Electrode and biosensor capable of measuring target analyte or uric acid with high sensitivity, and protein detection using electron from uric acid

Abstract

An electrode (10) according to the invention includes: a base section (12); a graphite-containing layer (14) arranged on the base section (12); and a multi-layered carbon nanotube-containing layer (16) arranged on the graphite-containing layer (14).

Description

Electrode and biosensor capable of measuring a target analyte or uric acid with high sensitivity, and protein detection using electrons from uric acid

[0001] The present disclosure relates to an electrode and a uric acid biosensor for measuring a target analyte (e.g., biomarker) or uric acid, and protein detection using electrons from uric acid.

[0002] Various biosensors for measuring biomarkers, which are target analytes in a living body, are known. In biomarker detection using an antibody or an aptamer, unlike detection using an enzyme such as a glucose sensor, a redox reaction cannot be directly utilized. Although it was possible to measure the concentration by integrating ferrocene, which is the reaction center, into the aptamer and using the oxidation electrons of ferrocene, long-term detection was difficult because ferrocene deteriorated quickly.

[0003] Also, when a marker is captured using a general antigen-antibody reaction with a dissociation constant of 10 -5 s -1 , when calculating the half-life in measurement at a general protein marker concentration of 1 nM range, it is about 20 hours. Therefore, for accurate continuous detection, a method for dissociating the protein marker from the antibody or aptamer in a short time is required.

[0004] On the other hand, uric acid is an antioxidant that always exists in the blood as the final product of purine nucleotide metabolism and is excreted from the kidneys into urine or serum. The normal range of uric acid in human serum is 178 - 400 μmol / L (3.0 - 6.8 mg / dL), and deviation from this range is associated with many medical conditions such as gout, hyperuricemia, kidney disease, cardiovascular disease, type 2 diabetes, Lesch-Nyhan syndrome, etc. On the other hand, a decrease in uric acid is associated with acute renal failure, urinary stones, and multiple sclerosis. Therefore, continuous monitoring of uric acid concentration is particularly important for patients suspected of having these diseases.

[0005] Highly conductive multiwalled carbon nanotubes (MWCNTs) possess excellent electrical conductivity and a large surface area, making them widely used for detecting target analytes in living organisms, facilitating the detection of low concentrations of these analytes. For example, Patent Document 1 discloses an electrode having a structure in which a layer of multiwalled carbon nanotubes dispersed in carboxymethylcellulose is formed on a metal substrate. However, the uric acid concentration measured by this electrode is between 0.65 mM and 2.4 mM, and there is a need for a sensor that can detect uric acid with higher accuracy.

[0006] WO2021 / 060097

[0007] One of the problems that this disclosure aims to solve is to provide an electrode that can be used in a sensor capable of detecting a target analyte or uric acid with high sensitivity, a sensor equipped with the electrode, a wearable microneedle sensor device, a method for detecting a target analyte or uric acid in a fluid, and a method for electrically dissociating a target analyte and molecules bound to the target analyte.

[0008] This disclosure includes, for example, the following subjects:

[0009] Item 1. An electrode comprising a base, a graphite-containing layer disposed on the base, and a multilayer carbon nanotube-containing layer disposed on the graphite. Item 2. The electrode according to Item 1, wherein the electrode comprises a microneedle array, a plate electrode, or a microwire. Item 3. The electrode according to Item 2, wherein the electrode comprises a microneedle array, and the microneedle array comprises a working electrode, a reference electrode, a counter electrode, and a potentiostat. Item 4. The electrode according to Item 1, wherein a molecule that binds to a target molecule is attached to the multilayer carbon nanotube-containing layer. Item 5. A sensor comprising an electrode according to any one of Items 1 to 3 and a molecule that binds to a target analyte attached to the multilayer carbon nanotube-containing layer. Item 6. The sensor according to Item 5, wherein the electrode comprises a microneedle array. Item 7. A wearable microneedle sensor device comprising a microneedle sensor unit having a body made of an electrically insulating material, an electrode according to Item 1, comprising a microneedle array disposed on the body, and a microfluidic device disposed below the electrode. Item 8. The microneedle sensor device according to Item 7, further comprising a plurality of detection and communication electrical circuits arranged in or on the main body, each of which is coupled to each of the microneedle arrays. Item 9. The microneedle sensor device according to Item 8, further comprising an electronic unit that electrically communicates with the plurality of detection and communication circuit connections, the electronic unit comprising a data processing unit and an electrical interface capable of contacting the plurality of detection and communication electrical circuits of the microneedle sensor unit. Item 10. The microneedle sensor unit according to Item 9, wherein the electrical interface electrically couples the microneedle sensor unit and the electronic unit.Item 11. A method for detecting uric acid, glucose, lactic acid, or ions in a fluid using the sensor described in Item 5, comprising: applying a sweep voltage to the electrode; measuring a current in response to the sweep voltage; and using the measured current to detect the presence or absence of uric acid, glucose, lactic acid, or ions in the fluid, or to measure the concentration of uric acid. Item 12. A method for detecting a target analyte in a fluid using the sensor described in Item 5, comprising: applying a sweep voltage to the electrode; measuring a current in response to the sweep voltage, wherein the measured value of the current reflects electrons generated by the oxidation of uric acid; and using the electrons generated by the oxidation of uric acid to measure the concentration of the target analyte in the fluid. Item 13. A method for electrically dissociating a target analyte and molecules bound to the target analyte using the sensor described in item 5, comprising: bringing a fluid containing uric acid and the target analyte into contact with the sensor to bind the target analyte and molecules bound to the target analyte attached to a layer containing multilayer carbon nanotubes of the electrode; and dissociating the target analyte and molecules bound to the target analyte by applying a sweep voltage to the electrode. Item 14. The method of item 13, further comprising detecting the electrical dissociation, wherein the fluid contains uric acid, and an increase in the uric acid concentration in the fluid during or after the application of the sweep voltage is an indicator of the dissociation of the target analyte and molecules bound to the target analyte.

[0010] This disclosure provides an electrode that can be used in a sensor capable of detecting a target analyte or uric acid with high sensitivity. Furthermore, it is possible to provide a sensor capable of detecting a target analyte or uric acid with high sensitivity and a wearable microneedle sensor device using such an electrode. Moreover, it is possible to provide a method for detecting a target analyte such as a biomarker with high sensitivity using such a sensor, and a method for accurately measuring the concentration by electrically dissociating the target analyte from a molecule that specifically binds to the target analyte.

[0011] Schematic diagram of a flat electrode according to one embodiment of the present invention. Photograph of a microneedle sensor device according to one embodiment of the present invention. Schematic diagram showing a microfluidic device for fluid collection located below the microneedle sensor. Photograph of a microneedle array sensor (right) and sensor holder (left) according to one embodiment of the present invention. Photograph of the microneedle array sensor housed in the sensor holder. Photograph of a microwire sensor according to one embodiment of the present invention. Photograph of the microwire array sensor housed in the sensor holder and connected to the electronic unit. (A) An example of a wearable microneedle sensor device comprising disposable sensor components and a reusable electronic unit. (B) Microneedle sensor device, (C) Schematic diagram showing the microneedle sensor device of Figure 6(A) attached to a target. Block diagram of an example of an electronic unit. Microneedle sensor IOMT system handling data measured by the microneedle sensor device of Figure 6(A). Electron microscope image of an example of a microneedle electrode of the present invention. Calibration curve of a uric acid detection sensor. Enlarged view of the horizontal axis of the graph in Figure 10A in the range of 0-1500 μm. Calibration curve for uric acid detection sensor. Continuous measurement (amperometry) results from uric acid detection sensor. Calibration curve using protein concentration detection with uric acid. The black circles on the vertical axis represent the detection current value at concentration 0 (BLANK). Antibody concentration: 10 μg / mL, uric acid concentration: 200 μM. DPV results using protein concentration detection with uric acid. Diagram of the electrical dissociation process of antigen-antibody complex. Detection of glucose at various concentrations. Continuous detection of glucose at varying concentrations. Schematic diagram explaining the capture of ferrocene by aptamers. Detection of cortisol at various concentrations.

[0012] In this specification, the singular form includes both singular and plural forms unless otherwise explicitly stated herein or the context clearly contradicts it.

[0013] In this specification, "contains" is a concept that also includes "substantially consists only of" and "consists only of."

[0014] In the numerical ranges described stepwise in this specification, the upper or lower limit of a numerical range in one step can be arbitrarily combined with the upper or lower limit of a numerical range in another step. Furthermore, in the numerical ranges described in this specification, the upper or lower limit of a numerical range may be replaced with values ​​shown in the examples or values ​​that can be uniquely derived from the examples. Moreover, in this specification, numbers connected by "~" mean a numerical range that includes the numbers before and after "~" as the lower and upper limits.

[0015] The embodiments included in this disclosure will be further described below with reference to the drawings. The embodiments described below are examples of typical embodiments of this disclosure and do not limit the scope of the invention.

[0016] This disclosure provides an electrode comprising a metal-containing base, a graphite-containing layer disposed on the base, and a multi-walled carbon nanotube-containing layer disposed on the graphite.

[0017] Figure 1 shows an electrode according to one embodiment of the present invention. The electrode 10 is a flat electrode and comprises a base portion 12, a graphite-containing layer 14 disposed on the base portion 12, and a multi-walled carbon nanotube-containing layer 16 disposed on the graphite-containing layer 14. The electrode 10 and the base portion 12, the base portion 12 and the graphite-containing layer 14, and the graphite-containing layer 14 and the multi-walled carbon nanotube-containing layer 16 are in direct contact.

[0018] The base 12 may also be called a substrate, and may be a metal substrate, a glass substrate, or a plastic substrate (i.e., a substrate containing synthetic resin, such as a PET substrate). In a preferred embodiment, the base 12 is a metal-containing substrate. Preferably, the substrate contains 60% or more, 70% or more, 80% or more, or 90% or more by mass of metal. The metal used in the metal-containing substrate may be a pure metal containing a single metallic element, or it may be an alloy. Examples of metals used in metal substrates include gold, platinum, iron, chromium, titanium, and stainless steel.

[0019] The multi-walled carbon nanotubes contained in layer 16 have excellent conductivity and a very large specific surface area, making them excellent as electrode materials. The diameter of the multi-walled carbon nanotubes is, for example, 100 to 1000 nm, which is the average particle size (50% particle size by wet laser scattering method) of the multi-walled carbon nanotubes.

[0020] The layer 16 containing the multi-walled carbon nanotube may have additional molecules attached to it that bind to the target analyte.

[0021] In this specification, "target analyte" can be used interchangeably with "test substance" and may be a target molecule. A "molecule that binds to a target analyte" binds to the target analyte more specifically or selectively to the target analyte than to any other substance.

[0022] The target analyte may be a target analyte contained in a fluid, such as body fluid. Body fluid may be body fluid collected from the subject. Examples of body fluids include, but are not limited to, blood (whole blood, serum, plasma, specific blood components), tissue fluid (interstitial fluid, intercellular fluid, interstitial fluid), body cavity fluid, lymph, cerebrospinal fluid, synovial fluid, and aqueous humor. Tissue fluids that are excreted outside the body, such as sweat, urine, tears, and saliva, are also included in body fluids.

[0023] Examples of target analytes include metabolites, electrolytes, proteins, amino acids, nucleic acids, lipids, liposomes, nanoparticles, and / or drugs, while examples of probes include antibodies, proteins that bind to target analytes such as aptamers, nucleic acids, etc.

[0024] In some embodiments, the target analyte is a biomarker. Examples of biomarkers include hormones or neurotransmitters. Examples of hormones or neurotransmitters include male hormones such as testosterone, dihydrotestosterone, and dihydroepiandrosterone; female hormones such as estrogen and progesterone; catecholamines such as norepinephrine, epinephrine, cortisol, aldosterol, and dopamine; serotonin, acetylcholine, and combinations thereof.

[0025] In some embodiments, the target analyte is a chemical substance that has an endocrine-disrupting effect in living organisms, i.e., an endocrine disruptor.

[0026] In some embodiments, the drug includes an antibiotic.

[0027] In some embodiments, the drug includes an anxiolytic.

[0028] In some embodiments, the drug includes an anticonvulsant.

[0029] In some embodiments, the drug includes a selective serotonin reuptake inhibitor (SSRI).

[0030] In some embodiments, the drug includes a drug with a narrow therapeutic index (NTI).

[0031] In some embodiments, the drug contains a metal.

[0032] In some embodiments, the drug is a blood component. Examples of blood components include insulin, transferrin, insulin-like growth factor I and II, human platelet-derived growth factor I and II, fibroblast growth factor, tissue-specific growth factors, and combinations thereof.

[0033] The electrode 10, to which molecules that bind to the target analyte are attached, can be used as a sensor for detecting the target analyte with high precision.

[0034] Such a sensor can qualitatively or quantitatively detect a target analyte by measuring the interaction or binding changes between the target analyte and molecules bound to it. For example, it can detect the presence of a target analyte in a body fluid or measure its concentration. Furthermore, such a sensor can also be used to qualitatively or quantitatively detect a substance other than the target analyte (e.g., uric acid) based on the interaction or binding changes between molecules bound to the target analyte.

[0035] Figure 2 is a photograph of a microneedle array sensor according to one embodiment of the present invention. The microneedle array sensor 20 comprises a support 21 made of an electrically insulating material, and a working electrode 22, a reference electrode 24, and a counter electrode 26, each independently supported by the support 21. The support 21 may also be called a base or substrate, and in this embodiment it is bowl-shaped with a bottom surface, through which the working electrode 22, the reference electrode 24, and the counter electrode 26 pass. The support 21 can be manufactured, for example, from resin or a resin composition using a 3D printer. The working electrode 22, the reference electrode 24, and the counter electrode 26 constitute a microneedle array and are each independent of each other.

[0036] In some embodiments, at least one of the working electrode 22, reference electrode 24, and counter electrode 26, preferably the working electrode 22, comprises a graphite-containing layer 14 disposed on a base and a multi-walled carbon nanotube-containing layer 16 disposed on the graphite-containing layer 14. In some embodiments, at least one of the working electrode 22, reference electrode 24, and counter electrode 26, preferably the reference electrode 24, may include gold, silver / silver chloride, platinum, or a combination of other materials that can function as a reference electrode. In some embodiments, at least one of the working electrode 22, reference electrode 24, and counter electrode 26, preferably the counter electrode 26, may include a metal (gold, platinum) or alloy, a carbon material (e.g., graphite, graphene, multi-walled carbon nanotube (MWCNT)), or a material that can function as a counter electrode.

[0037] At least one of the electrodes in these microneedle arrays is configured as an electrochemical sensor electrode for detecting electrical signals based on the interaction or change in binding between the target analyte and the molecules bound to the target analyte. For example, at least one of the electrodes in the microneedle array can generate an electrical signal related to the interaction or change in binding between the target analyte and the molecules bound to the target analyte, which is detectable by the electrochemical sensor electrode.

[0038] The microneedle array sensor 20 may further include fixing members 28 (e.g., resin adhesives) made of an electrically insulating material that are placed on a support 21 and surround each of the working electrode 22, reference electrode 24, and counter electrode 26, fixing the working electrode 22, reference electrode 24, and counter electrode 26 at separate positions.

[0039] A potentiostat (not shown) may be connected to the microneedle array of the microneedle array sensor 20. In some embodiments, the potentiostat provides the sweep voltage potential between the working electrode 22 and the reference electrode 24 of the microneedle array. In some embodiments, the potentiostat further measures the current flowing between the working electrode 22 and the counter electrode 26.

[0040] In some embodiments, the detection of the presence or absence of a target analyte in a bodily fluid of interest, or the measurement of its concentration, can be achieved based on a current measured between the working electrode 22 and the counter electrode 26. The current can be measured by generating a voltammogram. The microneedle array sensor 20 can operate as an electrochemical or electrophysiological electrode for various epidermal sensing applications by puncturing the epidermal layer of the skin of interest with the tips of the microneedle array.

[0041] In some embodiments, a microfluidic device for fluid collection may be located below the microneedle sensor. An example of such a configuration is shown in Figure 3. The microfluidic device 80 comprises a substantially rectangular body 81, a plurality of inlet ports 82 for the inflow of the test solution, a detection unit 83 connected to, coupled with or in contact with the microneedle array sensor 20 moved in the direction of the arrow, a connecting passage 84 connected to and united with each of the inlet ports 82 and connected to the detection unit 83, an outlet port 85 for the outflow of the test solution, and a connecting passage 86 connecting the detection unit 83 and the outlet port 85. The test fluid includes a reagent for detecting a fluid (e.g., sweat of the target) flowing into the microfluidic device 80 via the microneedle sensor 20. Such a reagent may include, but is not limited to, molecules that bind to a target analyte in the fluid. In this figure, the inlet ports 82, connecting passages 84, detection unit 83, connecting passages 86, and outlet ports 85 are all located inside the body 81, but at least a portion of each part may be located outside the body 81. Each of the multiple inlet sections 82 comprises a substantially cylindrical body 82a and an opening 82b provided in the body 82a. Each opening 82b opens onto the upper surface 81a of the body 81 and is configured to receive the test solution flowing in from outside the microfluidic device 80 for fluid collection. The detection section 83 comprises a substantially cylindrical body 83a and an opening 83b provided in the body 83a. The opening 83b opens onto the upper surface 81a of the body 81 and can receive the microneedle array sensor 20. The central axis of the body 82a and the central axis of the detection section 83 may be parallel to the thickness direction of the body 81. The discharge section 85 opens onto the lower surface 81b of the body 81 and is configured to discharge the test solution that flows out to the outside through the inside of the microfluidic device 80 for fluid collection. The test solution flowing in from the injection section 82 passes through the connection passage 84, the detection section 83, and the connection passage 86, and is discharged from the discharge section 85.

[0042] The fluid may be bodily fluid (e.g., sweat). By positioning a microfluidic device below the microneedle sensor 20 so that sweat from the subject wearing the microneedle array sensor flows into the microfluidic device 80 via the microneedles of the microneedle sensor 20, the detection unit 83 of the microfluidic device 80 can continuously detect biomarkers contained in the subject's sweat. Furthermore, using needle-shaped electrodes in the sensor in this way requires a very small volume or area of ​​the detection site, resulting in little to no time lag in measurement and enabling highly accurate measurements.

[0043] Figure 4A is a photograph of an example of a microneedle array sensor 30 (right) and a sensor holder 60 (left). The microneedle array sensor 30 comprises a support 31 made of an electrically insulating material, and a working electrode 32, a reference electrode 34, and a counter electrode 36, each independently supported by the support 31. The support 31 may also be called a base or substrate, and in this embodiment it is a flat plate. The working electrode 32, the reference electrode 34, and the counter electrode 36 each constitute a microneedle array, with a conical tip and a cylindrical body portion from the tip to the end. Conductive wires 37 extend from each of the working electrode 32, the reference electrode 34, and the counter electrode 36. The working electrode 32, the reference electrode 34, and the counter electrode 36 are positioned near one end of the support 31 in the short direction, with one end of each wire 37 connected to the working electrode 32, the reference electrode 34, and the counter electrode 36, respectively. The wires 37 extend along the longitudinal direction of the support 31 and terminate at the other end of the support 31 in the short direction. At least one of the working electrode 32, the reference electrode 34, and the counter electrode 36, preferably the working electrode 32, comprises a graphite-containing layer disposed on a base and a multi-walled carbon nanotube-containing layer disposed on the graphite. In this example, a substantially circular hole 38a is provided on the upper surface of the cover 38 that houses the support 31 and the working electrode 32, the reference electrode 34, and the counter electrode 36, and the cover 38 is absent above the working electrode 32, the reference electrode 34, and the counter electrode 36 to facilitate work. The microwire array sensor 30 can be used in contact with any object encompassing the object to be measured, or it can be used non-contact.

[0044] The micro needle array sensor 30 may be connected to a potentiostat (not shown). The sensor holder 60, which is substantially circular in plan view and disc-shaped, has a recess 61 for accommodating the micro needle array sensor 30. The sensor holder 60 can be manufactured, for example, from a resin or resin composition using a 3D printer. Since the shape of the recess 61 conforms to the outer shape of the micro needle array sensor 30, as shown in FIG. 4B, the micro needle array sensor 30 can be used in a state of being accommodated in the sensor holder 60. The sensor holder 60 enables easy replacement of the micro needle array sensor 30.

[0045] FIG. 5A is an example of the manufacture of a micro wire sensor. The micro wire array sensor 40 includes a support (not shown) having an electrically insulating material, and conductive wires 42, 44, 46 independently supported by the support. In this embodiment, it is flat and plate-shaped. The wires 42, 44, 46 act as electrodes. The wires 32, 34, 36 extend along the longitudinal direction of the support, and at least one of the wires 42, 44, 46 includes a layer containing graphite disposed on a base, and a layer containing multi-walled carbon nanotubes disposed on the graphite. A substantially circular hole 38a is provided in the upper surface of the cover 38 that accommodates both ends of the support and the wires 42, 44, 46, and the intermediate portions of the wires 42, 44, 46 extend outside the cover 38.

[0046] The micro-wire array sensor 40 may be connected to a potentiostat (not shown). Further, the micro-wire array sensor 40 can be used by contacting any object including the measurement target. The sensor holder 60 in the shape of a disk with a substantially circular contact plane view has a recess 61 for accommodating the micro-wire array sensor 40. Since the shape of the recess 61 conforms to the outer shape of the micro-needle array sensor 40, as shown in FIG. 5B, the micro-needle array sensor 40 can be used in a state of being accommodated in the sensor holder 60. The sensor holder 60 can be manufactured, for example, from a resin or a resin composition using a 3D printer or the like. The data and power supply adapter 70 is connected to the data transmission and analysis device main body 75 via the sensor holder 60.

[0047] As described above, the electrode may have any shape including a flat electrode, a micro-needle array, and a micro-wire. For example, a flat electrode or a micro-wire electrode can be attached to the skin and used as a wearable sensor for biomarkers in sweat. Alternatively, the micro-needle sensor can be used for detecting target analytes (markers) in interstitial skin fluid or sweat.

[0048] The present disclosure also provides a wearable micro-needle sensor device including a main body having an electrically insulating material, an electrode according to any of the above disposed on the main body, and a plurality of detection and communication electric circuits disposed in or on the main body, and a micro-needle sensor unit having a plurality of detection and communication electric circuits to which each of the micro-needles is coupled.

[0049] The micro-needle sensor device is an electronic unit that electrically communicates with a plurality of detection and communication electric circuits, and includes a data processing unit and an electrical interface capable of contacting the plurality of detection and communication electric circuits of the micro-needle sensor unit, and may further include an electronic unit. The electrical interface can electrically couple the micro-needle sensor unit and the electronic unit.

[0050] In some embodiments, the electrical interface includes conductive members such as conductive pins.

[0051] Figure 6(A) shows an example of a wearable, or attachable, microneedle sensor device. Figure 6(B) shows a portion of a disposable microneedle sensor device among the wearable microneedle sensor devices. Figure 6(C) is a schematic diagram showing the microneedle sensor device from Figure 6(A) attached to a subject.

[0052] The microneedle sensor device 100 may be a wearable sensor. As shown in Figure 6(C), the microneedle sensor device 100 may be attached to the skin of a subject 90 and configured to detect a target analyte in the bodily fluids of the subject 90. In some embodiments, the microneedles 54 of the microneedle sensor unit 50 of the microneedle sensor device 100 may penetrate the skin of the subject 90 once the microneedle sensor device 100 is attached to the skin of the subject 90, but may not penetrate. In some embodiments, the microneedle sensor device 100 is configured to be attached to the skin of the subject for about 1 to 30 days.

[0053] In Figure 6(A), the microneedle sensor device 100 comprises a holder 110 for housing a microneedle sensor unit 50 and an electronic unit 120 adjacent to the holder 110. The holder 110 is provided with a recessed housing portion 111 for housing the microneedle sensor unit 50, which is conformed to the external shape of the microneedle sensor unit 50. The microneedle sensor unit 50 shown in Figure 6(C) can be detachably housed in the housing portion 111 of the holder 110.

[0054] As shown in Figure 6(B), the microneedle sensor unit 50 comprises a main body 51 made of an electrically insulating material, an electrode 52 as described above disposed on the main body 51, and a plurality of detection and communication electrical circuits (not shown) disposed inside or on the main body 51. The electrode 52 comprises a support 53 and a plurality of (four in the figure) microneedles 54 constituting a microneedle array, which are independently supported and spaced apart from each other by the support 53. The electrical interconnections of each of the plurality of detection and communication electrical circuits are coupled to the plurality of microneedles 54 and to the contact terminal structure on the main body 51. The microneedles 54 may have the same structure as the working electrode 22, reference electrode 24, and counter electrode 26 shown in Figure 2, and the support 53 may have the same structure as the support 21 shown in Figure 2.

[0055] In some embodiments, at least one of the plurality of microneedles 54 comprises a graphite-containing layer disposed on a base and a multi-walled carbon nanotube-containing layer disposed on the graphite. In some embodiments, at least one of the plurality of microneedles 54 may be made from a material selected from metals (gold, silver, platinum, copper, palladium, nickel, iridium, rhodium, cobalt) or alloys, silicon materials, polymer materials, or carbon materials (e.g., graphite, graphene, multi-walled carbon nanotubes (MWCNTs)).

[0056] In some embodiments, the microneedles 54 are positioned to be in contact with or capable of contacting the skin without puncturing the epidermis. In some embodiments, the microneedles 54 may be adapted to penetrate the epidermis and descend into the superficial dermis to a depth of approximately 50 μm to 100 μm shallower than the length of the needle. In some embodiments, the microneedles 54 may be adapted to provide physical access to the dermal stroma (the fluid-filled space between skin cells).

[0057] In some embodiments, each needle of the plurality of microneedles 54 may independently have a cross-sectional shape selected from a tetragonal pyramidal shape, a conical shape, and a polygon (e.g., a triangle, rectangle, hexagon, octagon, etc., with tapered or straight walls).

[0058] In some embodiments, the multiple microneedles 54 include at least one of a working electrode, a reference electrode, and a counter electrode, and are coated with an electrode coating. The working electrode, reference electrode, and counter electrode can function for electrochemical sensing, thereby enabling the direct detection of a target analyte in a living organism.

[0059] In some embodiments, the microneedle 54, which functions as the working electrode, has a graphite layer and a MWCNT layer formed thereon. In some embodiments, the microneedle 54, which functions as the reference electrode 302, may include silver / silver chloride, platinum, and other materials that can function as a reference electrode. In some embodiments, the microneedle 54, which functions as the counter electrode, may include gold, platinum, carbon materials, and other materials that can function as a counter electrode.

[0060] As shown in Figure 6(A), the electronic unit 120, which electrically communicates with the multiple detection and communication electrical circuits of the microneedle sensor unit 50 via electrical interconnections, is reusable and comprises a control module (not shown) operably coupled to the microneedle sensor unit 50 (particularly the macroneedle array). The control module may include a potentiostat, a processor coupled to the potentiostat, and memory coupled to the processor.

[0061] In some embodiments, the control module's potentiostat provides the sweep voltage potential between the working electrode and the reference electrode of the microneedle array. In some embodiments, the potentiostat further measures the current flowing between the working electrode and the counter electrode.

[0062] In some embodiments, the processor of the control module may be configured to drive and / or control the potentiostat's measurements. In some embodiments, the processor analyzes the data from the potentiostat.

[0063] The control module's memory may include instructions for the processor to apply a sweep voltage to a potentiostat, to measure a current in response to the application of the sweep voltage, and to use the measured current to determine the presence, absence, or concentration of a target analyte, or the concentration of uric acid.

[0064] In some embodiments, the control module temporarily stores the data measured by the microneedle sensor unit 50 in memory. In some embodiments, the control module may further include a receiver and / or transmitter connected to a processor and configured to transfer data between the microneedle sensor unit 50 and one or more external devices, systems, or networks. In some embodiments, the transmitter is configured to transmit a signal to one or more external devices, systems, or networks based on the presence or absence of the target analyte, the measured current, or both. In some embodiments, the signal transmitted by the transmitter is based on the measured concentration of the target analyte.

[0065] In the embodiments shown in Figures 6(A) and (B), the microneedle sensor device 100 in Figure 6(A) is equipped with a disposable microneedle sensor unit 50 and an electronic unit 120 that can be separated. Alternatively, the configuration of the electronic unit 120 may be built into the main body 51 in Figure 6(B), so that the microneedle sensor unit 50 in Figure 6(B) performs the roles of both the microneedle sensor unit 50 and the electronic unit 120. In this case, the electronic unit 120 portion in Figure 6(A) can be omitted, and only the microneedle sensor unit 50 can be attached to the skin of the target 90.

[0066] Figure 7 shows a block diagram of an example of an electronic unit 120 of a wearable microneedle sensor device 100. As shown in this example, the electronic unit 120 includes a signal conditioning unit 125, a power supply 129, an output unit (which may be embodied as, for example, a wireless communication unit 127), and an electrical interface (which may include, for example, one or more electrical interconnections such as pins). The electronic unit 120 is configured to receive electrical signals acquired from the microneedle sensor unit 50 (for example, in the electrical interface 126) and process them at least partially (for example, in the signal conditioning unit 125). In some embodiments, as shown in Figure 7, the electronic unit 120 of the device 100 includes a data processing unit 124 for processing the at least partially processed signals as data, for example, in digital format. In some implementations, the data processing unit 124 includes a microcontroller and a multiplexer that manage data acquisition on the data channel from the electrodes. The electronic unit 120 is configured to output raw signals or partially processed electrical signals and / or processed data. For example, in some embodiments, the electrical interface 126 is configured to electrically couple to the output port of the microneedle sensor unit 50, which is electrically connected to a conductive member (e.g., a wire) in the substrate of the microneedle sensor unit 50. On the other hand, in some embodiments, for example, the electrical interface 126 may be configured as an array of electronic interface components such as pins, which electrically couple the array of electrodes to the signal conditioning unit 125 and / or other circuits of the electronic unit 120, and electrically couple to the corresponding array of termination portions of conductive members that connect to the array of electrodes of the microneedle sensor unit 50 (e.g., spike microneedles 111).

[0067] In some embodiments of the electronic unit 120, for example, the output unit may include electrical contacts that electrically interface with conductive members (e.g., conductive wires) to provide data to an external circuit or device. In some embodiments, the output unit may include a wireless communication unit 127, which may include a wireless transmitter or transceiver device such as an RF front end (RFE), capable of communicating with an external device to provide raw, partially processed, or fully processed data from the data processing unit 124. For example, in such exemplary embodiments, the RFE may manage the communication protocol of the radio signals transmitted and / or received by the antenna of the output unit. The power supply 129 may include a battery, fuel cell, or other power source to supply power to the components of the electronic unit 120 and / or the microneedle sensor unit 50.

[0068] In some embodiments of the electronic unit 120, the data processing unit 124 may include a processor 121 for processing data and a memory 122 that communicates with the processor 121 for storing and / or buffering data. For example, the processor 121 may include a central processing unit (CPU) or a microcontroller unit (MCU). For example, the memory 122 may contain and store processor-executable code that, when executed by the processor, configures the data processing unit 124 to perform various operations, such as receiving information, commands, and / or data, processing the information and data, and transmitting or providing the information / data to another device. To support the various functions of the data processing unit 124, the memory 122 may store information and data such as instructions, software, values, images, and other data processed or referenced by the processor 121. For example, the storage function of the memory 122 can be implemented using various types of random access memory (RAM), read-only memory (ROM), flash memory, and other suitable storage media.

[0069] In some implementations, the data processing unit 124 includes an input / output (I / O) unit 123 for interface the processor 121 and / or memory 122 with other modules, units, or devices associated with the mobile device 400 and / or other external devices.

[0070] In some embodiments, the processor 121, memory 122, and / or I / O unit 123 communicate with a wireless communication unit 127, such as a transmitter or transmitter / receiver unit. For example, in such embodiments, the I / O unit 123 can interface the processor 121 and memory 122 with the wireless communication unit 127 and can utilize various types of wireless interfaces compatible with typical data communication standards that can be used in communication of the data processing unit 124 with other devices, such as between one or more computers in a cloud and a user device. Data communication standards include, but are not limited to, Bluetooth, Bluetooth low energy (BLE), ZiGbee, IEEE 802.11, Wireless Local Area Network (WLAN), Wireless Personal Area Network (WPAN), Wireless Wide Area Network (WWAN), WiMAX, IEEE 802.16 (Worldwide Interoperability for Microwave Access (WiMAX)), 3G / 4G / LTE cellular communication methods, and parallel interfaces.

[0071] In some implementations, the data processing unit 124 can interface with other devices using a wired connection via the I / O unit 123. The data processing unit 120 can also interface with other external interfaces, data storage sources, and / or visual or audio display devices to retrieve and transfer data and information that can be processed by the processor 121, stored in memory 122, or presented on a mobile device 130 (e.g., a smartphone) or an output unit of an external device.

[0072] Figure 8 is a schematic diagram of a communication system 1000 that handles measurement data from the microneedle sensor device shown in Figure 6(A). The communication system 1000 in Figure 8 is an IOMT (Internet of Medical Things) system and comprises the computer systems of the microneedle sensor device 100, a portable electronic device 400, a cloud network 500 that communicates with the computer system of the portable electronic device 400 via a communication connection 200, a server 600 connected to the cloud network 500, a home 700, one or more facilities 800 such as a hospital, clinic, rehabilitation facility, or pharmacy, and an ambulance 900. One or more of the computer systems of the server 600, the home 700, one or more facilities 800, and the ambulance 900 may include an AI computer system. In some embodiments, the portable electronic device 400 is a mobile phone such as a smartphone, a smartwatch, or a tablet. The portable electronic device 400 includes a processing unit that processes data from the microneedle sensor device 100, a memory for storing various data, and a communication interface that enables sending and receiving data with other electronic devices. The communication interface can utilize any suitable communication technology known in the art, such as Bluetooth®, SMS messaging, or email. The communication connection 200 via the communication interface is, for example, a wireless communication connection. In some embodiments, data is transferred by an application to which additional data processing can be applied. In some embodiments, the application is a smartphone application. In some embodiments, it is also possible to communicate data directly over the LTD.

[0073] In the communication system 1000, current measurements, the presence or absence or concentration of a target analyte, or the concentration of uric acid in the body fluids of the target 90 detected or measured by the microneedle sensor device 100 are transmitted to and stored in the portable electronic device 400. The portable electronic device 400 transmits data such as current measurements, the presence or absence or concentration of a target analyte, or the concentration of uric acid to a computer system in at least one facility or mobile medium that can be selected from a group consisting of a server 600 connected to a cloud network 500 via a communication connection 200, a home 700, a facility such as a hospital 800, and an ambulance 900. The computer system in at least one facility or mobile medium that can be selected from a group consisting of a server 600, a home 700, a facility such as a hospital 800, and an ambulance 900 includes a processing unit for processing data from the portable electronic device 400, a memory for storing various data, and a communication interface that enables sending and receiving data with other electronic devices.

[0074] In some embodiments, in a communication system 1000, signals generated by the microneedle sensor device 100 in response to current measurements, the presence or absence or concentration of a target analyte, or the concentration of uric acid, or both, are transmitted by the microneedle sensor device 100 to a portable electronic device 400 (i.e., a mobile phone, smartwatch, or tablet). In some embodiments, the signals are further transmitted from the portable electronic device 400 via a cloud network 500 to a computer system in at least one facility or mobile medium that can be selected from a group consisting of servers 600 connected to the cloud network 500, homes 700, facilities such as hospitals 800, and ambulances 900, where they are viewed by the subject, the subject's family, and / or the subject's care providers, etc. Care providers include, for example, doctors, nurses, caregivers, hospitals, pharmacies, welfare facilities, government agencies, or a combination thereof.

[0075] In some embodiments, current measurements, the presence, absence, or concentration of a target analyte, or uric acid concentration may be determined by a computer system in at least one facility or mobile medium, or any combination thereof, which may be selected from the group consisting of a microneedle sensor device 100, a portable electronic device 400, and / or a server 600, a home 700, a facility such as a hospital 800, and an ambulance 900. In some embodiments, the subject's family, the subject's caregiver, or both may be able to communicate with the subject via a computer system in at least one facility or mobile medium, which may be selected from the group consisting of a server 600, a home 700, a facility such as a hospital 800, and an ambulance 900.

[0076] In some embodiments, if the concentration of the target analyte is greater than or less than a target-specific threshold for the concentration of the target analyte, or if the AI ​​determines that the change in the concentration of the target analyte differs from previous data, the microneedle sensor device 100 may transmit the concentration of the target analyte, an anomaly notification, or both. In some embodiments, the signal may be transmitted periodically. In some embodiments, the signal may be received by a computer system in at least one facility or mobile medium, which may be selected from the group consisting of a portable electronic device 400 and / or a server 600, a home 700, a facility such as a hospital 800, and an ambulance 900, where the signal may be accessed by the subject, the subject's family, and / or the subject's caregivers, or any combination thereof. In some embodiments, a computer system in at least one facility or mobile medium, which can be selected from the group consisting of a portable electronic device 400 and / or a server 600, a home 700, a facility such as a hospital 800, and an ambulance 900, can analyze the presence, absence, or concentration of a substance, the measured current, or both, to determine a pattern, recommend improvements to treatment, or both. In this way, by measuring target analytes derived from a subject in real time using a microneedle sensor device 100 and analyzing or determining them using a computer system in at least one facility or mobile medium, which can be selected from the group consisting of a portable electronic device 400 and a server 600, a home 700, a facility such as a hospital 800, and an ambulance 900, the subject, the subject's family, and / or the subject's caregivers can manage the subject's health, manage diseases, and / or detect diseases early.

[0077] This disclosure provides a method for detecting uric acid, glucose, lactic acid, or ions in a fluid using a sensor described in any of the above. Such a method includes applying a sweep voltage to the electrode, measuring a current in response to the sweep voltage, and using the measured current to detect the presence or absence of uric acid, glucose, lactic acid, or ions in the fluid, or to measure the concentration of uric acid, glucose, lactic acid, or ions. The ions include, for example, metal ions, and the metal ions include, for example, sodium ions.

[0078] This disclosure provides a method for detecting the concentration of a target analyte in a fluid using a sensor as described above. Such a method includes applying a sweep voltage to an electrode, measuring a current in response to the sweep voltage, and using the measured current to measure the concentration of the target analyte in the fluid. Since the measured current reflects electrons generated by the oxidation of the target analyte, this method can be considered to measure the concentration of the target analyte in the fluid using electrons generated by the oxidation of the target analyte. In certain embodiments, the fluid contains uric acid, and an increase in the uric acid concentration in the fluid during or after the application of the sweep voltage is an indicator of the dissociation of the target analyte and molecules bound to the target analyte.

[0079] This disclosure provides a method for electrically dissociating a target analyte and molecules bound to the target analyte using a sensor as described in any of the above. Such a method includes bringing a fluid containing the target analyte into contact with the sensor to bind the target analyte to molecules attached to a layer of the electrode containing multilayer carbon nanotubes, and dissociating the target analyte and molecules bound to the target analyte by applying a sweep voltage to the electrode. The method for electrically dissociating a target analyte and molecules bound to the target analyte may further include detecting the electrical dissociation. In this case, the fluid contains uric acid, and an increase in the uric acid concentration in the fluid during or after the application of the sweep voltage may be an indicator of the dissociation of the target analyte and molecules bound to the target analyte. The increase in uric acid concentration allows for the detection of the electrical dissociation of the target analyte and molecules bound to the target analyte.

[0080] In some embodiments, an increase in uric acid concentration in the fluid during or after the application of a sweep voltage is an indicator of dissociation between the target analyte and the probe.

[0081] Biological adhesion to the electrodes of electrochemical sensors has been a hindrance to long-term operation of these sensors due to the resulting decrease in sensor sensitivity. However, electrical dissociation of the electrodes has opened the way to long-term operation. Electrical dissociation allows the electrode surface to be reset, which is expected to extend the lifespan of the electrochemical sensor. Furthermore, because the target analyte and the probe can be dissociated, the concentration of the target analyte in the fluid can be measured repeatedly and continuously over a certain period (for example, between 2.5 seconds and 1 minute).

[0082] As described above, the inventors have invented electrodes (microneedles, plates, and microwires) that can measure uric acid concentration with extremely high precision by optimizing the metal surface to treat uric acid, which is abundant in bodily fluids such as sweat and interstitial skin fluid, as the reaction center. The electrodes are coated with graphite ink on the metal surface, and multi-walled carbon nanotubes are electrochemically deposited on top of it. As a result, they succeeded in detecting uric acid concentrations ranging from femtometers to nanomolars. The current that flows in response to the application of a sweep voltage to the electrode reflects the electrons generated by the oxidation of uric acid. Therefore, by utilizing the electrons from uric acid oxidation, the concentration of a target analyte can also be detected with high sensitivity.

[0083] All patent applications and document disclosures cited herein are incorporated herein by reference in their entirety.

[0084] The following examples are for illustrative purposes only and are not intended to limit the technical scope of the present invention in any way. Unless otherwise specified, reagents may be commercially available or obtained or prepared by methods commonly used in the art or by procedures in known literature.

[0085] Real-time detection of endogenous protein biomarkers using electrochemical methods is difficult to achieve and has been a poorly researched area. This study proposes a novel approach to measure the concentration of protein markers by utilizing the oxidation reaction of uric acid. When a marker is present, electrons do not reach the working electrode, resulting in a low current value. However, when a marker is absent, electrons reach the working electrode, resulting in a high current value. This method utilizes the interaction between oxidation electrons generated from uric acid, which is abundant in the body, and proteins captured by antibodies on the working electrode.

[0086] Example 1: Microneedle Manufacturing A layer containing graphite is coated onto the surface of a microneedle, and then multi-walled carbon nanotubes (MWCNTs) are electrochemically deposited onto it. Figure 9 is an electron microscope image (8500x magnification) of an example of the working electrode of the microneedle of the present invention. A layer containing graphite 14 and a layer containing multi-walled carbon nanotubes 16 placed on top of the graphite-containing layer 14 are observed.

[0087] Example 2: Uric Acid Detection Using a Microneedle Array This sensor consists of three stainless steel microneedles (0.17 mm in diameter) serving as the working electrode, counter electrode, and reference electrode. Commonly used graphite is deposited on the working electrode and counter electrode. Furthermore, multi-walled carbon nanotubes (MWCNTs) are electrochemically deposited on the working electrode. The reference electrode is fabricated by depositing Ag / AgCl paste onto stainless steel. These three needle electrodes are fixed to a 3D-printed holder and used for measurement. Differential pulsed voltammetry (DPV), which allows for high-resolution electrochemical analysis, was employed for measurement. The performance of this biosensor was investigated using uric acid concentrations in PBS solution ranging from 0.5 nM to 4000 nM.

[0088] The DPV profile showed a very high peak current, allowing for the creation of a reliable calibration curve for uric acid concentration detection (Figures 10A and 10B). The practical limit of detection (LOD) calculated using the 3σ / m method was found to be 0.053 μM.

[0089] Example 3: Continuous detection experiment of uric acid using a microneedle array. The same experiment as in Example 2 was performed continuously. The amperometry method was used as the measurement method, and the results were obtained by continuously flowing a uric acid solution through a microfluidic channel. As a result, detection current values ​​proportional to the concentration were obtained (Figure 11).

[0090] Example 4: Preliminary experiment using antigen-antibody. EDC, NHS, dendrimer, and experimental mouse antibody were modified on the working electrode of the microneedle of the uric acid detection sensor manufactured in Example 2 to create a substrate for protein detection, and measurements were performed (Figure 12). This experiment was conducted assuming the detection of a biomarker contained in living organisms (plasma). Therefore, it was assumed that there was sufficient uric acid in the region where this biomarker exists. At this time, it is utilized that when the biomarker is captured by the antibody, uric acid detection is inhibited and the detection current value decreases. It has already been proven that uric acid is present in human sweat and interstitial skin fluid between 100 μM and 500 μM.

[0091] Using the sensor fabricated in Example 2, preliminary experiments were conducted to immobilize mouse M2Flag monoclonal antibody onto the working electrode via goat anti-mouse IgG antibody and electrically dissociate it, and its usefulness was investigated. First, uric acid concentration detection with a LOD of 0.59 μM was achieved using the sensor and differential pulsed voltameometry (DPV) (Figure 13). This sensor has three microneedle electrodes (working electrode, counter electrode, and reference electrode) and is connected to a potentiostat.

[0092] Example 5: Uric Acid Detection Using Antigen-Antibody in a Microfluidic Channel Using the sensor manufactured in Example 2, goat anti-mouse IgG antibody was immobilized on MWCNTs, and the sensor was placed in a microfluidic channel. A uric acid solution containing mouse M2 Flag monoclonal antibody (Flag antibody) (adjusted to a sweat concentration of 200 μM) was injected into the channel, and the detection of its concentration and dissociation were evaluated. First, the concentration of the Flag antibody was measured using uric acid as the reaction standard, and it was confirmed that it was possible to detect a concentration of LOD: 55.9 fM (Figure 14).

[0093] A 67nM Flag antibody was captured on the working electrode, and a 0.3 V, 100 Hz AC voltage was applied between the working electrode and the counter electrode for 0.5 seconds. This process was repeated. After 1-5 applications of the AC voltage, a change in the antibody concentration in uric acid was observed. After repeating this process 5 times, the uric acid DPV current value recovered to a level where the Flag antibody was no longer attached (Figure 15: the graph corresponds to the DPV current value obtained in each experiment). This suggests that the Flag antibody dissociated. Subsequently, the same concentration (67nM) of Flag antibody was flowed into a microfluidic device and washed, causing the DPV value to decrease again. This is thought to be because the Flag antibody re-attached to the goat anti-mouse IgG antibody. These results demonstrate that the electrodissociation method is effective in electrochemical sensors aimed at continuous detection of protein markers.

[0094] By electrically dissociating the antigen-antibody reaction, which conventionally required a half-life of 20 hours, it becomes possible to perform continuous detection with higher accuracy and a greater number of trials over the same period compared to conventional methods.

[0095] Example 6 Individual and continuous glucose detection using microneedles After immobilizing MWCNTs on the surface of stainless steel microneedles, GOX (glucose oxidase) was immobilized using EDC / NHS. Then, glucose was dropped onto the surface of the microneedle sensor and detected. Figure 16A shows the detection of glucose concentrations at different concentrations, and Figure 16B shows the continuous detection of glucose. The LOD was 2.41 μM. This sensor is always highly sensitive, capable of detecting glucose in sweat (glucose concentration in sweat: 0.06-0.2 mM).

[0096] Example 7 Cortisol Measurement After immobilizing MWCNTs on the surface of a stainless steel microneedle, an aptamer that specifically binds to cortisol was immobilized using EDC / NHS (Figure 17A). Ferrocene, a source of electron oxide, was immobilized at the end of this aptamer. Then, cortisol was dropped onto the surface of the microneedle sensor and detected using DPV. Figure 17B shows the detection of cortisol concentrations at different concentrations. The LOD was 0.016 pM. The aptamer contains ferrocene, an electron donor, and when the target marker binds to the aptamer, electron transfer is inhibited, resulting in a decrease in the current value. Due to the rapid degradation of ferrocene, this sensor can be used as a disposable type.

[0097] 10…Electrode, 12…Base, 14…Graphite-containing layer, 16…Wall-layer carbon nanotube-containing layer

Claims

1. An electrode comprising a base, a graphite-containing layer disposed on the base, and a multilayer carbon nanotube-containing layer disposed on the graphite.

2. The electrode according to claim 1, wherein the electrode comprises a microneedle array, a plate electrode, or a microwire.

3. The electrode according to claim 2, wherein the electrode includes a microneedle array, and the microneedle array includes a working electrode, a reference electrode, a counter electrode, and a potentiostat.

4. The electrode according to claim 1, wherein a molecule that binds to a target molecule is attached to the layer containing the multilayer carbon nanotube.

5. A sensor comprising an electrode according to any one of claims 1 to 3 and a molecule that binds to a target analyte attached to the layer containing the multilayer carbon nanotube.

6. The sensor according to claim 5, wherein the electrode includes a microneedle array.

7. A wearable microneedle sensor device comprising a microneedle sensor unit having a body made of an electrically insulating material, an electrode according to claim 1, which includes a microneedle array disposed on the body, and a microfluidic device disposed below the electrode.

8. The microneedle sensor device according to claim 7, further comprising a plurality of detection and communication electrical circuits arranged in or on the main body, each of which is coupled to each of the microneedle arrays.

9. The microneedle sensor device according to claim 8, further comprising an electronic unit that electrically communicates with the plurality of detection and communication circuit connections, the electronic unit comprising a data processing unit and an electrical interface capable of contacting the plurality of detection and communication electrical circuits of the microneedle sensor unit.

10. The microneedle sensor unit according to claim 9, wherein the electrical interface electrically connects the microneedle sensor unit and the electronic unit.

11. A method for detecting uric acid, glucose, lactic acid, or ions in a fluid using the sensor described in claim 5, comprising: applying a sweep voltage to the electrode; measuring a current in response to the sweep voltage; and using the measured current to detect the presence or absence of uric acid, glucose, lactic acid, or ions in the fluid, or to measure the concentration of uric acid.

12. A method for detecting a target analyte in a fluid using the sensor described in claim 5, comprising: applying a sweep voltage to the electrode; measuring a current in response to the sweep voltage, wherein the measured value of the current reflects electrons generated by the oxidation of uric acid; and measuring the concentration of the target analyte in the fluid using the electrons generated by the oxidation of uric acid.

13. A method for electrically dissociating a target analyte and molecules bound to the target analyte using the sensor described in claim 5, comprising: bringing a fluid containing uric acid and the target analyte into contact with the sensor to bind the target analyte and molecules bound to the target analyte attached to a layer of the electrode containing multilayer carbon nanotubes; and dissociating the target analyte and molecules bound to the target analyte by applying a sweep voltage to the electrode.

14. The method according to claim 13, further comprising detecting the electrical dissociation, wherein the fluid contains uric acid, and an increase in the uric acid concentration in the fluid during or after the application of a sweep voltage is an indicator of the dissociation of the target analyte and the molecules bound to the target analyte.

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