Electrochemical temperature measurement using redox-active polymers

Abstract

A temperature sensing apparatus having an electrode with an associated redox-active polymer. The electrode is connected to circuitry which applies an interrogating electrical potential waveform. The circuitry measures an electrical current resulting from application of the electrical potential waveform. The measured electrical current is processed to determine the temperature of a solution contacting the electrode. The apparatus may further comprise an analyte sensing redox-active polymer for sensing the amount of an analyte in the solution.

Description

[0001] IRN20105094

[0002] ELECTROCHEMICAL TEMPERATURE MEASUREMENT USING REDOX- ACTIVE POLYMERS

[0003]

[0001] This invention was made with government support under EB022015 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004] FIELD

[0005]

[0002] The present disclosure relates generally to electrical temperature sensors. More particularly, the disclosure is directed to electrochemical sensors useful in measuring the temperature of a biological fluid.

[0006] BACKGROUND

[0007]

[0003] For many reasons it may be necessary to determine the temperature of a solution. As just one example, the temperature of a biological fluid in situ within an animal may be required to correct methods of determining the amount of a target analyte in the fluid for temperature deviations away from a reference temperature.

[0008]

[0004] Electrochemical sensors show significant promise in providing real time concentration of an analyte in the body of an animal. For example, the concentration of drug in the interstitial fluid of a diabetic individual may be continuously monitored so as to warn of any excursion outside of the safe and effective concentration range. Enzymatic and aptamer-based electrochemical sensors may be employed for such applications. Other clinically relevant analytes detectable by electrochemical methods include haemoglobin Ale (for diabetes), calcium (for kidney disease), troponin and cholesterol / lipids (for heart disease), prostate specific antigen (for prostate cancer), D-dimer (for clotting disorders), and C-reactive protein (for infection and inflammatory disorders).

[0009]

[0005] Accurate determination of analyte concentration by electrochemical means is reliant on an accurate determination of the temperature of the solution comprising the analyte. Any standard curve for an analyte generated using an electrochemical sensor will be relevant only for the temperature at which the curve was generated (the reference temperature). Thus, a standard curve generated at 37°C will allow for an accurate reading of analyte concentration only where the test solution is precisely at 37°C. The temperature of fluids, tissues and compartments within the body of an animal vary in the course of a day, and accordingly no single standard curve will necessarily be useful in determining analyte concentration. Accordingly, electrochemical methods utilize a number of standard curves, each generated at a slightly different temperature, covering the range of expected temperatures. Selection of the relevant standard curve is utilised based on the temperature, or estimated temperature, of the test fluid. Alternatively, analyte concentration may be IRN20105094 determined by reference to a continuous or discontinuous equation having temperature as an input.

[0010]

[0006] A problem arises in the critical need to accurately determine the temperature of the test fluid. Where the test fluid is interstitial fluid, a thermistor contacting the skin surface may be used. As the thermistor is not in direct contact with interstitial fluid, however, the temperature recorded by the thermistor can only ever be an approximation. The actual temperature of the fluid will certainly be different to that recorded at the skin surface, and accordingly an error will be inevitably introduced into the determination of analyte concentration.

[0011]

[0007] In any event, the addition of a thermistor or any other contrivance as a sensor separate to the analyte sensing functions and components of an electrochemical sensor introduces complexity, cost, weight and bulk.

[0012]

[0008] It is an aspect of the present disclosure to provide an improvement to prior art apparatus and methods for the determination of temperature in a solution or in a tissue or in some other sample environment. It is a further aspect of the present disclosure to provide a useful alternative to prior art temperature measurement apparatus and methods.

[0013]

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

[0014] SUMMARY

[0015]

[0010] In a first aspect, but not necessarily the broadest aspect, the present disclosure provides an apparatus for sensing thermal energy in a solution, the apparatus comprising: an electrically conductive element having a polymer associated therewith, the polymer being inherently redox-active or having a redox-active species associated therewith; circuitry in electrical connection with the electrically conductive element, the circuitry configured to apply an electrical potential waveform to the conductive element and to measure an electrical current resulting from application of the electrical potential waveform; an electronic processor in operable connection with the circuitry and configured to by way of program instructions process the measured electrical current so as to determine a thermal energy parameter of the solution; and a power source in operable connection with the circuitry.

[0016]

[0011] In one embodiment of the first aspect, the polymer has a three-dimensional conformation, and the three-dimensional conformation changes in response to a change in thermal energy thereabout, the conformational change in turn changing kinetics, including a rate of an electron transport between (i) the electrically conductive element and the IRN20105094 inherently redox-active polymer or (ii) the electrically conductive element and the redoxactive species.

[0017]

[0012] In one embodiment of the first aspect, the change in conformation causes:

[0018] (i) a redox-active portion of the inherently redox-active polymer to be more or less proximal to the electrically conductive element, or to be more or less proximal to the electrically conductive element for a greater portion of a time period; or

[0019] (ii) the redox-active species to be more or less proximal to the electrically conductive element, or to be more or less proximal to the electrically conductive element for a greater portion of a time period; or

[0020] (iii) the redox-active species to change the rate with which it becomes more or less proximal to the electrically conductive element.

[0021]

[0013] In one embodiment of the first aspect, the program instructions are configured to determine a temperature, or a change in temperature.

[0022]

[0014] In one embodiment of the first aspect, the circuitry is configured to apply a potential waveform at a first frequency (optionally a square wave frequency or an AC frequency) and a second frequency (optionally a square wave frequency or an AC frequency), and the thermal energy parameter is determined by the program instructions by reference to a relationship (optionally a ratio) of a current resulting from application of the potential waveform at the first frequency to a current resulting from the application of the potential waveform at the second frequency.

[0023]

[0015] In one embodiment of the first aspect, the electrically conductive element has a surface, and the polymer is connected to the surface.

[0024]

[0016] In one embodiment of the first aspect, the connection is, or comprises, a covalent chemical bond, a pseudo-covalent bond, or an electrostatic bond.

[0025]

[0017] In one embodiment of the first aspect, the polymer has a first terminus and a second terminus and the connection is at, or near, the first terminus and the second terminus is free.

[0026]

[0018] In one embodiment of the first aspect, the redox-active species is connected to the polymer.

[0027]

[0019] In one embodiment of the first aspect, the redox-active species is connected at, or near, the second terminus of the polymer.

[0028]

[0020] In one embodiment of the first aspect, the connection is, or comprises, a covalent chemical bond, a pseudo-covalent bond, or an electrostatic bond.

[0029]

[0021] In one embodiment of the first aspect, the polymer is an organic polymer, or an inorganic polymer, or a hybrid thereof.

[0030]

[0022] In one embodiment of the first aspect, the polymer is a biological polymer, or an analogue thereof.

[0031]

[0023] In one embodiment of the first aspect, the biological polymer or analogue thereof is selected from a DNA molecule, an RNA molecule, a xenonucleic acid (XNA) molecule, a peptide nucleic (PNA) molecule, a polypeptide, a lipid, and a polysaccharide. IRN20105094

[0032]

[0024] In one embodiment of the first aspect, the polymer is a non-biological polymer.

[0033]

[0025] In one embodiment of the first aspect, the non-biological polymer comprises a linear or a branched carbon chain.

[0034]

[0026] In one embodiment of the first aspect, the non-biological polymer is biologically compatible with a human or a non-human animal.

[0035]

[0027] In one embodiment of the first aspect, the non-biological polymer is incapable of instigating or facilitating a pathological biological response in a human or a non-human animal.

[0036]

[0028] In one embodiment of the first aspect, the pathological biological response is an immune response or an inflammatory response.

[0037]

[0029] In one embodiment of the first aspect, the non-biological polymer is substantially non-toxic to a human or a non-human animal, or substantially non-cell binding, or substantially non-protein binding, or substantially non-lipid binding, or substantially nonvolatile.

[0038]

[0030] In one embodiment of the first aspect, the non-biological polymer is hydrophilic overall.

[0039]

[0031] In one embodiment of the first aspect, the non-biological polymer is a poly(ethyleneglycol) or another polyether species.

[0040]

[0032] In one embodiment of the first aspect, the polymer is formed from about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more of the monomer, or where the polymer is a copolymer the copolymer is formed from a total of from about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more of the constituent monomers.

[0041]

[0033] In one embodiment of the first aspect, the apparatus comprises an analyte sensing element.

[0042]

[0034] In one embodiment of the first aspect, the analyte sensing element is associated with a surface of the electrically conductive element, or a surface of another electrically conductive element,

[0043]

[0035] In one embodiment of the first aspect, the another electrically conductive element is in operable connection with the circuitry, or another circuitry such that a potential can be applied to the another electrically conductive element and a current measured, and the processor, or another processor is configured by way of program instructions to determine the amount of the analyte.

[0044]

[0036] In one embodiment of the first aspect, the processor or another processor, or a further processor is configured by way of program instructions to determine the amount of the analyte by reference to the thermal energy parameter determined by the apparatus.

[0045]

[0037] In one embodiment of the first aspect, the analyte sensing element is an aptamer or a functional equivalent thereof. IRN20105094

[0046]

[0038] In one embodiment of the first aspect, the aptamer is a DNA aptamer, an RNA aptamer, an XNA aptamer, or a PNA aptamer.

[0047]

[0039] In one embodiment of the first aspect, the electrically conductive element and the another electrically conductive element are configured as electrodes in an electrochemical sensor.

[0048]

[0040] In one embodiment of the first aspect, the electrically conductive element and the another electrically conductive element is each a wire, a needle or a microneedle.

[0049]

[0041] In one embodiment of the first aspect, the solution is a biological fluid within or about a human or a non-human animal.

[0050]

[0042] In one embodiment of the first aspect, the biological fluid is an 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, or an exudate.

[0051]

[0043] In one embodiment of the first aspect, the biological fluid is in situ within the body of a human or a non-human animal.

[0052]

[0044] In one embodiment of the first aspect, the biological fluid is selected from blood, interstitial fluid, or a mixture of blood and interstitial fluid.

[0053]

[0045] In one embodiment of the first aspect, the electrically conductive element and the further electrically conductive element are each configured so as to pierce the skin of a human or a non-human animal such that the polymer and the analyte sensing element contact the blood and / or the interstitial fluid of the animal.

[0054]

[0046] In a second aspect, the present disclosure provides an electrochemical sensor comprising the apparatus of any embodiment of the first aspect.

[0055]

[0047] In one embodiment of the second aspect, the electrochemical sensor comprises a counter electrode in operable connection with the electrically conductive element.

[0056]

[0048] In one embodiment of the second aspect, the electrochemical sensor comprises a counter electrode in operable connection with the another electrically conductive element.

[0057]

[0049] In one embodiment of the second aspect, the electrochemical sensor comprises a reference electrode against which output of the electorally conductive element and / or the another electrically conductive element are referred.

[0058]

[0050] In a third aspect, the present disclosure comprises a method for sensing thermal energy in a solution, the method comprising: providing an electrically conductive element having a polymer associated therewith, the polymer being inherently redox-active or having a redox-active species associated therewith; applying an electrical potential waveform to the conductive element and measuring an electrical current resulting from application of the electrical potential waveform; and IRN20105094 using the measured electrical current to determine a thermal energy parameter of the solution.

[0059]

[0051] In one embodiment of the third aspect, the polymer has a three-dimensional conformation, and the three-dimensional conformation changes in response to a change in thermal energy thereabout, the conformational change in turn changing kinetics, including a rate, of an electron transport between (i) the electrically conductive element and the inherently redox-active polymer or (ii) the electrically conductive element and the redoxactive species.

[0060]

[0052] In one embodiment of the third aspect, the change in conformation causes:

[0061] (i) a redox-active portion of the inherently redox-active polymer to be more or less proximal to the electrically conductive element, or to be more or less proximal to the electrically conductive element for a greater portion of a time period; or

[0062] (ii) the redox-active species to be more or less proximal to the electrically conductive element, or to be more or less proximal to the electrically conductive element for a greater portion of a time period.

[0063]

[0053] In one embodiment of the third aspect, the processing step is configured to determine a temperature, or a change in temperature.

[0064]

[0054] In one embodiment of the third aspect, the method comprises applying a potential waveform at a first frequency and a second frequency, and determining the thermal energy parameter by reference to a ratio of a current resulting from application of the potential waveform at the first frequency to a current resulting from the application of the potential at the second frequency.

[0065]

[0055] In one embodiment of the third aspect, the electrically conductive element has a surface, and the polymer is connected to the surface.

[0066]

[0056] In one embodiment of the third aspect, the connection is, or comprises, a covalent chemical bond, a pseudo-covalent bond, or an electrostatic bond.

[0067]

[0057] In one embodiment of the third aspect, the polymer has a first terminus and a second terminus and the connection is at, or near, the first terminus and the second terminus is free.

[0068]

[0058] In one embodiment of the third aspect, the redox-active species is connected to the polymer.

[0069]

[0059] In one embodiment of the third aspect, the redox-active species is connected at, or near, the second terminus of the polymer.

[0070]

[0060] In one embodiment of the third aspect, the connection is, or comprises, a covalent chemical bond, a pseudo-covalent bond, or an electrostatic bond.

[0071]

[0061] In one embodiment of the third aspect, the polymer is an organic polymer, or an inorganic polymer, or a hybrid thereof.

[0072]

[0062] In one embodiment of the third aspect, the polymer is a biological polymer, or an analogue thereof. IRN20105094

[0073]

[0063] In one embodiment of the third aspect, the biological polymer or analogue thereof is selected from a DNA molecule, an RNA molecule, an XNA molecule, a PNA molecule, a polypeptide, a lipid, and a polysaccharide.

[0074]

[0064] In one embodiment of the third aspect, the polymer is a non-biological polymer.

[0075]

[0065] In one embodiment of the third aspect, the non-biological polymer comprises a linear or a branched carbon chain.

[0076]

[0066] In one embodiment of the third aspect, the non-biological polymer is biologically compatible with a human or a non-human animal.

[0077]

[0067] In one embodiment of the third aspect, the non-biological polymer is incapable of instigating or facilitating a pathological biological response in a human or a non-human animal.

[0078]

[0068] In one embodiment of the third aspect, the pathological biological response is an immune response or an inflammatory response.

[0079]

[0069] In one embodiment of the third aspect, the non-biological polymer is substantially non-toxic to a human or a non-human animal, or substantially non-cell binding, or substantially non-protein binding, or substantially non-lipid binding, or substantially nonvolatile.

[0080]

[0070] In one embodiment of the third aspect, the non-biological polymer is hydrophilic overall.

[0081]

[0071] In one embodiment of the third aspect, the non-biological polymer is a poly(ethylene glycol) or another polyether species.

[0082]

[0072] In one embodiment of the third aspect, the polymer is formed from about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more of the monomer, or where the polymer is a copolymer the copolymer is formed from a total of from about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more of the constituent monomers.

[0083]

[0073] In one embodiment of the third aspect, the method comprises use of an analyte sensing element.

[0084]

[0074] In one embodiment of the third aspect, the analyte sensing element is associated with a surface of the electrically conductive element, or a surface of another electrically conductive element.

[0085]

[0075] In one embodiment of the third aspect, a potential waveform is applied to another electrically conductive element and a current measured so as to determine the amount of the analyte.

[0086]

[0076] In one embodiment of the third aspect, the method comprises determining the amount of the analyte by reference to the thermal energy parameter determined, such as a temperature. IRN20105094

[0087]

[0077] In one embodiment of the third aspect, the analyte sensing element is an aptamer or a functional equivalent thereof.

[0088]

[0078] In one embodiment of the third aspect, the aptamer is a DNA aptamer, an RNA aptamer, an XNA aptamer, or a PNA aptamer.

[0089]

[0079] In one embodiment of the third aspect, the electrically conductive element and the another electrically conductive element are configured as electrodes in an electrochemical sensor.

[0090]

[0080] In one embodiment of the third aspect, the electrically conductive element and the another electrically conductive element is each a wire, a needle or a microneedle.

[0091]

[0081] In one embodiment of the third aspect, the solution is a biological fluid within or about a human or a non-human animal.

[0092]

[0082] In one embodiment of the third aspect, the biological fluid is an 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, or an exudate.

[0093]

[0083] In one embodiment of the third aspect, the biological fluid is in situ within the body of a human or a non-human animal.

[0094]

[0084] In one embodiment of the third aspect, the biological fluid is selected from blood, interstitial fluid, or a mixture of blood and interstitial fluid.

[0095]

[0085] In one embodiment of the third aspect, the method comprises piercing the skin of a human or a non-human animal with the electrically conductive element and the further electrically conductive element such that the polymer and the analyte sensing element contact the blood and / or the interstitial fluid of the animal.

[0096]

[0086] In one embodiment of the third aspect, the method is performed using the apparatus of any embodiment of the first aspect, or any embodiment of the electrochemical sensor of the second aspect.

[0097]

[0087] In a fourth aspect, the present disclosure provides a method for determining the amount of an analyte in a biological fluid by way of an electrochemical aptamer-based sensor, the method comprising determining a thermal energy parameter by the method of any embodiment of the third aspect, and mathematically transforming a current output of the sensor with the thermal energy parameter.

[0098]

[0088] In a fifth aspect, the present disclosure provides method for determining the amount of an analyte in a biological fluid by way of an electrochemical aptamer-based sensor, the method comprising determining a thermal energy parameter by the method of any embodiment of the third aspect, and determining an analyte amount by the sensor by reference to the determined thermal energy parameter.

[0099]

[0089] In a six aspect, the present disclosure provides a method for determining the amount of an analyte in a biological fluid by way of an electrochemical aptamer-based sensor, the IRN20105094 method comprising determining a thermal energy parameter by the method of any embodiment of the third aspect, and selecting a formula or a standard curve relating to a current output by the sensor to analyte amount by reference to the determined thermal energy parameter.

[0100] BRIEF DESCRIPTION OF THE FIGURES

[0101]

[0090] FIG. 1 illustrates highly diagrammatically an electrode having a temperature sensitive, redox-active polymers associated therewith.

[0102]

[0091] FIG. 2 illustrates highly diagrammatically a first electrode having a temperature sensitive, redox-active polymers associated therewith, being co-mounted with a second electrode having an analyte sensitive, redox-active elements associated therewith

[0103]

[0092] FIG. 3A illustrates highly diagrammatically an electrode having a mixture of (i) a temperature sensitive, redox-active polymer, and (ii) an analyte sensitive, redox-active element associated therewith, each of which is redox active at a distinct redox potential.

[0104]

[0093] FIG. 3B illustrates highly diagrammatically an electrode having two discrete surfaces, the first having temperature sensitive, redox-active polymers, and the second having an analyte sensitive, redox-active elements associated therewith.

[0105]

[0094] FIG. 3C illustrates highly diagrammatically an electrode having four discrete surfaces, each surface having either temperature sensitive, redox-active polymers, or analyte sensitive, redox-active elements associated therewith.

[0106]

[0095] FIG. 4 is a basic circuit diagram suitable for the electrode of FIG. 1, for temperature sensing only.

[0107]

[0096] FIG. 5 is a basic circuit diagram suitable for the electrode of FIG. 2, for temperature sensing and analyte sensing.

[0108]

[0097] FIG. 6 is a basic circuit diagram suitable for the electrode of FIG. 2, for temperature sensing and analyte sensing, utilising shared components for the two sensing functions.

[0109]

[0098] FIG. 7 is a graph of RT value against temperature for a temperature sensing electrode in whole bovine blood, and for three concentrations of the target analyte vancomycin. Several data points for each concentration are labelled to delineate each of the concentration data series.

[0110]

[0099] FIG. 8 is a graph of RT value against temperature for a temperature sensing electrode (interrogated at 100 / 150 Hz) and a vancomycin sensing electrode (interrogated at 30 / 100 Hz) in whole bovine blood.

[0111]

[0100] FIG. 9 is a graph of relative signal change against temperature for a temperature sensing electrode and a vancomycin sensing electrode in whole bovine blood, showing temperature correction for vancomycin concentration.

[0112]

[0101] FIG. 10A illustrates diagrammatically the structure of temperature sensitive construct LI 5. IRN20105094

[0113]

[0102] FIG. 1 OB is a graph of relative signal (as a percentage) against temperature for a working electrode functionalised with redox-modified construct LI 5. Interrogation was by square wave voltammetry and the frequencies indicated on the graph.

[0114]

[0103] FIG. 10C is a graph of KDM signal change (as a percentage) against temperature for a working electrode functionalised with redox-modified construct LI 5. Interrogation was by square wave voltammetry using the frequency pairs as indicated on the graph.

[0115]

[0104] FIG. 11A illustrates diagrammatically the structure of temperature sensitive construct L21.

[0116]

[0105] FIG. 1 IB is a graph of relative signal (as a percentage) against temperature for a working electrode functionalised with redox-modified construct L21. Interrogation was by square wave voltammetry and the frequencies indicated on the graph.

[0117]

[0106] FIG. 11C is a graph of KDM signal change (as a percentage) against temperature for a working electrode functionalised with redox-modified construct L21. Interrogation was by square wave voltammetry using the frequency pairs as indicated on the graph.

[0118]

[0107] FIG. 12A illustrates diagrammatically the structure of temperature sensitive construct L38.

[0119]

[0108] FIG. 12B is a graph of relative signal (as a percentage) against temperature for a working electrode functionalised with redox-modified construct L38. Interrogation was by square wave voltammetry and the frequencies indicated on the graph.

[0120]

[0109] FIG. 12C is a graph of KDM signal change (as a percentage) against temperature for a working electrode functionalised with redox-modified construct L38. Interrogation was by square wave voltammetry using the frequency pairs as indicated on the graph.

[0121]

[0110] FIG. 13A illustrates diagrammatically the structure of temperature sensitive construct L24-MM1.

[0122]

[0111] FIG. 13B is a graph of relative signal (as a percentage) against temperature for a working electrode functionalised with redox-modified construct L24-MM1. Interrogation was by square wave voltammetry and the frequencies indicated on the graph.

[0123]

[0112] FIG. 13C is a graph of KDM signal change (as a percentage) against temperature for a working electrode functionalised with redox-modified construct L24-MM1. Interrogation was by square wave voltammetry using the frequency pairs as indicated on the graph.

[0124]

[0113] FIG. 14A illustrates diagrammatically the structure of temperature sensitive construct L25-MM2.

[0125]

[0114] FIG. 14B is a graph of relative signal (as a percentage) against temperature for a working electrode functionalised with redox-modified construct L25-MM2. Interrogation was by square wave voltammetry and the frequencies indicated on the graph.

[0126]

[0115] FIG. 14C is a graph of KDM signal change (as a percentage) against temperature for a working electrode functionalised with redox-modified construct L25-MM2. Interrogation was by square wave voltammetry using the frequency pairs as indicated on the graph. IRN20105094

[0127]

[0116] FIG. 15 is a graph comparing KDM signal change (as a percentage) across a physiological temperature range of 30 to 40°C for a working electrode functionalised with one of redox-modified construct LI 5, L21, L38, L24-MM1 or L25-MM2. Interrogation was by square wave voltammetry at the frequencies indicated on the graph.

[0128]

[0117] FIG. 16A is a graph of relative signal (as a percentage) against time for a working electrode functionalised with redox-modified construct L24-MM1. Interrogation was by square wave voltammetry at the frequencies indicated on the graph.

[0129]

[0118] FIG. 16B is a graph of KDM signal change (as a percentage) against time for a working electrode functionalised with redox-modified construct L24-MM1. Interrogation was by square wave voltammetry at the frequency pairs indicated on the graph.

[0130]

[0119] 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.

[0131]

[0120] 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.

[0132] DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

[0133]

[0121] 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.

[0134]

[0122] 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.

[0135]

[0123] 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.

[0136]

[0124] In a first aspect, the present disclosure provides apparatus for sensing thermal energy in a solution, the apparatus comprising: an electrically conductive element having a polymer associated therewith, the polymer being inherently redox-active or having a redox-active species associated therewith; IRN20105094 circuitry in electrical connection with the electrically conductive element, the circuitry configured to apply an electrical potential waveform to the conductive element and to measure an electrical current resulting from application of the electrical potential waveform; an electronic processor in operable connection with the circuitry and configured to by way of program instructions process the measured electrical current so as to determine a thermal energy parameter of the solution; and a power source in operable connection with the circuitry.

[0137]

[0125] The present disclosure may be embodied alternatively in the form of a method for sensing thermal energy in a solution, the method comprising: providing an electrically conductive element having a polymer associated therewith, the polymer being inherently redox-active or having a redox-active species associated therewith; applying an electrical potential waveform to the conductive element and measuring an electrical current resulting from application of the electrical potential waveform; and using the measured electrical current to determine a thermal energy parameter of the solution.

[0138]

[0126] The present disclosure is predicated at least in part on the inventors’ discovery that a redox-active polymer coated onto the surface of an electrode of an electrochemical-based apparatus provides an output that is proportional or at least relatable to the temperature of the test solution to which it is contacted. After application of a potential to the electrode (typically via a circuit including a counter electrode contacting the same test solution), current passing through the polymer-coated electrode is measured. The measured current is then used to determine temperature.

[0139]

[0127] Without wishing to be limited by theory in any way it is proposed that in a plurality of redox-active polymers may be connected to the surface of an electrode, the combination of electrode and polymers form a temperature sensitive electrode. Each polymer has a redox active portion or a connected redox-active species. Each polymer is able to assume one or more conformations or ensembles of conformations. The kinetics of electron transport between the redox-active portions or the redox-active species and the surface of the electrode depends on the conformation assumed by the polymers. And the fraction of time the polymer spends in any given conformation or ensemble of conformations is related to the surrounding temperature. Because of this, the kinetics of electron transport between the redox-active portions or the redox-active species and the surface of the electrode depend on temperature in a manner that can be determined using standard electrochemical techniques.

[0140]

[0128] For many polymers, the magnitude of the conformational change or change in conformational ensembles can be adjusted by altering the design of the polymer. This is particularly true for biopolymers. For example, the lengthening of a DNA polymer that adopts a hairpin configuration that undergoes thermal unfolding will increase the mean IRN20105094 separation between an attached redox species and the electrode surface, which can enhance the change in electron transfer kinetics associated with changing temperature.

[0141]

[0129] The sensitivity of the conformational change or change in conformational ensembles to temperature can also be adjusted by altering the design of the polymer. That is, the fraction of the polymer in any given conformation can be made more or less sensitive to changes in temperature. For example, lengthening a DNA hairpin typically increases the change in heat capacity (ACP) associated with its conformational changes, rendering those conformational changes more sensitive to temperature. This can improve the precision with which temperature is measured.

[0142]

[0130] Aptamers may be designed for temperature sensitivity by modulating the enthalpy (delta H), entropy (delta S) and heat capacity change (delta Cp) of folding of the DNA. Each of these parameters may be exploited to adjust the steepness (sensitivity) of the response to temperature, and the temperature at which the transition midpoint falls.

[0143]

[0131] Structurally speaking, any one or more of the following parameters may be adjusted to tune temperature sensitivity: overall length (i.e. number of bases), stem structures (number of base pairs in a steam, number of stems), loop structures (number of base pairs in a loop, number of loops), and hairpin structures (number of base pairs in a hairpin, and number of hair pins).

[0144]

[0132] As will be appreciated other types of biological polymers and organic polymers are capable of self-association (and also dissociation) in a temperature dependent manner analogous to DNA. The self-association may result from base pairing, hydrogen bonding, strong and weak electrostatic interactions, hydrophobic effects and the like.

[0145]

[0133] Such self -association may result in a redox active part of the polymer or an attached redox reporter to become more or less proximal to the electrode surface. For example, at one temperature a protein may assume an unfolded conformation where a redox active part or a redox reporter is distal to the electrode surface, and at another temperature assume a folded conformation where the protein is more compact thereby bringing the redox active part or redox reported more proximal the electrode surface. The change in proximity causes measurable current changes through the working electrode upon interrogation.

[0146]

[0134] The interrogating potential for an electrochemical cell having a temperature sensitive working electrode may be applied in any suitable manner, including any means known in the prior art for analyte detection by electrochemical methods. Such means include square wave voltammetry, cyclic voltammetry, chronoamperometry, and electrochemical impedance spectroscopy to name several exemplary methods. In each method, a feature of the resulting current may be exploited which informs as to temperature. Such features may be investigated by the skilled person for suitability and include current magnitude, current decay rate, a ratio of two currents, an electron transfer rate determined from the current properties, and a peak current.

[0147]

[0135] Methods using a current ratio (such as a ratio obtained from two currents sampled at different times during the wave form, or two currents arising from the application of IRN20105094 potential waveform at two different frequencies) are preferred. Current ratios can be made substantially independent of the number of temperature-sensitive redox-active polymers associated with the electrode surface, and accordingly there is less or no requirement to take account of intra-batch variations, loss or degradation of polymer in storage or during use when determining temperature. This reduces or eliminates the requirement to calibrate individual sensors before use.

[0148]

[0136] Whichever current feature is exploited, a standard curve, equation, lookup table or other suitable means may be generated to relate, over a range, a current feature and a temperature. The standard curve, equation, or lookup table may be used at a later time to determine the temperature of a test solution, a tissue, or another sample matrix.

[0149]

[0137] In a preferred form of the disclosure a square wave voltammetry is used. As is understood in the art, a square wave potential overlaid on an upward sweeping potential is applied to interrogate the electrode, with current arising from the interrogation being measured. Interrogation of the electrode may be performed using two different square wave frequencies, and a current ratio generated for use in determining temperature.

[0150]

[0138] In some embodiments, kinetic differential measurements or other electrochemical approaches for removing sensor drift can be employed.

[0151]

[0139] In some embodiments, the present disclosure includes means for electrochemically detecting the amount of an analyte in the same solution as that being measured for temperature. Such an arrangement provides advantage in that the temperature output by the temperature measuring electrode may be used to more accurately determine analyte concentration in a test solution, tissue, or other sample matrix.

[0152]

[0140] The means for electrochemically detecting the amount of an analyte may be an electrode coated in an analyte recognition element (such as an aptamer) to forming an analyte sensing electrode. Such an arrangement may be configured such that the temperature measuring electrode and the analyte sensing electrode are very closely spaced, and therefore effectively contact the same sample matrix when in use so as to provide an accurate temperature for the solution in contact with the analyte sensing electrode. Advantageously, the two electrodes may be disposed physically within the same housing, and in that regard may automatically and contemporaneously contact the test solution.

[0153]

[0141] As a further advantage, the temperature-sensitive electrode and the working electrode may share components of an electrical circuit configured to interrogate the electrodes and accept output signals therefrom. For example, the electrodes may share any one or more of: a power supply, wiring, a processor (including program instructions), a voltage controlling device (for interrogation), a current measuring device, a potentiometer, and a user interface.

[0154]

[0142] In one embodiment, the polymer and an analyte recognition element may be associated with a single electrode. Such an electrode is bifunctional, being capable of sensing temperature and sensing analyte. As will be appreciated, any slight regional differences in temperature that may occur where two separate electrodes are used will be IRN20105094 overcome where the apparatus uses a single bifunctional electrode. In that embodiment, the interrogation and current measurement steps may be performed at different times, such that a current feature may be clearly attributed to either the temperature sensing function, or the analyte sensing function.

[0155]

[0143] The polymer used to form the temperature sensitive electrode may be selected on the basis of (i) having a portion that is inherently redox-active, or (ii) is capable of being associated with a redox active species. For example, the polymer may inherently comprise a redox-active functional group, or may have the ability to covalently bond to a redoxactive species, such as methylene blue.

[0156]

[0144] A further criterion will be the ability to, in a temperature dependant manner, change electron kinetics between the redox-active portion or redox-active species and the electrode surface. For example, the rate of electron flow may alter according to temperature. In that regard, a polymer that changes its three-dimensional confirmation or its ensemble of conformations according to temperature may be selected. The polymer may require some length (for example, 5 monomers long) in order for any significant change in confirmation to be available.

[0157]

[0145] In some embodiments, the electrode will contact the body of a living animal (such as a human, or another type of mammal), and in which case the criterion of biocompatibility may apply. Polymers such as DNA (preferably up to 60 bases), RNA (preferably up to 60 bases), peptides and the like may be suitable candidates given their biological origins.

[0158]

[0146] A biological polymer may be sensitive to temperature by way of a change in secondary structure or tertiary structure.

[0159]

[0147] Of course, biological molecules may nevertheless be toxic or otherwise contraindicated due to an ability to trigger an immune reaction or an inflammatory response. Toxic species of any origin will also be generally contraindicated.

[0160]

[0148] Polymers having a limited ability to flex along the backbone may be less preferred given the limited ability to alter conformation. Polymers having bulky side chains may that sterically inhibit flexing may be avoided.

[0161]

[0149] Polymers that are overall hydrophobic may be preferred given that many applications will be directed to biological fluids which are essentially aqueous solutions.

[0162]

[0150] Poly ethers provide a useful starting point for investigations, possessing many of the desirable characteristics described above. Polyethylene glycols for example, are well used in medical applications, and are available in a range of molecular weights.

[0163]

[0151] The polymer may be associated with the surface of electrode by any suitable means. Exemplary means are known in the art for the immobilization of aptamers such as physical adsorption, covalent bonding with use of self-assembled monolayers (SAMs), e.g., thiol- based or silane-based.

[0164]

[0152] Where a polymer not having inherent redox activity is selected, a redox active species, such as methylene blue, may be covalently connected to the polymer by any of the means known in the art. Methylene blue is a useful redox reporter for at least two reasons: IRN20105094

[0165] (1) its formal potential is within the stability window of the thiol-on-gold monolayer chemistry often used to attach DNA and other polymers to electrodes, and (2) its electrochemical behavior is similar to that used for electrochemical-aptamer based sensors. However, if it is desirable to determine the temperature of the electrode used in an EAB sensor, a different redox reporter to methylene blue, (such as anthraquinone, ferrocene, viologen, Atto MB2 or Nile blue) reporting at a distinct redox potential is used. Alternatively, a single reporter species could be used although with temporally separated interrogations being used to determine the thermal parameter and analyte amount.

[0166]

[0153] Reference is made to FIG. 1 showing a temperature sensing electrode (10) retained on a mounting portion (15). The electrode has a surface (20) to which is connected a plurality of polymers (25). Each polymer (25) is connected at one end to the electrode surface (20) and at the other end is tagged with methylene blue (30). Thus, the polymer (25) is immobilised, yet still free to move at its mid and terminal regions in response to changes in the temperature of the surrounding solution. The electrode (10) is connected to circuitry as will be further described below, and is capable of measuring temperature only.

[0167]

[0154] FIG. 2 shows a temperature sensing electrode (10) and a separate analyte sensing electrode (12) retained on a common mounting portion (15), such that both are readily simultaneously exposed to a test solution. The analyte sensing electrode (12) comprises a plurality of analyte recognition elements (17), each of which may be an aptamer, for example. Each element (17) is connected at one end to the electrode surface (22) and at the other end is tagged with methylene blue (30). Thus, the element (17) is immobilised, yet still free to move at its mid and terminal regions in response to changes in the amount of analyte in the surrounding solution. The electrode (12) is connected to circuitry as will be further described below. Given the mutual proximity of the electrodes (10, 12) the temperature sensed by electrode (12) will closely reflect that of the solution immediately surrounding electrode (10). Accordingly, the output of temperature sensing electrode (12) will be capable of improving the accuracy of the concentration of analyte as determined by the analyte sensing electrode (12)

[0168]

[0155] FIG. 3 A illustrates an embodiment of the disclosure using a single electrode (50) having a surface (55) with a mixture of temperature sensitive polymers (25) and analyte sensitive elements (17), each of which is tagged with a different redox reporter (60a and 60b respectively). In this embodiment, the two species (17, 25) are distributed randomly across the surface (55). In terms of interrogation, the two species (17, 25) are connected to a common conductor (the surface 55) and are therefore exposed to the same potential, and at the same time. The potential is adjusted to a first potential chosen so as to measure current from a first redox species but substantially not the second redox species. The potential is then adjusted to a second potential chosen so as to measure current from the second redox species but substantially not the first redox species. For example, if both the redox species are to be oxidised at the common conductor to yield a current flow, but require different potentials to be oxidised, then the first potential is chosen so as to only IRN20105094 oxidise a first redox species yielding a first current. When the first redox species is substantially oxidized, the potential is adjusted to a second potential that is sufficient to oxidize the second redox species. It should be noted that this second potential is also sufficient to oxidize the first redox species, should any be available in its reduced state, however application of the first potential has substantially oxidized all the reduced state first redox species, so it will contribute no substantial additional current when the second potential is applied. In this way current arising from the first and second redox species (17, 25) can be measured separately, and accordingly a temperature and analyte may be sensed contemporaneously.

[0169]

[0156] FIG. 3B shows a single electrode structure (70) having two mutually electrically isolated conductive surfaces (55a, 55b), the former having a plurality of analyte sensing elements (17), and the latter having a plurality of temperature sensing polymers (25). Each conductive surface (55a, 55b) is capable of independently conducting an interrogating potential and sensing a current feature, allowing for the simultaneous sensing of temperature and analyte concentration.

[0170]

[0157] FIG. 3C is a variation of the embodiment of FIG. 3B, having four mutually independent conductive surfaces.

[0171]

[0158] In terms of circuitry, reference is made firstly to FIG. 4, showing basic circuitry useful in the interrogation of the electrode of FIG. 1, for example. As will be appreciated, such circuitry is known in the field of electrochemical aptamer-based analyte sensors, and it has been found that such circuitry is applicable also to electrochemical temperature sensing apparatus as described herein. Moreover, such circuitry may be implemented in a similar manner as for electrochemical aptamer-based analyte sensors in so far as methods for interrogating and determining a current feature may be used.

[0172]

[0159] The ability to utilise the same or similar circuitry and methods as for aptamer-based sensors for the detection of analytes provides significant advantage in the manufacture of sensors. Especially where the sensor is wearable, it is generally desired to limit dimensions and weight. Where a sensor comprises both temperature sensing and an aptamer sensing functions, componentry for both functions may be shared.

[0173]

[0160] The advantage may be noted by a comparison of the circuitry for FIG. 5 and FIG.

[0174] 6. In FIG. 5, each of the temperature sensing function and the analyte sensing function has dedicated componentry, with temperature output by the temperature sensing function being passed (dashed line) as input to the analyte sensing function so as to improve accuracy. In the circuitry for FIG. 6, all common components are shared. The temperature sensing electrode and the analyte sensing electrode are switchable into and out of the main circuit. Thus, to sense temperature, the temperature sensing electrode in switched to connect to the main circuit, and the analyte sensing electrode is isolated. Conversely, to sense analyte, the analyte sensing electrode in switched to connect to the main circuit, and the temperature sensing electrode is isolated. Output from the electrode in the temperature sensing phase IRN20105094 is stored in RAM, and retrieved during the analyte sensing phase so as improve the accuracy of analyte concentration determination.

[0175]

[0161] The present disclosure will now be further described by reference to the following non-limiting examples.

[0176] EXAMPLE 1: Determining temperature in bovine blood

[0177]

[0162] A temperature sensing electrode was prepared in general accordance with that shown in FIG. 1. A methylene-blue-modified, 5-base DNA strand was used as the temperature sensing polymer. Using square wave voltammetry, the peak height in the voltammogram was measured at two square wave frequencies to generate an output (KDMRT) using the following equation:

[0178] .,n> ,

[0179] KDMT= — -

[0180] 0.5

[0181] RT=IOFT / ION at some reference temperature for the device, so as to correct for variations in the number of DNA strands on the electrode. Here IOFF is the current observed at the lower of the two potential waveform frequencies and ION is the signal at the higher of the two frequencies.

[0182]

[0163] Alternatively, the temperature can be related to the ratio of the two currents:

[0183] R(T) = IOff(T) / IOn(T), where IOJJ(T) and Ion(T) are the currents observed at the lower and higher of the two potential waveform frequencies.

[0184]

[0164] Reference is made to FIG. 7, showing that KDMRT and R(T) are sensitive to temperature and accordingly its value may be used as a measure of the temperature at the electrode surface. The value was calculated using square-wave frequencies of 100 and 150 Hz at three different concentrations of vancomycin (0 pM, 50 pM, lOOpM), demonstrating the target-independence of these measurements. Error regions are shown in dashed lines and represent standard deviations for multiple devices.

[0185]

[0165] KDMT and R are reproducible, monotonic functions of temperature, and thus determining either allows for the estimation of temperature by comparison to a calibration curve or calibration equation.

[0186] EXAMPLE 2: Matching of target analyte binding and redox modified polymer by selecting frequency pairs

[0187]

[0166] Reference is made to FIG. 8, showing that RT values can be matched for the target binding and the redox modified polymer by selecting frequency pairs. Shown here are the RT values for the T5 oligonucleotide sequence (black) interrogated at 100 / 150 Hz and a vancomycin target binding aptamer (blue) interrogated at 30 and 100 Hz. The graph shows IRN20105094 indistinguishable RT values across the physiological temperature range in the absence of vancomycin, in biological fluids (undiluted bovine blood). Error regions shown by the dashed lines represent standard deviations of independently fabricated devices.

[0188] EXAMPLE 3: Correction of temperature-related deviations in determined analyte concentration

[0189]

[0167] FIG. 9 demonstrates that RT-KDM (blue) successfully corrects for temperature related deviations in signal change from an analyte sensing electrode. In contrast, without temperature correction, the R-KDM (black) approach shows significant changes in the signal change across the physiological temperature range at a constant concentration (0 pM). By the R value of the vancomycin with the RT value of the redox modified polymer (as in RT KDM equation) at the frequency pair of 100 / 150 Hz, changes in the temperature were corrected for to maintain a constant signal change across the physiological temperature range. Error regions are shown in dashed lines and represent standard deviation.

[0190]

[0168] Specific frequency pairs can be selected for both the analyte binding aptamer and the redox modified polymer, such that the RT value for the polymer matches the R value (IOFF / ION when no target is present) for the sensor (FIG. 8). The RT value measured for the DNA temperature sensing sequence can be substituted into the R-KDM equation as follows:

[0191] RT * / QIW IOFF RyKDM = , ' , - -

[0192] 0.5 * (RT* I0N+ IQFF)

[0193] This can be used to effectively account for temperature induced, target independent, changes in signal gain (FIG. 9).

[0194] EXAMPLE 4: Effect of oligonucleotide structure on temperature sensitivity.

[0195]

[0169] Studies were performed to determine the temperature sensitivity of the five DNA oligonucleotides detailed below: IRN20105094

[0196]

[0170] A series of temperature sensing working electrodes was prepared. Each of the series of electrodes was functionalised with a single methylene-blue modified DNA construct (L15, L21, L38, L24 MM1 or L25 MM2).

[0197]

[0171] Each of the series of working electrodes was immersed in phosphate buffered saline and interrogated by square wave voltammetry across a temperature range, and at different frequencies as further detailed below.

[0198]

[0172] Each of the working electrodes was connected to a Ag|AgCl reference electrode, and a platinum counter electrode. The electrodes were secured with a Teflon lid an immersed in 25 mL of standard phosphate-buffered saline plus 870 pM MgCE to provide an electrochemical cell. The electrochemical cell was held in temperature controlled water bath to enable temperature ramping experiments. A high-precision thermometer was used to monitor the temperature of the solution inside of the electrochemical cell. The internal temperature within the cell was initially held around 23°C and then ramped incrementally by 1 °C via water bath temperature control.

[0199]

[0173] Electrochemical measurements employed a CHI- 1040c multipotentiostat connected to the electrodes via MiniGrabber™ connectors. Square wave voltammetry (SWV) was used to monitor signal response as a function of temperature and performed at degree increments over a potential window of -0.08 V to -0.42 V vs Ag / AgCl at SWV frequencies of 5, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 Hz. The pulse amplitude employed was 25 mV with a step increment of 3 mV for frequencies < 30 Hz and 1 mV for frequencies >30 Hz. Signal responses were normalized to the peak current collected at each SWV frequency at the starting temperature of each experiment to visualize signal response over the monitored range of temperature.

[0200]

[0174] Reference is made to FIG. 10A showing the structure and sequence of construct L15.

[0201]

[0175] FIG. 10B shows the results of electrochemical interrogation of the LI 5 construct across a temperature range of 25 to 50°C at the square wave frequencies indicated. Use of the LI 5 construct as an electrochemical temperature sensor reveals a broad range of melting behaviors as a function of temperature, presumably due to competing thermal effects on structure and on the electron transfer kinetics of the folded and unfolded conformations of the construct.

[0202]

[0176] FIG. 10C shows the difference in relative signal change between voltammetric peaks collected at pairs of square wave frequencies (200 / 10Hz, 200 / 20Hz or 200 / 30Hz) produces temperature-dependent signals varying in melting temperature and transition steepness, providing a means of tuning the sensitivity of the temperature measurement to match the optimal temperature range. IRN20105094

[0203]

[0177] The remaining constructs (L21, L38, L24-MM1 and L25-MM2) were represented structurally and tested analogously as for construct LI 5. Relevant Figures are detailed below:

[0204]

[0178] It will be noted that each of the constructs exhibited temperature dependent behavior. The current output of working electrodes functionalised with each of the constructs is therefore useful in determining temperature, of functioning as a temperature. A temperature value may be determine by reference to a previously generated standard curve, a formula defining a standard curve, or a lookup table, for example.

[0205]

[0179] Comparative analysis of the data provided by interrogation of the five constructs was performed across the temperature range of 30 to 40°C. That temperature range was selected because it includes the normal temperature of ISF in the human dermis and expected variations therefrom.

[0206]

[0180] Reference is made to FIG. 15 comparing KDM outputs for the five constructs at the frequency pairs indicated in the Table below.

[0207]

[0181] It will be noted from FIG. 15 that a generally linear relationship between KDM and temperature is demonstrated for all constructs. The graph for the L25-MM2 construct has the highest gradient, and therefore displays the greatest temperature sensitivity across the measured range.

[0208]

[0182] Construct L24-MM1 displayed useful drift correctability over a period of at least about 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. Reference is made to FIG. 16A

[0209]

[0183] Referring now to FIG. 16B, the L24MM1 construct exhibits pairs of frequencies that drift in concert when placed in undiluted bodily fluids (here undiluted whole blood) at 37 ° C but that respond to temperature differently. As illustrated in FIG. 16C, taking the difference between the relative peak currents obtained at 100 Hz and 30 Hz or 100 Hz and IRN20105094

[0210] 40 Hz produces a kinetic differential measurement drift corrected output that varies by less than 5% after 12 h. If the system is allowed to equilibrate in blood for 1 h before initiating measurements, a drift is less than 1% over 12 h is noted. Over the temperature range of most interest (30 - 40 °C; see FIG. 13C), 1% drift corresponds to <0.5 °C drift in the estimated temperature.

[0211]

[0184] The apparatus and methods and described herein may be deployed in part or in whole through one or more processors that execute computer software, program codes, and / or instructions on a processor. The processor may be part of a server, client, network infrastructure, mobile computing platform, stationary computing platform, or other computing platform. A processor may be any kind of computational or processing device capable of executing program instructions, codes, binary instructions and the like. The processor may be or may include a signal processor, digital processor, embedded processor, microprocessor or any variant such as a coprocessor (math co-processor, graphic coprocessor, communication co-processor and the like) and the like that may directly or indirectly facilitate execution of program code or program instructions stored thereon. In addition, the processor may enable execution of multiple programs, threads, and codes.

[0212]

[0185] The threads may be executed simultaneously to enhance the performance of the processor and to facilitate simultaneous operations of the application. By way of implementation, methods, program codes, program instructions and the like described herein may be implemented in one or more thread. The thread may spawn other threads that may have assigned priorities associated with them; the processor may execute these threads based on priority or any other order based on instructions provided in the program code. The processor may include memory that stores methods, codes, instructions and programs as described herein and elsewhere.

[0213]

[0186] Any processor or a mobile communication device or server may access a storage medium through an interface that may store methods, codes, and instructions as described herein and elsewhere. The storage medium associated with the processor for storing methods, programs, codes, program instructions or other type of instructions capable of being executed by the computing or processing device may include but may not be limited to one or more of a CD-ROM, DVD, memory, hard disk, flash drive, RAM, ROM, cache and the like.

[0214]

[0187] A processor may include one or more cores that may enhance speed and performance of a multiprocessor. In some embodiments, the processor may be a dual core processor, quad core processors, other chip-level multiprocessor and the like that combine two or more independent cores (called a die).

[0215]

[0188] The methods and systems described herein may be deployed in part or in whole through one or more hardware components that execute software on a server, client, firewall, gateway, hub, router, or other such computer and / or networking hardware. The software program may be associated with a server that may include a file server, print server, domain server, internet server, intranet server and other variants such as secondary IRN20105094 server, host server, distributed server and the like. The server may include one or more of memories, processors, computer readable media, storage media, ports (physical and virtual), communication devices, and interfaces capable of accessing other servers, clients, computers, and devices through a wired or a wireless medium, and the like. The methods, programs or codes as described herein and elsewhere may be executed by the server. In addition, other devices required for execution of methods as described in this application may be considered as a part of the infrastructure associated with the server.

[0216]

[0189] The server may provide an interface to other devices including, without limitation, clients, other servers, printers, database servers, print servers, file servers, communication servers, distributed servers and the like. Additionally, this coupling and / or connection may facilitate remote execution of program across the network. The networking of some or all of these devices may facilitate parallel processing of a program or method at one or more location without deviating from the scope of the disclosure. In addition, any of the devices attached to the server through an interface may include at least one storage medium capable of storing methods, programs, code and / or instructions. A central repository may provide program instructions to be executed on different devices. In this implementation, the remote repository may act as a storage medium for program code, instructions, and programs.

[0217]

[0190] The software program may be associated with a client that may include a file client, print client, domain client, internet client, intranet client and other variants such as secondary client, host client, distributed client and the like. The client may include one or more of memories, processors, computer readable media, storage media, ports (physical and virtual), communication devices, and interfaces capable of accessing other clients, servers, computers, and devices through a wired or a wireless medium, and the like. The methods, programs or codes as described herein and elsewhere may be executed by the client. In addition, other devices required for execution of methods as described in this application may be considered as a part of the infrastructure associated with the client.

[0218]

[0191] The client may provide an interface to other devices including, without limitation, servers, other clients, printers, database servers, print servers, file servers, communication servers, distributed servers and the like. Additionally, this coupling and / or connection may facilitate remote execution of program across the network. The networking of some or all of these devices may facilitate parallel processing of a program or method at one or more location without deviating from the scope of the disclosure. In addition, any of the devices attached to the client through an interface may include at least one storage medium capable of storing methods, programs, applications, code and / or instructions. A central repository may provide program instructions to be executed on different devices. In this implementation, the remote repository may act as a storage medium for program code, instructions, and programs.

[0219]

[0192] The methods and systems described herein may be deployed in part or in whole through network infrastructures. The network infrastructure may include elements such as IRN20105094 computing devices, servers, routers, hubs, firewalls, clients, personal computers, communication devices, routing devices and other active and passive devices, modules and / or components as known in the art. The computing and / or non-computing device(s) associated with the network infrastructure may include, apart from other components, a storage medium such as flash memory, buffer, stack, RAM, ROM and the like. The processes, methods, program codes, instructions described herein and elsewhere may be executed by one or more of the network infrastructural elements.

[0220]

[0193] The methods, program codes, calculations, algorithms, and instructions described herein may be implemented on a cellular network having multiple cells. The cellular network may either be frequency division multiple access (FDMA) network or code division multiple access (CDMA) network. The cellular network may include mobile devices, cell sites, base stations, repeaters, antennas, towers, and the like. The cell network may be a GSM, GPRS, 3G, 4G, EVDO, mesh, or other networks types.

[0221]

[0194] The methods, programs codes, calculations, algorithms and instructions described herein may be implemented on or through mobile devices. The mobile devices may include navigation devices, cell phones, mobile phones, mobile personal digital assistants, laptops, palmtops, netbooks, pagers, electronic books readers, music players and the like. These devices may include, apart from other components, a storage medium such as a flash memory, buffer, RAM, ROM and one or more computing devices. The computing devices associated with mobile devices may be enabled to execute program codes, methods, and instructions stored thereon.

[0222]

[0195] Alternatively, the mobile devices may be configured to execute instructions in collaboration with other devices. The mobile devices may communicate with base stations interfaced with servers and configured to execute program codes. The mobile devices may communicate on a peer-to-peer network, mesh network, or other communications network. The program code may be stored on the storage medium associated with the server and executed by a computing device embedded within the server. The base station may include a computing device and a storage medium. The storage device may store program codes and instructions executed by the computing devices associated with the base station.

[0223]

[0196] The computer software, program codes, and / or instructions may be stored and / or accessed on computer readable media that may include: computer components, devices, and recording media that retain digital data used for computing for some interval of time; semiconductor storage known as random access memory (RAM); mass storage typically for more permanent storage, such as optical discs, forms of magnetic storage like hard disks, tapes, drums, cards and other types; processor registers, cache memory, volatile memory, non-volatile memory; optical storage such as CD, DVD; removable media such as flash memory (e.g. USB sticks or keys), floppy disks, magnetic tape, paper tape, punch cards, standalone RAM disks. Zip drives, removable mass storage, off-line, and the like; other computer memory such as dynamic memory, static memory, read / write storage, mutable storage, read only, random access, sequential access, location addressable, file IRN20105094 addressable, content addressable, network attached storage, storage area network, bar codes, magnetic ink, and the like.

[0224]

[0197] The methods and systems described herein may transform physical and / or or intangible items from one state to another. The methods and systems described herein may also transform data representing physical and / or intangible items from one state to another.

[0225]

[0198] The elements described and depicted herein, including in flow charts and block diagrams throughout the figures, imply logical boundaries between the elements. However, according to software or hardware engineering practices, the depicted elements and the functions thereof may be implemented on computers through computer executable media having a processor capable of executing program instructions stored thereon as a monolithic software structure, as standalone software modules, or as modules that employ external routines, code, services, and so forth, or any combination of these, and all such implementations may be within the scope of the present disclosure.

[0226]

[0199] Furthermore, the elements depicted in any flow chart or block diagrams or any other logical component may be implemented on a machine capable of executing program instructions. Thus, while the foregoing drawings and descriptions set forth functional aspects of the disclosed systems, no particular arrangement of software for implementing these functional aspects should be inferred from these descriptions unless explicitly stated or otherwise clear from the context. Similarly, it will be appreciated that the various steps identified and described above may be varied, and that the order of steps may be adapted to particular applications of the techniques disclosed herein. All such variations and modifications are intended to fall within the scope of this disclosure. As such, the depiction and / or description of an order for various steps should not be understood to require a particular order of execution for those steps, unless required by a particular application, or explicitly stated or otherwise clear from the context.

[0227]

[0200] The methods and / or processes described above, and steps thereof, may be realized in hardware, software or any combination of hardware and software suitable for a particular application. The hardware may include a general-purpose computer and / or dedicated computing device or specific computing device or particular aspect or component of a specific computing device. The processes may be realized in one or more microprocessors, microcontrollers, embedded microcontrollers, programmable digital signal processors or other programmable device, along with internal and / or external memory. The processes may also, or instead, be embodied in an application specific integrated circuit, a programmable gate array, programmable array logic, or any other device or combination of devices that may be configured to process electronic signals. It will further be appreciated that one or more of the processes may be realized as a computer executable code capable of being executed on a computer readable medium.

[0228]

[0201] The Application software may be created using a structured programming language such as C, an object oriented programming language such as C++, or any other high-level or low-level programming language (including assembly languages, hardware description IRN20105094 languages, and database programming languages and technologies) that may be stored, compiled or interpreted to run on one of the above devices, as well as heterogeneous combinations of processors, processor architectures, or combinations of different hardware and software, or any other machine capable of executing program instructions.

[0229]

[0202] Thus, in one aspect, each method described above and combinations thereof may be embodied in computer executable code that, when executing on one or more computing devices, performs the steps thereof. In another aspect, the methods may be embodied in systems that perform the steps thereof, and may be distributed across devices in a number of ways, or all of the functionality may be integrated into a dedicated, standalone device or other hardware. In another aspect, the means for performing the steps associated with the processes described above may include any of the hardware and / or software described above. All such permutations and combinations are intended to fall within the scope of the present disclosure.

[0230]

[0203] The disclosure may be embodied in program instruction set executable on one or more computers. Such instruction sets may include any one or more of the following instruction types:

[0231]

[0204] Data handling and memory operations, which may include an instruction to set a register to a fixed constant value, or copy data from a memory location to a register, or vice-versa (a machine instruction is often called move, however the term is misleading), to store the contents of a register, result of a computation, or to retrieve stored data to perform a computation on it later, or to read and write data from hardware devices.

[0232]

[0205] Arithmetic and logic operations, which may include an instruction to add, subtract, multiply, or divide the values of two registers, placing the result in a register, possibly setting one or more condition codes in a status register, to perform bitwise operations, e.g., taking the conjunction and disjunction of corresponding bits in a pair of registers, taking the negation of each bit in a register, or to compare two values in registers (for example, to see if one is less, or if they are equal).

[0233]

[0206] Control flow operations, which may include an instruction to branch to another location in the program and execute instructions there, conditionally branch to another location if a certain condition holds, indirectly branch to another location, or call another block of code, while saving the location of the next instruction as a point to return to.

[0234]

[0207] Coprocessor instructions, which may include an instruction to load / store data to and from a coprocessor, or exchanging with CPU registers, or perform coprocessor operations.

[0235]

[0208] A processor of a computer of the present system may include "complex" instructions in their instruction set. A single "complex" instruction does something that may take many instructions on other computers. Such instructions are typified by instructions that take multiple steps, control multiple functional units, or otherwise appear on a larger scale than the bulk of simple instructions implemented by the given processor. Some examples of "complex" instructions include: saving many registers on the stack at IRN20105094 once, moving large blocks of memory, complicated integer and floating-point arithmetic (sine, cosine, square root, etc.), SIMD instructions, a single instruction performing an operation on many values in parallel, performing an atomic test-and-set instruction or other read-modify-write atomic instruction, and instructions that perform ALU operations with an operand from memory rather than a register.

[0236]

[0209] An instruction may be defined according to its parts. According to more traditional architectures, an instruction includes an opcode that specifies the operation to perform, such as add contents of memory to register-and zero or more operand specifiers, which may specify registers, memory locations, or literal data. The operand specifiers may have addressing modes determining their meaning or may be in fixed fields. In very long instruction word (VLIW) architectures, which include many microcode architectures, multiple simultaneous opcodes and operands are specified in a single instruction.

[0237]

[0210] Some types of instruction sets do not have an opcode field (such as Transport Triggered Architectures (TTA) or the Forth virtual machine), only operand(s). Other unusual "0-operand" instruction sets lack any operand specifier fields, such as some stack machines including NOSC.

[0238]

[0211] Conditional instructions often have a predicate field- several bits that encode the specific condition to cause the operation to be performed rather than not performed. For example, a conditional branch instruction will be executed, and the branch taken, if the condition is true, so that execution proceeds to a different part of the program, and not executed, and the branch not taken, if the condition is false, so that execution continues sequentially. Some instruction sets also have conditional moves, so that the move will be executed, and the data stored in the target location, if the condition is true, and not executed, and the target location not modified, if the condition is false. Similarly, IBM zJ Architecture has a conditional store. A few instruction sets include a predicate field in every instruction; this is called branch predication.

[0239]

[0212] The instructions constituting a program are rarely specified using their internal, numeric form (machine code); they may be specified using an assembly language or, more typically, may be generated from programming languages by compilers.

[0240]

[0213] The present disclosure refers primarily to the use of voltametric methods for sensor interrogation. Non-voltametric methods such as chronoamperometry and electrochemical impedance spectroscopy (as just two examples) are also operable.

[0241]

[0214] 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.

[0242]

[0215] 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

IRN20105094CLAIMS:

1. An apparatus for sensing thermal energy in a solution, the apparatus comprising: an electrically conductive element having a polymer associated therewith, the polymer being inherently redox-active or having a redox-active species associated therewith; circuitry in electrical connection with the electrically conductive element, the circuitry configured to apply an electrical potential waveform to the conductive element and to measure an electrical current resulting from application of the electrical potential waveform; an electronic processor in operable connection with the circuitry and configured by way of program instructions to process the measured electrical current so as to determine a thermal energy parameter of the solution; and a power source in operable connection with the circuitry.

2. The apparatus of claim 1, wherein the polymer has a three-dimensional conformation or ensemble of three-dimensional conformations, and these change in response to a change in thermal energy thereabout, the conformational change in turn changing the kinetics, including a rate of, electron transport between (i) the electrically conductive element and the inherently redox-active polymer or (ii) the electrically conductive element and the redox-active species.

3. The apparatus of claim 2, wherein the change in conformation causes:(i) a redox-active portion of the inherently redox-active polymer to be more or less proximal to the electrically conductive element, or to be more or less proximal to the electrically conductive element for a greater portion of a time period; or(ii) the redox-active species to be more or less proximal to the electrically conductive element, or to be more or less proximal to the electrically conductive element for a greater portion of a time period; or(iii) the rate with which the redox-active species approaches or moves away from the electrically conductive element changes.

4. The apparatus of any one of claims 1 to 3, wherein the program instructions are configured to determine a temperature, or a change in temperature.

5. The apparatus of any one of claims 1 to 4, wherein the circuitry is configured to apply a potential waveform at a first frequency and a second frequency, and the thermal energy parameter is determined by the program instructions by reference to a relationship of a current resulting from application of the potential waveform at the first frequency to a current resulting from the application of the potential at the second frequency.IRN201050946. The apparatus of any one of claims 1 to 5, wherein the electrically conductive element has a surface, and the polymer is connected to the surface.

7. The apparatus of claim 6, wherein the polymer has a first terminus and a second terminus and the connection is at, or near, the first terminus and the second terminus is free.

8. The apparatus of any one of claims 1 to78, wherein the redox-active species is connected to the polymer.

9. The apparatus of any one of claims 1 to 8, wherein the polymer is an organic polymer, or an inorganic polymer, or a hybrid thereof.

10. The apparatus of any one of claims 1 to 9, wherein the polymer is a biological polymer, or an analogue thereof.

11. The apparatus of claim 10, wherein the biological polymer or analogue thereof is selected from a DNA molecule, an RNA molecule, an XNA molecule, a PNA molecule, a polypeptide, a lipid, and a polysaccharide.

12. The apparatus of any one of claims 1 to 11, wherein the polymer is a non-biological polymer.

13. The apparatus of claim 12, wherein the non-biological polymer is hydrophilic overall.

14. The apparatus of claim 12 or claim 13, wherein the non-biological polymer is a poly(ethylene glycol) or another polyether species.

15. The apparatus of any one of claims 1 to 14, comprising an analyte sensing element.

16. The apparatus of claim 15, wherein the analyte sensing element is associated with a surface of the electrically conductive element, or a surface of another electrically conductive element.

17. The apparatus of claim 15 or claim 16, wherein the another electrically conductive element is in operable connection with the circuitry, or another circuitry such that a potential can be applied to the another electrically conductive element and a current measured, and the processor, or another processor is configured by way of program instructions to determine the amount of the analyte.IRN2010509418. The apparatus of claim 17, wherein the processor or another processor, or a further processor is configured by way of program instructions to determine the amount of the analyte by reference to the thermal energy parameter determined by the apparatus.

19. The apparatus of any one of claims 15 to 18, wherein the analyte sensing element is an aptamer or a functional equivalent thereof.

20. The apparatus of claim 19, wherein the aptamer is a DNA aptamer, an RNA aptamer, an XNA aptamer, or a PNA aptamer.

21. The apparatus of any one of claims 1 to 20, wherein the electrically conductive element and the another electrically conductive element are configured as electrodes in an electrochemical sensor.

22. The apparatus of any one of claims 1 to 21, wherein the electrically conductive element and the another electrically conductive element is each a wire, a needle or a microneedle.

23. The apparatus of any one of claims 1 to 22, wherein the electrically conductive element and the further electrically conductive element are each configured so as to pierce the skin of a human or a non-human animal such that the polymer and the analyte sensing element contact the blood and / or the interstitial fluid of the animal.

24. An electrochemical sensor comprising the apparatus of any one of claims 1 to 25.

25. A method for sensing thermal energy in a solution, the method comprising: providing an electrically conductive element having a polymer associated therewith, the polymer being inherently redox-active or having a redox-active species associated therewith; applying an electrical potential waveform to the conductive element and measuring an electrical current resulting from application of the electrical potential waveform; and using the measured electrical current to determine a thermal energy parameter of the solution.

Images