Aptamer screening method

Abstract

A method for identifying an aptamer suitable for use in an electrochemical sensor for sensing a target analyte. The method includes determining whether the target analyte competitively binds with the analyte recognition element. Competitive binding in favour of the target analyte is indicative of the suitability of the aptamer.

Description

APTAMER SCREENING METHODFIELD

[0001] . The present disclosure relates generally to screening methods for identification of aptamers capable of specific binding for a target analyte. An aptamer identified by the present methods may be useful in an aptamer-based sensor.BACKGROUND

[0002] . Aptamers are small (usually from 20 to 60 nucleotides) single-stranded RNA, DNA, or XNA oligonucleotides able to bind a target analyte with high affinity and specificity. Aptamers have been found particularly useful when implemented as an affinity element in electrochemical sensors. In such applications, the amount of analyte in a test sample can be continually monitored in real time in both in vivo and ex vivo settings.

[0003] . Aptamers may be bound to the working electrode of an electrochemical aptamerbased (EAB) sensor, with gold being commonly used as the electrode surface. Each aptamer has a bound redox-active species which acts as a reporter. Methylene blue is commonly used as a redox reporter in many applications. Upon interaction of the target analyte with the aptamer, the aptamer undergoes a conformational change, causing the redox reporter to become more accessible to the working electrode surface. This increase in accessibility increases electron transfer between the redox reporter and the electrode. The increase in electron transfer contributes to a change in Faradaic current that is detected by a potentiostat. In other instances, the redox reporter becomes less accessible to the working electrode surface upon target binding, thereby decreasing the electron transfer. Regardless, after application of an interrogation voltage, peak currents passing through the working electrode are measured, thereby informing of the amount of analyte in the environment about the working electrode.

[0004] . Aptamers for use in EAB sensors are usually selected from a highly diverse combinatorial library having a vast number (up to 1015) of different oligonucleotides. Selection of an aptamer that is specific for a given analyte may be facilitated by a process known as systematic evolution of ligands by exponential enrichment (SELEX). The process may be considered as two alternating stages. In the first stage, the library of oligonucleotides are amplified by a polymerase chain reaction (PCR) to the desired concentration. For theselection of RNA aptamers, the single -chained oligoribonucleotides are generated by in vitro transcription of double-stranded DNA with T7 RNA-polymerase. For DNA aptamers, a pool of single-stranded oligodeoxyribonucleotides is generated by strand separation of doublestranded PCR products. In the second stage, the products of amplification are incubated with a target analyte and oligonucleotides which bind the analyte are used in the next SELEX round.

[0005] . Separation of oligonucleotides with higher affinity for the target analyte and removal of unbound oligonucleotides are achieved through intense competition for binding sites. Selection pressure rises with every SELEX round. Maximum enrichment of the oligonucleotide pool with aptamers with the strongest affinity for the target analyte is usually achieved after 5 to 15 rounds of SELEX.

[0006] . While SELEX is undoubtedly a useful tool, significant time, effort, and expense is required to test aptamers of the enriched oligonucleotide pool for suitability in an analytespecific sensor. For example, biolayer interferometry kinetics experiments may be performed to identify preferred aptamers from the pool of enriched aptamers resulting from the SELEX process.

[0007] . It is an aspect of the present disclosure to provide an improvement to prior art aptamer selection methods, or an adjunct to such methods. It is a further aspect of the present disclosure to provide a useful alternative to prior art aptamer selection methods.

[0008] . 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

[0009] . In a first aspect, but not necessarily the broadest aspect, there is provided a method for identifying an analyte recognition element suitable for use in an electrochemical sensor for sensing a target analyte, the method comprising the step of determining whether the target analyte competitively binds with the analyte recognition element, wherein competitive binding in favour of the target analyte is indicative of the suitability of theanalyte recognition element for an electrochemical sensor configured to detect the target analyte.

[0010] . In one embodiment of the first aspect, competitive binding is determined by the steps of: providing a candidate analyte recognition element and a redox reporter species, allowing the redox reporter species to associate with the candidate analyte recognition element, exposing the candidate analyte recognition element to the target analyte, and determining any displacement of the redox reporter species from the candidate analyte recognition element, wherein displacement is indicative of the suitability of the analyte recognition element for an electrochemical sensor configured to detect the target analyte.[Oi l]. In one embodiment of the first aspect, competitive binding is determined by the steps of: providing a candidate analyte recognition element and a target analyte, allowing the target analyte to associate with the candidate analyte recognition element, exposing the candidate analyte recognition element to the redox reporter species, and determining any displacement of the target analyte from the candidate analyte recognition element, wherein displacement is indicative of the lack of suitability of the analyte recognition element for an electrochemical sensor configured to detect the target analyte.

[0012] . In one embodiment of the first aspect, the candidate analyte recognition element is separate to the redox reporter species.

[0013] . In one embodiment of the first aspect, the candidate recognition element is not bound or otherwise associated with a surface or a body.

[0014] . In one embodiment of the first aspect, the candidate recognition element is not bound or otherwise associated with a working electrode of an electrochemical cell.

[0015] . In one embodiment of the first aspect, the displacement is determined by a change in a characteristic of the redox reporter species or the candidate recognition element.

[0016] . In one embodiment of the first aspect, the characteristic is absorption and / or emission of electromagnetic energy.

[0017] . In one embodiment of the first aspect, the electromagnetic energy is light energy.

[0018] . In one embodiment of the first aspect, the light energy is ultraviolet light energy, visible light energy, or infrared light energy.

[0019] . In one embodiment of the first aspect, the characteristic is fluorescence of the redox reporter species.

[0020] . In one embodiment of the first aspect, (i) florescence of the redox reporter species is quenched when associated with the candidate analyte recognition element, and dequenched when displaced from the candidate analyte recognition element; or (ii) florescence of the redox reporter species is dequenched when associated with the candidate analyte recognition element, and quenched when displaced from the candidate analyte recognition element.

[0021] . In one embodiment of the first aspect, the displacement is determined by an electrochemical method.

[0022] . In one embodiment of the first aspect, the electrochemical method determines displacement by reference to a current through a working electrode having the candidate analyte recognition element connected thereto.

[0023] . In one embodiment of the first aspect, the displacement is determined by a method for investigating binding between the redox reporter species and the candidate analyte recognition element and / or the target analyte and the candidate analyte recognition element.

[0024] . In one embodiment of the first aspect, the redox reporter species is selected from: methylene blue, thioflavin T, viologen, Nile blue, ferrocene, vinylferrocene, anthraquinone, thionine, ethidium bromide, daunomycin, anthraquinone-C5, dabcyl, 2,6- dichlorophenal-indophenol, gallocyanine, ROX, pentamethylferrocene, ferrocene-C5, neutral red, and horseradish peroxidase, ruthenium, bis-pyridine, tris-pyridine, bis- imidizole, cytochrome c, cytochrome c’, and plastocyanin; or a functional equivalent thereof of any of the aforementioned redox reporter species.

[0025] . In one embodiment of the first aspect, the candidate analyte recognition element is a nucleic acid (including DNA and DNA) or an analogue thereof (including XNA), or a hybrid nucleic acid (including PNA).

[0026] . In one embodiment of the first aspect, the candidate analyte recognition element is an aptamer.

[0027] . In one embodiment of the first aspect, the step of determining any displacement is determined by reference to exposure of the candidate analyte recognition element to a solution comprising less analyte or zero analyte.

[0028] . In one embodiment of the first aspect, the change in a characteristic of the redox reporter species is determined by reference to exposure of the candidate analyte recognition element to a solution comprising less analyte or zero analyte.

[0029] . In one embodiment of the first aspect, the method is performed in a high-throughput method, including a microplate format, a random or ordered microarray format, or a multivessel array format, with a different candidate analyte recognition element being tested in each well of the microplate, random or ordered microarray spot, or each vessel of the array.

[0030] . In one embodiment of the first aspect, the candidate analyte recognition element is obtained from a selection method starting with a diverse pool of analyte recognition elements.

[0031] . In one embodiment of the first aspect, the selection method is based on the affinity of the candidate analyte recognition element for the target analyte.

[0032] . In one embodiment of the first aspect, the selection method is a sequential evolution of ligands by exponential enrichment (SELEX) method, or other enrichment method.

[0033] . In a second aspect, there is provided a method for screening a plurality of different analyte recognition elements for suitability in an electrochemical sensor for sensing a target analyte, the method comprising immobilizing each of the plurality of different analyte recognition elements and exposing the immobilized plurality of different analyte recognition elements to a target analyte and a redox reporter species so as to determine whether the target analyte competitively binds with the analyte recognition element, wherein competitive binding in favour of the target analyte is indicative of the suitability of the analyte recognition element for an electrochemical sensor configured to detect the target analyte.

[0034] . In one embodiment of the second aspect, competitive binding is determined by the method of embodiment of the first aspect.

[0035] . In one embodiment of the second aspect, each of the plurality of different analyte recognition elements is immobilized on a surface.

[0036] . In one embodiment of the second aspect, the plurality of different analyte recognition elements are arranged on the surface in a location-specific manner

[0037] . In one embodiment of the second aspect, the plurality of different analyte recognition elements are arranged on the surface in an array.

[0038] . In one embodiment of the second aspect, the surface is part of a flow cell, a microwell, a microarray, a microbead, or another analytical structure.

[0039] . In one embodiment of the second aspect, flow cell, the microwell, the microbead, the microarray, or another analytical structure is part of an automated fluidic system or a microfluidic system.

[0040] . In one embodiment of the second aspect, the plurality of different analyte recognition elements comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 100000, 1000000, or 10000000 different analyte recognition elements.

[0041] . In a third aspect, there is provided an apparatus for screening a plurality of different analyte recognition elements for suitability in an electrochemical sensor for sensing a target analyte, the apparatus comprising immobilizing each of the plurality of different analyte recognition elements immobilized thereon, one or more flow paths for exposing the immobilized plurality of different analyte recognition elements to a solution of target analyte and a solution of redox reporter species.

[0042] . In one embodiment of the third aspect, the apparatus comprises a reading device configured to determine directly or indirectly whether the target analyte competitively binds with the analyte recognition element.

[0043] . In one embodiment of the third aspect, the reading device is configured to detect absorption and / or emission of electromagnetic energy, including light energy, ultraviolet light energy, visible light energy, or infrared light energy.

[0044] . In one embodiment of the third aspect, the reading device comprises an energy source configured to excite a fluorescent compound.

[0045] . In a fourth aspect, there is provided an analyte recognition element identified as suitable for use in an electrochemical sensor by the method of any embodiment of the second aspect, or the apparatus of the fourth aspect.BRIEF DESCRIPTION OF THE FIGURES

[0046] . FIGS. 1 A, IB, and 1C, together illustrate diagrammatically a proposed mechanism by which a redox reporter “R” is displaced from a binding site on an aptamer by the target analyte “A” for which the aptamer is sensitive. In this embodiment, the aptamer and the redox reporter are free in solution.

[0047] . FIG. 1 A shows the aptamer in the absence of any proximal target analyte. The redox reporter is bound to the aptamer at a binding site. The open arrow indicates movement of a distal target analyte molecule toward the binding site.

[0048] . FIG. IB chronologically follows FIG. 1A, showing the target analyte molecule proximal to the aptamer and bound to the binding site previously occupied by the redox reporter. Having been displaced, the redox reporter moves away (indicated by the open arrow).

[0049] . FIG. 1C chronologically follows FIG. IB, showing the redox reporter located distal to the binding site.

[0050] . FIGS. 2 A, 2B, and 2C, together illustrate diagrammatically a proposed mechanism by which a redox reporter “R” is displaced from a binding site on an aptamer by the target analyte “A” for which the aptamer is sensitive. In this embodiment, the redox reporter is covalently bonded to the aptamer, and the redox reporter / aptamer complex is free in solution.

[0051] . FIG. 2 A shows the aptamer in the absence of any proximal target analyte. The redox reporter is bound to the aptamer at a binding site. The open arrow indicates movement of a distal target analyte molecule toward the binding site.

[0052] . FIG. 2B chronologically follows FIG. 2A, showing the target analyte molecule proximal to the aptamer and bound to the binding site previously occupied by the redox reporter. Having been displaced, the redox reporter moves away (indicated by the open arrow).

[0053] . FIG. 2C chronologically follows FIG. 2B, showing the redox reporter located distal to the binding site.

[0054] . FIG. 3A, 3B, and 3C, together illustrate diagrammatically an electrochemical method for detection of the displacement of a redox reporter “R” by the introduction of target analyte “A” about the working electrode of an EAB sensor. Changing position of the redox reporter arising from its displacement by target analyte causes changes in the electron transfer kinetics between the redox reporter and the working electrode surface. The change in kinetics alters the current passing through the working electrode, which in turn is informative of the displacement of the redox reporter.

[0055] . FIG. 3A shows the aptamer in the absence of any proximal target analyte. The redox reporter is bound to the aptamer at a binding site. The open arrow indicates movement of a distal target analyte molecule toward the binding site.

[0056] . FIG. 3B chronologically follows FIG. 3 A, showing the target analyte molecule proximal to the aptamer and bound to the binding site previously occupied by the redox reporter. Having been displaced, the redox reporter moves away (indicated by the open arrow).

[0057] . FIG. 3C chronologically follows FIG. 3B, showing the redox reporter located distal to the binding site.

[0058] . FIG. 4 illustrates diagrammatically a basic circuit of an electrochemical sensor that may be used to determine displacement of a redox reporter from its binding site on aptamer by a target analyte.

[0059] . FIG. 5 A is a graph showing that the introduction of vancomycin about free vancomycin-sensitive DNA aptamers in co-solution with free methylene blue redox reporter causes dequenching of methylene blue fluorescence.

[0060] . FIG. 5B is a graph showing that the introduction of vancomycin about free vancomycin-sensitive DNA aptamers in co-solution with free methylene blue redox reporter causes dequenching of methylene blue fluorescence in a concetration-dependent manner.

[0061] . FIG. 6A is a graph showing that the introduction of vancomycin about free vancomycin-sensitive DNA aptamers in co-solution with free thioflavin T redox reporter causes quenching of thioflavin T fluorescence

[0062] . FIG. 6B is a graph showing that the introduction of vancomycin about free vancomycin-sensitive DNA aptamers in co-solution with free thioflavin T redox reporter causes quenching of thioflavin T fluorescence.

[0063] ,

[0064] . FIG. 7A through FIG. 7D illustrates highly diagrammatically a high throughput screening system capable of contemporaneously determining the suitability of a plurality of candidate aptamers for use in an electrochemical sensor configured to detect a target analyte.

[0065] . 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.

[0066] . 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

[0067] . 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.

[0068] . 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.

[0069] . Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to the same embodiment, but may.

[0070] . The term “aptamer” is used conveniently throughout this specification. Unless the skilled person would understand any contrary intention, the term “aptamer” is intended to include DNA, RNA, and XNA aptamers, and also hybrid species such as peptide nucleic acid aptamers.

[0071] . In one aspect, the present disclosure provides a method for identifying an analyte recognition element for use in an electrochemical sensor for sensing a target analyte, the method comprising the steps of: providing a candidate analyte recognition element and a redox reporter species, allowing the redox reporter species to associate with the candidate analyte recognition element, exposing the candidate analyte recognition element to a solution comprising the target analyte, and determining any displacement of redox reporter species from the candidate analyte recognition element.

[0072] . The present disclosure identifies an aptamer as suitable for use in the context of an EAB sensor on the basis of its ability to bind with a redox reporter at first instance, and the subsequent displacement of the redox reporter from its bound position on the aptamer by the analyte concerned. The redox reporter may be free in solution about the candidate aptamer, although in other embodiments the redox reporter is covalently bonded to the candidate aptamer.

[0073] . Irrespective of the free or covalently bonded state of the redox reporter, the candidate aptamer may be free in solution or covalently bonded or non-covalently attached to a solid support. In some embodiments, the solid support is electrically non-conductive (such as a well in a microplate) while in other embodiments the solid support is electrically conductive. In relation to the latter, the solid support may be a working electrode of an EAB sensor.

[0074] . The present method allows for the identification of an aptamer suitable for use in an EAB sensor on the basis of its ability to competitively bind with a target analyte over a redox reporter. The ability of aptamer to competitively bind with the redox reporter may be determined by determining the ability of the target analyte to displace the redox reporter from a bound state with the aptamer. Such displacement is shown diagrammatically in FIG.1.

[0075] . As will be understood, the redox reporter and target analyte may be each repeatedly binding and releasing to the aptamer, with an equilibrium being established for each in relation to the bound / unbound state. In that circumstance, the equilibria favour the binding of the target analyte over that of the redox reporter.

[0076] . FIG. 1 A, FIG. IB and FIG. 1C shows a theoretical basis for one embodiment of the present method whereby all chemical elements are in free solution. A single-stranded aptamer (10) is co-incubated in solution with a redox reporter (20) and target analyte (15). As shown in FIG. 1A, when the aptamer (10) is distal to the target analyte (15), the redox reporter (20) binds to a binding site (25) on the aptamer (10). When the target analyte (15) becomes proximal to the aptamer (FIG. IB), the target analyte (15) displaces the redox reporter (20) from the binding site (25) due to its higher affinity for the binding site (25). As shown in FIG. 1C, the redox reporter may then freely move away from the binding site.

[0077] . This embodiment of the method may be implemented in a multi -well format. A different aptamer species may be dispensed into each well, and co-incubated with the redox reporter (FIG. 1A).

[0078] . Without wishing to be limited by theory, it is proposed that an aptamer that is suitable for use in an EAB sensor to detect a target analyte will demonstrate (i) the ability to bind a redox reporter, and (ii) the ability to also bind the target analyte (optionally in preference to the redox reporter). In the context of an EAB sensor, the alteration in electron flux between a redox reporter and a working electrode may be due to displacement of the redox reporter from a binding site on its covalently bonded aptamer by the target analyte. The alteration of electron flux (as measured by current flowing through the working electrode) is in turn indicative of the amount of target analyte about the working electrode.

[0079] . In identifying suitable aptamers in a microplate format, each candidate aptamer may be tested across two wells. A first well may comprise a solution of redox reporter with the candidate aptamer. A second well may comprise a solution of redox reporter, the candidate aptamer, and the target analyte. The contents of each well are assayed for displacement of the redox reporter by the target analyte. A suitable candidate aptamer may be identified where the assay results are different.

[0080] . Turning to a second embodiment of the method, reference is made to FIG. 2A. FIG. 2B and FIG. 2C, whereby the candidate aptamer (10) has a redox reporter (20) covalentlybonded to a terminus thereof. When the aptamer (10) is distal to target analyte (15), the redox reporter (20) binds to a binding site (25) on the aptamer (10). When the target analyte (15) becomes proximal to the aptamer (FIG. 2B), the target analyte (15) displaces the redox reporter (20) from the binding site (25) due to its higher affinity for the binding site (25). As shown in FIG. 2C, the redox reporter may then freely move away from the binding site. As an alternative to FIG. 2C, the redox reporter may associate with a region of the aptamer that is away from the binding site (25), or even a neighbouring aptamer attached to the electrode surface (30). In those circumstances, the redox reporter (20) is nevertheless displaced.

[0081] . In this second embodiment, a microplate format similar to that described above may be implemented. A first well may hold a solution of the aptamer / redox reporter complex, with a second well holding the aptamer / redox reporter complex and the target analyte. The contents of each well are assayed for displacement of the redox reporter by the target analyte. A suitable candidate aptamer may be identified where the assay results are different.

[0082] . The present methods require some means for detecting the displacement of the redox reporter from the candidate aptamer.

[0083] . In one embodiment, the detection of displacement relies on the redox reporter being optically active, with the optical activity changing according to whether the redox reporter is bound to or free from the aptamer. An exemplary form of optical activity is fluorescence. A redox reporter may be intrinsically fluorescent, with fluorescence changing upon binding to the aptamer. In some cases binding causes dequenching of fluorescence (manifesting as a detected increase in fluorescence) while in other cases binding leads to fluorescence quenching (manifesting as a detected decrease in fluorescence). Any change in fluorescence can be readily detected by eye and / or by a fluorescence reading apparatus such as a fluorescence microscope, fluorescence microarray scanner, or a fluorescence microplate reader.

[0084] . In some embodiments, the redox reporter has a chemical structure that interacts with aptamer nucleic acid, the interaction leading to a change in fluorescence. Such interaction includes intercalation (symmetric or asymmetric, preferably symmetric), majorgroove binding, and minor groove binding. The interaction may be partially or predominantly electrostatic in nature.

[0085] . In one embodiment, the redox reporter is methylene blue. Binding of a methylene blue redox reporter with the nucleic acid aptamer may be detected by the quenching of methylene blue fluorescence when the methylene blue molecule interacts with the nucleic acid aptamer. Methylene blue has an excitation peak of about 665 nm, and an emission of 682 nm, which is visible to the naked eye. The emission is quenched when methylene blue is associated with nucleic acid, in particular, the guanine bases. Such quenching is detectable by optical means, fluorescence microscopy, fluorescence microarray scanner, or a fluorescence microplate reader, for example, by means known to the skilled person. Whatever means are implemented, displacement of methylene blue from an aptamer can be inferred by increase in methylene blue fluorescence intensity upon addition of target analyte to a mixture of the aptamer and methylene blue.

[0086] . The present disclosure will be operable using redox reporters other than methylene blue. Species capable of exchanging electrons with a working electrode and also having fluorescence that is quenched or unquenched upon interaction with aptamer DNA, will be useful. An exemplary alternative to methylene blue is thioflavin T. Binding of a thioflavin T redox reporter may be detected by the increase of thioflavin T fluorescence when the thioflavin T molecule interacts with the nucleic acid aptamer. Thioflavin T has an excitation peak of about 330 nm, and two emission peaks at 450 nm and 485 nm, which are visible to the naked eye. The emission at 450 nm is quenched when thioflavin T is associated with nucleic acid, while the emission at 485 nm is enhanced when thioflavin T is associated with nucleic acid. Such quenching and enhancement is detectable by optical means, fluorescence microscopy, fluorescence microarray scanner, or a fluorescence microplate reader, for example, by means known to the skilled person. Whatever means isimplemented, displacement of thioflavin T from an aptamer can be inferred by a change in the fluorescence intensity upon addition of target analyte to a mixture of the aptamer and thioflavin T.

[0087] . Embodiments utilising a redox reporter exhibiting DNA-mediated fluorescence quenching enable a high throughput screening system for aptamers useful in EAB sensors. The only steps required are dispensing the test aptamer into a microplate, and reading anychange in fluorescence intensity upon addition of the target analyte using a plate reader having an excitation source.

[0088] . As an alternative to the use of the DNA-mediated fluorescence quenching embodiments described above, a further embodiment of the method may be read by reference to current generated by a aptamer tethered to an electrode surface, the aptamer having a redox reporter associated therewith. This current-read embodiment may provide a more faithful representation of the intended use of the aptamer in the context of an EAB sensor..

[0089] ,

[0090] . Reference is made to FIGS. 3A, 3B, and 3C, showing diagrammatically the detection of the displacement of the redox reporter by an electrochemical method, utilising the circuit of FIG. 4. Referring firstly to FIG. 3A, when the aptamer (10) is distal to target analyte (15), the redox reporter (20) binds to a binding site (25) on the aptamer (10). Electron transfer (indicated by the dashed arrow) is established between the redox reporter (20) and the surface (30) of the working electrode to which the aptamer (10) is attached. When the target analyte (15) becomes proximal to the aptamer (FIG. 3B), the target analyte (15) displaces the redox reporter (20) from the binding site (25) due to its higher affinity for the binding site (25). At that time, the kinetics of electron transfer are different to those in the circumstance shown in FIG. 3A.

[0091] . As shown in FIG. 3C, the redox reporter may then freely move away from the binding site, establishing yet further and different electron kinetics to those arising in FIGS. 3 A and 3B.

[0092] . As an alternative to FIG. 3C, the redox reporter may associate with a region of the aptamer that is away from the binding site (25), or even a neighbouring aptamer attached to the electrode surface (30). In that circumstance, the electron transfer kinetics will nevertheless differ from those in the circumstances of FIG. 3A and FIG. 3B.

[0093] . The embodiment of FIG. 3A, 3B and 3C may be implemented in the context of an electrochemical cell as shown in FIG. 4, comprising a working electrode, a counter electrode, a power source for interrogation, and optionally a reference electrode. In that implementation, displacement of methylene blue is detected by a change in Faradaic current flowing through the working electrode.

[0094] . The change in electron transfer kinetics manifests as a change in current that is detectable by the ammeter “A” in the circuit of FIG. 4. For example, where the working electrode is interrogated by square wave voltammetry, a current transient is generated in the circuit. The current transient is detectably different dependant on whether the redox reporter is bound to the aptamer binding site or is unbound after displacement by the target analyte.

[0095] . Of the various embodiments discussed above, it will be evident that the fluorescence-read embodiment is the most simple given the lack of any requirement for covalent bonding of redox porter to the aptamer, andthe lack of any need to connect the aptamer to a surface.

[0096] . As an alternative to the use of fluorescence intensity or current as described above, the present methods may exploit methods of determining molecular binding to detect displacement of a redox reporter from an aptamer by a target analyte. An exemplary method is Surface Plasmon Resonance (SPR). SPR is a label-free method capable of measuring real-time quantitative binding affinities, kinetics, and thermodynamic parameters of interacting molecules. SPR provides moderately high-throughput, while consuming relatively small quantities of sample.

[0097] . A further alternative to the use of fluorescence intensity or current is Isothermal Titration Calorimetry (ITC), which may be used for quantitative thermodynamic characterisation of a wide variety of biomolecular interactions by directly measuring the heat that is either released or absorbed during a biomolecular binding event. ITC requires no modification of binding partners, with fluorescent tags or through immobilisation, but it requires a significantly larger amount of sample compared with similar techniques.

[0098] . A further alternative to the use of fluorescence intensity or current is BioLayer Interferometry (BLI) which uses white light interferometry to quantify biomolecules which are typically adsorbed to the tips of optical fibres. White light travelling through an optical fibre is reflected at the fibre -biomolecular layer interface and at the biomolecular layerbuffer interface. The reflected beams interfere, generating a signal that directly depends on the amount of adsorbed molecules. Since the measurement is performed using optical fibres instead of using a fluidic system, it is possible to deliver the sample liquids to the sensor by dipping the optical fibres into well plates. Similar to SPR, immobilization of the ligandto the surface of the tip is required. BLI provides direct binding affinities and rates of association and dissociation as well as concentration measurements. Given that only substances bound to the sensor surface are detected, the medium surrounding the sensor does not influence the signal and thus no reference channel is needed.

[0099] . MicroScale Thermophoresis (MST) is performed using thin capillaries in free solution, which is comparable to ITC measurements. When performing an MST experiment, a microscopic temperature gradient is induced by an infrared laser, and the directed movement of molecules is detected by intrinsic fluorescence or in most cases, fluorescent labels of one interactant and quantified. The thermophoretic movement of molecules within the temperature gradient depends on size, charge, hydration shell or conformation that typically changes upon interaction. The thermophoresis signal is plotted against the ligand concentration to obtain a dose -response curve, from which the binding affinity can be deduced. The dynamic affinity range covers the pM to mM range, which is comparable to SPR measurements. MST requires a small amount of sample, and the technique is relatively easy to use.

[0100] . Given the benefit of the present disclosure, the skilled person may conceive of other means for detecting displacement of the redox reporter from the aptamer.

[0101] . It will be appreciated from the methods disclosed above that any redox reporter that is displaceable from the aptamer by the target analyte is contemplated to be operable in the present invention. This includes all presently known redox reporters and any redox reporters identified in the future. Redox reporters presently known (apart from methylene blue and thioflavin T) include viologen, Nile blue, ferrocene, vinylferrocene, anthraquinone, thionine, ethidium bromide, daunomycin, anthraquinone-C5, dabcyl, 2,6- dichlorophenal-indophenol, gallocyanine, ROX, pentamethylferrocene, ferrocene-C5, neutral red, and horseradish peroxidase, ruthenium, bis-pyridine, tris-pyridine, bis- imidizole, cytochrome c, cytochrome c’, and plastocyanin.

[0102] . Methods for fabricating a working electrode from an aptamer identified by a method as described herein are known to the skilled artisan, as are methods for assembling the working electrode into a functioning EAB sensor.

[0103] . EAB sensors incorporating aptamers identified by the present disclosure may be used in any application deemed suitable by the skilled artisan, including in vivo and ex vivo applications.

[0104] . For in vivo applications, the EAB sensor working electrode may be a needle or a microneedle configured to pierce the skin and contact the interstitial fluid of the subject. Alternatively, the working electrode may be a wire disposed in a blood vessel of a subject.

[0105] , Ex vivo applications may include pathology testing of a biological fluid taken from a subject and disposed in a container. The analysis of water and other environmental materials is further contemplated.

[0106] . The present disclosure will now be more fully described by reference to the following non-limiting example.EXAMPLE 1: Detection of displacement of methylene blue from a DNA aptamer in a fluorescence microplate assay format

[0107] . Experimental work was performed to demonstrate that a fluorescence microplate format could be used to detect displacement of methylene blue (“MB”) from a vancomycinsensitive aptamer by vancomycin. The method is based on the theoretical model of FIG. 1A, FIG. IB and FIG. 1C whereby the methylene blue and candidate aptamer are each free in solution.

[0108] . Chemicals and consumables

[0109] . Instruments

[0110] . Aptamers

[0111] . Experimental procedureFolding buffer preparationAdd 2 ml of 1 M MgCh into 1 L PBS. The final concentration of MgCh is 2 mM.Aptamers preparation a. Resuspend the aptamers using IDTE buffer to form a 100 pM solution. b. Dilute the aptamers solution to 12 pM working concentration in folding buffer. c. Fold the aptamers by heating them to 95 °C for 8 min, cooling down to 4°C for 10 min, and returning to 22°C for 15 min.Methylene blue preparationResuspend methylene blue using ultrapure water to form a 5 mM solution. Then further dilute it to 12 pM in folding buffer.Vancomycin preparationResuspend vancomycin using ultrapure water to form a 20 mM solution. Then further dilute it to 1.2 mM working solution in folding buffer.

[0112] . MB displacement assayTo investigate the ability of aptamers to quench MB fluorescence, mix 50 pF MB working solution with 50 pF aptamer working solution and 50 pF folding buffer. For target displacement experiments, mix 50 pF of vancomycin working solution with equal volumes of MB working solution and aptamer working solution.Measurements were conducted in triplicate in 96-well black plates using a plate reader (GM3000, Promega, Madison, US) to record MB fluorescence at excitation and emission wavelengths of 627 / 660-729 nm.

[0113] . Data analysis a. Calculate aptamer’s capacity to quenching MB.MB quenching = (fluorescence of free MB) - (fluorescence of the MB and aptamers mixture) MB quenching (%) = (MB quenching) / (fluorescence of free MB) x 100% b. Calculate target’s capacity to displace aptamer-binding MB.MB Dequenching = (fluorescence of the MB, aptamers and vancomycin mixture) - (fluorescence of the MB and aptamers mixture)MB dequenching / quenching (%) = MB Dequenching / MB quenching x 100%

[0114] . ResultsReference is made to FIG. 5A, demonstrating that PIK45 (a known vancomycin-sensitive aptamer) demonstrated a higher percentage of MB quenching compared to PIK45NC4 (a negative control), indicating stronger binding affinity of PIK45 to MB than for PIK45NC4. PIK45 also showed higher percentage of MB dequenching / quenching compared to PIK45NC4, demonstrating the ability of PIK45 to bind to vancomycin whereas the negative control aptamer PIK45NC4 lacks binding capacity to vancomycin. FIG. 5B demonstrates the concentration-dependence of Vanc45 (a known vancomycin-sensitive aptamer) to increasing concentrations of vancomycin, with higher concentrations causing increased % difference in MB fluorescence. These data support the successful establishment of a MB displacement assay to screen aptamers that can bind to both MB and vancomycin. These data further support the hypothesis that useful aptamers bind a redox reporter at first instance, with introduction of the relevant analyte causing displacement of the reporter from the aptamer.EXAMPLE 2: Detection of displacement of thioflavin T from a DNA aptamer in a fluorescence microplate assay format

[0115] . Experimental work was performed to demonstrate that a fluorescence microplate format could be used to detect displacement of thioflavin T (“ThT”) from a vancomycinsensitive aptamer by vancomycin. The method is based on the theoretical model of FIG. 1A, FIG. IB and FIG. 1C whereby the ThT and candidate aptamer are each free in solution.

[0116] . Chemicals and consumables

[0117] . Instruments

[0118] . AptamersExperimental procedure

[0119] . Folding buffer preparation2 ml of 1 M MgCh was added into 1 L PBS. The final concentration of MgCh was 2 mM.

[0120] . Aptamers preparationThe aptamers were resuspended using IDTE buffer to form a 100 pM solution.The aptamer solution was diluted to 12 pM working concentration in folding buffer.The aptamers were folded by heating to 95 °C for 8 minutes, and cooled down to 4°C for 15 minutes, and then returned to 22°C for 15 minutes.ThT preparation

[0121] . ThT was resuspended using ultrapure water to form a 5 mM solution. Then further diluted to 60 pM in folding buffer.

[0122] . V ancomycin preparationVancomycin was resuspended using ultrapure water to form a 20 mM solution. Then further diluted to 1.2 mM working solution in folding buffer.

[0123] . ThT displacement assayTo investigate the ability of aptamers to enhance ThT fluorescence, 50 pL ThT working solution was mixed with 50 pL aptamer working solution and 50 pL folding buffer. For vancomycin displacement experiments, 50 pL of vancomycin working solution was mixed with 50 pL of ThT working solution and 50 pL of aptamer working solution. Measurements were conducted in triplicate in 96-well black plates using a plate reader (GM3000, Promega, Madison, US) to record ThT fluorescence by excitation at 405 nm, and a bandpass emission filter with wavelengths 495-505 nm.

[0124] . Data analysisAnalysis was based on the aptamer’s capacity to enhance the ThT fluorescence, then how the vancomycin modulates the ThT fluorescence when added and displaces the ThT from the aptamer. This formula was utilized: ((Fluorescence intensity of ThT in the presence of vancomycin and aptamer) - (Fluorescence intensity of ThT in the presence of aptamer) ) / [(Fluorescence intensity of ThT in the presence of aptamer - fluorescence intensity of ThT only) x 100%]

[0125] . ResultsReference is made to FIG. 6A, demonstrating that PIK45 (a known vancomycin-sensitive aptamer) demonstrated a higher ThT fluorescence enhancement compared to PIK45NC4 (a vancomycin-insensitive negative control aptamer), indicating stronger binding affinity of ThT for PIK45 than ThT for PIK45NC4. PIK45 also showed higher percentage of ThT fluorescence quenching in the presence of vancomycin compared to PIK45NC4, demonstrating the ability of PIK45 to bind to vancomycin, thus displacing ThT, whereas the negative control aptamer PIK45NC4 lacks binding capacity to vancomycin. These dataindicate a ThT displacement assay is to screen aptamers that can bind to both ThT and vancomycin.

[0126] . FIG. 6B demonstrates the concentration-dependence of PIK45 to increasing concentrations of vancomycin, with higher concentrations causing increased quenching of fluorescence. These data further support the hypothesis that useful aptamers bind a redox reporter at first instance, with introduction of the relevant analyte causing displacement of the reporter from the aptamer.EXAMPLE 3: High throughput screening of candidate aptamer sequences for suitability in an EAB sensor

[0127] . The methods described herein are amenable to high throughput screening of candidate aptamers. The candidate aptamers may be a random library or a curated library of oligomers having at least a subpopulation likely to be useful in an EAB sensor configured to detect a target analyte.

[0128] . Reference is made to FIG. 7A showing an analytical surface (60) to which a plurality of candidate aptamers (65) are attached. Methylene blue (70) is attached to the free end of each candidate aptamer (65). For some of the candidate aptamer (65a, 65c) the methylene blue (70) is bound to a target analyte binding site of the aptamer. For candidate aptamer (65b) the methylene blue (70) is bound to the aptamer but away from a target analyte binding site. Some candidate aptamers (65d) are not bound to the aptamer at all.

[0129] . FIG. 7B is a plan view of the analytical surface (60) showing the relative light output of each aptamer (the light output being in fact the fluorescence of the methylene blue attached to the aptamer). For each of candidate aptamers (65s, 65b, 65c) the fluorescence is quenched by binding with the aptamer DNA. The methylene blue of candidate aptamer (65d) is unquenched, and therefore has a relatively high light output.

[0130] . At FIG 7C, the analytical surface is contacted with a solution of target analyte (70). The target analyte (70) binds to each of candidate aptamers (65a, 65b and 65d), but not candidate aptamer (65c).

[0131] . Referring to FIG. 7D, it will be clear that only candidate aptamer (65a) changes from low light output to a high light output. Candidate aptamer (65 a) is therefore the only aptamer where the methylene blue (70) is initially bound to a target analyte binding site,with that biding being disrupted by the addition of target analyte thereby leading to dequenching of the methylene blude. According to the present methods, only candidate aptamer (65a) is suitable for use in an EAB sensor, because that is the only aptamer that displaces methylene blue (70) from target analyte binding site so as to change the distance between the analytical surface (60) and the methylene blue (70). The analytical surface (60) is analogous to the surface of a working electrode in an EAB sensor, accordingly the movement of methylene blue (70) seen in this screening method will translate to a change in current passing through the working electrode of an EAB sensor upon interrogation. It is that change in current that is indicative of the presence of target analyte about the working electrode.

[0132] . The remaining candidate aptamers (65b, 65c, 65d) will provide no signal response, or an inferior signal response to the presence of target analyte about the working electrode of an EAB sensor when exposed to target analyte. For these aptamers exposure to target analyte fails to displace methylene blue (70) from the target analyte binding site, and accordingly the distance between the analytical surface (60) and the methylene blue (70) does not substantially change. Accordingly, when used to functionalise the working electrode of an EAB sensor, current passing through the electrode does not substantially change upon exposure to target analyte.

[0133] . Suitable candidate aptamer (65a) may be isolated and amplified by PCR to provide a pool of identical aptamers that may be used to functionalise a working electrode of an EAB sensor.

[0134] . Although this embodiment is described by reference to only four candidate aptamers, typical applications will screen thousands or millions of candidate aptamers contemporaneously .

[0135] . By this embodiment, immobilised candidate aptamers may be tested against different target analytes to assess specificity, affinity and other binding characteristics. Single analyte species or cocktails of analyte species may be tested. Non-target species (single and cocktails) may be tested, and optionally with one or more target analytes.

[0136] . An apparatus for the high throughput screening of candidate aptamers according to this Example (and also other applications of the present disclosure) may be constructed de novo. Alternatively, an apparatus may be repurposed for use in the context of the presentdisclosure. As one example, it is proposed the Agilent™ microarray system (CA, United States) is amenable to such modification. That apparatus comprises a solid surface on which a custom ordered array of nucleic acids, such as aptamers, are immobilised by any suitable method, such as by hybridisation to the array surface. Techniques such as spotting, printing with fine -pointed pins, ink-jet printing, photolithography and the like may be used. AS an alternative, chemical coupling by way of hydroxy’ carboxyl, NHS-ester, amine, PDITC, aldehyde, expoxy, azide and gold may be implemented. The aptamers may bind to the microarray surface at, or toward, the 5’ or 3’ terminus, or in a more central region. Indirect immobilization by, for example, biotinylated aptamers binding to a streptavidin- functionalised surface.. The arrayed nucleic acids can be contacted with fluids which contain the redox reporter and / or the target analyte and any other necessary reagents. The array can be interrogated by standard microarray fluorescence readers provided by Agilent™ or other vendors.

[0137] . As another example, it is proposed that the Miseq™ next-generation DNA sequencing apparatus of Illumina, Inc (CA, United States) is amenable to such modification. That apparatus comprises a flow cell having an analytical surface to which candidate aptamers may be immobilised randomly, and flow paths for delivering target analyte and any other necessary reagents. The apparatus further comprises a light source and a fluorescence reader. Firmware of the MiSeq™ apparatus may be modified so as to perform the method steps of the present disclosure. The flow cell is removable such that a separate fluidics system, excitation light source, and fluorescence reader may be used. Aptamers may be immobilised by any suitable method such as those detailed above for the Agilent™ system.EXAMPLE 4: High throughput screening of aptamer library for aptamer sensitive to vancomycin using MiSeq™ apparatus.

[0138] . Step 1: A vancomycin aptamer library comprised of 410 (1,048,576) unique sequences is generated by randomizing ten bases within a known vancomycin-sensitive aptamer sequence. This library is appended with the necessary flanking sequences (e.g. 'P5' and 'P7', and 'Index sequences') at both the 5' and 3' ends to enable next-generation DNA sequencing on the Illumina™ MiSeq™. The resulting library is synthesized by standard phosphoramidite-based DNA synthesis chemistry and purified by HPLC.

[0139] . Step 2: Upon receipt, the aptamer library is resuspended in 10 mM Tris, pH 8.0, 1 mM EDTA to a nominal concentration of 100 M. The resuspended aptamer library is quantified using the single-stranded DNA quantification kit on the Qubit™ fluorimeter. The Qubit™-measured concentration is used as the concentration for subsequent steps.

[0140] . Step 3: The aptamer library is prepared for high throughput sequencing by dilution and spiking with PhiX DNA as per the Illumina™-recommended protocol.

[0141] . Step 4: High throughput DNA sequencing of the aptamer library is executed on a modified MiSeq™ apparatus (having modified firmware) using the V3 150 MiSeq™ reagent kit as per the Illumina™ protocol.

[0142] . Step 5: After the DNA sequencing is completed, the chip is further treated as follows.

[0143] . Step 5 a: Blocking with dideoxy cytidine triphosphate (ddCTP): The distal ends of the DNA strands attached to the flow-cell are blocked with ddCTP by twice incubating the flow-cell for 45 minutes at 37°C with the following mixture: 16 pL terminal transferase enzyme (New England Biolabs, catalog # M0315L), 45 pL 2 mM ddCTP (Jena Bioscience, catalog # NU-1016L) , 40 pL 10X terminal transferase buffer (New England Biolabs, catalog # B0315SVIAL), 40 pL 2.5 mM C0CI2, and 259 pL water.

[0144] . Step 5b: Wash: the flow-cell is washed with 50 mM NaOH + 1% SDS, followed by binding buffer (IX phosphate buffered saline, pH 7.4, 2 mM MgCh), both at 22°C.

[0145] . Step 5c: Addition of complement strand: the flow-cell is incubated with 1 M complement strand in binding buffer for 15 minutes at 22 °C to attach the complement strand to the immobilized aptamer library by binding to the distal end.

[0146] . Step 5d: Endonuclease cleavage: the flow-cell is incubated with the endonucleaseDdel solution comprised of 10 pL Ddel enzyme (New England Biolabs, catalog # R0175L), 30 pL 10X rCutsmart buffer (New England Biolabs, catalog # NEB #B6004), and 260 pL water for 30 minutes at 37°C.

[0147] . Step 5e: Steps 5c and d are repeated (addition of complement strand and Ddel endonuclease digestion).

[0148] . Step 5f: Wash: the flow-cell is washed with 50 mM NaOH + 1% SDS, followed by binding buffer (IX phosphate buffered saline, pH 7.4, 2 mM MgCh), both at 22°C.

[0149] . Step 5g: End-labelling with 5-propargylamino-ddCTP-ATTO-MB2 (ddCTP-MB):The distal ends of the immobilized aptamer library are labelled with ddCTP-MB (Jena Bioscience, catalog # NU-850-MB2) by twice incubating the flow-cell for 45 minutes at 37°C with the following mixture: 16 pL terminal transferase enzyme, 45 L 2 mM ddCTP- MB, 40 pL 10X terminal transferase buffer, 40 pL C0CI2 buffer, and 259 pL water.

[0150] . Step 6: After this process (steps 5a through 5g) the DNA aptamers on the flow-cell are distally end-labelled with methylene blue.

[0151] . Step 7: A vancomycin titration is added to the flow-cell in step-wise increasing concentrations of 0, 0.5, 1, 2.5, 5, 10, 25, 50, and 100 pM vancomycin in binding buffer at 37 °C. Each concentration was incubated on the flow-cell for 5 minutes before the entire flow cell is imaged, the next higher concentration was added, etc. During this step the flowcell is imaged at each concentration using the Cy5 laser and emission filter set on board the MiSeq™, as those are the best excitation and emission wavelength match for methylene blue on that instrument.

[0152] . Step 8: The flow cell is washed with 50 mM NaOH + 1% SDS, followed by binding buffer (IX phosphate buffered saline, pH 7.4, 2 mM MgCh), both at 22°C.

[0153] . Step 9: Steps 7 and 8 (the addition of a step-wise vancomycin titration, using the same analyte concentrations, followed by washing) are repeated twice, for a total of three vancomycin titrations.

[0154] . Step 10: The images are analyzed to correlate the various aptamers within the aptamer library with their location on the flow-cell with their methylene blue fluorescence at the various vancomycin concentrations.

[0155] . Step 11: Those aptamers that showed the highest increase in methylene blue fluorescence across the three vancomycin titrations are identified as the best performing aptamers. In other words, increasing vancomycin concentrations leads to increased methylene blue fluorescence in the best aptamers.

[0156] . 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.

[0157] . 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

CLAIMS1. A method for identifying an analyte recognition element suitable for use in an electrochemical sensor for sensing a target analyte, the method comprising the step of determining whether the target analyte competitively binds with the analyte recognition element, wherein competitive binding in favour of the target analyte is indicative of the suitability of the analyte recognition element for an electrochemical sensor configured to detect the target analyte.

2. The method of claim 1, wherein competitive binding is determined by the steps of: providing a candidate analyte recognition element and a redox reporter species, allowing the redox reporter species to associate with the candidate analyte recognition element, exposing the candidate analyte recognition element to the target analyte, and determining any displacement of the redox reporter species from the candidate analyte recognition element, wherein displacement is indicative of the suitability of the analyte recognition element for an electrochemical sensor configured to detect the target analyte.

3. The method of claim 1, wherein competitive binding is determined by the steps of: providing a candidate analyte recognition element and a target analyte, allowing the target analyte to associate with the candidate analyte recognition element, exposing the candidate analyte recognition element to the redox reporter species, and determining any displacement of the target analyte from the candidate analyte recognition element, wherein displacement is indicative of the lack of suitability of the analyte recognition element for an electrochemical sensor configured to detect the target analyte.

4. The method of any one of claims 1 to 3, wherein the candidate analyte recognition element is separate to the redox reporter species.

5. The method of any one of claims 1 to 4, wherein the candidate recognition element is not bound or otherwise associated with a surface or a body.

6. The method of any one of claims 1 to 5, wherein the candidate recognition element is not bound or otherwise associated with a working electrode of an electrochemical cell.

7. The method of any one of claims 1 to 6, wherein the displacement is determined by a change in a characteristic of the redox reporter species or the candidate recognition element.

8. The method of claim 7, wherein the characteristic is absorption and / or emission of electromagnetic energy.

9. The method of claim 8, wherein the electromagnetic energy is light energy.

10. The method of claim 9, wherein the light energy is ultraviolet light energy, visible light energy, or infrared light energy.

11. The method of any one of claims 7 to 10, wherein the characteristic is fluorescence of the redox reporter species.

12. The method of claim 11, wherein (i) florescence of the redox reporter species is quenched when associated with the candidate analyte recognition element, and dequenched when displaced from the candidate analyte recognition element; or (ii) florescence of the redox reporter species is dequenched when associated with the candidate analyte recognition element, and quenched when displaced from the candidate analyte recognition element.

13. The method of any one of claims 1 to 12, wherein the displacement is determined by an electrochemical method.

14. The method of claim 13, wherein the electrochemical method determines displacement by reference to a current through a working electrode having the candidate analyte recognition element connected thereto.

15. The method of any one of claims 1 to 14, wherein the displacement is determined by a method for investigating binding between the redox reporter species and the candidate analyte recognition element and / or the target analyte and the candidate analyte recognition element.

16. The method of any one of claims 1 to 15, wherein the redox reporter species is selected from: methylene blue, thioflavin T, viologen, Nile blue, ferrocene, vinylferrocene, anthraquinone, thionine, ethidium bromide, daunomycin, anthraquinone -C5, dabcyl, 2,6- dichlorophenal-indophenol, gallocyanine, ROX, pentamethylferrocene, ferrocene -C5, neutral red, and horseradish peroxidase, ruthenium, bis-pyridine, tris-pyridine, bis-imidizole, cytochrome c, cytochrome c’, and plastocyanin; or a functional equivalent thereof of any of the aforementioned redox reporter species.

17. The method of any one of claims 1 to 16, wherein the candidate analyte recognition element is a nucleic acid (including DNA and DNA) or an analogue thereof (including XNA), or a hybrid nucleic acid (including PNA).

18. The method of any one of claims 1 to 17, wherein the candidate analyte recognition element is an aptamer.

19. The method any one of claims 1 to 18, wherein the step of determining any displacement is determined by reference to exposure of the candidate analyte recognition element to a solution comprising less analyte or zero analyte.

20. The method of any one of claims 7 to 19, wherein the change in a characteristic of the redox reporter species is determined by reference to exposure of the candidate analyte recognition element to a solution comprising less analyte or zero analyte.

21. The method of any one of claims 1 to 20, which is performed in a high-throughput method, including a microplate format, a random or ordered microarray format, or a multi-vessel array format, with a different candidate analyte recognition element being tested in each well of the microplate, random or ordered microarray spot, or each vessel of the array.

22. The method of any one of claims 1 to 21, wherein the candidate analyte recognition element is obtained from a selection method starting with a diverse pool of analyte recognition elements.

23. The method of claim 22, wherein the selection method is based on the affinity of the candidate analyte recognition element for the target analyte.

24. The method of claim 22 or claim 23, wherein the selection method is a sequential evolution of ligands by exponential enrichment (SELEX) method, or other enrichment method.

25. A method for screening a plurality of different analyte recognition elements for suitability in an electrochemical sensor for sensing a target analyte, the method comprising immobilizing each of the plurality of different analyte recognition elements and exposing the immobilized plurality of different analyte recognition elements to a target analyte and a redox reporter species so as to determine whether the target analyte competitively binds with the analyte recognition element, wherein competitive binding in favour of the target analyte is indicative of the suitability of the analyte recognition element for an electrochemical sensor configured to detect the target analyte.

26. The method of claim 25, wherein competitive binding is determined by the method of any one of claims 1 to 24.

27. The method of claim 25 or claim 26, wherein each of the plurality of different analyte recognition elements is immobilized on a surface.

28. The method of claim 27, wherein the plurality of different analyte recognition elements are arranged on the surface in a location-specific manner29. The method of claim 27 or claim 28, wherein the plurality of different analyte recognition elements are arranged on the surface in an array.

30. The method of any one of claims 25 to 29, wherein the surface is part of a flow cell, a microwell, a microarray, a microbead, or another analytical structure.

31. The method of any one of claims 25 to 30 wherein the flow cell, the microwell, the microbead, the microarray, or another analytical structure is part of an automated fluidic system or a microfluidic system.

32. The method of any one of claims 25 to 31, wherein the plurality of different analyte recognition elements comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 100000, 1000000, or 10000000 different analyte recognition elements.

33. An apparatus for screening a plurality of different analyte recognition elements for suitability in an electrochemical sensor for sensing a target analyte, the apparatus comprising immobilizing each of the plurality of different analyte recognition elements immobilized thereon, one or more flow paths for exposing the immobilized plurality of different analyte recognition elements to a solution of target analyte and a solution of redox reporter species.

34. The apparatus of claim 33, comprising a reading device configured to determine directly or indirectly whether the target analyte competitively binds with the analyte recognition element.

35. The apparatus of claim 34, wherein the reading device is configured to detect absorption and / or emission of electromagnetic energy, including light energy, ultraviolet light energy, visible light energy, or infrared light energy.

36. The apparatus of claim 35 wherein the reading device comprises an energy source configured to excite a fluorescent compound.

37. An analyte recognition element identified as suitable for use in an electrochemical sensor by the method of any one of claims 1 to 32, or the apparatus of any one of claims 33 to 36.

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