. In one embodiment of the second aspect, each analyte recognition element of the plurality of analyte recognition elements is associated with a single electrode.

[059]. In one embodiment of the second aspect, the analyte recognition elements of the plurality of analyte recognition elements are distributed across two or more electrodes, a single analyte recognition element species being associated with one or the other of the two or more working electrodes.

[060]. In one embodiment of the second aspect, the method comprises connecting together two or more electrodes, or mounting two or more electrodes on a single mounting portion.

[061]. In one embodiment of the second aspect, the analyte recognition elements of each of the analyte recognition element populations are contacted exclusively with a dedicated electrode associated with a dedicated electrical connection. According to this embodiment each working electrode can be separately interrogated by allowing connection between a measuring circuit and the dedicated electrical connection for that working electrode. Which working electrode connection is chosen as being the one that indicates the analyte concentration, can be based upon chosen criteria applied to the electrical signal arising from one or more of the plurality of working electrodes. [062]. In one embodiment of the second aspect, each of the analyte recognition element populations comprises respectively a analyte recognition element species having a different binding affinity for the target analyte.

[063]. In one embodiment of the second aspect, each respective analyte recognition element has a different base sequence composition and/or length.

[064]. In one embodiment of the second aspect, the plurality of analyte recognition elements are contacted with the electrode contemporaneously.

[065]. In one embodiment of the second aspect, the plurality of analyte recognition elements are contacted with the one or more electrodes by a co-deposition method.

[066]. In one embodiment of the second aspect, each of the first, second, third, fourth or more analyte recognition element populations are co-deposited such that each analyte recognition element species is present in substantially equal numbers.

[067]. In one embodiment of the second aspect, a substantially equal number is +/- about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of a mathematically equal number.

[068]. In one embodiment of the second aspect, each of the first, second, third, fourth or more analyte recognition element populations are co-deposited such that at least one analyte recognition element species is present in substantially different numbers to the other analyte recognition elements.

[069]. In one embodiment of the second aspect, the method comprises connecting each analyte recognition element of the plurality of analyte recognition elements at or about a first end thereof to a surface of the one or more electrodes.

[070]. In one embodiment of the second aspect, the connecting comprises forming one or more covalent bonds.

[071]. In one embodiment of the second aspect, the connecting consists of, or comprises, forming a thiol linkage.

[072]. In one embodiment of the second aspect, the method comprises associating each analyte recognition element of the plurality of analyte recognition elements with a redox reporter species.

[073]. In one embodiment of the second aspect, the method comprises covalently connecting the redox reporter species to each analyte recognition element. [074]. In one embodiment of the second aspect, the redox reporter species is methylene blue or a functional equivalent thereof.

[075]. In one embodiment of the second aspect, different redox reporter species with different E° values are each associated with a different analyte recognition element population.

[076]. In one embodiment of the second aspect, the method comprises connecting the redox reporter species to each analyte recognition element of the plurality of analyte recognition elements at or about a second end thereof.

[077]. In one embodiment of the second aspect, the target analyte is an inorganic species, a small molecule, an organic molecule, a therapeutic drug molecule or a metabolite thereof, or a molecule endogenous to an animal.

[078]. In one embodiment of the second aspect, the therapeutic drug molecule is vancomycin or another glycopeptide antibiotic.

[079]. In one embodiment of the second aspect, each analyte recognition element of the first population of analyte recognition elements is the analyte recognition element 4-Trunc, and each analyte recognition element of the second population of analyte recognition elements is the analyte recognition element 3-Trunc.

[080]. In one embodiment of the second aspect, the molecule endogenous to an animal is creatinine.

[081]. In one embodiment of the second aspect, each analyte recognition element of the first population of analyte recognition elements is the analyte recognition element Cre 1GC, and each analyte recognition element of the second population of analyte recognition elements is the analyte recognition element Cre OG.

[082]. In one embodiment of the second aspect, each analyte recognition element of the plurality of analyte recognition elements is capable of detecting the target analyte selectively amongst one, more than one, or all non-target analytes present in a biological fluid.

[083]. In one embodiment of the second aspect, the biological fluid is selected from interstitial fluid, blood, saliva, a lacrimal secretion, a lactational secretion, a nasal secretion, a tracheal secretion, a bronchial secretion, an alveolar secretion, a gastric secretion, a gastric content, a glandular secretion, a vaginal secretion, a uterine secretion, a prostate secretion, semen, urine, sweat, cerebrospinal fluid, a glomerular filtrate, a hepatic secretion, bile, and an exudate.

[084]. In one embodiment of the second aspect, the biological fluid is interstitial fluid or blood present in situ in the body of an animal.

[085]. In one embodiment of the second aspect, the one or more electrodes is each a wire, a needle, or a microneedle.

[086]. In a third aspect, the present disclosure provides an electrochemical sensor comprising the one or more working electrodes of any embodiment of the first aspect.

[087]. In one embodiment of the third aspect, the sensor is an electrochemical aptamerbased sensor.

[088]. In one embodiment of the third aspect, the sensor further comprises a reference electrode.

[089]. In one embodiment of the third aspect, the sensor further comprises a reference electrode and a power supply.

[090]. In a fourth aspect, the present disclosure provides a method for determining the concentration of a target analyte in a fluid, the method comprising the step of contacting the one or more working electrodes of any embodiment of the first aspect to the fluid.

[091]. In one embodiment of the fourth aspect, the fluid is a biological fluid.

[092]. In one embodiment of the fourth aspect, step of contacting the one or more electrodes to the biological fluid is performed in vivo, ex vivo or in vitro.

[093]. In one embodiment of the fourth aspect, the biological fluid is selected from interstitial fluid, blood, saliva, a lacrimal secretion, a lactational secretion, a nasal secretion, a tracheal secretion, a bronchial secretion, an alveolar secretion, a gastric secretion, a gastric content, a glandular secretion, a vaginal secretion, a uterine secretion, a prostate secretion, semen, urine, sweat, cerebrospinal fluid, a glomerular filtrate, a hepatic secretion, bile, and an exudate.

[094]. In one embodiment of the fourth aspect, the target analyte is an inorganic species, a small molecule, an organic molecule, a therapeutic drug molecule or a metabolite thereof, or a molecule endogenous to an animal.

[095]. In one embodiment of the fourth aspect, the therapeutic drug molecule is vancomycin or another glycopeptide antibiotic. [096]. In one embodiment of the fourth aspect, the method is a therapeutic drug monitoring method.

BRIEF DESCRIPTION OF THE FIGURES

[097]. FIGS. 1A to ID illustrate highly diagrammatically various optional spatial arrangements for first and second aptamer species on the surface of one or more electrodes. FIG. 1A shows an arrangement where the first and second aptamer species are mutually intermingled on a single electrode. FIG. IB shows an arrangement where the first and second aptamer species are arranged in two discrete groups on a single electrode. FIG. 1C shows an arrangement where the first and second aptamer species are arranged as discrete groups, the discrete groups being mutually intermingled on a single electrode. FIG. ID shows two discrete electrodes that are mounted on a mounting portion. One of the electrodes is coated exclusively with the first aptamer species, and the other of the electrodes is coated exclusively with the second aptamer species.

[098] . FIG. 2 is a graph showing the relationship between kinetic differential measurement (KDM) signal change and vancomycin concentration for a working electrode coated with 4-Trunc aptamer (red), a working electrode coated with 3-Trunc aptamer (blue), and a working electrode coated with a 1: 1 mixture of 4-Trunc and 3-Trunc aptamers (purple).

[099]. FIG. 3 is a graph showing the relationship between KDM signal change and creatinine concentration for a working electrode coated with Cre 1GC aptamer (blue), a working electrode coated with Cre OG aptamer (red), and a working electrode coated with a 1: 1 mixture of Cre 1GC and Cre OG aptamers (purple).

[100]. FIG. 4 illustrates an upper perspective view of a microneedle embedding apparatus of the present disclosure. The embodiment relies on the user to provide the motive force for insertion of the microneedles into the skin. The arm is shown in the first position as it is presented to the user, and before embedment of the microneedles in the skin.

[101]. FIG. 5A illustrates a lower perspective view of the embodiment of FIG. 4.

[102]. FIG. 5B illustrates an upper perspective view of the embodiment of FIG. 4.

[103]. FIG. 6 illustrates a lower perspective view of the embodiment of FIG. 4 more completely showing the removable flexible layer that is removed to expose the dermatologically acceptable adhesive. [104]. FIG. 7 illustrates, in lower perspective view, the microneedle embedding apparatus of FIG. 6 having the removable flexible layer removed to expose the dermatologically acceptable adhesive.

[105]. FIG. 8 illustrates, in lower perspective view, the microneedle embedding apparatus of FIG. 7 with the microneedles in an extended position, as required for embedment in the skin of a subject.

[106]. 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.

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

DETAILED DESCRIPTION OF THE DISCLOSURE AND PREFERRED EMBODIMENTS THEREOF

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

[109]. 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.

[110]. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. [111]. The present inventors have discovered, through the use of EAB sensors, that plasma concentrations of a target analyte, which may be an inorganic species, a small molecule, an organic molecule, a therapeutic drug molecule or a metabolite thereof, or a molecule endogenous to an animal, may span a large numerical range. EAB sensors are capable of continuous real-time monitoring of such target analytes in the blood or other bodily fluid, allowing for the measurement of the true peak concentration. Conventionally, blood samples are drawn from a subject at intervals and assayed for the molecule in a laboratory. As will be appreciated, the peak concentration (C_max) in a concentration curve will typically occur between sampling times and will therefore be missed. For example, continuous drug measurements have shown that the concentration range between a trough drug concentration and a peak drug concentration may be sufficiently large so as to extend beyond the useful dynamic range of a conventional EAB sensor. Although the problem of insufficient dynamic range in EAB sensors has arisen in the context of therapeutic drug monitoring, the present disclosure is in no way limited to such applications.

[112]. In one aspect, the present disclosure provides one or more working electrodes for use with an electrochemical analyte recognition element-based sensor, the one or more working electrodes having associated therewith a plurality of analyte recognition elements specific for a target analyte, the plurality of analyte recognition elements comprising a first population of analyte recognition elements allowing for quantitation of the target analyte across a first concentration range, and a second population of analyte recognition elements allowing for quantification of the target analyte across a second concentration range, the first concentration range being different to the second concentration range. The analyte recognition elements will typically be aptamers (such as DNA and RNA aptamers), but other elements such as PNA or another XNA and, indeed, other biological or organic molecules are contemplated to be useful.

[113]. It has been found that a single working electrode can be formed by association with two or more different aptamer species that bind the same target but with differing affinities. Each aptamer may be attached to the same working electrode/gold surface, and each having a methylene blue molecule covalently bonded at the free terminal end. Each of the aptamer species possess a different useful dynamic range, and accordingly the working electrode as a whole has an extended useful dynamic range. These embodiments have the advantage of simplicity of the resulting device as they only require a single working electrode to achieve an extended concentration range. However, these embodiments may degrade the accuracy of the sensing response, as part of the signal that is measured at any analyte concentration can be non-responsive or minimally responsive to changes in analyte concentration. This issue may be overcome by using redox reporter species with different E° values associated with the different aptamer species, such that the electrical signals arising from the different aptamer species can be measured separately by adjusting the potential applied to the working electrode.

[114]. An alternative approach to increasing the dynamic concentration range of a sensing device is to associate binding elements with different affinities each to their unique working electrode, or plurality of working electrodes, where the current arising from the working electrode or plurality of working electrodes associated with the same binding element affinity type can be measured separately. According to this embodiment, dynamic concentration range of the device can be extended without the accuracy of the sensing response being degraded due to mixing of highly concentration-responsive signals with non-responsive or minimally responsive signals. This embodiment may require more working electrodes to cover a concentration range than alternative embodiments. The embodiment chosen for a particular application will be dependent upon such factors as the analyte concentration range that is required to be covered, the ease and cost of incorporating multiple working electrodes in the device, the access to multiple suitable redox reporter species, the intrinsic sensitivity of the analyte recognition elements to changes in the concentration of the analyte and the required accuracy of the sensing response.

[115]. In this approach it is desirable to be able to choose which working electrode signal would optimally quantify the analyte concentration in the fluid that is being measured. In one embodiment this choice is made by determining the signals from the different working electrodes and comparing them to the expected maximum or minimum signal expected from that electrode when it is saturated by the analyte. The electrode chosen may be the one where the signal produced is within a pre -determined fractional range of the expected maximum or minimum signal. For example, the factional range may be within the range 0% to 90%, 10% to 80%, 20% to 70%, 20% to 60% of the expected maximum or minimum signal, or other suitable fraction ranges, based upon the expected sensitivity of the signal response to changes in concentration of analyte at different points in the possible range of signals.

[116]. Reference is made to FIG. 1, showing various non -limiting ways in which two different aptamer species (6a or 6b) may be spatially arranged on a single electrode (FIG. 1A, FIG. IB, FIG. 1C) or across two electrodes (FIG. ID). In each case, the aptamer (6a or 6b) is connected to the electrode (2) on its surface by art-conventional means. Similarly, each aptamer (6a or 6b) is modified to have a redox reporter (8) at its free terminus. The arrangement shown in FIG. 1A will likely be the most easily implemented given that it does not require any higher level of aptamer organisation, given that aptamers (6a and 6b) are co-deposited and therefore randomly distributed across the electrode surface (4). As will be appreciated, third, fourth and more different aptamer species can be arranged according to basic schemas drawn at FIG. 1A, FIG. IB, and FIG. 1C.

[117]. FIG. ID shows an arrangement whereby two electrodes (2a and 2b) are used, each of which is exclusively coated on its surface (4a or 4b) with one species of aptamer only (6a or 6b). The electrodes (2a and 2b) are connected together with a mounting portion (9).

[118]. Reference is made to FIG. 2 showing the result of depositing two vancomycin sensitive aptamer species (4-Trunc and 3-Trunc) to form a single working electrode having an extended dynamic range, as described further described in the Examples herein.

[119]. As an alternative to the use of a single electrode, a first aptamer species may be associated with a first electrode, and a second aptamer species (having a different affinity for the target analyte compared with the first aptamer species) is associated with a second electrode (as shown in the schema of FIG. ID), the first and second electrodes being incorporated into a single EAB sensor. Such a sensor will have two working electrodes, with the currents through each electrode being informative of different concentration ranges of vancomycin. Overall, the sensor will possess a greater dynamic range than a sensor having a working electrode coated with the first or second aptamer species.

[120]. As will be appreciated, if the dynamic ranges of the two aptamer species are sufficiently far apart, a non-responsive concentration region between two responsive regions may result. A non-responsive region may not be detrimental in some applications, such as in therapeutic drug monitoring. In that application, there is some clinical importance attached to a peak drug concentration attained after or during drug administration, and also a trough concentration (i.e., a minimum concentration arising after the peak caused by metabolism or clearance of the drug from the body). For applications requiring a continuous dynamic range, a third (or more) aptamer species may be implemented to cover any non-responsive concentration range.

[121]. As will be appreciated, the present disclosure requires first and second aptamer species, and in one embodiment the first aptamer species has a higher affinity for the target analyte than the second (or vice versa). Exemplary methods for identifying aptamers useful in that context are discussed below.

[122]. Aptamers specific for a target analyte are typically selected from a combinatorial library having a vast number (of order 10¹⁴ or larger) of different oligonucleotides.

[123]. Selection of an aptamer that is selective for a given target analyte may be facilitated by a repetitive process known as SELEX (Systematic Evolution of Ligands by Exponential enrichment). The SELEX process may be considered as three successive stages within a single ‘round’ of SELEX. In the first stage, the oligonucleotide library is presented to the molecular target analyte. Those oligonucleotides that interact with the molecular target are physically separated from those oligonucleotides that do not interact with the molecular target. Separation of oligonucleotides with higher affinity for the target analyte and removal of unbound oligonucleotides are achieved through intense competition for binding sites. The selection stringency rises with every SELEX round. In the second stage, the DNA oligonucleotides that interact with the target are amplified by a polymerase chain reaction (PCR) to the desired concentration. For the selection of RNA aptamers, the singlechained oligoribonucleotides are first reverse transcribed into single-stranded DNA oligonucleotides, which are then amplified by PCR. generated by. In the third stage, for DNA aptamers, a pool of single oligodeoxyribonucleotide strands (although intra-chain hybridization may cause double-stranded regions in a single strand) is generated by strand separation of the double-stranded PCR products. The unwanted complementary DNA strand is removed from the DNA aptamer pool. For RNA aptamers, the single-stranded RNA pool is generated by in vitro transcription of double-stranded DNA PCR product with T7 RNA-polymerase. For both DNA and RNA aptamer pools the single-stranded products are used as the input for the next round of SELEX. [124]. Maximum enrichment of the oligonucleotide pool with aptamers with the strongest affinity for the target molecule is usually achieved after 5 to 15 SELEX rounds.

[125]. As discussed elsewhere herein, the two aptamer species possessing different dynamic ranges for an analyte may have different affinities for the analyte. Obtaining relatively high and relatively low affinity aptamers in the context of the SELEX process (and similar process) may be achieved by modifications to the process. Capture steps in SELEX may be modified to alter stringency, with higher stringencies favoring the capture of high affinity aptamers, and low stringencies favoring low affinity aptamers.

[126]. As will be appreciated, the stringency of capture in a SELEX method may be adjusted by any one or more of the following means: molecular target concentration, the presence of competing molecules of similar structure, incubation temperature, incubation time, ionic strength, detergent concentration, pH, and the like. Details of SELEX methods and means for adjusting stringency are found in Kohlberger et al; Biotechnol Appl Biochem. 2022 Oct; 69(5): 1771-1792, published online 2021 Sep 3. doi: 10. 1002/bab. 2244.

[127]. Alternatives to the identification of aptamers useful in the context of the present disclosure may be used. For example, an existing aptamer species having a useful dynamic range at low concentrations may be modified to provide a second aptamer species having a useful dynamic range at higher concentrations. Modifications such as substitutions, deletions and insertions may be made randomly or rationally to the first aptamer species, with the resultant (second) modified aptamer being tested for dynamic range that is different to that of the first. Where the second aptamer species is found to possess a dynamic range that is different to that of the first, both aptamer species may be incorporated into a sensor (optionally deposited on the same working electrode) to provide a sensor having a desired useful dynamic range.

[128]. As a further alternative, one or more aptamer species may be generated randomly or rationally from single nucleotides, and/or nucleic acid fragments (2-bases, 3-bases, 4- bases etc.). Testing of resultant aptamers to determine dynamic range may be implemented as described above.

[129]. As yet a further alternative, aptamer species that are known, and are characterised or tested to demonstrate different dynamic ranges may be used. [130]. The present disclosure will be more fully described by reference to the following non-limiting examples.

EXAMPLE 1: In vitro demonstration of extended dynamic range working electrode having two vancomycin-specific aptamer species deposited thereon

DNA Aptamer Pair 4-Trimc and 3-Trunc

[131]. This experiment demonstrates that vancomycin-sensitive aptamer species (4-Trunc and 3-Trunc) differ in affinity for vancomycin, and when used together in an EAB sensor function to increase the dynamic range.

[132]. The following aptamer sequences were used in this experiment:

[133]. The 4-Trunc aptamer has a higher affinity for vancomycin that does the 3-Trunc aptamer.

[134]. EAB sensors were fabricated in general accordance established protocols for the attachment of single aptamer species to the surface of a working electrode. Briefly, segments of bare gold wire (200 pm diameter) were cut (5 cm in length), and the insulated body of the wires coated using two layers of heat-shrink polyolefin tubing. To facilitate connection with the potentiostat, a gold pin was soldered to one end of the electrode and this contact was further coated with insulating connector paint (MG Chemicals, Burlington, ON, Canada). Finally, the uninsulated end of the electrodes was cut to a final length of 6 mm prior to electrochemical cleaning with the following protocol: (1) 999 cycles between -1 and -1. 8 V in a solution of 0.5 M NaOH at IVs^-1 to remove any residual thiol/organic contaminants on the electrode surface.

[135]. Working electrodes were functionalized with thiol-modified 4-Trunc, thiol-modified

3-Trunc, and a 1: 1 mixture of thiol-modified 4-Trunc / thiol-modified 3-Trunc). The DNA aptamers (each at a concentration of 100 pM) were reduced by treatment at 1 h in a solution of 10 mM TCEP at room temperature in the dark. Each was then dissolved in phosphate buffered saline (PBS) to a final concentration of 500 nM and then combined together in a 1: 1 solution to achieve a solution with 250 nM of each DNA aptamer. The electrochemically cleaned gold wire electrodes were then immersed in 400 pL of this solution for 1 h in the dark. Following this, the electrode surface was incubated overnight in the PBS containing 10 mM 6-mercaptohexanol at room temperature in the dark.

[136]. In vitro testing of the functionalized working electrodes was conducted against a range of vancomycin concentrations. More specifically, a series of solutions across a range of vancomycin concentrations (0 to 10^-3M) were prepared. Each of the working electrodes were incorporated into a sensor circuit having a counter electrode and a reference electrode, and immersed into each of the series of solutions. For each solution, each working electrode was interrogated by square wave voltammetry. As appreciated by the skilled person, square wave voltammetry is a voltammetric technique for measuring Faradaic current that ignores as far as possible current arising from non-Faradaic processes. For interrogation, a square potential waveform is applied to a working electrode and current measured at or toward the end of each forward and reverse potential pulse after allowing time for non-Faradaic currents to decay exponentially. KDM values are determined, the values being proportional to the concentration of target analyte about the working electrode.

[137]. Reference is made to FIG. 2 showing the response of each electrode (in terms of fractional increase in KDM) to increasing concentrations of vancomycin.

[138]. The curve for the 1:1 mixture of both aptamer species deposited on the same electrode (purple) spans the combined dynamic range of both aptamers 4-Trunc and 3- Trunc. Combining both aptamers on the same working electrode thus expands the overall dynamic range of the sensor.

EXAMPLE 2: In vitro demonstration of extended dynamic range working electrode having two creatinine-specific aptamer species deposited thereon

DNA Aptamer Pair Cre OG and Cre 1GC

[139]. This experiment demonstrates that depositing two aptamers (Cre OG and Cre 1GC) on a single electrode can effectively increase the dynamic range of an EAB sensor in vitro. The aptamer sequences used are as follows:

[140]. EAB sensors sensitive to creatinine, with a single working electrode coated with both

Cre OG and Cre 1GC, were fabricated generally in accordance with the protocol described at Example 1 herein.

[141]. A series of solutions across a range of creatinine concentrations (IO^-6 to 10^-3M) were prepared. Three working electrodes were prepared (Cre OG alone, Cre 1GC alone, and a 1: 1 mixture of Cre OG and Cre 1GC), each electrode being incorporated into a sensor circuit having a counter electrode and a reference electrode, and immersed into each of the series of solutions. For each solution, each working electrode was interrogated by square wave voltammetry. KDM values were determined, the values being proportional to the concentration of target analyte about the working electrode.

[142]. Reference is made to FIG. 3 showing KDM signal change (expressed as a percentage) for each of the three sensor types: relatively high affinity for creatinine (Cre 1GC aptamer, blue), relatively low affinity for vancomycin (Cre OG, red) and mixed (Cre OG and Cre 1GC, purple) for each creatinine concentration tested. The curve for the 1: 1 mixture of both aptamer species deposited on the same electrode (purple) spans the combined dynamic range of both aptamers Cre OG and Cre 1GC. Combining both aptamers on the same working electrode thus expands the overall dynamic range of the sensor.

[143]. The one or more working electrodes of the present disclosure may be implemented in the form of a wearable EAB sensor. EAB sensors typically incorporate a circuit having a working electrode and a reference electrode. The reference electrode is the site of a known chemical reaction that has a known redox potential. For example, a reference electrode based on the silver-silver chloride (Ag|AgCl) redox pair has a fixed and known potential forming the point against which the redox potential of the working electrode is measured. Also typically included in the circuit is a counter electrode which functions as a cathode or an anode to the working electrode. Because the applied voltage bias does not pass through the reference electrode (due to an impedance of the potentiostat), any potential generated is attributed to the working electrode. Current is measured as potential of the interrogating electrode versus the stable potential of the reference electrode. The difference in potential produces the current in the circuit thereby generating an output signal. The resulting signal change is ideally monotonically related to target binding, thus enabling the ready quantification of the target from the signal.

[144]. The sensor may have the electrodes configured as microneedles, one of which is coated with two or more redox modified aptamer species thereby functions as a working electrode having an extended dynamic range. This arrangement provides a minimally invasive platform for real-time, continuous in vivo drug detection, which is sufficiently sensitive and selective for monitoring the concentration of the drug in the body of a subject over time.

[145]. Aptamers and needles may be exploited together in the form of an EAB biosensor, whereby an aptamer-loaded needle is inserted through the skin so as to contact a biological fluid. The needle functions essentially as a working electrode which detects analyte in the biological fluid. Typically, a second needle is used as a counter electrode, with a third needle functions as a reference electrode.

[146]. The working electrode (and any other electrodes) described herein may be configured as microneedles and incorporated into a wearable sensor apparatus, and exemplary type being shown in FIG. 4, FIG. 5A, FIG. 5B, FIG. 6, FIG. 7, and FIG. 8.

[147]. The apparatus comprises an upper housing portion (25) and a skin contacting portion (30). Also provided is a removable flexible layer (90) being graspable by way of the tab (95), the removal of which exposes a dermatologically acceptable adhesive on the skin contacting surface (35). The adhesive is for the purpose of retaining the apparatus on the subject’s skin for an extended period. The flexible layer (90) functions to prevent curing or drying of the adhesive, prevent contamination of the adhesive layer before use and/or premature attachment of the adhesive to packaging, or to other surfaces. In a particularly preferred embodiment, in addition to covering the adhesive layer, the flexible layer (90) extends over the spaces (45) to prevent contamination of the microneedles (15) and also help prevent unintended needle-stick injuries to a user. [148]. The apparatus may have a retaining portion functioning to retain the apparatus on the skin such that the projecting portions remain in contact with a biological fluid of the subject. The retaining portion may be dedicated to that function, or may perform another function.

[149]. In many circumstances, a retaining portion being or comprising a dermatologically acceptable adhesive will be useful. Adhesives allow for simplicity in application of the apparatus by a user, often requiring only the removal of a protective backing sheet to expose the adhesive and then contacting the exposed adhesive to the skin. This method of application is similar to the application of a sticking plaster, and is therefore already a familiar process to users.

[150]. As an alternative to the use of adhesives, the retaining portion may be some mechanical means for maintaining the apparatus in the required position on the skin. For example, the apparatus may comprise a dedicated strap that engages about limb that is adjustable so as to keep the apparatus firmly applied to the subject. As an alternative, the apparatus may be incorporated into a wearable item such as a glove or a shirt, or an item of jewellery such as a ring which functions to retain the apparatus in position. The apparatus may be configured to engage with a discrete wearable item (such as by complimentary hook-and-loop means), or may have the wearable item integral therewith.

[151]. In some embodiments, the apparatus is retained simply by the wearable item bearing against the housing. For example, the retaining portion may be a snug-fitting elasticised glove which is worn over the apparatus.

[152]. In some embodiments, the retaining portion is any surface or part of the apparatus which contacts the skin of the subject, with a feature of the subject being at least partially responsible for maintaining the apparatus in place on the subject. For example, the apparatus may be configured to be retained between two parts of the body normally in close apposition, or within an existing anatomical structure. The apparatus may be shaped and/or dimensioned to be retained between the toes, the buttocks, in the groin, in the buccal cavity, in a nostril, in the ear canal, or in the umbilicus.

[153]. In other embodiments the apparatus housing is shaped and/or dimensioned to snugly fit over a digit, a toe, or an ear, for example. The apparatus housing may be elastically deformable, composed of a rubberised material for example, and configured to be stretched over any anatomical part (such as a finger).

[154]. Each of the aforementioned embodiments is considered to be a retaining portion in the context of the present disclosure.

[155]. The apparatus further comprises a releasing member (100) having a grasping portion (105) and a wedging portion (110), the function of which will be more fully described infra.

[156]. Turning now to the exploded views of FIG. 5A and FIG. 5B components that are analogous to those in earlier figures will be immediately apparent.

[157]. In this embodiment, the motive force responsible for moving the arm (205) thereby urging the microneedles (15) into the underlying skin is provided by the user. In use, the user places a finger on the upper housing (25) and pushes downwardly. Furthermore, the arm (205) is movable by way of a hinging arrangement.

[158]. The hinging arrangement is provided by way of opposing lugs (115) extending from skin contacting portion (30), each lug comprising an aperture. The arm (205) comprises opposing laterally extending discs (120), each of which seats into an aperture of the lugs (115). It will be apparent that the arm (205) is able to hinge relative to skin contacting portion (30) to allow movement from the first position to the second position.

[159]. The arm (205) is presented to the user having the arm in the first position. The arm

(205) is maintained in the first position by the wedging portion (110) of the releasing member (100). Before removal of the releasing member (100) the wedging portion inserts between the skin contacting portion (30) and the arm (205), thereby keeping the microneedles within the apparatus.

[160]. When intending to apply the apparatus to the subject’s skin, the user removes the flexible layer (90) by pulling on the tab (95) to expose the adhesive layer on the skin contacting surface (35). The apparatus is then applied to the skin, with the adhesive maintaining it in situ for an extended period.

[161]. Once the apparatus has been applied to the skin, the user grasps the grasping portion

(105) and pulls laterally to the left (as drawn), so as to completely remove the releasing member (100). The releasing member (100) has no further function and is discarded at this juncture. By removal of the releasing member (100) the arm (205) is released from the first position and permitted to move (under a downward force exerted by the user) into the second position whereby the lower face of arm (205) contacts the upper face of the skin contacting portion (30). In the second position, the microneedles (15) extend through the spaces (45) and into the underlying skin.

[162]. As will be appreciated, the releasing member (100) may be configured to prevent the upper housing (25) of the apparatus from closing to the skin contacting portion (30) when not intended by the user. The releasing member (100) is inserted or otherwise juxtaposed between the upper housing (25) and the skin contacting portion (30) to prevent closure of the upper housing (25) towards the skin contacting portion (30) sufficient to allow the tips of the microneedles (i.e., projecting portions) to protrude from the base of the holes in the skin contacting portion (30). Preventing closure also prevents movement of the arm (205) from the first position to the second position. Thus, when the releasing member (100) is in place, the tips of the microneedles cannot be inadvertently accessed to cause microneedle contamination or injury. In using the apparatus, the user removes the releasing member (100) as a step in the use process. In a preferred embodiment of apparatus use, the user first adheres the apparatus to the subject’s skin and then removes the releasing member (100), prior to pressing the upper housing (25) to insert the microneedles into the skin.

[163]. Prior to removal by the user, the releasing member (100) can be kept in place by any one of a variety of features. In one example the releasing member (100) comprises protrusions that fit into recesses in either the upper housing (25), the skin contacting portion (30) or both the upper housing (25) and the skin contacting portion (30) to assist in retaining it in place until intentionally removed. In another example the releasing member (100) is designed to be slidably assembled to the skin contacting portion (30) or upper housing (25), such that friction between the releasing member (100) and either the upper housing (25) or the skin contacting portion (30) assists in keeping it in place until intentionally removed. In yet another example magnetic force may be used to assist in keeping the releasing member (100) in place. In a one embodiment of the disclosure, a magnet mounted within the releasing member (100) is positioned so as to be proximal to a Hall effect sensor positioned in either the upper housing (25) or the skin contacting portion (30), when the releasing member (100) is in place. According to this embodiment, when the releasing member (100) is removed by the user, the Hall effect sensor detects the removal of the magnet and causes the apparatus to take some action, such as powering up the electronic circuitry ready for use, converting it from sleep mode to active mode. It is to be understood that the above are examples of possible methods for assisting in retaining the releasing member (100) in place prior to intentional removal that may be used alone or in combination and that other methods as known in the art can also be used alone or in combination with the examples given.

[164]. In some embodiments of the disclosure, the releasing member (100) can also function as a covering element that is used to cover the microneedles after the apparatus has been removed from the subject. In a preferred example of this embodiment the locking element is located on the upper housing (25), extending down towards the skin contacting portion (30). The releasing member (100) comprises a groove that allows the releasing member (100) to slide past the locking element when the releasing member (100) is being withdrawn from the apparatus, while keeping the face of the releasing member (100) facing the upper surface of the skin contacting portion (30) continuous. In use, a releasing member (100) according to this preferred embodiment is removed by the user prior to pressing the upper housing (25) to insert the microneedles into the subject’s skin and retained by the user. After the apparatus is removed from the subject post use, the user is instructed to adhere the releasing member (100) to the adhesive layer on the lower surface of the skin contacting portion (30) to cover the protruding microneedles. In another example of this embodiment, the releasing member (100) is flexibly attached to the apparatus such that the releasing member (100) can remain attached to the apparatus after it has been withdrawn by the user and then repositioned to cover the protruding microneedles after the apparatus has been removed from the subject post use. In yet another example of this embodiment, the releasing member (100) and the upper housing (25) are designed such that the releasing member (100) can be slidably or otherwise engaged with the upper housing (25) once it has been removed, where it is intended that the releasing member (100) be stored while the apparatus is in use and removed to be used as a covering element after the apparatus has been removed from the subject.

[165]. In some embodiments, of the apparatus is configured to facilitate the user in removing the apparatus from the subject. As will be appreciated, the use of an adhesive layer may result in difficulty in removal of the apparatus from the skin. Examples of such configuration include leaving a portion of the skin contacting surface (35) uncoated with adhesive, such that a gap is present between the subject’s skin and the surface (35), wherein the user uses the gap as a leverage point to assist in pulling the apparatus away from the skin by breaking the adhesive bond. In another example, a leverage mechanism not located on the skin contacting surface is incorporated to allow a taller gap than that created by the absence of adhesive on a portion of the skin contacting surface. In yet another example, a tab extending beyond at least one edge of the skin contacting portion (30) and attached to the adhesive layer can be incorporated, where the user pulls on the tab with sufficient force to cause the adhesive layer to stretch and yield, further causing the adhesive to delaminate from the skin contacting surface (35) and the skin.

[166]. In some embodiments of the disclosure, the apparatus is designed such that the releasing member (100) is locked into place in its position prior to apparatus use unless pressure is applied to the upper housing (25). This embodiment is intended to further ameliorate the risk of the releasing member (100) being prematurely withdrawn. In an example of this embodiment, there are features on the releasing member (100) and on at least one of the upper housing (25) and skin contacting portion (30) that are lockably engaged when the upper housing (25) is not being pressed. When the upper housing (25) is depressed, the feature on at least one of the upper housing (25) and skin contacting portions (30) is distorted, so as to disengage the releasing member (100) and allow it to be withdrawn.

[167]. In yet other embodiments, the releasing member (100) need not be removed from the apparatus by the user. According to these embodiments, the releasing member (100) comprises a flexible element of sufficiently high stiffness that it does not substantially deflect when subjected to closing forces likely to be present on the apparatus during manufacture, storage and in the user’s hands prior to application to the subject, but flexible enough that it deflects when the user intentionally applies a closing force to the apparatus when it is applied to the subject’s skin. In so flexing, the releasing member (100) is deflected, allowing the upper housing (25) to close towards the skin contacting portion (30). In these embodiments, the releasing member (100) could also function as the locking element, or the releasing member (100) could be separate from a locking portion. In some of these embodiments, a feature such as that labelled as (220) in FIG. 5A, FIG. 5B, forms the releasing member (100).

[168]. Each space (45) of the apparatus is dimensioned such that a microneedle can extend through it clearly, with at least a tapered part of the microneedle not impacting the sides of the hole during insertion. In some embodiments the holes may be of sufficient cross-section such that no part of the microneedle will contact the sides of the space during insertion. In other embodiments, at least a part of the hole along its length will have a cross-section such that a portion of the length of the microneedle contacts the sides of the hole during insertion. According to this embodiment the hole functions to help support a portion of the length of the microneedle to assist in preventing bending of the microneedle as it is inserted.

[169]. In some embodiments of the apparatus, the skin contacting portion (30) comprises further spaces or depressions configured to accept protrusions on the releasing member, to assist in retaining the releasing member until it is removed by the user. In addition, or alternatively, the skin contacting portion (30) comprises protrusions designed to be accepted into recesses in the releasing member to assist in retaining the releasing member in place until deliberate removal by the user.

[170]. The apparatus comprises a locking portion in the form of a latch (220) which permanently locks the arm (205) in the second position preventing the arm (205) from any hinging movement. In the drawn embodiment, the latch (220) is a simple unitary member capable of deflecting in response to movement of the arm (205) toward the closed position, but then returning to its original position when the arm (205) is in the second position (205b), thereby locking the arm (205) in place.

[171]. Rather than act on the arm (205 ), the locking portion may act on another component of the apparatus, that component in turn locking the arm in place. For example, the locking portion may act on the upper housing (25), with the upper housing (25) in turn retaining the arm (205) in the second position. In a further alternative the locking portion may act on the PCB (65), with the PCB (65) in turn retaining the arm (205) in the second position.

[172]. In other embodiments, the locking portion comprises a recess into which a protrusion on the upper housing (25) is inserted to lock the upper housing (25) in a closed position (i.e., with the arm (205) in the second position). In one embodiment, the locking portion comprises a flexible element that is designed to allow the locking portion to move when impinged upon by the upper housing (25), so at to allow the housing (25) to close relative to the skin contacting portion (30) and whereby once the upper housing (25) has closed, allows the locking portion to move to lock in place the upper housing (25) in the closed position. In one embodiment, the apparatus comprises a protrusion on the upper housing (25), designed to be inserted into a recess in the locking portion, the protrusion comprising a flexible element to allow the protrusion to move, allowing the upper housing (25) to close relative to the skin contacting portion (30) and whereafter the housing (25) has closed relative to the skin contacting portion (30) the protrusion moves to be inserted in the recess in the locking portion, so as to lock the upper housing (25) in the closed position. The flexible element may comprise a shaft that is sufficiently deformable to allow the upper housing (25) to close without yielding of the shaft, so that the flexible element will try to return to its original position post the upper housing (25) closing. In a less preferred, but nonetheless functional embodiment, the flexible element comprises a coil spring.

[173]. A flexible element of the locking portion may be fabricated from any suitable material having the necessary stiffness and yield point. Examples of suitable material include non-crystalline plastics, crystalline plastics, sprung steel, unsprung steel, stainless steel, or other materials as are known if the art with suitable mechanical properties.

[174]. In a preferred embodiment of the disclosure, the locking portion is fabricated from the same material as the skin contacting portion (30), to facilitate the fabrication of a skin contacting portion with an integral locking portion.

[175]. In a particularly preferred embodiment of the disclosure, the force required to deflect or otherwise move the flexible element is designed to be large enough that the pressure the user needs to supply to deform the flexible element and thus cause the upper housing (25) to close towards the skin contacting portion, is sufficient to insert the microneedles into the skin. According to this embodiment, the flexible element of the locking portion is used to set the force necessary to close the apparatus (thereby causing the arm to assume the second position) and ensure that the force is sufficient to insert the microneedles in their intended position embedded in the skin.

[176]. In other embodiments, the locking portion comprises at least one adhesive region located on at least one of the lower surfaces of the upper housing (25) and the upper surface of the skin contacting surface (35). When the apparatus is closed, the one or more adhesive regions adhere the upper housing (25) to the skin contacting portion (30), locking the apparatus in the closed position.

[177]. In another embodiment of the disclosure, the locking portion can assume three different stable states. In a first state, the locking portion is in a disengaged configuration, before the upper housing (25) is pushed downwardly towards the skin contacting portion (30) to close the apparatus. In a second state, the locking portion is in a first engaged position. When the locking portion is in the first engaged position it serves to lock the microneedles (15) in the embedded position in the skin (i.e., the arm (205) being in the second position). In a third state, the locking portion is in a second engaged position. In this state, the locking portion locks the apparatus in the open position (i.e., with the arm (205) in the first position) with the microneedles withdrawn into the apparatus to ameliorate the possibility of needle-stick injury resulting from microneedles protruding after apparatus use. In an example of this embodiment, the locking portion comprises a user engagement portion, that can be gripped or otherwise engaged by the user, for example by engaging a fingernail under an overhanging ledge, so that the user can deflect the flexible portion of the locking portion. According to this example, to close the apparatus the user presses on the upper housing (25) and locks it in place, as in other embodiments disclosed herein. When it is desired to remove the apparatus from the subject, the user engages with the locking portion and deflects it in a first direction, so as to unlock the upper housing (25) from the skin contacting portion (25), and then deflect the locking portion in a second direction, to lock the apparatus in the open position (i.e., with the arm in the first position) with the microneedles in the withdrawn position. In a preferred embodiment of this example, in the first direction, the locking portion is moved is away from the body of the apparatus, and in the second direction, is towards the body of the apparatus. When deflected sufficiently in the second direction, the locking portion is designed, for example, to be stably engaged in a recess so as to prevent closure of the apparatus without intentionally doing so.

[178]. In some embodiments of the disclosure, a downward force on the microneedles when inserted into the skin is provided via the flexible element of the locking portion applying a downward force when the apparatus is locked in the closed position (i.e., with the movable arm in the second position). In some embodiments, effective locking of the movable arm in the second position is provided by a dedicated spring or other suitable biasing means. In other embodiments, the spiring or other biasing means is not dedicated to a locking function and may, for example, act also as a motive force in the movement of the arm from the first position to the second position. For example, a torsion spring may apply a closing torque at a pivot point (where present). In yet another example a flat, disk or coil spring is mounted to the rear of microneedles, such that when the apparatus is closed the spring is distorted or compressed so as to apply a downward force on the microneedles when the apparatus is in the closed position.

[179]. Although not an essential feature of the disclosure, the PCB (65) will be required for many applications where the microneedles are for the purpose of conducting electrical current to, from or through the skin. In that regard, the PCB may carry a microprocessor, and/or volatile electronic memory (such as RAM) and/or non-volatile electronic memory (such as ROM) and/or a wireless networking module (such as a Bluetooth™ module). The apparatus will of course comprise a power source, typically by way of a button battery.

[180]. 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. While the present disclosure is discussed mainly reference to aptamers (and particularly DNA aptamers) it will be understood that other types of analyte recognition elements may be operable. In accordance with the present disclosure, 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.

[181]. An aptamer is an exemplary form of analyte recognition element. Aptamers are small (usually from 20 to 60 nucleotides) RNA, DNA or XNA 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 and other, less regular structures. 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, basestacking, and intercalation.

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

[183]. 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.

[184]. 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.

[185]. 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'-0-methyl RNA, locked nucleic acid (LNA), threose nucleic acid (TNA), and PNA. Collectively, these non-natural backbone oligomers are referred to as xeno nucleic acids (XNA).

[186]. 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. [187]. 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.

[188]. 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.

[189]. As for polynucleotides, proteins may also be subject to modification. For example, backbone modification may be used to improve proteolytic stability of the peptide. Such backbone modification includes the substitution of L-amino acids by D-amino acids, insertion of methyl-amino acids, and the incorporation of beta-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.

[190]. 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, benzyloxytyrosine, and beta-phenylalanine are commonly commercially available, and can be conveniently used to chemically modify the peptide side chain during peptide synthesis.

[191]. 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, amino- or carboxytermini, or side -chains for stabilization of secondary structures may be pursued.

[192]. 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. [193]. 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. One or more working electrodes for use with an electrochemical analyte recognition element-based sensor, the one or more working electrodes having associated therewith a plurality of analyte recognition elements specific for a target analyte, the plurality of analyte recognition elements comprising a first population of analyte recognition elements allowing for quantitation of the target analyte across a first concentration range, and a second population of analyte recognition elements allowing for quantification of the target analyte across a second concentration range, the first concentration range being different to the second concentration range.

2. The one or more working electrodes of claim 1 , wherein the plurality of analyte recognition elements is independently selected from: an aptamer, an XNA (incorporating non- naturally occurring nucleotides) or another aptamer analogue, including a PNA (peptide nucleic acid) or a hybrid polymer including a hybrid species comprised of two or more monomer types.

3. The one or more working electrodes of claim 1 or claim 2, wherein the first concentration range is defined as the range between a lower chosen fraction of the upper saturation limit and an upper chosen fraction of the upper saturation limit for the first population of analyte recognition elements, the second concentration range is defined as the range between the lower chosen fraction of the upper saturation limit and a chosen upper fraction of the upper saturation limit for the second population of analyte recognition elements.

4. The one or more working electrodes of any one of claims 1 to 3, wherein the first and second concentration ranges are discrete, abutting, or overlapping.

5. The one or more working electrodes of any one of claims 1 to 4, wherein the midpoint of the first concentration range is lower than the midpoint of the second concentration range.

6. The one or more working electrodes of any one of claims 1 to 5, wherein the first and second populations of analyte recognition elements respond differently to the same change in concentration of target analyte, with one of the populations having a different relative response to the change than the other population.

7. The one or more working electrodes of claim 6, the different relative response to the change is a greater or lesser change in a measured electrochemical output.

8. The one or more working electrodes of any one of claims 1 to 7, wherein the plurality of analyte recognition elements comprise a third, a fourth or more analyte recognition element populations, each of which allows for improved quantification of the target analyte across a third, a fourth, or more concentration ranges.

9. The one or more working electrodes of any one of claims 1 to 8, wherein the second, third, fourth or more populations of analyte recognition elements allow for improved quantitation of the target analyte across a wider range of concentrations compared with that allowed for by the first population of analyte recognition elements.

10. The one or more working electrodes of any one of claims 1 to 9, wherein each analyte recognition element of the plurality of analyte recognition elements is associated with a single working electrode.

11. The one or more working electrodes of any one of claims 1 to 10, wherein the analyte recognition elements of the plurality of analyte recognition elements are distributed across two or more working electrodes, a single analyte recognition element species being associated with one or the other of the two or more working electrodes.

12. The one or more working electrodes of claim 11, wherein the two or more electrodes are mutually connected, or are mutually connectable, or are mounted on a single mounting portion.

13. The one or more working electrodes of any one of claims 1 to 12, wherein the analyte recognition elements of each of the analyte recognition element populations are associated exclusively with a dedicated working electrode associated with a dedicated electrical connection.

14. The one or more working electrodes of any one of claims 1 to 13, wherein each of the analyte recognition element populations comprises respectively a analyte recognition element species having a different binding affinity for the target analyte.

15. The one or more working electrodes of claim 14, wherein each respective analyte recognition element species has a different base sequence composition and/or length.

16. The one or more working electrodes of any one of claims 1 to 15, wherein the plurality of analyte recognition elements were associated with the working electrode contemporaneously.

17. The one or more working electrodes of any one of claims 1 to 16, wherein the plurality of analyte recognition elements were associated with the one or more working electrodes by codeposition onto a surface of the working electrode.

18. The one or more working electrodes of claim 17, wherein each of the first, second, third, fourth or more analyte recognition element populations are co-deposited such that each analyte recognition element species is present in substantially equal numbers.

19. The one or more working electrodes of claim 18, wherein a substantially equal number is +/- about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of a mathematically equal number.

20. The one or more working electrodes of claim 17, wherein each of the first, second, third, fourth or more analyte recognition element populations are co-deposited such that at least one analyte recognition element species is present in substantially different numbers to the other analyte recognition elements.

21. The one or more working electrodes of any one of claims 1 to 20, wherein each analyte recognition element of the plurality of analyte recognition elements is connected at or about a first end thereof to a surface of the one or more working electrodes.

22. The one or more working electrodes of claim 21, wherein the connection comprises one or more covalent bonds, or the connection consists of, or comprises, a thiol linkage.

23. The one or more working electrodes of any one of claims 1 to 22, wherein each analyte recognition element of the plurality of analyte recognition elements is associated with a redox reporter species.

24. The one or more working electrodes of claim 23, wherein the redox reporter species is covalently connected to each analyte recognition element.

25. The one or more working electrodes of claim 23 or claim 24, wherein the redox reporter species is methylene blue or a functional equivalent thereof.

26. The one or more working electrodes of claim 23 or claim 24, wherein different redox reporter species with different E° values are each associated with a different analyte recognition element population.

27. The one or more working electrodes of any one of claims 23 to 26, wherein the redox reporter species is connected to each analyte recognition element of the plurality of analyte recognition elements at or about a second end thereof.

28. The one or more working electrodes of any one claims 1 to 27, wherein the target analyte is an inorganic species, a small molecule, an organic molecule, a therapeutic drug molecule or a metabolite thereof, or a molecule endogenous to an animal.

29. The one or more working electrodes of claim 28, wherein the therapeutic drug molecule is vancomycin or another glycopeptide antibiotic.

30. The one or more working electrodes of claim 29, wherein each analyte recognition element of the first population of analyte recognition elements is the analyte recognition element 4-Trunc, and each analyte recognition element of the second population of analyte recognition elements is the analyte recognition element 3-Trunc.

31. The one or more working electrodes of claim 28, wherein the molecule endogenous to an animal is creatinine.

32. The one or more working electrodes of claim 31, wherein each analyte recognition element of the first population of analyte recognition elements is the analyte recognition element Cre 1GC, and each analyte recognition element of the second population of analyte recognition elements is the analyte recognition element Cre OG.

33. The one or more working electrodes of any one of claims 1 to 32, wherein each analyte recognition element of the plurality of analyte recognition elements is capable of detecting the target analyte selectively amongst one, more than one, or all non-target analytes present in a biological fluid.

34. The one or more working electrodes of claim 33, wherein the biological fluid is selected from interstitial fluid, blood, saliva, a lacrimal secretion, a lactational secretion, a nasal secretion, a tracheal secretion, a bronchial secretion, an alveolar secretion, a gastric secretion, a gastric content, a glandular secretion, a vaginal secretion, a uterine secretion, a prostate secretion, semen, urine, sweat, cerebrospinal fluid, a glomerular filtrate, a hepatic secretion, bile, and an exudate.

35. The one or more working electrodes of claim 33 or claim 34, wherein the biological fluid is interstitial fluid or blood present in situ in the body of an animal.

36. The one or more working electrodes of any one of claims 1 to 35 that is each a wire, or a needle, or a microneedle.

37. A method for producing a working electrode for use in an electrochemical analyte recognition element-based sensor, the method comprising the steps of: providing one or more electrodes; providing a first population of analyte recognition elements allowing for quantitation of the target analyte across a first concentration range, and a second population of analyte recognition elements allowing for quantification of the target analyte across a second concentration range, the first concentration range being different to the second concentration range; and contacting the one of more electrodes with the first and second populations of analyte recognition elements under conditions allowing the analyte recognition elements of the first and second populations to associate with a surface of the one of more electrodes.

38. The method of claim 37, wherein the analyte recognition elements are independently selected from: an aptamer, a XNA (incorporating non-naturally occurring nucleotides) or another aptamer analogue, or a PNA (peptide nucleic acid) or a hybrid polymer including a hybrid species comprised of two or more monomer types.

39. The method of claim 37 or claim 38, wherein the first concentration range is defined as the range between a lower chosen fraction of the upper saturation limit and an upper chosen fraction of the upper saturation limit for the first population of analyte recognition elements, the second concentration range is defined as the range between the lower chosen fraction of the upper saturation limit and a chosen upper fraction of the upper saturation limit for the second population of analyte recognition elements.

40. The method of any one of claims 37 to 39, wherein the first and second concentration ranges are discrete, abutting, or overlapping.

41. The method of any one of claims 37 to 40, wherein the midpoint of the first concentration range is lower than the midpoint of the second concentration range.

42. The method of any one of claims 37 to 41, wherein upon exposure to a fixed concentration of target analyte and application of an interrogating potential waveform, the first population of analyte recognition elements provides a different response sensitivity to that of the second population of analyte recognition elements.

43. The method of claim 42, wherein the different response sensitivity is a greater or lesser change in measured electrochemical output. .

44. The method of any one of claims 37 to 43, wherein the plurality of analyte recognition elements comprise a third, a fourth or more analyte recognition element populations, each of which allows for improved quantification of the target analyte across a third, a fourth, or more concentration ranges.

45. The method of any one of claims 37 to 44, wherein the second, third, fourth or more populations of analyte recognition elements allow for improved quantitation of the target analyte across a wider range of concentrations compared with that allowed for by the first population of analyte recognition elements.

46. The method of any one of claims 37 to 45, wherein each analyte recognition element of the plurality of analyte recognition elements is contacted with a single electrode.

47. The method of any one of claims 37 to 46, wherein the analyte recognition elements of the plurality of analyte recognition elements are distributed across two or more electrodes, a single analyte recognition element species being associated with one or the other of the two or more working electrodes.

48. The method of claim 47, comprising connecting together two or more electrodes, or mounting two or more electrodes on a single mounting portion.

49. The method of any one of claims 37 to 48, wherein the analyte recognition elements of each of the analyte recognition element populations are contacted exclusively with a dedicated electrode associated with a dedicated electrical connection.

50. The method of any one of claims 37 to 49, wherein each of the analyte recognition element populations comprises respectively a analyte recognition element species having a different binding affinity for the target analyte.

51. The method of claim 50, wherein each respective analyte recognition element species has a different base sequence composition and/or length

52. The method of any one of claims 37 to 51, wherein the plurality of analyte recognition elements are contacted with the electrode contemporaneously.

53. The method of any one of claims 37 to 52, wherein the plurality of analyte recognition elements are contacted with the one or more electrodes by a co-deposition method.

54. The method of claim 53, wherein each of the first, second, third, fourth or more analyte recognition element populations are co-deposited such that each analyte recognition element species is present in substantially equal numbers.

55. The method of claim 54, wherein a substantially equal number is +/- about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of a mathematically equal number.

56. The method of claim 53, wherein each of the first, second, third, fourth or more analyte recognition element populations are co-deposited such that at least one analyte recognition element species is present in substantially different numbers to the other analyte recognition elements.

57. The method of any one of claims 37 to 56, comprising connecting each analyte recognition element of the plurality of analyte recognition elements at or about a first end thereof to a surface of the one or more electrodes.

58. The method of claim 57, wherein the connecting comprises forming one or more covalent bonds or the connecting consists of, or comprises, forming a thiol linkage.

59. The method of any one of claims 37 to 58, comprising associating each analyte recognition element of the plurality of analyte recognition elements with a redox reporter species.

60. The method of claim 59, comprising covalently connecting the redox reporter species to each analyte recognition element.

61. The method of claim 59 or claim 60, wherein the redox reporter species is methylene blue or a functional equivalent thereof.

62. The method of claim 59 or claim 60, wherein different redox reporter species with different E° values are each associated with a different analyte recognition element population.

63. The method of any one of claims 59 to 62, comprising connecting the redox reporter species to each analyte recognition element of the plurality of analyte recognition elements at or about a second end thereof.

64. The method of any one claims 37 to 63, wherein the target analyte is an inorganic species, a small molecule, an organic molecule, a therapeutic drug molecule or a metabolite thereof, or a molecule endogenous to an animal.

65. The method of claim 64, wherein the therapeutic drug molecule is vancomycin or another glycopeptide antibiotic.

66. The method of claim 65, wherein each analyte recognition element of the first population of analyte recognition elements is the analyte recognition element 4-Trunc, and each analyte recognition element of the second population of analyte recognition elements is the analyte recognition element 3-Trunc.

67. The method of claim 64, wherein the molecule endogenous to an animal is creatinine.

68. The method of claim 65, wherein each analyte recognition element of the first population of analyte recognition elements is the analyte recognition element Cre 1 GC, and each analyte recognition element of the second population of analyte recognition elements is the analyte recognition element Cre OG.

69. The method of any one of claims 37 to 68, wherein each analyte recognition element of the plurality of analyte recognition elements is capable of detecting the target analyte selectively amongst one, more than one, or all non-target analytes present in a biological fluid.

70. The method of claim 69, wherein the biological fluid is selected from interstitial fluid, blood, saliva, a lacrimal secretion, a lactational secretion, a nasal secretion, a tracheal secretion, a bronchial secretion, an alveolar secretion, a gastric secretion, a gastric content, a glandular secretion, a vaginal secretion, a uterine secretion, a prostate secretion, semen, urine, sweat, cerebrospinal fluid, a glomerular filtrate, a hepatic secretion, bile, and an exudate.

71. The method of claim 69 or claim 70, wherein the biological fluid is interstitial fluid or blood present in situ in the body of an animal.

72. The method of any one of claims 37 to 71, wherein the one or more electrodes is each a wire, a needle, or a microneedle.

73. An electrochemical sensor comprising the one or more working electrodes of any one of claims 1 to 36.

74. The sensor of claim 73, that is an electrochemical aptamer-based sensor.

75. The sensor of claim 73 or claim 74, further comprising a reference electrode.

76. The sensor of any one of claims 73 to 75, further comprising a reference electrode and a power supply.

77. A method for determining the concentration of a target analyte in a fluid, the method comprising the step of contacting the one or more working electrodes of any one of claims 1 to 36 to the fluid.

78. The method of claim 77, wherein the fluid is a biological fluid.

79. The method of claim 78, wherein the step of contacting the one or more electrodes to the biological fluid is performed in vivo, ex vivo or in vitro.

80. The method of claim 78 or claim 79, wherein the biological fluid is selected from interstitial fluid, blood, saliva, a lacrimal secretion, a lactational secretion, a nasal secretion, a tracheal secretion, a bronchial secretion, an alveolar secretion, a gastric secretion, a gastric content, a glandular secretion, a vaginal secretion, a uterine secretion, a prostate secretion, semen, urine, sweat, cerebrospinal fluid, a glomerular filtrate, a hepatic secretion, bile, and an exudate.

81. The method of any one claims 77 to 80, wherein the target analyte is an inorganic species, a small molecule, an organic molecule, a therapeutic drug molecule or a metabolite thereof, or a molecule endogenous to an animal.

82. The method of claim 81, wherein the therapeutic drug molecule is vancomycin or another glycopeptide antibiotic.

83. The method of any one of claims 77 to 82, that is a therapeutic drug monitoring method.