Background interference mitigation for high-sensitivity ketone detection by accumulation mode detection at low working electrode potentials.

Abstract

This disclosure relates to a method for improving the sensitivity of detecting ketones, comprising the steps of: i) providing a ketone detection electrode comprising a ketone-responsive enzyme and a redox mediator; and ii) providing a background detection electrode comprising a redox mediator but not comprising a ketone-responsive enzyme; and providing a steady state by applying a potential of less than +40 mV. The ketone detection electrode and the background detection electrode may be disconnected from the circuit simultaneously or sequentially to allow charge accumulation over a set period of time. After sufficient charge has been accumulated, both electrodes can be reconnected to the circuit. The ketone signal can be measured by subtracting the signal obtained from the background detection electrode from the signal obtained from the ketone detection electrode. This disclosure further relates to a ketone sensor comprising a first detection electrode for detecting ketones and a second detection electrode for detecting background.

Description

[Technical Field]

[0001] This invention relates to background interference mitigation for highly sensitive ketone detection by accumulation mode detection at low working electrode potentials. [Background technology]

[0002] Glucose management is a primary focus of diabetes treatment, and continuous ketone monitoring can be beneficial for diabetic patients, especially those with type 1 diabetes. Individuals with type 1 diabetes may experience a potentially fatal complication known as diabetic ketoacidosis (DKA). Continuous ketone detection may help prevent DKA. Ketone concentrations also correlate with heart failure metrics. For example, patients with elevated ketone concentrations above 300 μM are at risk of heart failure. While such measurements are important, in vivo ketone concentrations tend to be relatively low.

[0003] Furthermore, ascorbates, uric acid, and other compounds naturally found in interstitial fluid generate background interference signals to the biosensor, which can reduce the accuracy of analyte measurements. At ketone concentrations above 1 mM, this background interference signal is significantly lower than the ketone signal, allowing for accurate determination of ketone concentrations. However, as ketone concentrations decrease to below 1 mM, the ratio of background interference to ketone signal increases, hindering accurate ketone measurement.

[0004] Therefore, there is a need for a biosensor that can provide accurate and continuous in vivo ketone monitoring by improving ketone sensitivity and reducing background interference signals. [Overview of the project] [Means for solving the problem]

[0005] This disclosure may provide a method for improving the sensitivity of detecting ketones, comprising: i) providing a ketone-detecting electrode comprising a ketone-responsive enzyme and a redox mediator; and ii) providing a background-detecting electrode comprising a redox mediator and not comprising a ketone-responsive enzyme; and providing a steady state by applying a potential of less than +40 mV. The ketone-detecting electrode and the background-detecting electrode can be disconnected from the circuit simultaneously or sequentially to accumulate charge over a set period of time. The ketone signal can be measured by subtracting the signal obtained from the background-detecting electrode from the signal obtained from the ketone-detecting electrode. This disclosure may further provide a ketone sensor comprising a first detection electrode for detecting ketones and a second detection electrode for detecting background.

[0006] This disclosure also relates to a method for detecting ketones, the method relating to a biological fluid containing ketones: a) A first detection electrode containing a ketone-responsive enzyme and a redox mediator; and b) Contacting a second detection electrode containing a redox mediator but not a ketone-responsive enzyme, The first and second sensing electrodes are connected to the circuit, and a potential of less than +40mV is applied to both electrodes to provide a steady state. Disconnecting the first and second detection electrodes from the circuit, Accumulating the charge generated by the biological fluid that is in contact with the first and second detection electrodes over a set period of time, After the set period, the first and second detection electrodes are connected to the circuit (for example, reconnected), This includes measuring a ketone signal by subtracting the signal obtained from the second detection electrode from the signal obtained from the first detection electrode.

[0007] This disclosure also relates to a method for detecting ketones, the method being (a) Contacting first and second detection electrodes with a biological fluid containing ketones, wherein the first detection electrode contains a ketone-responsive enzyme and a redox mediator, and the second detection electrode contains a redox mediator but does not contain a ketone-responsive enzyme, (b) Connecting the first sensing electrode to the circuit and applying a potential of less than +40mV to provide a steady state, (c) Disconnecting the first detection electrode from the circuit and connecting the second detection electrode to the circuit, and applying a potential of less than +40mV to provide a steady state, (d) During the first set period, accumulate the charge derived from the biological fluid that reacts with the first detection electrode, (e) After the first setting period, the first detection electrode is connected to the circuit (for example, reconnected), and the second detection electrode is disconnected from the circuit. (f) During the second set period, accumulate the charge derived from the biological fluid that reacts with the second detection electrode, (g) After the second setting period, the second detection electrode is connected to the circuit (for example, reconnected), (h) Measuring a ketone signal by subtracting the signal obtained from the second detection electrode from the signal obtained from the first detection electrode.

[0008] In some aspects of this method, the cutting and connecting in steps (b) and (c) are simultaneous. In any of these embodiments, the setting period, the first setting period, the second setting period, or any combination thereof is 30 seconds or longer.

[0009] In any of these embodiments, the applied potential is approximately +5mV to approximately -250mV. In some embodiments, the applied potential is approximately -80mV. In any of these embodiments, the first detection electrode includes a working electrode and a ketone detection layer on a portion of the working electrode, the ketone detection layer including a ketone-responsive enzyme and a redox mediator.

[0010] In any of these embodiments, the ketone-responsive enzyme is 3-hydroxybutyrate dehydrogenase. In any of these embodiments, the first detection electrode further comprises NAD(P)H oxidoreductase and nicotinamide adenine dinucleotide phosphate (NAD(P)+) or a derivative thereof.

[0011] In any of these embodiments, the ketone-responsive enzyme is bound to a redox mediator. In any of these embodiments, the first detection electrode further comprises albumin.

[0012] In any of these embodiments, the first detection electrode further includes a pH buffer. In any of these embodiments, the second sensing electrode includes a working electrode and a background sensing layer on a portion of the working electrode, the background sensing layer including a redox mediator. In one embodiment, the redox mediators in the first and second sensing electrodes are made of the same material.

[0013] In any of these embodiments, the redox mediator comprises a polymer and an electron transfer agent. In some embodiments, the polymer comprises poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene). In some embodiments, the polymer comprises a polymer or copolymer repeating unit comprising at least one pendant pyridinyl group, an imidazolyl group, or both a pyridinyl group and an imidazolyl group. In some embodiments, the electron transfer agent comprises a transition metal complex. In some embodiments, the transition metal complex comprises osmium, ruthenium, iron, cobalt, or a combination thereof. In some embodiments, the transition metal complex is an osmium transition metal complex comprising one or more ligands, at least one of which comprises a nitrogen-containing heterocycle.

[0014] In any of these embodiments, the redox mediator comprises an osmium complex bonded to a poly(vinylpyridine) polymer. In some embodiments, the polymer is crosslinked with a crosslinking agent. In some embodiments, the crosslinking agent is a polyepoxide, cyanuryl chloride, N-hydroxysuccinimide, imide ester, epichlorohydrin, or a combination thereof. In some embodiments, the crosslinking agent is polyethylene glycol diglycidyl ether (PEGDGE).

[0015] In some of these embodiments, the ketone detection layer or the background detection layer is continuous, or both detection layers are continuous. In some embodiments, the ketone detection layer or the background detection layer is discontinuous, or both detection layers are discontinuous.

[0016] In some embodiments, the membrane is overcoated with at least a ketone detection layer, at least a background detection layer, or both. In some embodiments, the membrane comprises poly(4-vinylpyridine).

[0017] In any of these embodiments, the first and second sensing electrodes are part of a sensor comprising a housing. In some embodiments, the sensor further comprises a sensor tail configured for implantation into tissue, and the first and second sensing electrodes are disposed on the sensor tail. In some embodiments, the sensor further comprises a reference electrode, a counter electrode, or both a reference electrode and a counter electrode. In some embodiments, the sensor further comprises at least one insulating layer. In some embodiments, the sensor further comprises at least one substrate, on which the first or second sensing electrode is disposed, or on which both sensing electrodes are disposed.

[0018] This disclosure further relates to ketone sensors, including: A first detection electrode comprising a first working electrode and a ketone detection layer on a portion of the first working electrode, wherein the ketone detection layer comprises a ketone-responsive enzyme and a redox mediator; and A second detection electrode comprising a second working electrode and a background detection layer on a portion of the second working electrode, wherein the background detection layer contains a redox mediator but does not contain a ketone-responsive enzyme.

[0019] In some embodiments, the ketone-responsive enzyme is 3-hydroxybutyrate dehydrogenase. In some embodiments, the first detection electrode further comprises NAD(P)H oxidoreductase and nicotinamide adenine dinucleotide phosphate (NAD(P)+) or a derivative thereof. In some embodiments, the first detection electrode further comprises albumin. In some embodiments, the first detection electrode further comprises a pH buffer.

[0020] In some embodiments, ketone-responsive enzymes are bound to redox mediators. In one embodiment, the redox mediators in the first and second sensing electrodes are made of the same material. In some embodiments, the redox mediator comprises a polymer and an electron transfer agent.

[0021] In some embodiments, the polymer comprises poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene). In some embodiments, the polymer comprises a polymer or copolymer repeating unit comprising at least one pendant pyridinyl group, an imidazolyl group, or both a pyridinyl group and an imidazolyl group. In some embodiments, the polymer is crosslinked with a crosslinking agent. In some embodiments, the crosslinking agent is a polyepoxide, cyanuryl chloride, N-hydroxysuccinimide, imide ester, epichlorohydrin, or a combination thereof. In some embodiments, the crosslinking agent is polyethylene glycol diglycidyl ether (PEGDGE).

[0022] In some embodiments, the electron transfer agent comprises a transition metal complex. In some embodiments, the transition metal complex comprises osmium, ruthenium, iron, cobalt, or a combination thereof. In some embodiments, the transition metal complex is an osmium transition metal complex comprising one or more ligands, at least one of which comprises a nitrogen-containing heterocycle. In some embodiments, the redox mediator comprises an osmium complex bonded to a poly(vinylpyridine) polymer.

[0023] In some embodiments, the ketone detection layer or the background detection layer is continuous, or both detection layers are continuous on the working electrode. In some embodiments, the ketone detection layer or the background detection layer is discontinuous, or both detection layers are discontinuous on the working electrode.

[0024] In some embodiments, the sensor further includes a film that overcoats at least a ketone detection layer, at least a background detection layer, or both. In some embodiments, the film comprises poly(4-vinylpyridine).

[0025] In some embodiments, the sensor further comprises a housing. In some embodiments, the sensor further comprises a sensor tail configured for embedding in tissue, and first and second sensing electrodes are disposed on the sensor tail. In some embodiments, the sensor further comprises a reference electrode, a counter electrode, or both a reference electrode and a counter electrode. In some embodiments, the sensor further comprises at least one insulating layer. In some embodiments, the sensor further comprises at least one substrate, and either the first sensing electrode or the second sensing electrode is disposed on the substrate, or both sensing electrodes are disposed on the substrate.

[0026] Additional aspects and benefits of this disclosure may be described in part in the following description, derived from the description, or learned through the implementation of this disclosure. Please understand that the above summary and the following detailed explanation are illustrative and descriptive only, and do not limit the scope of the claims. [Brief explanation of the drawing]

[0027] [Figure 1] An example of a detection system that may incorporate the analyte sensor of this disclosure is shown in the diagram. [Figure 2A] This shows a cross-sectional view of an analyte sensor containing a single detection layer. [Figure 2B] This shows a cross-sectional view of an analyte sensor containing a single detection layer. [Figure 2C] This shows a cross-sectional view of an analyte sensor containing a single detection layer. [Figure 3A] A cross-sectional view of an analyte sensor containing two detection layers is shown. [Figure 3B] A cross-sectional view of an analyte sensor containing two detection layers is shown. [Figure 3C] A cross-sectional view of an analyte sensor containing two detection layers is shown. [Figure 4] A cross-sectional view of an analyte sensor containing two detection layers is shown. [Figure 5A] A perspective view of an analyzer sensor containing two detection layers on separate working electrodes is shown. [Figure 5B] A perspective view of an analyzer sensor containing two detection layers on separate working electrodes is shown. [Figure 5C] A perspective view of an analyzer sensor containing two detection layers on separate working electrodes is shown. [Figure 6] The graph shows the sensor current (nA) of a blank (background) sensor and a standard (ketone) sensor, each with different detection potentials of +40mV or -80mV relative to the Ag / AgCl reference, plotted against time (in units of time). [Figure 7]This figure plots the sensor current (nA) of a blank (background) sensor and a standard (ketone) sensor at -80mV against time (in units of time) relative to the Ag / AgCl reference. The large double-headed arrow represents the standard baseline of 190 μM ketone in serum + background signal. The smaller double-headed arrow represents the blank baseline for background signal only. [Figure 8A] The figure shows the sensor current (nA) of an exemplary ketone sensor of this disclosure using accumulation mode detection plotted against time (in units of time). [Figure 8B] The integrated charge for each peak is shown, with a large double-headed arrow indicating the standard baseline for 190 μM ketone in serum + background signal, and a smaller double-headed arrow indicating the blank baseline for background signal only. [Figure 9A] The figure shows the sensor current (nA) of an exemplary ketone sensor of this disclosure using cumulative detection ("Sensor 1") and a second ketone sensor ("Sensor 2") measuring the ketone concentration for Ag / AgCl using a standard amperometry method at -80mV, plotted against time (in units of time). [Figure 9B] The graph shows the sensor current (nA) from the time window between 4:00 PM and 12:00 AM plotted against time (in units of time). [Figure 10] The figure shows the sensor current (nA) of the exemplary ketone sensor (Sensor 1) of this disclosure after background subtraction, plotted against time (in units of time). [Figure 11A] The figure shows the sensor current (nA) of exemplary ketone sensors (sensors 1 and 2) of this disclosure using cumulative detection, plotted against time (in units of time). [Figure 11B] The figure shows the sensor current (maximum 10 nA) of exemplary ketone sensors (sensors 1 and 2) of this disclosure using cumulative detection, plotted against time (in units of time). [Figure 12](1) A figure showing the exemplary ketone sensors of this disclosure (sensors 1 and 2) after background subtraction, and (2) a figure showing the sensor current (nA) of the blood ketone test strip measurement plotted against time (in units of time). [Modes for carrying out the invention]

[0028] The headings provided herein are not intended to limit the various aspects of this disclosure, but can be defined by referring to this disclosure as a whole. Furthermore, since the scope of this disclosure is limited only by the appended claims, it should be understood that the terms used herein are intended solely to describe and not to limit specific aspects.

[0029] definition For convenience, the meanings of some terms and phrases used in this specification, the examples, and the appended claims are provided below. Unless otherwise specified or implied by the context, the following terms and phrases have the meanings set forth below. Since the scope of this art is limited only by the claims, the definitions are provided to help describe specific aspects and do not limit the claimed art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this art belongs. In the event of any apparent inconsistency between the use of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0030] The articles “a,” “an,” and “the” are used herein to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “an element” means one or more elements.

[0031] As used herein, the term “about” means within an acceptable range of error for a particular value, as determined by those skilled in the art, which depends in part on how that value is measured or determined (i.e., the limits of the measuring system). For example, “about” could mean within 3 or more standard deviations, according to convention in the art. Alternatively, “about” could mean within a range of up to 10% (e.g., up to 5% or up to 1%) of a given value.

[0032] The term "at least" preceding a number or a set of numbers is understood, as is clear from the context, to include the number related to the term "at least," and all subsequent numbers or integers that may logically be included. When "at least" precedes a set of numbers or a range, it is understood that "at least" can modify each of the numbers in that set or range. For example, "at least 3" means at least 3, at least 4, at least 5, etc. When "at least" precedes a component in a method step, that component is included in the step, but any additional components are optional.

[0033] As used herein, terms such as “comprises,” “comprising,” “having,” “including,” and “containing” are open-ended terms meaning “including, but not limited to.” To the extent that a given aspect disclosed herein “includes” certain elements, it should be understood that this disclosure also specifically intends to and disclose aspects that “essentially consist of” those elements and aspects that “consist of” those elements.

[0034] As used herein, terms such as "consists essentially of" and "consisting essentially of" should be interpreted as semi-closed terms meaning that no other components that substantially affect the basic and novel features of a particular aspect are included.

[0035] As used herein, terms such as "consists of," "consisting of," etc., should be interpreted as restrictive terms such that a particular set of elements "consisting of" excludes any elements, steps, or components not specified in that embodiment.

[0036] As used herein, “a set period of time” is the amount of time required to perform a particular step (e.g., a step of bringing a biological fluid into contact with a sensing electrode, a step of bringing one or more sensing electrodes into contact with a circuit, or a step of accumulating charge to provide a signal output that can be measured and quantified for a given analyte (e.g., a ketone)). The various set periods described herein may be the same or different. Typically, as will be discussed in detail elsewhere herein, a set period may be about 1 second or more (e.g., about 5 seconds or more, about 10 seconds or more, or about 30 seconds or more) and about 30 minutes or less (e.g., about 20 minutes or less, about 10 minutes or less, about 5 minutes or less, about 3 minutes or less, or about 1 minute or less).

[0037] As used herein, the term "accumulation mode sensing" refers to the accumulation of electrons generated from the oxidation of an analyte, where oxidation occurs in or on the sensing element of a working electrode that is not connected to a circuit, thereby resulting in the accumulation of electrons.

[0038] As used herein, “analyte” refers to the enzyme substrate that is being measured or detected. The analyte may be derived from, for example, a biological fluid and may be tested in vivo, ex vivo, or in vitro. In most embodiments herein, the analyte is a ketone.

[0039] As used herein, the terms “background interfering substance” or “interfering substance” refer to substances in the sample being assayed that may prevent the accurate measurement of the desired analyte (e.g., ketones). Common interfering substances include, for example, ascorbic acid, uric acid, homovanillic acid, 5-hydroxytryptamine, catecholamines (e.g., dopamine, norepinephrine and their major metabolites (e.g., 3,4-dihydroxyphenylacetic acid, 3-methoxytyramine)), indoleamines, drug metabolites, fibrinogen, proteins, cells (e.g., leukocytes, erythrocytes), metal ions (e.g., copper ions, mercury ions), and combinations thereof.

[0040] As used herein, “biofluid” refers to any body fluid or derivative of body fluid in which the analyte can be measured. Examples of biofluids include, for example, skin fluid, subcutaneous fluid, interstitial fluid, plasma, blood (e.g., venous or vascular), lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, sweat, or tears. In certain embodiments, the biofluid is skin fluid or interstitial fluid.

[0041] As used herein, the phrase "configured to accumulate charge" refers to an arrangement of working electrodes and circuits that allows for the accumulation of electrons generated from the oxidation of an analyte (e.g., a ketone). Oxidation occurring on or in the sensing element of the working electrode is not connected to the circuit, thereby resulting in the accumulation of electrons.

[0042] As used herein, the term “counter electrode” refers to an electrode paired with a working electrode through which a current equal in magnitude and opposite in sign to the current passing through the working electrode passes. In the context of aspects of this disclosure, unless otherwise indicated, the term “counter electrode” includes both a) a counter electrode and b) a counter electrode that also functions as a reference electrode (i.e., a pair / reference electrode).

[0043] As used herein, the term “crosslinking agent” refers to a molecule containing at least two (e.g., 2, 3, or 4) reactive groups (e.g., terminal functional groups) that can link at least two molecules together (intermolecular crosslinking) or at least two parts of the same molecule together (intramolecular crosslinking). Crosslinking agents having more than two reactive groups can perform both intermolecular and intramolecular crosslinking simultaneously.

[0044] As used herein, the term “electrolysis” refers to the electro-oxidation or electro-reduction of a compound, either directly at an electrode or via one or more electron-transfer agents. As used herein, the term "electron transfer agent" refers to a compound that carries electrons between the analyte and the working electrode, either directly or in cooperation with other electron transfer agents. An example of an electron transfer agent is a redox mediator.

[0045] As used herein, a component is “immobilized” or “adhered” to a polymer and / or sensor, for example, when the component is captured on or within a component of the polymer, sol-gel matrix, membrane, and / or sensor, covalently bonded to, ionically bonded to, electrostatically bonded to, or coordinately bonded to, a component of the polymer and / or sensor, thereby reducing or eliminating its mobility.

[0046] As used herein, the terms “non-leachable” compound or “non-leachable” compound mean a compound that is immobilized on the sensor so as not to substantially diffuse from the sensing layer of the working electrode over the period the sensor is used (e.g., the period the sensor is implanted in a patient or a sample is measured).

[0047] As used herein, the term “patient” refers to a living animal and therefore includes, for example, living mammals and living humans. The term “user” may be used herein as a term encompassing the term “patient.”

[0048] As used herein, the term “precursor polymer” refers to the starting polymer before various modifying groups are attached to form a modified polymer. As used herein, the term “reactive group” refers to a functional group of a molecule (e.g., polymer, crosslinker, enzyme) that can react with another compound to couple at least a portion of the other compound (e.g., another reactive group) to the molecule. Reactive groups include carboxy, activated esters, sulfonyl halides, sulfonic acid esters, isocyanates, isothiocyanates, epoxides, aziridines, halides, aldehydes, ketones, amines, acrylamides, thiols, acyl azides, acyl halides, hydrazines, hydroxylamines, alkyl halides, imidazoles, pyridines, phenols, alkyl sulfonates, halotriazines, imide esters, maleimides, hydrazides, hydroxy, and photoreactive azidoaryl groups. As understood in the art, activated esters generally include succinimidyl, benzotriazolyl, or aryl esters substituted with electron-withdrawing groups (e.g., sulfo, nitro, cyano, or halo groups); or carboxylic acids activated with carbodiimides.

[0049] As used herein, the term “redox mediator” means an electron transfer agent for transporting electrons between the analyte, the enzyme into which the analyte is reduced or the enzyme into which the analyte is oxidized, and the electrode, either directly or via one or more further electron transfer agents. Redox mediators containing a polymer backbone may also be referred to as “redox polymers.”

[0050] As used herein, the term “reference electrode” includes, unless otherwise indicated, both a) a reference electrode and b) a reference electrode that also functions as a counter electrode (i.e., a counter / reference electrode).

[0051] As used herein, the term “sensing layer” refers to a component of a sensor that includes components that facilitate the electrolysis of an analyte. The sensing layer may include components such as a redox mediator (e.g., an electron transfer agent or redox polymer), a catalyst (e.g., an analyte-specific enzyme) that catalyzes the reaction of the analyte to produce a response at the working electrode, or both an electron transfer agent and a catalyst. In some embodiments of this disclosure, the sensor includes a sensing layer that is non-leachingly disposed in close proximity to or on the working electrode.

[0052] As used herein, the term “detection element” refers to an application or region of an analyte-specific enzyme that is arranged with the detection layer. Thus, the detection element can interact with the analyte. The detection layer may have two or more detection elements constituting an analyte detection region positioned on the working electrode. In some embodiments, the detection element comprises an analyte-specific enzyme and an electron transfer agent (e.g., an electron transfer agent). In some embodiments, the detection element comprises an analyte-specific enzyme, a redox mediator, and a crosslinking agent.

[0053] As used herein, the term “sensor” refers to a device configured to detect the presence of an analyte in a sample and / or measure its level via electrochemical oxidation and reduction reactions on the sensor. These reactions are converted into electrical signals that may correlate (e.g., be proportional) to the amount, concentration, or level of the analyte in the sample.

[0054] As used herein, the term “continuous” in relation to a continuous analyte sensor (e.g., a “continuous ketone sensor”) refers to a sensor configured to perform one or more measurements of an analyte (e.g., a ketone) over a period of time. A continuous sensor can perform measurements sequentially according to its sampling frequency. For example, one or more measurements may be performed approximately every 1 ms, every 10 ms, every 100 ms, every 1 s, every 10 seconds, every 30 seconds, every 1 minute, every 5 minutes, every 10 minutes, every 30 minutes, or every 1 hour. Measurements may be performed continuously, for example, over a continuous period of at least 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, or longer. A continuous ketone sensor is typically in continuous contact with a sample, such as a biological fluid. For example, a continuous ketone sensor may include an implantable part or component as defined herein, which, during use, comes into continuous contact with a biological fluid such as skin fluid or interstitial fluid, and as a result, can perform measurements continuously or periodically over a continuous period of time according to the sampling frequency of the sensor.

[0055] As used herein, the term “substituted” functional group (e.g., substituted alkyl, alkenyl, alkoxy, aryl) includes, for example, halo, alkoxy, mercapto, aryl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxy, amino, alkylamino, dialkylamino, trialkylammonium, alkanoylamino, arylcarboxamide, hydrazino, alkylthio, alkenyl, and at least one substituent (e.g., 1, 2, 3, 4, or 5) which may be a reactive group.

[0056] As used herein, the term “working electrode” refers to the electrode on which the analyte or background interfering substance is electrooxidized or electroreduced, with or without the action of an electron transfer agent.

[0057] When used herein, "C 6~30The term "aryl" refers to aromatic compounds containing monocyclic, bicyclic, or tricyclic carbocyclic systems having one, two, or three aromatic rings, such as phenyl, naphthyl, anthracenyl, or biphenyl. Aromatic compounds generally contain, for example, 6 to 30 carbon atoms, 6 to 18 carbon atoms, 6 to 14 carbon atoms, or 6 to 10 carbon atoms. The term "aryl" is understood to include a carbocyclic moiety that is planar and contains 4n+2π electrons (where n=1, 2, or 3) according to Hückel's rule.

[0058] As used herein, the term "halo" refers to a halogen radical (i.e., F, Cl, Br, or I). When used herein, "C 1~6 The term "alkyl" refers to a linear or branched alkyl substituent containing, for example, about 1 to about 6 carbon atoms, for example, about 1 to about 4 carbon atoms, or about 1 to about 3 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, and n-hexyl. This definition also means that "alkyl" is, for example, C 1~6 This applies whenever it exists as part of a group such as a haloalkyl group (e.g., trifluoromethyl(-CF3)).

[0059] When used herein, "C 2~6 The term "alkenyl" refers to a linear alkenyl substituent containing, for example, 2 to about 6 carbon atoms (branched alkenyls have about 3 to about 6 carbon atoms). In one embodiment, the alkenyl group is C 2~4 It is an alkenyl. Examples of alkenyl groups include, but are not limited to, ethenyl, allyl, 2-propenyl, 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, and 1-hexenyl.

[0060] When used herein, "C 2~6The term "alkynyl" refers to, for example, a straight-chain alkynyl substituent containing from 2 to about 6 carbon atoms (branched alkynyls have from about 3 to about 6 carbon atoms). According to one embodiment, the alkynyl group is C 2~4 alkynyl. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 1-hexynyl, and the like.

[0061] As used herein, the term "hydroxy" refers to -OH. As used herein, the term "nitro" refers to -NO2. As used herein, the term "cyano" refers to -CN.

[0062] As used herein, the term "amino" refers to -NH2. Mono- and di-C 1~6 The term "alkylamino" refers to nitrogen bonded to one or two C 1~6 alkyl groups, i.e., -NHR or -NRR' (where R and R' are the same or different C 1~6 alkyl groups).

[0063] As used herein, "C 1~6 alkoxy" refers to a C 1~6 alkyl group bonded to oxygen, i.e., -OR (where R is a C 1~6 alkyl group). As used herein, "C 6~10 aryloxy" refers to an aryl group bonded to oxygen, i.e., -O(Ar) (where Ar is a C 6~10 aryl group).

[0064] As used herein, the term "aralkoxy" refers to the group -OR(Ar), where R is a C 1~6 alkyl group and Ar is a C 6~10 aryl group. As used herein, the term "carboxy" refers to -C(O)OH.

[0065] When used herein, "C 1~6 The term "alkylcarboxy" refers to a hydrogen atom bonded to a carboxyl group. 1~6 A carboxyl group replaced by an alkyl group, i.e., -C(O)OR(where R is C) 1~6 This refers to an alkyl group.

[0066] As used herein, the term "amide" refers to the structure -C(O)NH or -NHC(O). 1-6 The term "alkylamide" refers to -C(O)NR or -NRC(O), where R is C. 1-6 It is alkyl.

[0067] When used herein, "C 1~6 The term "haloalkylamide" is C 1~6 C is a C molecule in which the alkyl group is substituted with one, two, or three halo groups as described herein. 1~6 This refers to an alkylamide group.

[0068] As used herein, the term “heteroaryl” refers to aromatic compounds, such as those described herein, that contain a five- or six-membered ring in which one or two carbon atoms are replaced by nitrogen, sulfur, and / or oxygen. Examples of heteroaryls include, but are not limited to, pyridinyl, furanyl, pyrrolyl, quinolinyl, thiophenyl, indolyl, oxazolyl, isoxazolyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, 1,3,4-thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, and triazinyl.

[0069] As used herein, the term “heterocycloalkyl” refers to a monocyclic, bicyclic, or spirocyclic system containing 3 to 7 carbon atom ring members and 1, 2, or 3 other atoms selected from nitrogen, sulfur, and / or oxygen. Examples of such heterocycloalkyl rings include, but are not limited to, azilidinyl, oxylanil, thiazolinyl, imidazolidinyl, piperazinyl, homopiperazinyl, pyrrolidinyl, pyrrolidinyl, tetrahydrofuranil, tetrahydrothiofuranil, pyranyl, tetrahydropyranil, piperidinyl, and morpholinil.

[0070] Methods, sensors, and compositions disclosed herein Before further detailing the analyte sensors and their components of this disclosure, an overview of suitable in vivo analyte sensor configurations and sensor systems using such analyte sensors is provided to better understand aspects of this disclosure. Figure 1 shows a diagram of an exemplary detection system into which the analyte sensors of this disclosure may be incorporated. As shown, the detection system 100 includes a sensor control device 102 and a reader device 120 configured to communicate with each other via a local communication path or link 140, which may be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. The reader device 120 may, according to certain embodiments, constitute an output medium for viewing analyte concentrations and alerts or notifications determined by the sensor 104 or an associated processor, and for enabling one or more user inputs. The reader device 120 may be a multipurpose smartphone or a dedicated electronic reader device. Although only one reader device 120 is shown, there may, in some cases, be multiple reader devices 120. The reader device 120 can also communicate with the remote terminal 170 and / or the trusted computer system 180 via communication paths / links 141 and / or 142, respectively, which can also be wired or wireless, one-way or two-way, and encrypted or unencrypted. The reader device 120 can also, or alternatively, communicate with the network 150 (e.g., a cellular network, the internet, or a cloud server) via communication path / link 151. The network 150 can be further communicated to the remote terminal 170 via communication path / link 152 and / or the trusted computer system 180 via communication path / link 153. Alternatively, the sensor 104 can communicate directly with the remote terminal 170 and / or the trusted computer system 180 without the presence of the intervening reader device 120.For example, but not limited to, sensor 104 can communicate with remote terminal 170 and / or trusted computer system 180 via a direct communication link to network 150, according to certain embodiments, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is incorporated herein by reference in its entirety. Any suitable electronic communication protocol, such as Near Field Communication (NFC), Radio Frequency Identification (RFID), Bluetooth® or Bluetooth® Low Energy protocol, or Wi-Fi®, may be used for each of the communication paths or links. Remote terminal 170 and / or trusted computer system 180 may be accessible by individuals other than the primary user who are interested in the user's analyte level, according to certain embodiments. The reader device 120 may include a display 122 and an optional input component 121. The display 122 may include a touchscreen interface, according to several embodiments.

[0071] The sensor control device 102 includes a sensor housing 103 that can house the circuitry and power supply for operating the sensor 104. Optionally, the power supply and / or active circuitry can be omitted. A processor (not shown) can be communicatively connected to the sensor 104, and the processor is physically located within the sensor housing 103 or the reader device 120. The sensor 104 protrudes from the underside of the sensor housing 103 and extends through an adhesive layer 105, which, according to one embodiment, is adapted to adhere the sensor housing 103 to a tissue surface such as skin.

[0072] The sensor 104 is adapted to be at least partially inserted into the target tissue, such as within the dermis or subcutaneous layer of the skin. The sensor 104 may include a sensor tail of sufficient length to be inserted to a desired depth within the given tissue. The sensor tail may include at least one working electrode. In some configurations, the sensor tail may include a sensing layer for detecting an analyte (e.g., ketones). A counter electrode may be present in combination with at least one working electrode. Specific electrode configurations on the sensor tail are described in more detail below.

[0073] The sensing layer can be configured to detect a specific analyte (e.g., a ketone). For example, but not limited to, the disclosed analyte sensor includes at least one sensing layer configured to detect an analyte (e.g., a ketone).

[0074] In certain embodiments of this disclosure, an analyte (e.g., ketone) may be monitored in any biological fluid of interest, such as skin fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, or amniotic fluid. In certain embodiments, the analyte sensor of this disclosure may be adapted to assay skin fluid or interstitial fluid in vivo to determine the concentration of one or more analytes. In certain embodiments, the biological fluid is interstitial fluid.

[0075] Referring further to Figure 1, the sensor 104 can automatically transfer data to the reader device 120. For example, but not limited to, analyte concentration data (i.e., glucose concentration) can be communicated automatically and periodically, such as at a specific frequency when data is acquired or after a specific period of time has elapsed, and the data is stored in memory until transmission (e.g., every minute, every 5 minutes, or at other predetermined intervals). In some other embodiments, the sensor 104 may communicate with the reader device 120 non-automatically and without following a set schedule. For example, but not limited to, data can be communicated from the sensor 104 using RFID technology when the sensor electronics are brought within the communication range of the reader device 120. The data may remain stored in the sensor 104's memory until it is communicated to the reader device 120. Therefore, the user does not need to maintain constant proximity to the reader device 120, but can instead upload data at their convenience. In some other embodiments, a combination of automatic and non-automatic data transfer can be implemented. For example, rather than being limited, data transfer can automatically continue until the reader device 120 is no longer within the communication range of the sensor 104.

[0076] To facilitate the introduction of the sensor 104 into the tissue, an introducer may be present temporarily. In certain exemplary embodiments, the introducer may include a needle or a similar sharp object. As will be readily apparent to those skilled in the art, other types of introducers, such as a sheath or blade, may be present in alternative forms. More specifically, the needle or other introducer may be present temporarily in close proximity to the sensor 104 prior to tissue insertion, and then subsequently withdrawn. While present, the needle or other introducer can facilitate the insertion of the sensor 104 into the tissue by opening an access path for the sensor 104 to follow. For example, according to one or more embodiments, not as an limitation, the needle facilitates penetration of the epidermis as an access path to the dermis, thereby enabling the implantation of the sensor 104. After opening the access path, the needle or other introducer may be withdrawn so that the sharp object does not pose a danger. In certain embodiments, a suitable needle may be solid or hollow, beveled or unbeveled, and / or have a round or non-round cross-section. In a more specific embodiment, a suitable needle may be comparable to an acupuncture needle in terms of its cross-sectional diameter and / or tip design, having a cross-sectional diameter of approximately 250 microns. However, a suitable needle may have a larger or smaller cross-sectional diameter if required for a particular application.

[0077] In one embodiment, the tip of the needle (while present) can be angled across the end of the sensor 104 so that the needle first penetrates the tissue and opens an access path for the sensor 104. In another embodiment, the sensor 104 is positioned within the lumen or groove of the needle, and the needle can similarly open an access path for the sensor 104. In either case, the needle is subsequently withdrawn after facilitating the insertion of the sensor.

[0078] Sensor configurations featuring a single sensing layer configured for the detection of a single analyte can utilize two-electrode or three-electrode sensing motifs, as further described herein with reference to Figures 2A to 2C. Sensor configurations featuring two different sensing layers for detecting different analytes, either on separate working electrodes or on the same working electrode, are described separately below with reference to Figures 3A to 5C. Sensor configurations with multiple working electrodes may be particularly advantageous for incorporating two different sensing layers within the same sensor tail, as the signal contribution from each sensing layer can be more easily determined.

[0079] When a single working electrode is present within the analyte sensor, a three-electrode sensor configuration may include a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration may include a working electrode and a second electrode, in which case the second electrode may function as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). The various electrodes may be stacked at least partially on top of each other and / or separated laterally from each other on the sensor tail. A suitable sensor configuration may be substantially flat, substantially cylindrical, or any other suitable shape. In any of the sensor configurations disclosed herein, the various electrodes may be electrically insulated from each other by a dielectric material or similar insulator.

[0080] An analyte sensor featuring multiple working electrodes may similarly include at least one additional electrode. If one additional electrode is present, that one additional electrode can function as a pair / reference electrode for each of the multiple working electrodes. If two additional electrodes are present, one of the additional electrodes can function as a pair electrode for each of the multiple working electrodes, and the other additional electrode can function as a reference electrode for each of the multiple working electrodes.

[0081] Figure 2A shows an exemplary two-electrode analyte sensor configuration suitable for use in the disclosure herein. As shown, the analyte sensor 200 includes a substrate 212 positioned between a working electrode 214 and a counter / reference electrode 216. Alternatively, the working electrode 214 and the counter / reference electrode 216 may be positioned on the same side of the substrate 212, with a dielectric material inserted between them (configuration not shown). A sensing layer 218 is positioned as at least one layer on at least a portion of the working electrode 214. The sensing layer 218 may include multiple spots or a single spot configured for the detection of an analyte (e.g., a ketone), as will be discussed further herein.

[0082] Referring further to Figure 2A, the membrane 220 overcoats at least the detection layer 218. In certain embodiments, the membrane 220 may also overcoat part or all of the working electrode 214 and / or the pair / reference electrode 216, or the entire analyte sensor 200. One or both sides of the analyte sensor 200 may be overcoated with the membrane 220. The membrane 220 may comprise one or more polymer membrane materials having the ability to limit the analyte flux to the detection layer 218 (i.e., the membrane 220 is a mass transport limiting membrane with some permeability to the analyte of interest). In some embodiments, the membrane 220 is not crosslinked, as further described below. The analyte sensor 200 may be operable to assay an analyte (e.g., a ketone) by any of the electrochemical detection techniques such as coulometry, amperometry, voltammetry, or potentiometry.

[0083] Figures 2B and 2C illustrate exemplary three-electrode analyte sensor configurations, which are also suitable for use in the disclosure herein. The three-electrode analyte sensor configurations may be similar to those shown for analyte sensor 200 in Figure 2A, except that they include an additional electrode 217 within analyte sensors 201 and 202 (Figures 2B and 2C). The additional electrode 217 allows the pair / reference electrode 216 to function as either the pair electrode or the reference electrode, and the additional electrode 217 to perform other electrode functions that would otherwise not be considered. The working electrode 214 continues to perform its original function. The additional electrode 217 may be positioned above either the working electrode 214 or electrode 216, with a dielectric material isolation layer in between. For example, but not limited to, as shown in Figure 2B, dielectric layers 219a, 219b, and 219c isolate electrodes 214, 216, and 217 from each other and provide electrical insulation. Alternatively, at least one of electrodes 214, 216, and 217 may be positioned on opposing surfaces of the substrate 212, as shown in Figure 2C. Thus, in certain embodiments, electrodes 214 (working electrode) and 216 (counter electrode) may be located on opposite surfaces of the substrate 212, and electrode 217 (reference electrode) may be located above one of electrodes 214 or 216, separated from it by a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, and the location of the reference material layer 230 is not limited to those depicted in Figures 2B and 2C. Similar to the sensor 200 shown in Figure 2A, the sensing layer 218 in analyte sensors 201 and 202 may include multiple spots or a single spot. Furthermore, the analyte sensors 201 and 202 may be operable to assay the analyte by any of the electrochemical detection techniques, such as coulometry, amperometry, voltammetry, or potentiometry.

[0084] Similar to the analyte sensor 200, the film 220 can also overcoat the sensing layer 218 within the analyte sensors 201 and 202, as well as other sensor components, thereby acting as a mass transfer limiting film. In certain embodiments, an additional electrode 217 can be overcoated with film 220. Figures 2B and 2C depict electrodes 214, 216, and 217 being overcoated with film 220, but it should be noted that in certain embodiments, only the working electrode 214 is overcoated. Furthermore, the thickness of film 220 on each of electrodes 214, 216, and 217 may be the same or different. As in the two-electrode analyte sensor configuration (Figure 2A), one or both sides of the analyte sensors 201 and 202 may be overcoated with film 220 in the sensor configurations of Figures 2B and 2C, or the entire analyte sensors 201 and 202 may be overcoated. Therefore, the three-electrode sensor configuration shown in Figures 2B and 2C is not intended to limit the embodiments disclosed herein, and alternative electrode and / or layer configurations should be understood to remain within the scope of this disclosure.

[0085] Figure 3A shows an exemplary configuration of a sensor 203 having a single working electrode on which two different sensing layers are arranged. Figure 3A is similar to Figure 2A except that there are two sensing layers on the working electrode 214, namely a first sensing layer 218a and a second sensing layer 218b, the first sensing layer 218a and the second sensing layer 218b respond to different analytes and are spaced laterally apart from each other on the surface of the working electrode 214. The sensing layers 218a and 218b may include multiple spots or a single spot configured for the detection of each analyte. The composition of the film 220 may differ or be compositionally the same in the sensing layers 218a and 218b. The first sensing layer 218a and the second sensing layer 218b may be configured to detect their corresponding analytes at different working electrode potentials, as will be discussed further below.

[0086] Figures 3B and 3C show cross-sectional views of exemplary three-electrode sensor configurations for sensors 204 and 205, respectively, featuring a single working electrode having a first sensing layer 218a and a second sensing layer 218b positioned thereon. Figures 3B and 3C are otherwise similar to Figures 2B and 2C and can be better understood by referring to Figures 2B and 2C. As with Figure 3A, the composition of the film 220 may be different or compositionally the same in the sensing layers 218a and 218b.

[0087] Exemplary sensor configurations having multiple working electrodes, specifically two working electrodes, are described in more detail with reference to Figures 4-5C. While the following description primarily concerns sensor configurations with two working electrodes, it should be understood that more than two working electrodes can be incorporated through extensions of the disclosure herein. Additional working electrodes can be used to impart additional detection capabilities to the analyte sensor, for example, for the detection of a third and / or fourth analyte, in addition to the detection capabilities of the first and second analytes.

[0088] Figure 4 shows a cross-sectional view of an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode, suitable for use in the disclosure herein. As shown, the analyte sensor 300 includes working electrodes 304 and 306 disposed on both sides of a substrate 302. A first sensing layer 310a is disposed on the surface of the working electrode 304, and a second sensing layer 310b is disposed on the surface of the working electrode 306. The counter electrode 320 is electrically insulated from the working electrode 304 by a dielectric layer 322, and the reference electrode 321 is electrically insulated from the working electrode 306 by a dielectric layer 323. Outer dielectric layers 330 and 332 are disposed on the reference electrode 321 and the counter electrode 320, respectively. A film 340 can, according to various embodiments, overcoat at least the sensing layers 310a and 310b, and other components of the analyte sensor 300 or the entire analyte sensor 300 may optionally be overcoated with film 340.

[0089] Similar to analyte sensors 200, 201, and 202, analyte sensor 300 may be capable of assaying analytes (e.g., ketones) by any of the electrochemical detection techniques such as coulometry, amperometry, voltammetry, or potentiometry.

[0090] Alternative sensor configurations having multiple working electrodes, different from the configuration shown in Figure 4, may feature the pair / reference electrodes instead of separate pair and reference electrodes 320, 321, and / or feature different layer and / or film arrangements than those explicitly shown. For example, but not limited to, the arrangement of pair electrode 320 and reference electrode 321 may be reversed from that shown in Figure 4. In addition, working electrodes 304 and 306 do not necessarily have to be located on opposing surfaces of the substrate 302 in the manner shown in Figure 4.

[0091] While a suitable sensor configuration may feature electrodes that are essentially planar, it should be understood that sensor configurations featuring non-planar electrodes may be advantageous and particularly suitable for use in the disclosure herein. In particular, substantially cylindrical electrodes arranged concentrically with respect to each other can facilitate the deposition of mass transfer limiting films, as described below. Figures 5A–5C show perspective views of an analyte sensor featuring two working electrodes arranged concentrically with respect to each other. It should be understood that sensor configurations having a concentric electrode arrangement but lacking a second working electrode are also possible in this disclosure.

[0092] Figure 5A shows a perspective view of an exemplary sensor configuration in which multiple electrodes are substantially cylindrical and arranged concentrically with respect to one another around a central substrate. As shown, the analyte sensor 400 includes a central substrate 402, around which all electrodes and dielectric layers are arranged concentrically with respect to one another. In particular, the working electrode 410 is located on the surface of the central substrate 402, and the dielectric layer 412 is located on a portion of the working electrode 410 distal to the sensor tip 404. The working electrode 420 is located on the dielectric layer 412, and the dielectric layer 422 is located on a portion of the working electrode 420 distal to the sensor tip 404. The counter electrode 430 is located on the dielectric layer 422, and the dielectric layer 432 is located on a portion of the counter electrode 430 distal to the sensor tip 404. The reference electrode 440 is located on the dielectric layer 432, and the dielectric layer 442 is located on a portion of the reference electrode 440 distal to the sensor tip 404. Therefore, the exposed surfaces of the working electrode 410, the working electrode 420, the counter electrode 430, and the reference electrode 440 are spaced apart from each other along the longitudinal axis B of the analyte sensor 400.

[0093] Referring further to Figure 5A, the first sensing layer 414a and the second sensing layer 414b, which respond to different or the same analytes, are positioned on the exposed surfaces of the working electrodes 410 and 420, respectively, thereby allowing contact with the fluid to occur for detection. Although the sensing layers 414a and 414b are depicted as three separate spots in Figure 5A, it should be understood that fewer than three or more spots, including a continuous layer of sensing layers, may exist in alternative sensor configurations.

[0094] In Figure 5A, the sensor 400 is partially coated with film 450 over the working electrodes 410 and 420 and the sensing layers 414a and 414b placed thereon. Figure 5B shows an alternative sensor configuration in which substantially the entire sensor 401 is overcoated with film 450. Film 450 may be the same or have different compositions in the sensing layers 414a and 414b.

[0095] It should be further understood that the positioning of the various electrodes in Figures 5A and 5B may differ from those explicitly shown. For example, the positions of the counter electrode 430 and the reference electrode 440 can be reversed from the configurations shown in Figures 5A and 5B. Similarly, the positions of the working electrodes 410 and 420 are not limited to those explicitly depicted in Figures 5A and 5B. Figure 5C shows an alternative sensor configuration to that shown in Figure 5B, where the sensor 405 includes counter electrodes 430 and 440 positioned more proximal to the sensor tip 404, as well as working electrodes 410 and 420 positioned more distal to the sensor tip 404. A sensor configuration in which the working electrodes 410 and 420 are positioned more distal to the sensor tip 404 may be advantageous by providing a larger surface area for the deposition of the sensing layers 414a and 414b (five distinct sensing spots illustrated in Figure 5C), thereby facilitating an increase in signal intensity in some cases. Similarly, the central substrate 402 may be omitted in any concentric sensor configuration disclosed herein, and instead, the innermost electrodes may support the subsequently deposited layers.

[0096] Some parts of the sensor are described further below. This disclosure relates to a ketone sensor and method for detecting ketones with high sensitivity, enabling the measurement of lower concentrations of ketones. Surprisingly, it has been found that sensitivity can be improved by reducing the applied potential, subtracting background interference, and accumulating charge.

[0097] While not bound by theory, cumulative detection involves alternating connection and disconnection of the sensing electrode, and in some embodiments, it may be possible to use a single channel that switches between background and ketone detection chemistry systems (e.g., switching between WE1 (standard chemistry) and WE2 (blank chemistry) approximately every 2.5 minutes). Thus, the redox mediator can act as a "data storage medium" when accumulating charge, resulting in a simplified design of the electronics by requiring only one channel for background storage rather than two. This setup may introduce a time offset between background (blank) and ketone (standard) measurements, which could complicate background subtraction, but this offset can be short enough to prevent significant changes in analyte and interference concentrations between measurements. Alternatively, a single channel can be connected to the first working electrode (WE1) for a short time (e.g., a few seconds, such as about 15 seconds), then to the second working electrode (WE2) for a short time (e.g., a few seconds, such as about 15 seconds), and then disconnected from both channels for a longer period (e.g., a few minutes, such as about 2 minutes), providing a peak current similar to the previous example, but reducing the time offset between channels from a few minutes (e.g., about 2.5 minutes) to a few seconds (e.g., about 15 seconds).

[0098] In some embodiments of the ketone detection method, both a ketone detection electrode and a background detection electrode are connected to a circuit and a potential is applied. The period for which both detection electrodes are connected to the circuit may be any suitable period that allows for the detection of ketone and / or background signals. Typically, the duration for which both sensing electrodes are connected (e.g., connection setup time) can range from approximately 1 second or more (e.g., approximately 2 seconds or more, approximately 3 seconds or more, approximately 4 seconds or more, approximately 5 seconds or more, approximately 10 seconds or more, approximately 20 seconds or more, approximately 30 seconds or more, approximately 40 seconds or more, approximately 50 seconds or more, approximately 1 minute or more, approximately 2 minutes or more, approximately 3 minutes or more, approximately 4 minutes or more, approximately 5 minutes or more, approximately 6 minutes or more, approximately 7 minutes or more, approximately 8 minutes or more, or approximately 10 minutes or more) to approximately 30 minutes or less (e.g., approximately 25 minutes or less, approximately 20 minutes or less, approximately 15 minutes or less, approximately 10 minutes or less, approximately 8 minutes or less, approximately 7 minutes or more, approximately 6 minutes or more, approximately 5 minutes or more, approximately 4 minutes or less, approximately 3 minutes or more, approximately 2 minutes or more, approximately 1 minute or less, approximately 50 seconds or less, approximately 40 seconds or less, approximately 30 seconds or less, approximately 20 seconds or less, approximately 10 seconds or less, or approximately 5 seconds or less). In some embodiments, the connection period is approximately 1 second to 2 minutes or approximately 5 seconds to 1 minute. Afterward, both electrodes are disconnected from the circuit to allow charge accumulation. Once sufficient charge has accumulated, both electrodes can be reconnected to the circuit, and both the ketone signal (i.e., the ketone signal + background signal measurement) and the background signal (i.e., the background signal measurement only) are measured. The ketone concentration can be correlated with the measured ketone signal minus the measured background signal.

[0099] In one embodiment, the present disclosure relates to a method for detecting ketones, the method being: The method involves contacting a biological fluid containing ketones with a first detection electrode containing a ketone-responsive enzyme and a redox mediator, and a second detection electrode containing a redox mediator but not a ketone-responsive enzyme. The first and second sensing electrodes are connected to the circuit, and a potential of less than +40mV is applied to both electrodes to provide a steady state. Disconnecting the first and second detection electrodes from the circuit, The charge derived from the biological fluid that reacts with the first and second detection electrodes is accumulated over a set period (e.g., an accumulation set period), After the aforementioned set period, the first and second detection electrodes are connected to the circuit (for example, reconnected), This includes measuring a ketone signal by subtracting the signal obtained from the second detection electrode from the signal obtained from the first detection electrode.

[0100] In some embodiments of the ketone detection method, the connecting step and the disconnecting / accumulating step are performed alternately between the ketone detection electrode and the background detection electrode. In particular, this disclosure relates to a method for detecting ketones, the method being: (a) Contacting first and second detection electrodes with a biological fluid containing ketones, wherein the first detection electrode contains a ketone-responsive enzyme and a redox mediator, and the second detection electrode contains a redox mediator but does not contain a ketone-responsive enzyme, (b) Connecting the first sensing electrode to the circuit and applying a potential of less than +40mV to provide a steady state, (c) Disconnecting the first detection electrode from the circuit and connecting the second detection electrode to the circuit, and applying a potential of less than +40mV to provide a steady state, (d) During the first set period, accumulate the charge derived from the biological fluid that reacts with the first detection electrode, (e) After the first set period, the first detection electrode is connected to the circuit (for example, reconnected), and the second detection electrode is disconnected from the circuit. (f) During the second set period, accumulate the charge derived from the biological fluid that reacts with the second detection electrode, (g) After the second setting period, the second detection electrode is connected to the circuit (for example, reconnected), (h) Measuring a ketone signal by subtracting the signal obtained from the second detection electrode from the signal obtained from the first detection electrode.

[0101] In some embodiments of the ketone detection method, both a ketone detection electrode and a background detection electrode are brought into contact with a biological fluid containing ketones. The period during which both detection electrodes are in contact with the fluid may be any suitable period that allows for the detection of ketones and / or background signals. Typically, the period during which both sensing electrodes are in contact with the biological fluid (e.g., the contact setting period) can range from approximately 1 second or more (e.g., approximately 2 seconds or more, approximately 3 seconds or more, approximately 4 seconds or more, approximately 5 seconds or more, approximately 10 seconds or more, approximately 20 seconds or more, approximately 30 seconds or more, approximately 40 seconds or more, approximately 50 seconds or more, approximately 1 minute or more, approximately 2 minutes or more, approximately 3 minutes or more, approximately 4 minutes or more, approximately 5 minutes or more, approximately 6 minutes or more, approximately 7 minutes or more, approximately 8 minutes or more, or approximately 10 minutes or more) to approximately 30 minutes or less (e.g., approximately 25 minutes or less, approximately 20 minutes or less, approximately 15 minutes or less, approximately 10 minutes or less, approximately 8 minutes or less, approximately 7 minutes or less, approximately 6 minutes or less, approximately 5 minutes or less, approximately 4 minutes or less, approximately 3 minutes or less, approximately 2 minutes or more, approximately 1 minute or less, approximately 50 seconds or less, approximately 40 seconds or less, approximately 30 seconds or less, approximately 20 seconds or less, approximately 10 seconds or less, or approximately 5 seconds or less). In some embodiments, the contact period is approximately 1 second to 2 minutes or approximately 5 seconds to 1 minute.

[0102] In some embodiments, the measurement step may include determining the concentration of ketones in a fluid (e.g., biological fluid), which can be correlated by subtracting the signal obtained from a second sensing electrode (second signal) from the signal obtained from a first sensing electrode (first signal). In some embodiments, the ketone concentration (mM) can be determined by the following formula: (Ketone signal (nA) - Blank signal (nA)) / Ketone response (nA / mM).

[0103] By adding a known ketone concentration to a control sample, the ketone response value (nA / mM) of the sensor can be determined. The disconnection and connection in step (b) and step (c) may be simultaneous or sequential. In some embodiments of this alternating method, the disconnection and connection in step (b) and step (c) are simultaneous.

[0104] The accumulation setting period, including the first and second setting periods, can be any suitable period that allows sufficient charge to accumulate to detect ketones and / or background when the circuit is reconnected. Typically, the setting time can be about 30 seconds or longer (e.g., 1 minute or longer, 2 minutes or longer, 3 minutes or longer, 4 minutes or longer, 5 minutes or longer, 6 minutes or longer, 7 minutes or longer, 8 minutes or longer, 10 minutes or longer, 15 minutes or longer, 20 minutes or longer, 25 minutes or longer). Generally, the setting time may be about 30 minutes or less (e.g., 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 8 minutes or less, 7 minutes or less, 6 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, or 1 minute or less) to allow for the complete reaction of all ketones present at the ketone detection electrode, the reaction of interfering substances at the background detection electrode, or both. For example, the setting time may be approximately 30 seconds or more and approximately 30 minutes or less, approximately 1 to approximately 20 minutes, approximately 1 to approximately 15 minutes, approximately 1 to approximately 10 minutes, approximately 1 to approximately 8 minutes, approximately 1 to approximately 5 minutes, approximately 2 to approximately 8 minutes, approximately 2 to approximately 5 minutes, or approximately 30 seconds or more. The first setting period may be the same as or different from the second setting period. In some embodiments, the first setting period may be the same as the second setting period. In other embodiments, the first setting period may be different from the second setting period. In any of these embodiments, the setting period, the first setting period, the second setting period, or any combination thereof may be 30 seconds or more.

[0105] Surprisingly, it was discovered that lowering the detection potential reduces the interaction between the detection electrode and background interfering material (e.g., electroactive interfering material), thereby reducing the interference signal. In some aspects, the potential applied to the ketone sensor is, relative to Ag / AgCl, less than +40mV to approximately -250mV (less than +40mV to approximately -225mV, less than +40mV to approximately -200mV, less than +40mV to approximately -175mV, less than +40mV to approximately -150mV, less than +40mV to approximately -125mV, approximately +30mV to approximately -250mV, approximately +30mV to approximately -225mV, approximately +30mV to approximately -200mV, approximately +30mV to approximately -175mV, approximately +30mV to approximately -150mV, approximately +30mV to approximately -125mV, approximately +20mV to approximately -250mV, approximately +20mV to approximately -225mV, +20mV to -200mV, +20mV to -175mV, +20mV to -150mV, +20mV to -1 25mV, approximately +10mV to approximately -250mV, approximately +10mV to approximately -225mV, approximately +10mV to approximately -200mV, approximately +10mV to approximately -175mV The ranges may include approximately +10mV to approximately -150mV, approximately +10mV to approximately -125mV, approximately +5mV to approximately -250mV, approximately +5mV to approximately -225mV, approximately +5mV to approximately -200mV, approximately +5mV to approximately -175mV, approximately +5mV to approximately -150mV, and approximately +5mV to approximately 125mV. In some embodiments, the applied potentials may be approximately +5mV to approximately -125mV, approximately -5mV to approximately -100mV, approximately -10mV to approximately -90mV, or approximately -20mV to approximately -80mV, respectively, relative to the Ag / AgCl reference. According to some accounts, the applied potentials are approximately +35mV, +30mV, +25mV, +20mV, +15mV, +10mV, +5mV, -5mV, -10mV, -15mV, -20mV, -30mV, -40mV, -50mV, -60mV, and -70mV, respectively, relative to the Ag / AgCl reference. It could be approximately -80mV, approximately -90mV, approximately -100mV, approximately -110mV, approximately -120mV, approximately -130mV, approximately -140mV, approximately -150mV, approximately -160mV, approximately -170mV, approximately -180mV, approximately -190mV, approximately -200mV, approximately -210mV, approximately -220mV, approximately -230mV, approximately -240mV, or approximately -250mV.In some specific embodiments, the applied potential may be approximately -80mV for Ag / AgCl.

[0106] In any of these embodiments, the sensing electrode may include a working electrode and either a ketone sensing layer or a background sensing layer on a portion of the working electrode. In a ketone sensor, the working electrode (e.g., the working electrode in the first sensing electrode, the working electrode in the second sensing electrode) may be any suitable conductive material. Examples of suitable conductive materials include, for example, aluminum, carbon (including graphite), cobalt, copper, gallium, gold, indium, iridium, iron, lead, magnesium, mercury (as amalgam), nickel, niobium, osmium, palladium, platinum, rhenium, rhodium, selenium, silicon (e.g., doped polycrystalline silicon), silver, tantalum, tin, titanium, tungsten, uranium, vanadium, zinc, zirconium, mixtures thereof, and alloys, oxides, or metallic compounds of these elements. In some embodiments, the working electrode (e.g., the working electrode in the first sensing electrode, the working electrode in the second sensing electrode) may include carbon.

[0107] In the first detection electrode, a ketone detection layer positioned on at least a portion of the working electrode detects ketones and includes a ketone-responsive enzyme and a redox mediator. In some embodiments, the detected ketone may be an endogenous ketone. For example, the detected ketone may be acetone, acetoacetic acid, acetacetate, β-hydroxybutyrate (β-HBA), or any combination thereof. Although β-hydroxybutyrate (β-HBA) is technically a carboxylic acid and not a ketone, it is commonly referred to as a blood ketone in this field. In some embodiments, the detected ketone may be acetacetate, β-HBA, β-hydroxybutyrate (β-HB), or any combination thereof. In some embodiments, the detected ketone may be β-hydroxybutyrate (β-HB).

[0108] The ketone detection layer contains a ketone-responsive enzyme that functions as a catalyst for electron transfer. In some embodiments, the ketone-responsive enzyme may be 3-hydroxybutyrate dehydrogenase (3-HBDH), glucose dehydrogenase, alcohol dehydrogenase, or a combination thereof. In some embodiments, the ketone-responsive enzyme may be 3-hydroxybutyrate dehydrogenase (3-HBDH).

[0109] In some embodiments, the first detection electrode may include an NAD(P)H oxidoreductase such as diaphorase. If necessary, one or more cofactors may be included with the ketone-responsive enzyme or NAD(P)H oxidoreductase enzyme. Suitable cofactors include, for example, nicotinamide adenine dinucleotide, either oxidized (NAD) or reduced (NADH), or derivatives thereof. While we do not wish to be bound by theory, it is thought that ketone-responsive enzymes such as 3-HBDH cannot readily transfer electrons directly to the redox mediator. Therefore, an NAD(P)H oxidoreductase (e.g., diaphorase) and an optional cofactor (e.g., NAD) may be added to allow electrons to be transferred back and forth from the ketone-responsive enzyme to the redox mediator. In any of these embodiments, the first detection electrode may include a ketone-responsive enzyme (e.g., 3-HBDH), NAD(P)H oxidoreductase, and nicotinamide adenine dinucleotide phosphate (NAD(P)+) or a derivative thereof.

[0110] In some embodiments, the ketone detection layer may further contain albumin, which can act as an enzyme stabilizer. In one embodiment, the albumin may be serum albumin, such as bovine serum albumin (BSA) or human serum albumin (HSA). In certain embodiments, the detection layer may contain human serum albumin.

[0111] In a particular embodiment, the detection layer may include albumin stabilizer to enzyme (e.g., 3-HBDH) ratios of approximately 40:1 to 1:40, for example, approximately 35:1 to 1:35, approximately 30:1 to 1:30, approximately 25:1 to 1:25, approximately 20:1 to 1:20, approximately 15:1 to 1:15, approximately 10:1 to 1:10, approximately 9:1 to 1:9, approximately 8:1 to 1:8, approximately 7:1 to 1:7, approximately 6:1 to 1:6, approximately 5:1 to 1:5, approximately 4:1 to 1:4, approximately 3:1 to 1:3, approximately 2:1 to 1:2, or approximately 1:1. In a particular embodiment, the detection layer may include albumin stabilizer to enzyme ratios of approximately 1:1 to 1:10, for example, approximately 1:1 to 1:9, approximately 1:1 to 1:8, approximately 1:1 to 1:7, approximately 1:1 to 1:6, approximately 1:1 to 1:5, approximately 1:2 to 1:9, approximately 1:3 to 1:8, approximately 1:3 to 1:7, or approximately 1:4 to 1:6.

[0112] In certain cases, β-hydroxybutyrate dehydrogenase can convert β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD+) to acetoacetate and reduced nicotinamide adenine dinucleotide (NADH), respectively. Enzyme cofactors NAD and NADH can assist in promoting the concerted enzymatic reactions disclosed herein. NADH can then be reduced diaphorase-mediated, and the electrons transferred during this process provide a basis for ketone detection at the working electrode. The resulting electrochemical signal can then be correlated with the amount of ketone initially present in the sample at the time of measurement. Thus, there is a 1:1 molar correspondence between the amount of electrons transferred to the working electrode and the amount of converted β-hydroxybutyrate, thereby providing a basis for ketone detection and quantification based on the current measured at the working electrode. The transfer of electrons from NADH reduction to the working electrode may occur via electron transfer agents such as osmium (Os) compounds, as described herein. Albumin may be present as an enzyme stabilizer, if necessary.

[0113] Ketone-responsive enzymes may be present in any suitable amount, including approximately 1% to 50% by weight relative to the redox mediator (e.g., approximately 1% to 40% by weight, approximately 1% to 30% by weight, approximately 1% to 20% by weight, approximately 1% to 15% by weight, approximately 1% to 10% by weight, or approximately 1% to 5% by weight).

[0114] NAD(P)H oxidoreductase (e.g., diaphorase) may be present in the ketone detection layer in any suitable amount, including about 0.01% to 10% by weight of the total enzyme composition (e.g., about 0.05% to about 9.5% by weight, about 0.1% to about 9% by weight, about 0.5% to about 8.5% by weight, about 1% to about 8% by weight, or about 2% to about 7% by weight).

[0115] In any of the multiple embodiments, the ketone detection layer may include a pH buffer. The buffer may be any suitable composition that is water-soluble and controls (i.e., maintains) the pH of the detection composition within a range of about 5 to about 8 (e.g., maintaining a pH of about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, or about 8). In some embodiments, the pH can be controlled to be within the range of about 6 to about 8. For example, the buffer may contain phosphates (e.g., monobasic and dibasic sodium phosphate), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS), carbonates (e.g., sodium carbonate; carbonates and carbonates such as sodium carbonate and sodium bicarbonate), or citrates (e.g., citrate and citrates such as trisodium citrate). The buffer may optionally contain one or more (e.g., 1, 2, 3, or 4) additional salts (e.g., Group I or Group II halide salts, e.g., sodium chloride, potassium chloride, magnesium chloride). In one embodiment, the buffer may be phosphate-buffered saline (PBS) containing disodium hydrogen phosphate, sodium chloride, and optionally potassium chloride and potassium dihydrogen phosphate. In another embodiment, the buffer may be an MES or phosphate buffer, which may contain phosphate, sodium chloride, potassium chloride, and / or magnesium chloride.

[0116] In some embodiments, the buffer may typically be an aqueous buffer. In other embodiments, a non-aqueous solvent such as an alcohol (e.g., ethanol) may be present. In some embodiments, the buffer may contain water as the sole solvent. In other embodiments, the buffer may contain water and at least one (e.g., 1, 2, or 3) non-aqueous solvents in any suitable ratio, such as a volume ratio of non-aqueous solvent to water in the range of 99.9:0.1 to 0.1:99.9. In some embodiments, the volume ratio of non-aqueous solvent to water may be about 1:99, about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5, or about 99:1, etc. In certain examples, ethanol (EtOH) and water may be used in volume ratios of EtOH:H2O ranging from 50:50 to 90:10 (e.g., 70:30, about 75:25, about 80:20, about 85:15, or about 90:10, etc.).

[0117] In some embodiments, the second detection electrode is a background detection electrode comprising a working electrode and a background detection layer on a portion of the working electrode. As described herein, the background detection layer detects background interference and contains a redox mediator but does not contain a ketone-responsive enzyme. The redox material in the first and second detection electrodes may be the same or different. In one embodiment, the redox mediator in the first and second detection electrodes is the same material. In one embodiment, the background detection layer does not contain 3-HBDH. Any components such as NAD(P)H oxidoreductase (e.g., diaphorase), one or more cofactors, albumin (e.g., human serum albumin (HSA)), and pH buffers may be present in the background detection layer as described herein. In some embodiments, the background detection layer may contain albumin (e.g., HSA), NAD(P)H oxidoreductase (e.g., diaphorase), and oxidized (NAD) or reduced (NADH) nicotinamide adenine dinucleotide, or derivatives thereof. In some embodiments, the background detection layer may include human serum albumin, diaphorase, and oxidized nicotinamide adenine dinucleotide (NAD).

[0118] In some embodiments, the background detection layer contains a ketone-responsive enzyme (e.g., the ketone-responsive enzyme is the sole enzyme) but does not contain any other detection components such as albumin (e.g., HSA), NAD(P)H oxidoreductase (e.g., diaphorase), nicotinamide adenine dinucleotide in either its oxidized (NAD) or reduced (NADH) form, or its derivatives, or redox mediators. In some embodiments, the background detection layer consists solely of the ketone-responsive enzyme.

[0119] In some embodiments, the background detection layer does not contain albumin (e.g., HSA). In some embodiments, the background detection layer does not contain diaphorase. In some embodiments, the background detection layer does not contain added NAD or NADH. In some embodiments, the background detection layer contains ambient NAD and NADH in the complete or substantial absence of added NAD and / or NADH. In some embodiments, the background detection layer does not contain redox mediators.

[0120] In some embodiments, the background detection layer does not contain ketone-responsive enzymes or albumin (e.g., HSA). In some embodiments, the background detection layer does not contain ketone-responsive enzymes or diaphorase. In some embodiments, the background detection layer does not contain ketone-responsive enzymes or added NAD or NADH. In some embodiments, the background detection layer does not contain ketone-responsive enzymes or redox mediators.

[0121] In some embodiments, the background detection layer does not contain ketone-responsive enzymes, albumin (e.g., HSA), or diaphorase. In some embodiments, the background detection layer does not contain ketone-responsive enzymes, diaphorase, or added NAD. In some embodiments, the background detection layer does not contain ketone-responsive enzymes, albumin (e.g., HSA), or diaphorase. In some embodiments, the background detection layer does not contain ketone-responsive enzymes, diaphorase, or added NAD.

[0122] The ketone detection layer and / or background detection layer may be arranged continuously or discontinuously on at least a portion of each working electrode. Discontinuous application means that the detection layer may form distinct shapes on the working electrode, such as spots, lines, or multiple (e.g., array-like) spots and / or lines. The number of spots or lines is not considered particularly limited but may range from 2 to about 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, including about 3 to about 8, or about 4 to about 6). In any embodiment of this specification, the ketone detection layer, the background detection layer, or both may be continuous on each working electrode. In other embodiments, the detection layer, the background detection layer, or both may be discontinuous on each working electrode.

[0123] The total size of the creatinine detection layer(s) (e.g., the combined area of ​​all spots, layers, or active regions) is at least approximately 0.05 mm². 2 It could be, and about 100mm 2 It can be up to 0.05 mm. In some embodiments, the total size is approximately 0.05 mm. 2 ~approximately 100mm 2 , about 0.05mm 2 ~about 75mm 2 , about 0.05mm 2 ~approximately 50mm 2 , about 0.05mm 2 ~40mm 2 , about 0.05mm 2 ~about 30mm 2 , about 0.05mm 2 ~about 25mm 2 , about 0.05mm 2 ~about 15mm 2 , about 0.05mm 2 ~about 10mm 2 , about 0.05mm 2 ~about 5mm 2 , about 0.05mm 2 ~about 1mm 2 , or approximately 0.05 mm 2 ~about 0.1mm 2It is possible. In certain embodiments, the total size of one or more detection layers is approximately 0.05 to approximately 0.1 mm. 2 Approximately 0.05 to 100 mm 2 Approximately 0.1 to 50 mm 2 , about 0.5~about 30mm 2 , about 1~20mm 2 , or approximately 1 to 15 mm 2 It is within the range of [the specified range].

[0124] Each of the one or more detection layers, including a ketone detection layer, a background detection layer, or both, typically has a thickness in the range of about 0.1 to 10 μm. For example, each detection layer may have a thickness of 0.1 μm or more (e.g., 0.2 μm or more, 0.3 μm or more, 0.5 μm or more, 0.8 μm or more, 1 μm or more, 2 μm or more, 3 μm or more, 5 μm or more, or 8 μm or more), and typically has a thickness of 10 μm or less (e.g., 8 μm or less, 5 μm or less, 3 μm or less, 2 μm or less, 1 μm or less, 0.8 μm or less, 0.5 μm or less, 0.3 μm or less, or 0.2 μm or less). In one example, each layer present may have a thickness of about 0.1 to about 10 μm, about 0.2 to about 8 μm, about 0.5 to about 5 μm, about 1 to about 4 μm, or about 2 μm.

[0125] In some embodiments, conductive materials such as carbon nanotubes, graphene, or metal nanoparticles can be combined within one or more sensing layers, including a ketone sensing layer, a background sensing layer, or both, to facilitate the rapid achievement of a steady-state current. The conductive material may be included in each sensing layer in a range of about 0.1% to about 50% (pbw) (e.g., about 1 to about 50 pbw, about 1 to about 10 pbw, or about 0.1 to about 10 pbw).

[0126] Both the first detection electrode (e.g., a ketone detection layer) and the second detection electrode (e.g., a background detection layer) include a redox mediator. Each redox mediator may be the same or different. In some embodiments, the redox mediator may be the same for both the first and second detection electrodes.

[0127] In one embodiment, the redox mediator may include a polymer and an electron transfer agent. In some embodiments, the polymer in the redox mediator can be any suitable polymer that enables the transfer of electrons between the electron transfer agent and the working electrode. For example, the polymer may be polyvinylpyridine (e.g., poly(4-vinylpyridine; PVP)), polyvinylimidazole (e.g., poly(1-vinylimidazole; PVI)), poly(aniline), poly(pyrrole), poly(acetylene), poly(acrylic acid), styrene / maleic anhydride copolymer, methyl vinyl ether / maleic anhydride copolymer, poly(vinylbenzyl chloride), poly(allylamine), poly(lysine), poly(acrylamide-co-1-vinylimidazole), poly(4-vinylpyridine) quaternized with carboxypentyl groups, or poly(sodium 4-styrenesulfonate). These polymers can be considered precursor polymers in that they are further modified to immobilize (e.g., bind) electron transfer complexes. In some embodiments, the polymer may include a backbone comprising poly(4-vinylpyridine), poly(1-vinylimidazole), poly(styrene), poly(thiophene), poly(aniline), poly(pyrrole), poly(acetylene), or any combination thereof. In other embodiments, the polymer may include polymer or copolymer repeating units that contain at least one (e.g., 1, 2, 3, 4, 5, or 6) pendant pyridinyl groups, imidazolyl groups, or both pyridinyl and imidazolyl groups. Suitable polymers, for example, may include partially or fully quaternized poly(4-vinylpyridine) and poly(1-vinylimidazole), where the quaternized pyridine and imidazole groups may be used to form spacers by reaction with an electron transfer agent (e.g., complexation with an electron transfer agent), respectively.

[0128] In some embodiments, the electron transfer agent in the redox mediator may include a transition metal complex. The transition metal in the transition metal complex may be any suitable transition metal that can be effectively reduced and oxidized in the methods described herein. For example, the transition metal complex may include osmium, ruthenium, iron, cobalt, vanadium, or a combination thereof. In some embodiments, the transition metal may be ruthenium or osmium, particularly osmium. In some embodiments, suitable electron transfer agents include low-potential osmium complexes, for example, those described in U.S. Patent Nos. 6,134,461, 6,605,200, 6,736,957, 7,501,053, and 7,754,093, each of which is incorporated herein by reference in whole. Other suitable examples of electron transfer mediators and polymer-bonded electron transfer mediators are those described in U.S. Patent No. 8,444,834, No. 8,268,143, and No. 6,605,201, which are incorporated herein by reference in their entirety.

[0129] The transition metal complex may further contain at least one ligand that can be monodentate or polydentate (e.g., bidentate, tridentate, or tetradentate). Typically, the complex contains enough ligands to provide a complete coordination sphere. In some embodiments, at least one ligand (e.g., 1, 2, 3, 4, 5, or 6) may contain a nitrogen-containing heterocycle.

[0130] Monodentate ligands include, for example, -F, -Cl, -Br, -I, -CN, -SCN, -OH, NH3, alkylamines, dialkylamines, trialkylamines, alkoxys, heterocyclic compounds, compounds containing such groups, solvent molecules (e.g., H2O, EtOH), or reactive groups. For example, alkyl ligands (e.g., C 1~12 , C 1~6 , C 1~4 , C 1~3The ) or aryl (e.g., phenyl, benzyl, naphthyl) moieties may be substituted with F, Cl, Br, I, alkylamino, dialkylamino, trialkylammonium (excluding the aryl moiety), alkoxy, alkylthio, or aryl. Suitable examples of heterocyclic monodentate ligands include imidazole, pyrazole, oxazole, thiazole, pyridine, and pyrazine, each of which may or may not be substituted as described herein (e.g., with at least one reactive group (e.g., one, two, three, or four reactive groups)).

[0131] Examples of suitable bidentate ligands include, for example, 1,10-phenanthroline, amino acids, oxalic acid, acetylacetone, diaminoalkanes, ortho-diaminoarenes, 2,2'-biimidazole, 2,2'-bioxazole, 2,2'-bithiazole, 2-(2-pyridyl)imidazole, and 2,2'-bipyridine, each of which may be unsubstituted or substituted as described herein (e.g., substituted with at least one reactive group such as 1, 2, 3, or 4 reactive groups). Particularly suitable bidentate ligands for electron transfer complexes include substituted and unsubstituted 2,2'-biimidazole, 2-(2-pyridyl)imidazole, and 2,2'-bipyridine. Examples of suitable tridentate ligands include, for example, diethylenetriamine, 2,2',2''-terpyridine, and 2,6-bis(N-pyrazolyl)pyridine, each of which may be substituted or unsubstituted (e.g., substituted with one or more alkyl groups such as methyl, or one or more reactive groups).

[0132] A suitable 2,2'-biimidazole ligand may be one that follows formula (I):

[0133] [ka]

[0134] In equation (I), R 1 and R2 These are identical or different, and are substituted or unsubstituted alkyl, alkenyl, or aryl atoms, respectively. Generally, R 1 and R 2 These are either identical or different, and each is a non-substituted C. 1~12 Alkyl (for example, C 1~4 It is alkyl. In some embodiments, R 1 and R 2 Both are methyl.

[0135] In equation (I), R 3 , R 4 , R 5 and R 6 These are identical or different, and each is H, F, Cl, Br, I, NO2, CN, CO2H, SO3H, SH, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, alkanoylamino, arylcarboxamide, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkyl, alkenyl, or aryl. Or, R 3 and R 4 together, or R 5 and R 6 Together, and independently, they form saturated or unsaturated 5-membered or 6-membered rings (e.g., benzo). Typically, the alkyl and alkoxy moieties are C 1~12 The alkyl or aryl portion of any substituent may be optionally substituted with one or more substituents (e.g., 1, 2, 3, 4, 5, or 6) such as F, Cl, Br, I, amino, alkylamino, dialkylamino, trialkylammonium (excluding the aryl portion), alkoxy, alkylthio, aryl, or reactive groups (e.g., CO2H). Generally, R 3 , R 4 , R 5 and R 6 They are either identical or different, and each is either H or unsubstituted C. 1~12 Alkyl (for example, C 1~4is (alkyl). In some embodiments, R 3 , R 4 , R 5 and R 6 are all H.

[0136] Suitable 2-(2-pyridyl)imidazole ligands can be ligands according to formula (II):

[0137]

Chemical formula

[0138] In formula (II), R 1 is substituted or unsubstituted alkyl, alkenyl, or aryl. Generally, R 1 is unsubstituted C 1~12 alkyl (e.g., C 1~4 alkyl) or C 1~12 alkyl optionally substituted with a reactive group. In some embodiments, R 1 is methyl.

[0139] In formula (II), R 3’ , R 4’ , R a , R b , R c and R d are the same or different and each is H, F, Cl, Br, I, NO2, CN, CO2H, SO3H, SH, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, alkanoylamino, arylcarboxamide, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkyl, alkenyl or aryl. Alternatively, R 3’ and R 4’ are taken together, or R a , R b , R c and R d [[ID=6 four]]two adjacent substituents of (e.g., R a and R b, R b and R c , or R c and R d ) combine to independently form saturated or unsaturated 5-membered or 6-membered rings (e.g., benzo). Typically, the alkyl and alkoxy moieties are C 1~12 The alkyl or aryl portion of any substituent may be optionally substituted with one or more substituents (e.g., 1, 2, 3, 4, 5, or 6) such as F, Cl, Br, I, amino, alkylamino, dialkylamino, trialkylammonium (excluding the aryl portion), alkoxy, alkylthio, aryl, or reactive groups (e.g., CO2H). Generally, R 3’ , R 4’ , R a , R b , R c and R d They are either identical or different, and each is either H or unsubstituted C. 1~12 Alkyl (for example, C 1~4 It is alkyl. In some embodiments, R 3’ , R 4’ , R a , R b , R c and R d All of them are H.

[0140] A suitable 2,2'-bipyridine ligand may be one that follows formula (III):

[0141] [ka]

[0142] In equation (III), R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 and R 23These are identical or different, and each is one of the following: F, Cl, Br, I, NO2, CN, CO2H, SO3H, SH, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, alkanoylamino, arylcarboxamide, hydrazino, alkylhydrazino, hydroxylamino, alkoxyamino, alkylthio, alkyl, alkenyl, or aryl. Typically, the alkyl and alkoxy portions are C 1~12 The alkyl or aryl portion of any substituent may be optionally substituted with one or more substituents (e.g., 1, 2, 3, 4, 5, or 6) such as F, Cl, Br, I, amino, alkylamino, dialkylamino, trialkylammonium (excluding the aryl portion), alkoxy, alkylthio, aryl, or a reactive group (e.g., CO2H).

[0143] A concrete example of an appropriate combination is R 16 and R 23 Both are H or both are methyl, and / or R 17 and R 23 Both are H or both are methyl, and / or R 18 and R 21 Both are H or both are methyl, and / or R 19 and R 20 This includes combinations where both substituents are H or both are methyl. Alternative combinations include one or more adjacent pairs of substituents (e.g., R 16 and R 17 , R 17 and R 18 , R 18 and R 19 , R 23 and R 22 , R 22 and R 21 , or R 21 and R 20 This is the case when these compounds combine to form a saturated or unsaturated five-membered or six-membered ring (e.g., benzo).

[0144] In one embodiment, one or more ligands are 4,4'-dimethyl-2,2'-bipyridine, mono-, di-, or polyalkoxy-2,2'-bipyridine (e.g., 4,4'-dimethoxy-2,2'-bipyridine), 4,7-dimethyl-1,10-phenanthroline, mono-, di-, or polyalkoxy-1,10-phenanthroline (e.g., 4,7-dimethoxy-1,10-phenanthroline), or any combination thereof.

[0145] In some embodiments, transition metal complexes may contain counterions (X) to balance the charge of the transition metal. Typically, there may be 1 to 5 counterions (i.e., 1, 2, 3, 4, or 5). Multiple counterions in a complex are not necessarily all the same. Suitable examples of counterions include anions such as halides (e.g., fluorides, chlorides, bromides, or iodides), sulfates, phosphates, hexafluorophosphates, and tetrafluoroborates, as well as cations such as lithium, sodium, potassium, tetraalkylammonium, and ammonium (e.g., monovalent cations). In some embodiments, the counterion is a halide such as a chloride.

[0146] In one embodiment, the transition metal complex may be an osmium transition metal complex that may contain one or more ligands, at least one (e.g., 1, 2, 3, 4, 5, or 6) ligands may be nitrogen-containing heterocycles (e.g., imidazole, pyrazole, oxazole, thiazole, pyridine, and pyrazine). In some embodiments, the osmium transition metal complex may contain one or more ligands selected from 4,4'-dimethyl-2,2'-bipyridine, mono-, di-, or polyalkoxy-2,2'-bipyridine (e.g., 4,4'-dimethoxy-2,2'-bipyridine), 4,7-dimethyl-1,10-phenanthroline, mono-, di-, or polyalkoxy-1,10-phenanthroline (e.g., 4,7-dimethoxy-1,10-phenanthroline).

[0147] In one embodiment, the redox mediator may include an osmium complex bonded to a poly(1-vinylimidazole) or poly(4-vinylpyridine) polymer or copolymer. The poly(4-vinylpyridine) polymer is a prepolymer modified to bond an osmium complex (e.g., a poly(biimididyl)osmium complex), as shown in the following structure:

[0148] [ka]

[0149] In the formula, n can be 2, n' can be 17, and n'' can be 1. Other reactive groups and / or spacer groups can be used. In one embodiment, the electron redox mediator may contain an osmium-containing poly(4-vinylpyridine) polymer, as shown below.

[0150] [ka]

[0151] In the equation, n is 2, n' is 17, and n'' is 1. In some embodiments, the electron transfer agent may be bonded (e.g., non-leaching and / or covalently) to the polymer in the redox mediator. For example, covalent bonding of the electron transfer agent to the polymer may occur by polymerizing monomer units having the covalently bonded electron transfer agent, or the electron transfer agent may react separately with the polymer after the polymer has already been synthesized.

[0152] In some embodiments, a first reactive group is reactive with the polymer (e.g., a functional group capable of quaternizing a pyridine nitrogen atom or an imidazole nitrogen atom), and a second reactive group is reactive with the electron transfer agent (e.g., a functional group reactive with a ligand that coordinates a metal ion), and a bifunctional spacer can be used to bond (e.g., covalently bond) the electron transfer agent to the polymer in the redox mediator. Typically, a covalent bond is formed between the two reactive groups, resulting in linkage. Suitable reactive groups include, for example, activated esters (e.g., succinimidyl, benzotriazolyl, or aryl substituents having one or more electron-withdrawing groups such as sulfo, nitro, cyano, halo), acrylamides, acyl azides, acyl halides, carboxy(-COO- or -CO2H), aldehydes, ketones, alkyl halides, alkyl sulfonates, anhydrides, azilidino, epoxy, halotriadinyl, imide esters, isocyanates, isothiocyanates, maleimides, sulfonyl halides, aminos, thiols(-SH), hydroxy, pyridinyl, imidazolyl, and hydroxyaminos. The reaction between two reactive groups can form a covalent bond between a transition metal complex and a polymer, which is a carboxamide, thioether, hydrazonyl, oxymyl, alkylamino, ester, carboxylic acid ester, imidazolium, pyridinium, ether, thioether, aminotriazinyl, triazinyl ether, amidinyl, urea, uretanyl, thiourea, thioether, sulfonamide, or any combination thereof. In addition to reactive groups, bifunctional spacers are typically alkylenyl (i.e., -(CH2) n -) and / or ethyleneoxy (i.e., -(CH2CH2O) m - and in the formula, n and m can each independently further include integers from 1 to 12 (for example, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2).

[0153] In some embodiments, the redox mediator may further contain a crosslinking agent. Generally, the crosslinking agent is any suitable polyfunctional (e.g., bifunctional) short-chain molecule that enables the electron transfer agent to bond (e.g., covalently) to the polymer of the redox mediator. For example, crosslinking agents may include polyepoxides (e.g., polyethylene glycol diglycidyl ether (PEGDGE), ethylene glycol diglycidyl ether (EGDGE), resorcinol diglycidyl ether, 1,2,7,8-diepoxyoctane, Gly3), cyanuryl chloride, N-hydroxysuccinimide, imide esters, epichlorohydrins, or combinations thereof. In one embodiment, the crosslinking agent is polyethylene glycol diglycidyl ether (PEGDGE) of the following formula:

[0154] [ka]

[0155] In the formula, n is an integer between 1 and approximately 50 (for example, 1 to approximately 45, 1 to approximately 40, 1 to approximately 35, 1 to approximately 30, 1 to approximately 25, approximately 5 to approximately 50, approximately 5 to approximately 45, approximately 5 to approximately 40, approximately 5 to approximately 35, or approximately 5 to approximately 30).

[0156] In certain examples, PEGDGE may be PEGDGE200, PEGDGE400 (where n is 10), PEGDGE500, PEGDGE600, PEGDGE1000, or PEGDGE2000, where the number represents the average molecular weight (M n This shows that in one embodiment, the crosslinking agent may be PEGDGE400.

[0157] The redox mediator can be applied to the working electrode using any suitable technique, such as spray coating, painting, inkjet printing, stencil, roller coating, dipping coating, or any combination thereof. In some embodiments, the redox mediator can be applied by dipping at least a portion of the working electrode into a solution of the redox mediator. One or more applications can be applied (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 applications). In some embodiments, the redox mediator can be applied in 1, 2, 3, or 4 applications (e.g., a pass). In some embodiments, the redox mediator can be applied in 1 or 2 applications (e.g., a pass).

[0158] In some embodiments, one or more enzymes in the ketone detection layer or background detection layer can bind (e.g., covalently or unleachably bound) to the polymer portion of the redox mediator in the first detection electrode. In some embodiments, one or more enzymes can covalently bind to the polymer portion of the redox mediator. Covalent binding of one or more enzymes to the redox mediator (e.g., polymer) can occur via crosslinking agents and reaction sites on the enzymes as described herein. Thus, in such cases, the enzymes can be electronically "wired" to the working electrode via the redox mediator. In one embodiment, a hydrogel can be formed by crosslinking the enzymes and their wires on the electrode. In another embodiment, at least a portion of the enzymes can diffuse into the polymer and / or hydrogel and adhere to the polymer, but do not necessarily need to be covalently bound.

[0159] In one embodiment, the first detection electrode, the second detection electrode, or both may include a membrane that overcoats at least a ketone detection layer and / or a background detection layer and optionally other components. The overcoating forms an outer membrane that provides stability to detection reagents (e.g., ketone-responsive enzymes, redox mediators), restricts mass transfer, biocompatibility, and / or prevents electrode fouling. The membrane may optionally coat all or part of the working electrode, and optionally any counter electrode or reference electrode that may be present. In one embodiment, the membrane covers (e.g., encapsulates) the entire detection system (e.g., a sensor tail) including the first and second detection electrodes having their respective detection layers, and any counter electrode, reference electrode, and / or substrate that may be present.

[0160] The membrane may include one or more polymer membrane materials having a physical structure that allows analyte flow to the sensing layer (i.e., the membrane is a mass transfer limiting membrane). The composition of the membrane (e.g., degree of hydrophobicity and / or degree of crosslinking) may be modified to facilitate the desired flow of ketone(s) to the sensing electrode, as further described herein, thereby providing the desired signal intensity and stability. In one embodiment, the membrane may be permeable to at least one ketone to be measured.

[0161] Coating of a film covering at least a ketone detection layer, at least a background detection layer, or both can be carried out by any suitable technique. Typically, the film can be coated by spray coating, paint, inkjet printing, roller coating, dipping coating, or any combination thereof. The coating step can be carried out once or multiple times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times), which affects the thickness of the film coating. In one embodiment, the coating step can be carried out twice to form a bilayer.

[0162] Generally, when multiple coatings are applied, the first coating is dried before the next coating is applied. The amount of time between coating steps will vary depending on the type of film, working electrode, and sensing layer, as well as atmospheric conditions. Generally, drying times can be 1 minute or more (e.g., 2 minutes or more, 3 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, or 20 minutes or more). Once the film coating is applied, the coating can be cured. In one embodiment, the coating can be cured for 12 hours or more (e.g., 18 hours or more, 24 hours or more, 30 hours or more, 36 hours or more, 42 hours or more, or 48 hours or more). Curing can occur at room temperature (i.e., about 20°C) or at slightly elevated temperatures (e.g., below 100°C, below 80°C, below 70°C, below 60°C, below 50°C, below 40°C, below 30°C, or below 25°C). Generally, curing does not occur below about 20°C.

[0163] Typically, the film has a thickness in the range of about 1 μm to about 100 μm. For example, in some embodiments, the film can have a thickness of about 1 μm or more (e.g., about 5 μm or more, about 10 μm or more, about 15 μm or more, about 20 μm or more, about 25 μm or more, about 30 μm or more, about 35 μm or more, about 40 μm or more, about 50 μm or more, about 60 μm or more, about 70 μm or more, about 80 μm or more, or about 90 μm or more), and typically has a thickness of about 100 μm or less (e.g., about 90 μm or less, about 80 μm or less, about 70 μm or less, about 60 μm or more, about 50 μm or more, about 45 μm or less, about 40 μm or less, about 35 μm or less, about 30 μm or less, about 25 μm or less, about 20 μm or less, about 15 μm or less, about 10 μm or less, or about 5 μm or less). In one example, the film may have a thickness of approximately 5 to 85 μm, approximately 10 to 65 μm, approximately 20 to 50 μm, approximately 20 to 40 μm, approximately 25 to 35 μm, or approximately 30 μm.

[0164] In one embodiment, the membrane may optionally include a crosslinked poly(4-vinylpyridine), poly(vinyl alcohol), poly(acrylic acid), poly(methacrylic acid), tetrafluoroethylene-perfluoro-3,6-dioxa-4-methyl-7-octensulfonic acid copolymer-based membrane (e.g., NAFION® membrane), polyurethane, or a combination thereof. In some embodiments, the mass transfer limiting membrane may include at least a poly(4-vinylpyridine) homopolymer or copolymer (e.g., poly(4-vinylpyridine)-co-polystyrene sulfonate (PVP-co-PSS)), where the poly(4-vinylpyridine) may optionally be crosslinked.

[0165] Suitable poly(4-vinylpyridine) copolymers for inclusion in mass transfer limiting membranes may contain up to approximately 25% comonomers (based on the total amount of monomers in the copolymer), such as approximately 0.1% to approximately 5%, or approximately 5% to approximately 15%, or approximately 15% to approximately 25%, or approximately 1% to approximately 10%. Suitable comonomers are not particularly limited, as long as the mass transfer limiting membrane provides sufficient ketone permeability to provide analyte sensitivity of approximately 1 nA / mM or higher when exposed to ketones. In some embodiments, the mass transfer limiting membrane may optionally contain crosslinked poly(4-vinylpyridine)-co-polystyrene sulfonate (PVP-co-PSS).

[0166] In some embodiments, the film may include multiple layers, each having a different composition and / or degree of crosslinking. For example, the film coating may be a bilayer film including a first layer which may include a poly(4-vinylpyridine) homopolymer or copolymer, and a second layer which may include a crosslinked poly(4-vinylpyridine) homopolymer or copolymer (e.g., crosslinked with PEGDGE). In some embodiments, the film may include a bilayer of (i) a poly(4-vinylpyridine) homopolymer crosslinked with a crosslinking agent and (ii) a poly(4-vinylpyridine) copolymer optionally crosslinked with a crosslinking agent (e.g., a high molecular weight (e.g., 400 g / mol) poly(ethylene glycol) diglycidyl ether), as described herein. In some embodiments, the second layer may include a crosslinked polyvinylpyridine-costyrene polymer. In some embodiments, the second layer may include a polyvinylpyridine-costyrene polymer in which some of the pyridine nitrogen atoms are functionalized with uncrosslinked poly(ethylene glycol) tails and some of the pyridine nitrogen atoms are functionalized with alkyl sulfonic acid groups.

[0167] Coating of a film covering at least the ketone detection layer and / or background detection layer can be carried out using any suitable technique. In some embodiments, the film may be coated by spray coating, painting, inkjet printing, roller coating, dipping coating, or any combination thereof. In one embodiment, the coating includes dipping a ketone sensor, including a ketone detection layer and a background detection layer (e.g., sensor tail), into a solution containing a polymer and a solvent to provide a dipping ketone sensor. The coating step may be carried out once or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times), which affects the thickness of the film coating. In one embodiment, the coating step may be carried out twice to form a double layer. In one embodiment, the coating step may be dipping coating. In one embodiment, the coating step may be dipping coating carried out 2 to 6 times (i.e., 2, 3, 4, 5, or 6 times).

[0168] Typically, the film has a thickness in the range of about 1 μm to about 100 μm. For example, in some embodiments, the film can have a thickness of about 1 μm or more (e.g., about 5 μm or more, about 10 μm or more, about 15 μm or more, about 20 μm or more, about 25 μm or more, about 30 μm or more, about 35 μm or more, about 40 μm or more, about 50 μm or more, about 60 μm or more, about 70 μm or more, about 80 μm or more, or about 90 μm or more), and typically has a thickness of about 100 μm or less (e.g., about 90 μm or less, about 80 μm or less, about 70 μm or less, about 60 μm or more, about 50 μm or more, about 45 μm or less, about 40 μm or less, about 35 μm or less, about 30 μm or less, about 25 μm or less, about 20 μm or less, about 15 μm or less, about 10 μm or less, or about 5 μm or less). In one example, the film can have a thickness of approximately 5 to 80 μm, approximately 10 to 60 μm, approximately 20 to 50 μm, approximately 20 to 40 μm, approximately 25 to 35 μm, or approximately 30 μm.

[0169] In some embodiments, the first and second electrodes are part of a sensor (e.g., a ketone sensor) and may be contained within a sensor housing configured for adhesion to tissue (e.g., skin). Optionally, the sensor housing may include an adhesive layer to enable adhesion to the desired tissue. The sensor housing may hold all necessary components of the sensor, such as the circuitry and power supply for operating the sensor. In some embodiments, the power supply (e.g., a coin cell battery) and / or active circuitry are not housed within the sensor housing. A processor may be communicatively coupled to the sensor and may be physically located within the sensor housing or a reader device. The power supply may include one or more batteries, which may be rechargeable or single-use disposable batteries. Power management circuitry may coordinate battery charging and power monitoring, boost power, or perform DC conversion.

[0170] In some embodiments, the sensor may comprise a sensor tail (e.g., an insertion tip) configured to penetrate (e.g., implant into) tissue. The sensor tail may comprise at least a first sensing electrode and a second sensing electrode. A counter electrode may be present in combination with one or both working electrodes. The various electrodes may be stacked (layered) at least partially on top of each other and / or spaced laterally apart from each other on the sensor tail. Generally, the sensor tail may be of sufficient size and shape to be positioned below the tissue surface (e.g., penetrating the skin (dermis)), in the subcutaneous space, and in contact with the wearer's biological fluids, such as interstitial fluid. A suitable sensor configuration may be substantially flat, substantially cylindrical, or any other suitable shape. In one example, the sensor tail may be about 5 mm long, about 0.6 mm wide, and about 0.25 mm thick. Suitable tissues include, for example, skin, including the dermis, interstitial layer, and / or subcutaneous layer. In any of the sensor configurations disclosed herein, the various electrodes can be electrically isolated from each other by a dielectric material or a similar insulator.

[0171] In some embodiments, the sensor may include a reference electrode, a counter electrode, or both, as part of a first sensing electrode, a second sensing electrode, or both. In one embodiment, the counter electrode may be carbon (e.g., screen-printed carbon), and the reference electrode may be Ag / AgCl. In some embodiments, the working electrode and a second electrode (i.e., a counter / reference electrode) that functions as both a counter electrode and a reference electrode may be used as part of a first sensing electrode, a second sensing electrode, or both.

[0172] In one example, the electrode contacts are positioned on a first portion of the sensor located above the skin surface and may extend to a position within the sensor tail. The first working electrode, reference electrode, and counter electrode may be located on a second portion of the sensor, and the second working electrode, reference electrode, and counter electrode may be located on a third portion of the sensor, and the second and third portions may typically be located on the bottom portion of the sensor tail. The first working electrode may include a ketone detection layer, and the second working electrode may include a background detection layer, each of which is described herein.

[0173] In some embodiments, the sensor may comprise at least one insulating (e.g., dielectric) layer as part of a first sensing electrode, a second sensing electrode, or both. In some embodiments, the insulating layer may be composed of a suitable dielectric material that can form a solid. In some examples, the insulating layer may be formed from porcelain (ceramic), mica, glass, barium strontium titanate, plastic (e.g., polystyrene, polytetrafluoroethylene, polyethylene terephthalate, polyethylene, polypropylene, polymethyl methacrylate, polysulfone, polydimethylsiloxane, polyvinyl chloride, or a combination thereof), or metal oxide (e.g., silica, alumina, titania, zirconia, tantalum oxide, etc.).

[0174] In some embodiments, the sensor may include a substrate, and the first and second sensing electrodes may be arranged on the substrate. The substrate can be formed from any suitable inert material. In some embodiments, the substrate may be biocompatible. Examples of suitable substrates include titanium, carbon-based substrates (e.g., cellulose, polylactic acid), and plastic substrates (e.g., polyethylene terephthalate, polyethylene, polypropylene, polymethyl methacrylate, polysulfone, polydimethylsiloxane, polyvinyl chloride, etc.). In some embodiments, the substrate may be placed between the working electrode and the counter electrode and / or reference electrode as part of the first sensing electrode, the second sensing electrode, or both.

[0175] The sensor may be part of a system that comprises a first sensing electrode (e.g., a ketone sensing electrode), a second sensing electrode (e.g., a background sensing electrode), and circuits configured to connect to and disconnect the first and second sensing electrodes. In one embodiment, the system may be a ketone sensor comprising a first sensing electrode including a first working electrode, a ketone sensing layer, and a redox mediator; a second sensing electrode including a second working electrode, a redox mediator, and a background sensing layer that does not contain a ketone-responsive enzyme; and a membrane containing PVP that overcoats at least the ketone sensing layer and the background sensing layer. The ketone sensing layer may contain 3-HBDH, diaphorase, nicotinamide adenine dinucleotide phosphate (NAD(P)+) or a derivative thereof, optionally albumin, and an osmium-containing poly(4-vinylpyridine) polymer as a redox mediator. The background detection layer may contain diaphorase, nicotinamide adenine dinucleotide phosphate (NAD(P)+) or its derivatives, albumin, and osmium-containing poly(4-vinylpyridine) polymers as redox mediators.

[0176] In the presence of a biological fluid containing the analyte, a first detection electrode (ketone detection electrode) oxidizes the analyte, and the amount of oxidation is measured as the amount of electron charge produced from the reaction. A second detection electrode (background detection electrode) oxidizes background interfering substances (e.g., electroactive interfering substances) present in the biological fluid, and the amount of oxidation is measured as the amount of electron charge produced from the reaction. Unless the first and second detection electrodes are connected to another electrode, the charge from the redox reaction continues to accumulate on their respective electrodes. Accumulating charge (electrons) over a set period allows low concentrations of ketones and background to yield a signal output that is easier to measure and quantify compared to other known methods. After the set period for charge accumulation, the first and / or second detection electrodes can be connected to at least one (e.g., one, two, three, or four) other electrodes, such as a counter electrode and / or a reference electrode, to form a circuit. Once the circuit is connected, the electrons accumulated on the first and / or second detection electrodes can be emitted as an electrical signal, the amplitude of which can be measured and correlated with the amount of ketone present at the detection electrode. By subtracting the measured background signal (i.e., the measured value of the background signal only) from the measured ketone signal (i.e., the measured value of the ketone signal + background signal), a ketone-only signal proportional to the ketone concentration can be obtained.

[0177] Detection of analyte (A) relies on electrically "wired" an oxidoreductase (AOx) to the working electrode of the sensor via a redox mediator. During normal amperometric detection, the electrode is maintained at a certain potential (voltage) so that the analyte reacts at a constant rate proportional to the analyte concentration. + In the case of (a), electrons are transferred from the analyte (A) to the analyte-specific enzyme (AOx) and to the redox mediator (e.g., Os 3+) flows at a constant rate to the working electrode, generating a steady-state current. When the working electrode is disconnected from the circuit, the flow of electrons from the redox polymer to the working electrode stops, and no current flows in the circuit. However, the analyte still undergoes enzymatic oxidation, and as a result, the redox mediator is reduced (e.g., Os 3+ from OS 2+ (e) This is the electrons (e) from the analyte. - ) is stored in the redox mediator, so over time the reduced form of the redox mediator (e.g., Os) 2+ This leads to the accumulation of a charge. When the working electrode is reconnected to the circuit so that it equilibrium with its original potential (voltage), the reduced accumulation of the redox mediator is oxidized, resulting in a large current spike. Then, when the redox system reaches a steady state again, the current decays back to its original amperometric current. This two-step process forms the basis of accumulation-mode detection: firstly, the working electrode of the sensor is disconnected from or not connected to the circuit for a set period (also called the accumulation time), allowing the charge from the analyte to accumulate in the redox polymer; and secondly, the working electrode of the sensor is connected to the circuit after the accumulation time, allowing the accumulated charge to discharge and be measured as a sharp peak.

[0178] In some embodiments, in the ketone sensors used herein, a potential (voltage) sufficient to drive the redox reaction and reduce background interference is less than +40mV for Ag / AgCl (e.g., less than +30mV, less than +20mV, less than +10mV, less than +5mV, less than 0mV, less than -5mV, less than -10mV, less than -20mV, less than -30mV, less than -40mV, less than -50mV). It may be less than -60mV, less than -70mV, less than -80mV, less than -90mV, less than -100mV, less than -110mV, less than -120mV, less than -130mV, less than -140mV, less than -150mV, less than -160mV, less than -170mV, less than -180mV, less than -190mV, less than -200mV, less than -210mV, less than -220mV, less than -230mV, or less than -240mV.

[0179] In some embodiments, the ketone signal and background signal can be measured at different times. In some embodiments, the ketone signal and background signal can be obtained simultaneously through a first channel and a second channel.

[0180] Since ketones exist in biological fluids (e.g., serum) at relatively low concentrations, this system is designed to detect low concentrations of analytes by accumulating them on an enzyme biosensor. In the context of detecting ketones, low concentrations can range from approximately 1 mM or less (e.g., approximately 900 μM or less, approximately 800 μM or less, approximately 700 μM or less, approximately 600 μM or less, approximately 500 μM or less, approximately 400 μM or less, approximately 300 μM or less, approximately 200 μM or less, or approximately 100 μM or less) to approximately 10 μM or more (e.g., approximately 20 μM or more, approximately 30 μM or more, approximately 40 μM or more, approximately 50 μM or more, approximately 60 μM or more, approximately 70 μM or more, approximately 80 μM or more, approximately 90 μM or more, approximately 100 μM or more, approximately 120 μM or more, approximately 140 μM or more, or approximately 150 μM or more). For example, the ketone concentration in the analyte may be approximately 10 μM to 1 mM, 50 to 400 μM, 60 to 300 μM, or 70 to 200 μM.

[0181] In some embodiments, the sensor is exposed to biological fluids within a living organism. Generally, the method can be used in an in vivo monitoring system that uses a system such as those disclosed herein (e.g., a ketone sensor) to measure the concentration of ketones, and while positioned in vivo within a user (e.g., a patient such as a human), comes into contact with the user's biological fluids and detects the ketones contained therein. The in vivo monitoring system may include one or more reader devices that receive analyte data detected from a sensor control device. The reader devices can process and / or display the detected analyte data or sensor data in any number of forms. In some embodiments, the reader device may optionally be a mobile communication device such as a computer system, a mobile phone (e.g., a WiFi® or internet-enabled smartphone), a tablet, a personal digital assistant (PDA), or a dedicated reader device configured to communicate with a sensor control device, such as a mobile communication device that works in conjunction with a computer system, a mobile phone (e.g., a WiFi® or internet-enabled smartphone), a tablet, a personal digital assistant (PDA), or a mobile smart wearable electronic device assembly (e.g., smart glasses, multiple smart glasses, a watch, a bracelet, or a necklace). Configuring a reader device into an in vivo monitoring system is described, for example, in U.S. Patent No. 11,371,957, the disclosure of which is incorporated herein by reference in its entirety.

[0182] A reader device typically includes input components, a display, and processing circuitry, the processing circuitry of which may include one or more processors, microprocessors, controllers, and / or microcontrollers, each of which may be a discrete chip or distributed across several different chips (and parts thereof). The processing circuitry may include a communications processor with onboard memory and an application processor with onboard memory. The reader device may further include radio frequency (RF) communications circuitry coupled to an RF antenna, memory, a multifunction circuitry with one or more associated antennas, a power supply, power management circuitry, and / or a clock. It will be recognized that other hardware and functions may be included in the reader device.

[0183] In addition to the aforementioned detection methods using sensors (e.g., ketone sensors), this disclosure relates to ketone sensors including: A first detection electrode comprising a first working electrode and a ketone detection layer on a portion of the first working electrode, wherein the ketone detection layer comprises a ketone-responsive enzyme and a redox mediator; and A second detection electrode comprising a second working electrode and a background detection layer on a portion of the second working electrode, wherein the background detection layer contains a redox mediator but does not contain a ketone-responsive enzyme.

[0184] The ketone-responsive enzyme is as described herein. In some embodiments, the ketone-responsive enzyme may be 3-hydroxybutyrate dehydrogenase. In some embodiments, the first detection electrode may include NAD(P)H oxidoreductase and nicotinamide adenine dinucleotide phosphate (NAD(P)+) or a derivative thereof. In some embodiments, the first detection electrode may include albumin as described herein. In some embodiments, the first detection electrode may include a pH buffer as described herein.

[0185] In some embodiments, the ketone-responsive enzyme may be bound to a redox mediator as described herein. In some embodiments, the redox mediator may include polymers and electron transfer agents, each as described herein.

[0186] In some embodiments, the polymer may include poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene). In some embodiments, the polymer may include a polymer or copolymer repeating unit comprising at least one pendant pyridinyl group, an imidazolyl group, or both a pyridinyl group and an imidazolyl group. In some embodiments, the polymer may be crosslinked with a crosslinking agent as described herein. In some embodiments, the crosslinking agent may be a polyepoxide, cyanuryl chloride, N-hydroxysuccinimide, imide ester, epichlorohydrin, or a combination thereof. In some embodiments, the crosslinking agent may be polyethylene glycol diglycidyl ether (PEGDGE) as described herein.

[0187] In some embodiments, the electron transfer agent may include a transition metal complex as described herein. In some embodiments, the transition metal complex may include osmium, ruthenium, iron, cobalt, or a combination thereof. In some embodiments, the transition metal complex may be an osmium transition metal complex comprising one or more ligands, at least one of which comprises a nitrogen-containing heterocycle. In some embodiments, the redox mediator may include an osmium complex bonded to a poly(vinylpyridine) polymer.

[0188] In some embodiments, as described herein, the ketone detection layer or the background detection layer may be continuous, or both detection layers may be continuous on the working electrode. In some embodiments, as described herein, the ketone detection layer or the background detection layer may be discontinuous, or both detection layers may be discontinuous on the working electrode.

[0189] In some embodiments, the sensor may include a film described herein that overcoats at least a ketone detection layer, at least a background detection layer, or both. In some embodiments, the film comprises poly(4-vinylpyridine), which is optionally crosslinked.

[0190] In some embodiments, the sensor may comprise a housing as described herein. In some embodiments, the sensor may comprise a sensor tail configured for implantation into tissue as described herein, and first and second sensing electrodes may be disposed on the sensor tail. In some embodiments, the sensor may include a reference electrode, a counter electrode, or both a reference electrode and a counter electrode, as described herein, respectively. In some embodiments, the sensor may comprise at least one insulating layer as described herein. In some embodiments, the sensor may comprise at least one substrate as described herein, and either a first sensing electrode or a second sensing electrode is disposed on the substrate, or both sensing electrodes are disposed on the substrate.

[0191] In some embodiments, a ketone sensor or a method of using it can provide approximately three times or more (e.g., approximately four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times, or fifteen times or more) improved sensor accuracy compared to a ketone sensor that does not include one, two, or all three of the following features: a detection potential of less than +40mV for Ag / AgCl (e.g., a detection potential at -80mV), background subtraction, and accumulation mode detection.

[0192] In some embodiments, a ketone sensor or a method of using it can provide a signal increase of approximately 3 times or more (e.g., approximately 4 times or more, approximately 5 times or more, approximately 6 times or more, approximately 7 times or more, approximately 8 times or more, approximately 9 times or more, approximately 10 times or more, approximately 11 times or more, approximately 12 times or more, approximately 13 times or more, approximately 14 times or more, or approximately 15 times or more) compared to a ketone sensor that does not have one, two or all three of the following features: detection potential less than +40mV for Ag / AgCl (e.g., detection potential at -80mV), background subtraction, and accumulation mode detection.

[0193] In some embodiments, the ketone sensor can provide accurate ketone measurements (e.g., within approximately 20%, 18%, 15%, 12%, 10%, 8%, 5%, 4%, 3%, 2%, or 1%). In some embodiments, the ketone sensor can provide ketone measurements within approximately 10% of the actual value (e.g., relative to a control). In any of these embodiments, the ketone sensor can provide accurate ketone measurements over a period of one day or more (e.g., two days or more, three days or more, four days or more, five days or more, six days or more, seven days or more, eight days or more, nine days or more, ten days or more, eleven days or more, twelve days or more, thirteen days or more, fourteen days or more, fifteen days or more, sixteen days or more, seventeen days or more, eighteen days or more, nine days or more, ten days or more, eleven days or more, twelve days or more, thirty days or more, fifteen days or more, sixteen days or more, seventeen days or more, eighteen days or more, nineteen days or more, twenty days or more, or twenty-one days or more). In some embodiments, the ketone sensor can provide accurate ketone measurements over a period of 7 days or more. In some embodiments, the ketone sensor can provide accurate ketone measurements over a period of 14 days or more. In some embodiments, the ketone sensor can provide accurate ketone measurements over a period of 21 days or more. [Examples]

[0194] The embodiments shown below are provided for illustrative purposes only, and the embodiments described herein should not be construed as being limited to these embodiments. Rather, the embodiments should be construed as encompassing all possible variations that become apparent as a result of the teachings provided herein.

[0195] Example 1 To determine the relationship between the detection potential and background interference, ketone sensors having a first detection electrode and a second detection electrode were prepared according to Tables 1 and 2. Membrane solutions were prepared according to Table 3.

[0196] [Table 1]

[0197]

Table 2

[0198]

Table 3

[0199] The sensor was tested in single donor serum having a measured ketone (D-β-hydroxybutyrate) concentration of 190 μM using standard amperometry either at +40 mV or -80 mV versus Ag / AgCl. Aliquots of ketone (D-β-hydroxybutyrate at +100 μM, +100 μM, +300 μM, and +500 μM respectively) were added cumulatively to the serum every 15 minutes. The total ketone (D-β-hydroxybutyrate) concentration was 290 μM after the first 100 μM aliquot addition, 390 μM after the second 100 μM aliquot addition, 690 μM after the third 300 μM aliquot addition, and 1,190 μM after the fourth 500 μM aliquot addition. Time (in hours) and current were recorded at a 5-minute data acquisition (DAQ) rate using a multi-channel potentiostat (Figure 6).

[0200] As seen in Figure 6, as a result of the signal from ketones in the serum, it was observed that the standard sensing chemistry showed a higher signal than the blank sensing chemistry. However, the blank sensing chemistry showed a much lower background at -80 mV compared to +40 mV, indicating that the decrease in the sensing potential significantly decreased the background signal. This then enabled more accurate ketone measurements.

[0201] Figure 7 shows a magnified (focused view) of the added ketone aliquot portion of Figure 6. As seen in Figure 7, the initial standard sensor signal represents the sum of the signal from ketones present in the serum and background interference from the serum. The blank sensor signal represents only the serum background interference. The signal from ketones alone can be determined by subtracting the blank signal from the standard signal. However, because ketone concentrations are very low, this difference cannot be accurately determined due to noise associated with the electronic equipment. This background noise will be more pronounced in sensors that are worn and used.

[0202] Example 2 To determine the effectiveness of the accumulation mode detection, the same sensor as in Example 1 was used. In the accumulation detection mode, the blank electrode and the ketone electrode were left uncharged / open-circuited for 2.5 minutes, and then connected at -80mV for 2.5 minutes. This cycle was continuously repeated, with aliquots of ketones (+100μM, +100μM, +300μM, and +500μM D-β-hydroxybutyrate, respectively) added cumulatively to the serum every 30 minutes. The total ketone (D-β-hydroxybutyrate) concentration was 290μM after the first 100μM aliquot addition, 390μM after the second 100μM aliquot addition, 690μM after the third 300μM aliquot addition, and 1,190μM after the fourth 500μM aliquot addition.

[0203] Figure 8A shows current (nA) versus time (in units of time). Figure 8B shows the integrated charge of each peak. As seen in Figure 8A, the peak height is >5nA, which is much higher than the 0.1–0.3nA observed in previous experiments using standard amperometry (Figure 6). This demonstrates that cumulative detection increases submM ketone signals to levels similar to other analytes such as glucose sensors by providing an acceptable signal-to-noise ratio.

[0204] Based on these measurements, ketone concentrations were determined as shown in Table 4.

[0205] [Table 4]

[0206] As shown in Table 4, predicted ketones without background subtraction significantly overestimated serum ketone concentrations (approximately 232% overestimate), while values ​​using background subtraction were significantly more accurate (approximately 16% overestimate).

[0207] Example 3 Two ketone sensors ("Sensor 1" and "Sensor 2"), each having a first and second sensing electrode, were prepared according to Tables 5 and 6 to test whether in vivo low-concentration ketone monitoring was enhanced by detection potential reduction, background subtraction, and accumulation modes. As shown in Tables 5 and 6, sensing chemistry was deposited onto the sensing electrode in a 5-drop width × 10-drop height pattern using a non-contact piezoelectric dispensing system (first pass). Each droplet was approximately 0.6 nL, and the droplets were connected to each other, forming a "slot" pattern. The sensing chemistry was dried, and then dispensed again in a 5 × 10 pattern (second pass).

[0208] A membrane solution was prepared according to Table 7. The working electrode was coated by immersion five times in the prepared membrane solution at a rate of 5 mm / second to deposit the film. The electrode was then calcined at 56°C for three days to form a ketone sensor.

[0209] [Table 5]

[0210] [Table 6]

[0211]

Table 7

[0212] Sensors 1 and 2 were placed on both arms of the subject, and ketone concentration was continuously monitored starting at 4:00 PM for 16 hours. The subject ingested incremental amounts (2 mL, 4 mL, 8 mL, 16 mL, and 32 mL) of a ketone beverage (KETONE-IQ (registered trademark)) every hour. Sensor 1 measured ketone concentration using cumulative detection (the blank and ketone electrodes were left open-circuit without applied potential for 2.5 minutes and then connected at -80 mV for 2.5 minutes). Sensor 2 measured ketone concentration using a standard amperometry method at -80 mV versus Ag / AgCl.

[0213] As shown in FIGS. 9A-9B, it was observed that cumulative detection (i.e., Sensor 1) increased the signal by about 10-fold and decreased the noise compared to the standard amperometry method (Sensor 2). The signal-to-background ratio was also significantly higher in Sensor 1 than in Sensor 2. And as seen in FIG. 10, background subtraction stabilized the cumulative detection signal, especially in the more noisy regions. The reduced detection potential (-80 mV) also reduced background interference, as seen in the relatively low signal from the background channel.

[0214] Example 4 The consistency and sensitivity of ketone readings for ketone concentrations below 1 mM were confirmed using blood ketone test strip measurements. Sensors 1 and 2 were placed on both arms of the subject, and ketone concentration was continuously monitored. Sensors 1 and 2 measured ketone concentration using cumulative detection (the blank and ketone electrodes were left open-circuit without applied potential for 2.5 minutes and then connected at -80 mV for 2.5 minutes).

[0215] Participants were given vitamin C (2g) at 8:30 AM, followed by a ketone beverage (100mL) at 10:00 AM, and ketone concentrations were continuously monitored throughout the day using sensors 1 and 2. Reference measurements were also performed using blood ketone test strips. As shown in Figures 11A-11B, sensors 1 and 2 showed high signals and low noise at ketone concentrations of 1 mM or less. Background signals were less significant at -80mV than at +40mV in sensors 1 and 2, but the background channel detected vitamin C (used as a background interferant). The signals from sensors 1 and 2, plotted against blood ketone strip measurements after background subtraction, confirmed that sensors 1 and 2 provided consistently high-sensitivity ketone readings at ketone concentrations of 1 mM or less (Figure 12).

[0216] It should be understood that the section describing embodiments for carrying out the invention, rather than the sections describing the summary and abstract of the invention, is intended to be used to interpret the claims. The sections describing the "summary" and "abstract" may describe one or more exemplary embodiments of the invention as contemplated by the inventors(s), but may not describe all exemplary embodiments, and are therefore not intended to limit the scope of the invention and the appended claims.

[0217] This disclosure is described above using functional components that demonstrate the implementation of specific functions and their relationships. The boundaries of these functional components are arbitrarily defined herein for the sake of clarity. Alternative boundaries may be defined, provided that the identified functions and their relationships are adequately performed.

[0218] The foregoing descriptions of specific embodiments fully illustrate the general nature of the invention, so others can readily modify and / or adapt such specific embodiments for various uses without excessive experimentation and without departing from the general concept of the invention, by applying knowledge within the scope of the art of the art. Such adaptations and modifications are therefore intended to be within the meaning and scope of equivalents of the disclosed embodiments, based on the teachings and guidance presented herein. It should be understood that the expressions and terms herein are for illustrative purposes only, not limiting purposes, and therefore should be interpreted by those skilled in the art in light of the teachings and guidance.

[0219] The breadth and scope of the present invention should not be limited by any of the exemplary embodiments described above, but should be defined solely in accordance with the following claims and their equivalents.

[0220] The claims in this application are different from the claims of the parent application or any other related application. Accordingly, the applicant withdraws any disclaimers relating to the claims made in the parent application or any prior application in connection with this application. Accordingly, the examiner should be aware that it may be necessary to reconsider such past disclaimers and the prior art cited to circumvent them. Furthermore, the examiner should also be reminded that any disclaimers made in this application should not be read into or interpreted in relation to the parent application.

Claims

1. A method for detecting ketones: The method is Body fluids containing ketones, i) A first detection electrode comprising a ketone-responsive enzyme and a redox mediator; and ii) Contacting a second detection electrode that contains a redox mediator but does not contain a ketone-responsive enzyme, The first and second sensing electrodes are connected to the circuit, and a potential of less than +40 mV is applied to both electrodes to provide a steady state. Disconnecting the first and second detection electrodes from the circuit, Accumulating the charge derived from the biological fluid that reacts with the first and second detection electrodes over a set period of time, After the aforementioned set period, the first and second detection electrodes are connected to the circuit. The ketone signal is measured by subtracting the signal obtained from the second detection electrode from the signal obtained from the first detection electrode, Methods that include...

2. A method for detecting ketones: The method is (a) Contacting first and second detection electrodes with a biological fluid containing ketones, wherein the first detection electrode contains a ketone-responsive enzyme and a redox mediator, and the second detection electrode contains a redox mediator but does not contain a ketone-responsive enzyme, (b) Connecting the first sensing electrode to the circuit and applying a potential of less than +40 mV to provide a steady state, (c) Disconnecting the first detection electrode from the circuit and connecting the second detection electrode to the circuit, and applying a potential of less than +40 mV to provide a steady state, (d) During the first set period, charge derived from the biological fluid that reacts with the first detection electrode, (e) After the first setting period, the first detection electrode is connected to the circuit and the second detection electrode is disconnected from the circuit, (f) During the second set period, accumulate the charge derived from the biological fluid that reacts with the second detection electrode, (g) Connecting the second detection electrode to the circuit after the second setting period, (h) Measuring the ketone signal by subtracting the signal obtained from the second detection electrode from the signal obtained from the first detection electrode, Methods that include...

3. The method according to claim 2, wherein the cutting and connecting in step (b) and step (c) are performed simultaneously.

4. The method according to any one of claims 1 to 3, wherein the setting period, the first setting period, the second setting period, or any combination thereof is 30 seconds or more.

5. The method according to any one of claims 1 to 4, wherein the applied potential is approximately +5 mV to approximately -250 mV.

6. The method according to any one of claims 1 to 5, wherein the applied potential is approximately -80 mV.

7. The method according to any one of claims 1 to 6, wherein the first detection electrode comprises a working electrode and a ketone detection layer on a portion of the working electrode, and the ketone detection layer comprises the ketone-responsive enzyme and the redox mediator.

8. The method according to claim 7, wherein the ketone detection layer or the background detection layer is continuous, or both detection layers are continuous.

9. The method according to claim 7 or 8, wherein the ketone detection layer or the background detection layer is discontinuous, or both detection layers are discontinuous.

10. The method according to any one of claims 7 to 9, further comprising a film that overcoats at least the ketone detection layer, at least the background detection layer, or both.

11. The method according to claim 10, wherein the film comprises poly(4-vinylpyridine).

12. The method according to any one of claims 1 to 11, wherein the ketone-responsive enzyme is 3-hydroxybutyrate dehydrogenase.

13. The method according to any one of claims 1 to 12, wherein the first detection electrode further comprises NAD(P)H oxidoreductase and nicotinamide adenine dinucleotide phosphate (NAD(P)+) or a derivative thereof.

14. The method according to any one of claims 1 to 13, wherein the ketone-responsive enzyme is bound to the redox mediator.

15. The method according to any one of claims 1 to 14, wherein the first detection electrode further comprises albumin.

16. The method according to any one of claims 1 to 15, wherein the first detection electrode further comprises a pH buffer.

17. The method according to any one of claims 1 to 12, wherein the second sensing electrode includes a working electrode and a background sensing layer on a portion of the working electrode, and the background sensing layer includes the redox mediator.

18. The method according to any one of claims 1 to 16, wherein the redox mediator comprises a polymer and an electron transfer agent.

19. The method according to claim 18, wherein the polymer comprises poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene).

20. The method according to claim 19, wherein the polymer comprises a polymer or copolymer repeating unit comprising at least one pendant pyridinyl group, an imidazolyl group, or both a pyridinyl group and an imidazolyl group.

21. The method according to any one of claims 18 to 20, wherein the electron transfer agent comprises a transition metal complex.

22. The method according to claim 21, wherein the transition metal complex comprises osmium, ruthenium, iron, cobalt, or a combination thereof.

23. The method according to claim 21 or 22, wherein the transition metal complex is an osmium transition metal complex comprising one or more ligands, and at least one ligand comprises a nitrogen-containing heterocycle.

24. The method according to any one of claims 18 to 23, wherein the polymer is crosslinked with a crosslinking agent.

25. The method according to claim 24, wherein the crosslinking agent is a polyepoxide, cyanuryl chloride, N-hydroxysuccinimide, imide ester, epichlorohydrin, or a combination thereof.

26. The method according to claim 24 or 25, wherein the crosslinking agent is polyethylene glycol diglycidyl ether (PEGDGE).

27. The method according to any one of claims 1 to 26, wherein the redox mediator comprises an osmium complex bonded to a poly(vinylpyridine) polymer.

28. The method according to any one of claims 1 to 27, wherein the first and second sensing electrodes are part of a sensor comprising a housing.

29. The method according to claim 28, wherein the sensor further comprises a sensor tail configured for implantation in tissue, and the first and second sensing electrodes are arranged on the sensor tail.

30. The method according to claim 28 or 29, wherein the sensor further includes a reference electrode, a counter electrode, or both a reference electrode and a counter electrode.

31. The method according to any one of claims 28 to 30, wherein the sensor further comprises at least one insulating layer.

32. The method according to any one of claims 28 to 31, further comprising at least one substrate, wherein the first detection electrode or the second detection electrode is disposed on the substrate, or both detection electrodes are disposed on the substrate.

33. It is a ketone sensor: A first detection electrode comprising a first working electrode and a ketone detection layer on a portion of the first working electrode, wherein the ketone detection layer comprises a ketone-responsive enzyme and a redox mediator; and A second detection electrode comprising a second working electrode and a background detection layer on a portion of the second working electrode, wherein the background detection layer contains a redox mediator but does not contain a ketone-responsive enzyme, A ketone sensor equipped with [the necessary components].

34. The ketone sensor according to claim 33, wherein the ketone-responsive enzyme is 3-hydroxybutyrate dehydrogenase.

35. The ketone sensor according to claim 33 or 34, wherein the first detection electrode further comprises NAD(P)H oxidoreductase and nicotinamide adenine dinucleotide phosphate (NAD(P)+) or a derivative thereof.

36. The ketone sensor according to any one of claims 33 to 35, wherein the ketone-responsive enzyme is bound to the redox mediator.

37. The ketone sensor according to any one of claims 33 to 36, wherein the first detection electrode further comprises albumin.

38. The ketone sensor according to any one of claims 33 to 37, wherein the first detection electrode further comprises a pH buffer.

39. The ketone sensor according to any one of claims 33 to 38, wherein the redox mediator comprises a polymer and an electron transfer agent.

40. The ketone sensor according to claim 39, wherein the polymer comprises poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene).

41. The ketone sensor according to claim 40, wherein the polymer comprises a polymer or copolymer repeating unit comprising at least one pendant pyridinyl group, an imidazolyl group, or both a pyridinyl group and an imidazolyl group.

42. The ketone sensor according to any one of claims 39 to 41, wherein the polymer is crosslinked with a crosslinking agent.

43. The ketone sensor according to claim 42, wherein the crosslinking agent is a polyepoxide, cyanuryl chloride, N-hydroxysuccinimide, imide ester, epichlorohydrin, or a combination thereof.

44. The ketone sensor according to claim 42 or 43, wherein the crosslinking agent is polyethylene glycol diglycidyl ether (PEGDGE).

45. The ketone sensor according to any one of claims 39 to 44, wherein the electron transfer agent comprises a transition metal complex.

46. The ketone sensor according to claim 45, wherein the transition metal complex includes osmium, ruthenium, iron, cobalt, or a combination thereof.

47. The ketone sensor according to claim 45 or 46, wherein the transition metal complex is an osmium transition metal complex comprising one or more ligands, and at least one ligand comprises a nitrogen-containing heterocycle.

48. The ketone sensor according to any one of claims 33 to 47, wherein the redox mediator comprises an osmium complex bonded to a poly(vinylpyridine) polymer.

49. The ketone sensor according to any one of claims 33 to 48, wherein the ketone detection layer or the background detection layer is continuous, or both detection layers are continuous on the working electrode.

50. The ketone sensor according to any one of claims 33 to 48, wherein the ketone detection layer or the background detection layer is discontinuous, or both detection layers are discontinuous on the working electrode.

51. The ketone sensor according to any one of claims 33 to 50, further comprising a film that overcoats at least the ketone detection layer, at least the background detection layer, or both.

52. The ketone sensor according to claim 51, wherein the membrane contains poly(4-vinylpyridine), and the poly(4-vinylpyridine) is crosslinked as necessary.

53. A ketone sensor according to any one of claims 33 to 52, further comprising a housing.

54. The ketone sensor according to claim 53, further comprising a sensor tail configured for implantation into tissue, wherein the first and second sensing electrodes are arranged on the sensor tail.

55. The ketone sensor according to claim 53 or 54, further comprising a reference electrode, a counter electrode, or both a reference electrode and a counter electrode.

56. The ketone sensor according to any one of claims 53 to 55, further comprising at least one insulating layer.

57. The ketone sensor according to any one of claims 53 to 56, further comprising at least one substrate, wherein the first detection electrode or the second detection electrode is disposed on the substrate, or both detection electrodes are disposed on the substrate.

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