Background interference mitigation through high sensitivity ketone sensing of cumulative mode sensing at low working electrode potential

Abstract

The present disclosure relates to a method of increasing the sensitivity of sensing ketones, the method comprising providing: i) a ketone sensing electrode comprising a ketone-responsive enzyme and a redox medium; and ii) a background sensing electrode comprising a redox medium and not comprising a ketone-responsive enzyme, and applying a potential of less than + 40 mV to provide homeostasis. The ketone sensing electrode and the background sensing electrode may be simultaneously or sequentially disconnected from the circuit to allow charge accumulation for a set period of time. After sufficient charge has been accumulated, the two electrodes may be reconnected to the circuit. A ketone signal may be measured by subtracting a signal from the background sensing electrode from a signal from the ketone sensing electrode. The disclosure further relates to a ketone sensor comprising a first sensing electrode sensing a ketone and a second sensing electrode sensing a background.

Description

[0001] BACKGROUND

[0002] While glucose management is the primary focus of diabetes therapy, continuous ketone monitoring can be beneficial for diabetic patients, particularly type 1 diabetic patients. Type 1 diabetic patients can experience a potentially fatal complication called diabetic ketoacidosis (DKA). Continuous ketone sensing holds promise for preventing DKA. Ketone concentrations are also correlated with indicators of heart failure. For example, patients with elevated ketone concentrations greater than 300 μΜ are at risk for developing heart failure. While such measurements are important, ketone concentrations in vivo tend to be relatively low.

[0003] Further, naturally occurring ascorbate, urate, and other compounds in interstitial fluid produce a background interference signal for the biosensor, which can reduce the accuracy of analyte measurements. At ketone concentrations greater than 1 mM, this background interference signal is significantly lower than the ketone signal, and ketone concentrations can be accurately determined. However, as the ketone concentration decreases below 1 mM, the ratio of background interference to ketone signal increases, hindering accurate ketone measurements.

[0004] Accordingly, there is a need for a biosensor with higher ketone sensitivity and that can reduce background signal interference to provide accurate and continuous in vivo ketone monitoring.

[0005] BRIEF SUMMARY

[0006] The present disclosure can provide a method of increasing the sensitivity of sensing ketones, the method comprising: providing i) a ketone sensing electrode comprising a ketone-responsive enzyme and a redox mediator; and ii) a background sensing electrode comprising a redox mediator and not comprising a ketone-responsive enzyme, and applying a potential of less than +40 mV to provide a steady state. The ketone sensing electrode and the background sensing electrode can be disconnected from the circuit simultaneously or sequentially for a set period of time to allow charge to accumulate. The ketone signal can be measured by subtracting the signal from the background sensing electrode from the signal from the ketone sensing electrode. The present disclosure can further provide a ketone sensor comprising a first sensing electrode that senses ketones and a second sensing electrode that senses background.

[0007] The present disclosure also relates to a method for sensing ketones, the method comprising contacting a biological fluid comprising ketones with:

[0008] a) a first sensing electrode comprising a ketone-responsive enzyme and a redox mediator; and

[0009] b) a second sensing electrode comprising a redox mediator and not comprising a ketone-responsive enzyme;

[0010] connecting the first and second sensing electrodes to the circuit and applying a potential of less than +40 mV to both electrodes to provide a steady state;

[0011] disconnecting the first and second sensing electrodes from the circuit;

[0012] accumulating charge from the biological fluid reacting with the first and second sensing electrodes for a first set period of time;

[0013] after the first set period of time, connecting (e.g., reconnecting) the first and second sensing electrodes to the circuit; and

[0014] measuring a ketone signal by subtracting the signal from the second sensing electrode from the signal from the first sensing electrode.

[0015] The present disclosure also relates to a method for sensing a ketone, the method comprising:

[0016] (a) contacting first and second sensing electrodes with a biological fluid comprising a ketone, wherein the first sensing electrode comprises a ketone-responsive enzyme and a redox mediator and the second sensing electrode comprises a redox mediator and does not comprise a ketone-responsive enzyme;

[0017] (b) connecting the first sensing electrode to a circuit and applying a potential of less than +40 mV to provide a steady state;

[0018] (c) disconnecting the first sensing electrode from the circuit and connecting the second sensing electrode to the circuit and applying a potential of less than +40 mV to provide a steady state;

[0019] (d) accumulating charge from the biological fluid reacting with the first sensing electrode for a first set period of time;

[0020] (e) after the first set period of time, connecting (e.g., reconnecting) the first sensing electrode to the circuit and disconnecting the second sensing electrode from the circuit;

[0021] (f) accumulating charge from the biological fluid reacting with the second sensing electrode for a second set period of time;

[0022] (g) after the second set period of time, connecting (e.g., reconnecting) the second sensing electrode to the circuit; and

[0023] (h) measuring a ketone signal by subtracting the signal from the second sensing electrode from the signal from the first sensing electrode.

[0024] In certain aspects of the method, the disconnecting and connecting of step (c) and step (b) are simultaneous.

[0025] In any of these aspects, the set time period, the first set time period, the second set time period, or any combination thereof is 30 seconds or more.

[0026] In any of these aspects, the applied potential is about +5 mV to about -250 mV. In certain aspects, the applied potential is about -80 mV.

[0027] In any of these aspects, the first sensing electrode comprises a working electrode and a ketone sensing layer located on a portion of the working electrode, wherein the ketone sensing layer comprises the ketone-responsive enzyme and the redox mediator.

[0028] In any of these aspects, the ketone-responsive enzyme is 3-hydroxybutyrate dehydrogenase.

[0029] In any of these aspects, the first sensing electrode further comprises an NAD(P)H oxidoreductase enzyme and nicotinamide adenine dinucleotide phosphate (NAD(P)+) or a derivative thereof.

[0030] In any of these aspects, the ketone-responsive enzyme is linked to the redox mediator.

[0031] In any of these aspects, the first sensing electrode further comprises albumin.

[0032] In any of these aspects, the first sensing electrode further comprises a pH buffer.

[0033] In any of these aspects, the second sensing electrode comprises a working electrode and a background sensing layer located on a portion of the working electrode, wherein the background sensing layer comprises a redox mediator. In one aspect, the redox mediator in the first and second sensing electrodes is the same material.

[0034] In any of these aspects, the redox mediator comprises a polymer and an electron transfer reagent. In certain aspects, the polymer comprises a poly(vinylpyridine), a poly(thiophene), a poly(aniline), a poly(pyrole), or a poly(acetylene). In certain aspects, the polymer comprises a polymeric or copolymeric repeat unit comprising at least one pendant pyridyl group, imidazolyl group, or both a pyridyl and imidazolyl group. In certain aspects, the electron transfer reagent comprises a transition metal complex. In certain aspects, the transition metal complex comprises osmium, ruthenium, iron, cobalt, or a combination thereof. In certain aspects, the transition metal complex is an osmium transition metal complex comprising one or more ligands, wherein at least one ligand comprises a nitrogen-containing heterocycle.

[0035] In any of these aspects, the redox mediator comprises an osmium complex bonded to a poly(vinylpyridine)-based polymer. In certain aspects, the polymer is crosslinked with a crosslinking agent. In certain aspects, the crosslinking agent is a polyepoxide, cyanuric chloride, N-hydroxysuccinimide, imido ester, epichlorohydrin, or a combination thereof. In certain aspects, the crosslinking agent is polyethylene glycol diglycidyl ether (PEGDGE).

[0036] In some of these aspects, the ketone sensing layer or the background sensing layer is continuous, or both sensing layers are continuous. In certain aspects, the ketone sensing layer or the background sensing layer is discontinuous, or both sensing layers are discontinuous.

[0037] In certain aspects, a membrane covers at least the ketone sensing layer, at least the background sensing layer, or both. In certain aspects, the membrane comprises poly(4-vinylpyridine).

[0038] In any of these aspects, the first and second sensing electrodes are part of a sensor comprising a housing. In certain aspects, the sensor further comprises a sensor tail configured for implantation in tissue, wherein the first and second sensing electrodes are disposed on the sensor tail. In certain aspects, the sensor further comprises a reference electrode, a counter electrode, or both a reference electrode and a counter electrode. In certain aspects, the sensor further comprises at least one insulating layer. In certain aspects, the sensor further comprises at least one substrate, wherein the first sensing electrode or the second sensing electrode is disposed on the substrate, or both sensing electrodes are disposed on the substrate.

[0039] The present disclosure further relates to a ketone sensor comprising

[0040] a first sensing electrode comprising a first working electrode and a ketone sensing layer disposed on a portion of the first working electrode, wherein the ketone sensing layer comprises a ketone-responsive enzyme and a redox mediator; and

[0041] a second sensing electrode comprising a second working electrode and a background sensing layer disposed on a portion of the second working electrode, wherein the background sensing layer comprises a redox mediator and does not comprise a ketone-responsive enzyme.

[0042] In certain aspects, the ketone-responsive enzyme is 3-hydroxybutyrate dehydrogenase. In certain aspects, the first sensing electrode further comprises an NAD(P)H oxidoreductase and a nicotinamide adenine dinucleotide phosphate (NAD(P)+) or a derivative thereof. In certain aspects, the first sensing electrode further comprises albumin. In certain aspects, the first sensing electrode further comprises a pH buffer.

[0043] In certain aspects, the ketone-responsive enzyme is linked to the redox mediator.

[0044] In one aspect, the redox mediator in the first and second sensing electrodes is the same material. In certain aspects, the redox mediator comprises a polymer and an electron transfer reagent.

[0045] In certain aspects, the polymer comprises a poly(vinylpyridine), a poly(thiophene), a poly(aniline), a poly(pyrole), or a poly(acetylene). In certain aspects, the polymer comprises a polymer or copolymer repeat unit comprising at least one pendant pyridyl group, imidazolyl group, or both a pyridyl and imidazolyl group. In certain aspects, the polymer is crosslinked with a crosslinking agent. In certain aspects, the crosslinking agent is a polyepoxide, cyanuric chloride, N-hydroxysuccinimide, imidate, epichlorohydrin, or a combination thereof. In certain aspects, the crosslinking agent is polyethylene glycol diglycidyl ether (PEGDGE).

[0046] In certain aspects, the electron transfer reagent comprises a transition metal complex. In certain aspects, the transition metal complex comprises osmium, ruthenium, iron, cobalt, or a combination thereof. In certain aspects, the transition metal complex is an osmium transition metal complex comprising one or more ligands, wherein at least one ligand comprises a nitrogen-containing heterocycle. In certain aspects, the redox mediator comprises an osmium complex bonded to a poly(vinylpyridine)-based polymer.

[0047] In certain aspects, the ketone sensing layer or the background sensing layer is continuous on the working electrode, or both sensing layers are continuous. In certain aspects, the ketone sensing layer or the background sensing layer is discontinuous on the working electrode, or both sensing layers are discontinuous.

[0048] In certain aspects, the sensor further comprises a membrane covering at least the ketone sensing layer, at least the background sensing layer, or both. In certain aspects, the membrane comprises a poly(4-vinylpyridine).

[0049] In certain aspects, the sensor further comprises a housing. In certain aspects, the sensor further comprises a sensor tail configured for implantation into tissue, wherein the first and second sensing electrodes are disposed on the sensor tail. In certain aspects, the sensor further comprises a reference electrode, a counter electrode, or both a reference electrode and a counter electrode. In certain aspects, the sensor further comprises at least one insulating layer. In certain aspects, the sensor further comprises at least one substrate, wherein the first sensing electrode or the second sensing electrode is disposed on the substrate, or both sensing electrodes are disposed on the substrate.

[0050] Other aspects and advantages of the present disclosure will be in part apparent and in part pointed out in the description in which reference is made to the accompanying drawings.

[0051] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the claims. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 A diagram shows an exemplary sensor system that can include an analyte sensor of the present disclosure.

[0054] Figures 2A-2C A cross-sectional view of an analyte sensor including a single sensing layer is shown.

[0055] Figures 3A-3C A cross-sectional view of an analyte sensor including two sensing layers is shown.

[0056] Figure 4 A cross-sectional view of an analyte sensor including two sensing layers is shown.

[0057] Figures 5A-5C A perspective view of an analyte sensor including two sensing layers on separate working electrodes is shown.

[0058] Figure 6 A plot of sensor current (nA) vs. time (hours) for a blank (background) sensor and a standard (ketone) sensor with different sensing potentials applied (+40 mV or -80 mV, each vs. Ag / AgCl reference electrode) is shown.

[0059] Figure 7 A plot of sensor current (nA) vs. time (hours) for a blank (background) sensor and a standard (ketone) sensor at -80 mV (vs. Ag / AgCl reference electrode) is shown. The higher double arrow is the standard baseline for 190 μΜ ketones in serum + background signal. The lower double arrow is the blank baseline for background signal only.

[0060] Figure 8A A plot of sensor current (nA) vs. time (hours) for one exemplary ketone sensor of the present disclosure using cumulative mode sensing is shown. Figure 8B A plot of integrated charge for each peak is shown, where the higher double arrow indicates the standard baseline for 190 μΜ ketones in serum + background signal, and the lower double arrow indicates the blank baseline for background signal only.

[0061] Figure 9AThe diagram shows a graph of the sensor current (nA) of an exemplary ketone sensor (“Sensor 1”) using cumulative detection and a second ketone sensor (“Sensor 2”) measuring ketone concentration at -80 mV (relative to Ag / AgCl) using standard amperometry, as a function of time (hours). Figure 9B The graph shows the change of sensor current (nA) over time (hours) within a time window from 4:00 PM to 12:00 AM.

[0062] Figure 10 A graph showing the variation of sensor current (nA) over time (hours) of an exemplary ketone sensor (sensor 1) of this disclosure after background subtraction is displayed.

[0063] Figure 11A A graph showing the sensor current (nA) of an exemplary ketone sensor (sensor 1 and 2) of this disclosure using cumulative detection is shown as a function of time (hours). Figure 11B A graph showing the sensor current (up to 10 nA) of an exemplary ketone sensor (sensor 1 and 2) of this disclosure using cumulative detection is shown as a function of time (hours).

[0064] Figure 12 The diagram shows (1) the exemplary ketone sensors (sensors 1 and 2) of this disclosure after background subtraction; and (2) a graph showing the change of sensor current (nA) measured by a blood ketone test strip over time (hours).

[0065] Detailed description

[0066] The headings provided herein are not intended to limit the various aspects of this disclosure, which are defined by reference to the entire specification. It should also be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting, as the scope of this disclosure will be defined solely by the appended claims.

[0067] definition

[0068] For convenience, the meanings of certain terms and phrases used in the specification, embodiments, and appended claims are provided below. Unless otherwise stated or implied in the context, the following terms and phrases include the meanings provided below. These definitions are provided to aid in the description of particular aspects and are not intended to limit the claimed technology, as its scope is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the technology pertains. If there is a significant difference between the use of a term in the art and its definition provided herein, the definition provided in the specification shall prevail.

[0069] The articles “a,” “an,” and “the” are used in this text to indicate one or more (i.e., at least one) grammatical object of the article. For example, “an element” refers to one element or more than one element.

[0070] As used herein, the term “about” means within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, depending in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within three or more standard deviations according to practice in the art. Alternatively, “about” can mean a range up to 10% of a given value (e.g., up to 5% or up to 1%).

[0071] The term "at least" preceding a number or series of numbers should be understood to include the number associated with the term "at least," as well as any subsequent numbers or integers that are logically included and which are clearly apparent from the context. When "at least" appears before a series of numbers or a range, it should be understood that "at least" can modify each number in that series or range. For example, "at least 3" means at least 3, at least 4, at least 5, etc. When "at least" precedes a component of a method step, that component is included in that step, while other components are optional.

[0072] The terms “comprising,” “having,” “including,” “containing,” etc., used herein are open-ended terms meaning “including, but not limited to”. With respect to the fact that a given aspect disclosed herein “comprising” certain elements, it should be understood that this disclosure also specifically considers and discloses aspects that “essentially consist of those elements” and “comprise those elements.”

[0073] The terms “consists essentially of” and “consisting essentially of” used in this paper should be interpreted as semi-closed terms, meaning that they do not include other components that substantially affect the basic and novel features of a particular aspect.

[0074] The terms “consists of” and “consisting of” used in this document should be interpreted as closed terms. Therefore, an aspect that “consists of a specific set of elements” excludes any element, step, or component not specified in that aspect.

[0075] As used herein, a “set time period” is the amount of time required to perform a specific step, such as contacting a biofluid with a sensing electrode, contacting one or more sensing electrodes with a circuit, or accumulating electronic charge to provide a sufficient signal output for measurement and quantification of a given analyte (e.g., a ketone). The various set time periods described herein may be the same or different. Typically, and as discussed in detail elsewhere herein, set time periods can be about 1 second or longer (e.g., about 5 seconds or longer, about 10 seconds or longer, or about 30 seconds or longer) and about 30 minutes or shorter (e.g., about 20 minutes or shorter, about 10 minutes or shorter, about 5 minutes or shorter, about 3 minutes or shorter, or about 1 minute or shorter).

[0076] As used herein, the phrase “accumulation mode sensing” refers to the accumulation of electrons resulting from the oxidation of the analyte, which occurs at or on a sensing element that is not connected to a circuitry working electrode, thereby generating the accumulation of electrons.

[0077] As used herein, "analyte" refers to an enzyme substrate to be measured or detected. The analyte may be derived from, for example, biological fluids and may be tested in vivo, in vitro, or ex vivo. In most respects herein, the analyte is a ketone.

[0078] As used herein, the term "background interference" or "interference" refers to substances in a sample that can prevent the accurate measurement of a desired analyte (e.g., ketone). Common interferences include, for example, ascorbic acid, uric acid, homooxalic acid, 5-hydroxytryptamine, catecholamines (e.g., dopamine, norepinephrine and their major metabolites, such as 3,4-dihydroxyphenylacetic acid, 3-methoxytyramine), indoleamine, drug metabolites, fibrinogen, proteins, cells (e.g., leukocytes, erythrocytes), metal ions (e.g., copper ions, mercury ions), and combinations thereof.

[0079] As used herein, "biofluid" refers to any bodily fluid or bodily fluid derivative in which an analyte can be measured. Examples of biofluids include, for example, dermal fluid, subcutaneous fluid, interstitial fluid, plasma, blood (e.g., from veins or blood vessels), lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, sweat, or tears. In some respects, the biofluid is dermal fluid or interstitial fluid.

[0080] The phrase “configured to accumulate charge” as used herein refers to an arrangement of the working electrode and circuitry that allows the accumulation of electrons generated from the oxidation of the analyte (e.g., a ketone). Oxidation occurring at or above the sensing element of the working electrode is not connected to the circuitry, thus resulting in the accumulation of electrons.

[0081] As used herein, the term "counter electrode" refers to an electrode that is paired with the working electrode, through which the current flowing is equal in magnitude and opposite in sign to the current flowing through the working electrode. For certain aspects of this disclosure, the term "counter electrode" includes a) a true counter electrode and b) a counter electrode that is also used as a reference electrode (i.e., counter electrode / reference electrode), unless otherwise indicated.

[0082] 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 link at least two parts of the same molecule together (intramolecular crosslinking). Crosslinking agents having more than two reactive groups can be capable of both intermolecular and intramolecular crosslinking simultaneously.

[0083] As used in this article, the term "electrolysis" refers to the electro-oxidation or electro-reduction of a compound at an electrode, either directly or via one or more electron transfer reagents.

[0084] As used herein, the term "electron transfer reagent" refers to a compound that transfers electrons directly or in synergistic with other electron transfer reagents between the analyte and the working electrode. An example of an electron transfer reagent is a redox medium.

[0085] The components used in this article are “fixed” or “attached” to polymers and / or sensors, for example, when the components are trapped on, trapped in, covalently bonded, ionicly bonded, electrostatically bonded or coordinately bonded to polymers, sol-gel matrices, membranes and / or sensor components, thereby reducing or preventing flowability.

[0086] As used herein, the terms “non-leaching” compound or “non-leaching set” compound are intended to define a compound fixed on a sensor such that it does not diffuse significantly from the sensing layer of the working electrode during sensor use (e.g., when the sensor is implanted in a patient or during sample measurement).

[0087] As used herein, the term "patient" refers to a living animal and therefore encompasses, for example, living mammals and living humans. In this document, the term "user" may be used as a term that encompasses the term "patient".

[0088] As used in this article, the term "precursor polymer" refers to the starting polymer prior to the attachment of various modifying groups to form the modified polymer.

[0089] As used herein, the term "reactive group" refers to a functional group of a molecule (e.g., a polymer, crosslinking agent, enzyme) that can react with another compound to attach at least a portion of that other compound (e.g., another reactive group) to the molecule. Reactive groups include carboxyl groups, activated esters, sulfonyl halides, sulfonates, isocyanates, isothiocyanates, epoxides, aziridines, halides, aldehydes, ketones, amines, acrylamides, thiols, acyl azides, acyl halides, hydrazides, hydroxylamines, alkyl halides, imidazoles, pyridines, phenols, alkyl sulfonates, halotriazines, imido esters, maleimides, acyl hydrazides, hydroxyl groups, and photoreactive azidoaryl groups. Activated esters as understood in the art generally include esters of succinimide, benzotriazole, or aryl groups substituted with electron-withdrawing groups such as sulfonyl, nitro, cyano, or halogen groups; or carboxylic acids activated with carbodiimides.

[0090] As used herein, the term "redox medium" refers to an electron transfer agent used to transfer electrons between an analyte, a reduced or oxidized analyte, an enzyme, and an electrode. This transfer can be direct or via one or more other electron transfer agents. Redox media comprising a polymer backbone can also be referred to as "redox polymers."

[0091] As used herein, the term “reference electrode” includes a) a reference electrode and b) a reference electrode that is also used as a counter electrode (i.e., counter / reference electrode), unless otherwise indicated.

[0092] As used herein, the term "sensing layer" refers to a component of a sensor that includes components that facilitate the electrolysis of the analyte. The sensing layer may include components such as a redox medium (e.g., an electron transfer reagent or redox polymer), a catalyst (e.g., an analyte-specific enzyme), etc., which catalyzes the reaction of the analyte to produce a response at the working electrode, or may include both an electron transfer reagent and a catalyst. In some aspects of this disclosure, the sensor includes a sensing layer disposed non-leachingly in proximity to or on the working electrode.

[0093] As used herein, the term "sensing element" refers to the application or region of an analyte-specific enzyme disposed together with the sensing layer. Therefore, the sensing element is capable of interacting with the analyte. The sensing layer may have more than one sensing element constituting an analyte detection region disposed on the working electrode. In some aspects, the sensing element includes an analyte-specific enzyme and an electron transfer reagent (e.g., an electron transfer agent). In other aspects, the sensing element includes an analyte-specific enzyme, a redox medium, and a cross-linking agent.

[0094] As used herein, the term "sensor" refers to a device configured to detect the presence and / or measure the level of an analyte in a sample via electrochemical oxidation and reduction reactions on the sensor. These reactions are converted into electrical signals that can be correlated (e.g., proportionally) to the amount, concentration, or level of the analyte in the sample.

[0095] As used herein, the term "continuous" in relation to continuous analyte sensors (e.g., "continuous ketone sensors") means a sensor configured to perform one or more measurements of an analyte (e.g., ketones) over a period of time. Continuous sensors can perform continuous measurements depending on their sampling frequency. For example, measurements can be performed once or more every approximately 1 ms, approximately every 10 ms, approximately every 100 ms, approximately every 1 s, approximately every 10 seconds, approximately every 30 seconds, approximately every minute, approximately every 5 minutes, approximately every 10 minutes, approximately every 30 minutes, or approximately every hour. These measurements can be performed continuously, for example, over a continuous time 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. Continuous ketone sensors are typically in continuous contact with a sample (such as a biological fluid). For example, a continuous ketone sensor may include an implantable portion or component as defined herein, which is in continuous contact with a biological fluid (such as dermal fluid or interstitial fluid) during use, thereby enabling continuous or periodic measurements over a continuous time period depending on the sensor’s sampling frequency.

[0096] As used herein, the term "substituted" functional group (e.g., substituted alkyl, alkenyl, alkoxy, aryl) includes at least one substituent (e.g., 1, 2, 3, 4, or 5), which may be, for example, halogen, alkoxy, mercapto, aryl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxyl, amino, alkylamino, dialkylamino, trialkylammonium, alkylacylamino, arylcarboxylamino, hydrazyl, alkylthio, alkenyl, and reactive groups.

[0097] As used in this article, the term "working electrode" refers to an electrode that performs electro-oxidation or electro-reduction of analytes or background interferences with or without the presence of electron transfer reagents.

[0098] The term "C" used in this article 6-30 "Aryl" refers to an aromatic compound comprising a monocyclic, bicyclic, or tricyclic carbocyclic system having one, two, or three aromatic rings, such as phenyl, naphthyl, anthracene, or biphenyl. Such aromatic compounds typically contain, for example, 6 to 30 carbon atoms, 6 to 18 carbon atoms, 6 to 14 carbon atoms, or 6 to 10 carbon atoms. It should be understood that the term aryl includes a carbocyclic portion that is planar and contains 4n + 2π electrons (according to Hückel's rule), where n = 1, 2, or 3.

[0099] As used in this article, the term "halogenated" refers to a residue of a halogen (i.e., F, Cl, Br, or I).

[0100] The term "C" used in this article 1-6 "alkyl" refers to a straight-chain or branched alkyl substituent containing, for example, about 1 to about 6 carbon atoms (e.g., 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, n-hexyl, etc. This definition also applies when "alkyl" appears as part of a group, such as, for example, C 1-6 Halogenated alkyl groups (e.g., -trifluoromethyl(-CF3)).

[0101] The term "C" used in this article 2-6 "Alkenyl" indicates a linear alkenyl substituent containing, for example, 2 to 6 carbon atoms (branched alkenyl groups contain about 3 to 6 carbon atoms). According to one aspect, the alkenyl group is C... 2-4 Alkenyl. Examples of alkenyl groups include, but are not limited to, vinyl, allyl, 2-propenyl, 1-butenyl, 2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, etc.

[0102] The term "C" used in this article 2-6 "Alynyl" indicates a straight-chain alkynyl substituent containing, for example, 2 to 6 carbon atoms (branched alkynyl groups contain about 3 to 6 carbon atoms). According to one aspect, 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, etc.

[0103] The term "hydroxyl" as used in this article refers to -OH.

[0104] The term “nitro” as used in this article refers to -NO2.

[0105] The term "cyano" as used in this article refers to -CN.

[0106] The term "amino" as used in this article refers to -NH2. The terms mono- and di-C are also used. 1-6 Alkylamino means with one or two C atoms respectively. 1-6 Nitrogen groups bonded to alkyl groups, i.e., -NHR or -NRR. , where R and R Are they the same or different C? 1-6 Alkyl groups.

[0107] The term "C" used in this article 1-6 "Alkoxy" indicates a C that is bonded to oxygen.1-6 Alkyl groups, i.e., -OR, where R is C 1-6 Alkyl groups.

[0108] The term "C" used in this article 6-10 "Aryloxy group" represents an aryl group bonded to oxygen, i.e., -O(Ar), where Ar is C. 6-10 Aryl group.

[0109] As used in this article, the term "arylalkoxy" refers to the group -OR(Ar), where R is C. 1-6 alkyl group and Ar is C 6-10 Aryl group.

[0110] The term "carboxyl group" as used in this article refers to -C(O)OH.

[0111] The term "C" used in this article 1-6 "alkylcarboxyl" indicates a carboxyl group in which the hydrogen atom bonded to the carboxyl group has been converted to carbon dioxide. 1-6 Alkyl group substitution, i.e., -C(O)OR, where R is C 1-6 Alkyl groups.

[0112] As used herein, the term "acylamino" signifies the structure -C(O)NH or -NHC(O). The term "C 1-6 "alkylamide" represents -C(O)NR or -NRC(O), where R is C 1-6 alkyl.

[0113] The term "C" used in this article 1-6 "Haloalkylamide" indicates C 1-6 alkyl amide group, wherein C 1-6 The alkyl group is replaced by one, two, or three halogen groups as described herein.

[0114] As used herein, the term "heteroaryl" refers to an aromatic compound as described herein containing a 5- or 6-membered ring, wherein one or two carbons have been replaced by nitrogen, sulfur, and / or oxygen. Examples of heteroaryl groups include, but are not limited to, pyridyl, furanyl, pyrroleyl, quinolinyl, thiophenyl, indolyl, oxazolyl, isoxazolyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, 1,3,4-thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, and triazinyl.

[0115] As used herein, the term "heterocyclic alkyl" refers to a monocyclic, bicyclic, or spirocyclic system containing 3 to 7 carbon ring members and 1, 2, or 3 other atoms selected from nitrogen, sulfur, and / or oxygen. Examples of such heterocyclic alkyl rings include, but are not limited to, aziropropyl, oxopropyl, thiazolinyl, imidazoalkyl, piperazinyl, homopiperazinyl, pyrrolinyl, pyrrolalkyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyranyl, tetrahydropyranyl, piperidinyl, and morpholinyl.

[0116] The methods, sensors, and compositions disclosed herein

[0117] Before describing the analyte sensors and their components in more detail, a brief overview of suitable in vivo analyte sensor configurations and sensor systems employing analyte sensors will be provided to facilitate a better understanding of the various aspects of this disclosure. Figure 1A simplified diagram of an exemplary sensing system for an analyte sensor that may include the contents of this disclosure is shown. As shown, the sensing system 100 includes a sensor control device 102 and a readout device 120, which are configured to communicate with each other via a local communication path or link 140, which may be wired or wireless, one-way or two-way, encrypted or unencrypted. Depending on some aspects, the readout device 120 may serve as an output medium for viewing analyte concentrations and alarms or notifications determined by the sensor 104 or its associated processor, and for allowing input from one or more users. The readout device 120 may be a multi-functional smartphone or a dedicated e-reader. Although only one readout device 120 is shown, multiple readout devices 120 may be present in some cases. The readout device 120 may also communicate with a remote terminal 170 and / or a trusted computer system 180 via communication paths / links 141 and / or 142, which may also be wired or wireless, one-way or two-way, encrypted or unencrypted. The readout device 120 may also, or alternatively, communicate with the network 150 (e.g., a mobile phone network, the Internet, or a cloud server) via communication path / link 151. The network 150 may further communicate with the remote terminal 170 via communication path / link 152, and / or with the trusted computer system 180 via communication path / link 153. Alternatively, the sensor 104 may communicate directly with the remote terminal 170 and / or the trusted computer system 180 without the presence of an intermediate readout device 120. For example, but not limited to, according to certain aspects, the sensor 104 may communicate with the remote terminal 170 and / or the trusted computer system 180 via a direct communication link with the network 150, as described in U.S. Patent Application Publication 2011 / 0213225, which is incorporated herein by reference in its entirety. For each communication path or link, any suitable electronic communication scheme may be used, such as Near Field Communication (NFC), Radio Frequency Identification (RFID), Bluetooth® or Bluetooth Low Energy® schemes, WiFi, etc. According to some aspects, individuals interested in the user's analytics level, in addition to the primary user, may also access remote terminal 170 and / or trusted computer system 180. Readout device 120 may include display 122 and optional input components 121. According to some aspects, display 122 may include a touchscreen interface.

[0118] The sensor control device 102 includes a sensor housing 103 that houses circuitry and a power supply for operating the sensor 104. Optionally, the power supply and / or active circuitry may be omitted. A processor (not shown) may be communicatively connected to the sensor 104, physically located within the sensor housing 103 or the readout device 120. According to some aspects, the sensor 104 protrudes from the bottom of the sensor housing 103 and passes through an adhesive layer 105 adapted to bond the sensor housing 103 to a tissue surface, such as skin.

[0119] Sensor 104 is adapted to be at least partially inserted into target tissue, such as the dermis or subcutaneous layer of skin. Sensor 104 may include a sensor tail long enough to be inserted to a desired depth in a 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., a ketone). 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.

[0120] The sensing layer can be configured to detect a specific analyte (e.g., ketone). For example, but not by limitation, disclosed analyte sensors include at least one sensing layer configured to detect an analyte (e.g., ketone).

[0121] In some aspects of this disclosure, analytes (e.g., ketones) can be monitored in any biofluid of interest, such as dermal fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, etc. In certain specific aspects, the analyte sensor of this disclosure can be used to measure dermal fluid or interstitial fluid to determine the concentration of one or more analytes in vivo. In some aspects, the biofluid is interstitial fluid.

[0122] Still referencing Figure 1Sensor 104 can automatically forward data to readout device 120. For example, but not limited to, it can automatically and periodically transmit analyte concentration data (i.e., glucose concentration), such as at a certain frequency or after a certain period of time when data is acquired, and store the data in memory until transmission (e.g., every minute, every five minutes, or other predetermined time intervals). In some other aspects, sensor 104 can communicate with readout device 120 in a non-automatic manner, rather than according to a set schedule. For example, but not limited to, RFID technology can be used to transmit data from sensor 104 when the sensor electronics enter the communication range of readout device 120. Data can be stored in the memory of sensor 104 until transmitted to readout device 120. Therefore, the user does not need to remain constantly near readout device 120, but can upload data at convenient times. In some other aspects, a combination of automatic and non-automatic data transmission can be implemented. For example, and not limited to, data transmission can continue automatically until readout device 120 is no longer within the communication range of sensor 104.

[0123] The introducer may be present temporarily to facilitate the introduction of sensor 104 into the tissue. In some exemplary aspects, the introducer may include a needle or similar sharp object. Those skilled in the art will readily recognize that other types of introducers (such as sheaths or blades) may appear in other ways. More specifically, the needle or other introducer may be temporarily proximate to sensor 104 before tissue insertion and then withdrawn after tissue insertion. When present, the needle or other introducer may facilitate the insertion of sensor 104 into the tissue by opening a channel for sensor 104 to enter the tissue. For example, and not in a limiting manner, according to one or more aspects, the needle may facilitate penetration of the epidermis as a channel into the dermis, thereby allowing implantation of sensor 104. After opening the access channel, the needle or other introducer may be withdrawn to avoid the risk of sharps injury. In some aspects, suitable needles may be solid or hollow, beveled or non-beveled, and / or have a circular or non-circular cross-section. In more specific aspects, suitable needles may be comparable to acupuncture needles in cross-sectional diameter and / or needle tip design, such that acupuncture needles may have a cross-sectional diameter of about 250 micrometers. However, if certain applications require it, the appropriate needle can have a larger or smaller cross-sectional diameter.

[0124] In some respects, the tip of the needle (when present) may be tilted above the end of the sensor 104, such that the needle first penetrates the tissue and opens an access channel for the sensor 104. In other respects, the sensor 104 may be located within the lumen or groove of the needle, with the needle again opening an access channel for the sensor 104. In either case, the needle is subsequently withdrawn after facilitating sensor insertion.

[0125] The characteristic is that the sensor configuration of a single sensing layer used to detect a single analyte can employ a two-electrode or three-electrode detection mode, as referenced in this article. Figures 2A-2C Further description. See below for reference. Figures 3A-5C The description is characterized by a sensor configuration with two distinct sensing layers that detect different analytes, located on different or the same working electrode. Sensor configurations with multiple working electrodes are particularly advantageous for integrating two distinct sensing layers within the same sensor tail, as the signal contribution of each sensing layer can be more easily determined.

[0126] When a single working electrode is present in 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, wherein the second electrode may simultaneously serve as both a counter electrode and a reference electrode (i.e., counter / reference electrode). The electrodes may at least partially stack (layer) and / or be laterally spaced from each other at the sensor's tail. Suitable sensor configurations may be substantially flat, substantially cylindrical, or any other suitable shape. In any sensor configuration disclosed herein, the electrodes may be electrically isolated from each other by a dielectric material or similar insulator.

[0127] An analyte sensor characterized by multiple working electrodes can similarly include at least one additional electrode. When one additional electrode is present, it can serve as a counter / reference electrode for each of the multiple working electrodes. When two additional electrodes are present, one can serve as a counter electrode for each of the multiple working electrodes, and the other can serve as a reference electrode for each of the multiple working electrodes.

[0128] Figure 2A A simplified diagram of an exemplary dual-electrode analyte sensor configuration is shown, which is compatible with the uses disclosed herein. As shown, the analyte sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter electrode / reference electrode 216. Alternatively, the working electrode 214 and the counter electrode / reference electrode 216 may be located on the same side of the substrate 212, with a dielectric material (configuration not shown) sandwiched between them. A sensing layer 218 is configured as at least one layer on at least a portion of the working electrode 214. The sensing layer 218 may include multiple points or a single configuration point configured for detecting an analyte (e.g., a ketone), as discussed further herein.

[0129] Still referencing Figure 2AMembrane 220 at least covers sensing layer 218. In some aspects, membrane 220 may also cover part or all of the working electrode 214 and / or counter electrode / reference electrode 216, or the entire analyte sensor 200. One or both surfaces of analyte sensor 200 may be covered by membrane 220. Membrane 220 may comprise one or more polymer membrane materials capable of limiting the analyte flux to sensing layer 218 (i.e., membrane 220 is a mass transfer-limiting membrane with some permeability to the target analyte). In some aspects and as further described below, membrane 220 is not cross-linked. Analyte sensor 200 can determine analytes (e.g., ketones) by any of coulometric, amperometric, voltammetric, or potentiochemical detection techniques.

[0130] Figure 2B and 2C A simplified diagram of an exemplary three-electrode analyte sensor configuration is shown, which is also compatible with the uses disclosed herein. The three-electrode analyte sensor configuration can be used with... Figure 2A The configuration shown for analyte sensor 200 is similar, but in analyte sensors 201 and 202 ( Figure 2B and 2C The electrode 216 includes an additional electrode 217. With the addition of the additional electrode 217, the counter electrode / reference electrode 216 can function as either a counter electrode or a reference electrode, and the additional electrode 217 can perform other previously unconsidered electrode functions. The working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed on either the working electrode 214 or the electrode 216, with a dielectric material insulating layer between them. For example, but not limited to, such as... Figure 2B As shown, dielectric layers 219a, 219b, and 219c space electrodes 214, 216, and 217 apart from each other and provide electrical isolation. Alternatively, as Figure 2C As shown, at least one of electrodes 214, 216, and 217 can be located on opposite surfaces of substrate 212. Therefore, in some respects, electrode 214 (working electrode) and electrode 216 (counter electrode) can be located on opposite surfaces of substrate 212, while electrode 217 (reference electrode) is located on one of electrodes 214 or 216 and separated from them by a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) can be located on electrode 217, and the location of the reference material layer 230 is not limited to... Figure 2B and 2C The location shown. (And) Figure 2A Similar to the sensor 200 shown, the sensing layer 218 in analyte sensors 201 and 202 may include multiple points or a single point. Furthermore, analyte sensors 201 and 202 can determine analytes using any of the following methods: coulometric, amperometric, voltammetric, or potentiochemical detection techniques.

[0131] Similar to analyte sensor 200, membrane 220 can also cover sensing layer 218 and other sensor components in analyte sensors 201 and 202, thereby acting as a mass transfer confinement membrane. In some respects, additional electrode 217 can be covered by membrane 220. Although Figure 2B and 2C Electrodes 214, 216, and 217 are depicted as being covered by membrane 220; however, it should be recognized that, in some respects, only the working electrode 214 is covered. Furthermore, the thickness of membrane 220 at each of electrodes 214, 216, and 217 can be the same or different. (Comparison with dual-electrode analyte sensor configuration) Figure 2A Similarly, in Figure 2B and 2C In the sensor configuration, one or both surfaces of the analyte sensors 201 and 202 can be covered by the membrane 220, or the entire analyte sensors 201 and 202 can be covered by the membrane 220. Therefore, Figure 2B and 2C The three-electrode sensor configuration shown should be understood as non-limiting to the aspects disclosed herein, and alternative electrode and / or layer configurations remain within the scope of this disclosure.

[0132] Figure 3A An exemplary configuration of sensor 203 is shown, which has a single working electrode on which two different sensing layers are disposed. Figure 3A and Figure 2A Similarly, the difference lies in the presence of two sensing layers on the working electrode 214: a first sensing layer 218a and a second sensing layer 218b, which respond to different analytes and are laterally spaced apart from each other on the surface of the working electrode 214. Sensing layers 218a and 218b may comprise multiple points or a single point configured to detect each analyte. The composition of the membrane 220 at sensing layers 218a and 218b may be different or the same. The first sensing layer 218a and the second sensing layer 218b may be configured to detect their respective analytes at different working electrode potentials, as discussed further below.

[0133] Figure 3B and 3C Cross-sectional views of exemplary three-electrode sensor configurations of sensors 204 and 205 are shown, each sensor characterized by a single working electrode on which a first sensing layer 218a and a second sensing layer 218b are disposed. Figure 3B and 3C and Figure 2B and 2C They are basically similar and can be used as a reference. Figure 2B and 2C To understand better. With Figure 3ASimilarly, the composition of membrane 220 at sensing layers 218a and 218b may be different or the same.

[0134] refer to Figures 4-5C An exemplary sensor configuration with multiple working electrodes (particularly two working electrodes) is described in more detail. While the following description is primarily directed to a sensor configuration with two working electrodes, it should be understood that more than two working electrodes can be incorporated by expanding the disclosure herein. In addition to the first and second analytes, additional working electrodes can be used to impart additional sensing capabilities to the analyte sensor, for example, for detecting a third and / or fourth analyte.

[0135] Figure 4 A cross-sectional view of an exemplary analyte sensor configuration is shown, having two working electrodes, a reference electrode, and a counter electrode, compatible with the uses disclosed herein. As shown, the analyte sensor 300 includes working electrodes 304 and 306 disposed on opposite surfaces 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. A counter electrode 320 is electrically isolated from the working electrode 304 by a dielectric layer 322, and a reference electrode 321 is electrically isolated from the working electrode 306 by a dielectric layer 323. Outer dielectric layers 330 and 332 are located on the reference electrode 321 and the counter electrode 320, respectively. Depending on various aspects, a membrane 340 may at least cover sensing layers 310a and 310b, and other components of the analyte sensor 300 or the entire analyte sensor 300 may optionally be covered by the membrane 340.

[0136] Similar to analyte sensors 200, 201, and 202, analyte sensor 300 can determine analytes (e.g., ketones) using any of the following methods: coulometric, amperometric, voltammetric, or potentiochemical detection techniques.

[0137] It has multiple working electrodes and is Figure 4 Alternative sensor configurations with different configurations shown may be characterized by a counter electrode / reference electrode instead of separate counter and reference electrodes 320, 321, and / or by different layer and / or film arrangements than those explicitly shown in the figures. For example, but not limited to, the positions of the counter electrode 320 and reference electrode 321 may be different from those shown in the figures. Figure 4 The positions shown are reversed. Furthermore, working electrodes 304 and 306 do not necessarily need to be... Figure 4 The arrangement shown is located on the opposite surface of substrate 302.

[0138] While a suitable sensor configuration may be characterized by substantially planar electrodes, it should be understood that sensor configurations characterized by non-planar electrodes can be advantageous and are particularly suitable for use in the disclosure herein. Specifically, 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 A perspective view of an analyte sensor is shown, characterized by two working electrodes arranged concentrically relative to each other. It should be understood that a sensor configuration with a concentric electrode arrangement but lacking a second working electrode is also possible in this disclosure.

[0139] Figure 5A A perspective view of an exemplary sensor configuration is shown, in which a plurality of electrodes are substantially cylindrical and concentrically arranged relative to each other around a central substrate. As shown, the analyte sensor 400 includes a central substrate 402, and all electrodes and dielectric layers are concentrically arranged relative to each other around the central substrate 402. Specifically, a working electrode 410 is disposed on a surface of the central substrate 402, and a dielectric layer 412 is disposed on a portion of the working electrode 410 remote from the sensor tip 404. A working electrode 420 is disposed on the dielectric layer 412, and a dielectric layer 422 is disposed on a portion of the working electrode 420 remote from the sensor tip 404. A counter electrode 430 is disposed on the dielectric layer 422, and a dielectric layer 432 is disposed on a portion of the counter electrode 430 remote from the sensor tip 404. A reference electrode 440 is disposed on the dielectric layer 432, and a dielectric layer 442 is disposed on a portion of the reference electrode 440 remote from the sensor tip 404. Therefore, the exposed surfaces of the working electrode 410, working electrode 420, counter electrode 430 and reference electrode 440 are spaced apart from each other along the longitudinal axis B of the analyte sensor 400.

[0140] Still referencing Figure 5A A first sensing layer 414a and a second sensing layer 414b, responsive to different or the same analytes, are respectively disposed on the exposed surfaces of the working electrodes 410 and 420, thereby allowing contact with the fluid for sensing. Although in Figure 5A The sensing layers 414a and 414b are depicted as three discrete points, but it should be understood that in alternative sensor configurations there may be fewer or more than three points, including continuous layers of sensing layers.

[0141] exist Figure 5A In the sensor 400, a film 450 is partially coated on the working electrodes 410 and 420, and sensing layers 414a and 414b are disposed thereon. Figure 5B An alternative sensor configuration is shown, in which sensor 401 is almost entirely covered by membrane 450. Membrane 450 may be identical or different in composition at sensing layers 414a and 414b.

[0142] It should also be understood that, Figure 5A and 5B The positions of the various electrodes may differ from those explicitly shown in the figure. For example, the positions of the counter electrode 430 and the reference electrode 440 may differ from those shown in the figure. Figure 5A and 5B The configurations shown are interchangeable. Similarly, the positions of the working electrodes 410 and 420 are not limited to those shown. Figure 5A and 5B The location is clearly shown in the text. Figure 5C Showing with Figure 5B The different sensor configurations shown include sensor 405 containing a counter electrode 430 and a reference electrode 440 closer to the sensor tip 404, and working electrodes 410 and 420 further away from the sensor tip 404. The sensor configuration where the working electrodes 410 and 420 are located further away from the sensor tip 404 can become advantageous as it provides a larger surface area for depositing sensing layers 414a and 414b (in...). Figure 5C Five discrete sensing points are illustrated in the example, thereby improving signal strength in some cases. Similarly, in any concentric sensor configuration disclosed herein, the central substrate 402 may be omitted, whereby the innermost electrode may instead support subsequently deposited layers.

[0143] The sensor is further described below in several parts.

[0144] This disclosure relates to a ketone sensor and a method for detecting ketones with high sensitivity, enabling the measurement of low concentrations of ketones. Surprisingly, it was found that sensitivity can be improved by reducing the applied potential, subtracting background interference, and by accumulating charge.

[0145] We do not wish to be bound by theory, as cumulative detection involves the alternating connection and disconnection of sensing electrodes. In some respects, it might be possible to use a single channel to switch between background and ketone sensing chemistry (e.g., switching between WE1 (standard chemistry) and WE2 (blank chemistry) approximately every 2.5 minutes). Therefore, the redox medium can act as a "data storage medium" during charge accumulation, simplifying the design of the electronics, as only one channel is needed for background storage instead of two. This setup can introduce a time shift between background (blank) measurements and ketone (standard) measurements, which complicates background subtraction, but this shift can be small enough to prevent significant variations in analyte and interfering concentrations between measurements. Alternatively, a single channel can be connected to the first working electrode (WE1) for a short period of time (e.g., a few seconds, such as about 15 seconds), connected to the second working electrode (WE2) for a short period of time (e.g., a few seconds, such as about 15 seconds), and then disconnected from both channels for a longer period of time (e.g., a few minutes, such as about 2 minutes) to provide a peak current similar to that in the previous embodiment, 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).

[0146] In some aspects of ketone sensing methods, both the ketone sensing electrode and the background sensing electrode are connected to a circuit and a potential is applied. The time period during which the two sensing electrodes are connected to the circuit can be any suitable time period that allows for the detection of ketones and / or background signals. Typically, the time period during which the two sensing electrodes are connected (e.g., a set connection time period) can be about 1 second or longer (e.g., about 2 seconds or longer, about 3 seconds or longer, about 4 seconds or longer, about 5 seconds or longer, about 10 seconds or longer, about 20 seconds or longer, about 30 seconds or longer, about 40 seconds or longer, about 50 seconds or longer, about 1 minute or longer, about 2 minutes or longer, about 3 minutes or longer, about 4 minutes or longer, about 5 minutes or longer, about 6 minutes or longer, about 7 minutes or longer, about 8 minutes or longer). The connection time is from about 10 minutes or longer to about 30 minutes or less (e.g., about 25 minutes or less, about 20 minutes or less, about 15 minutes or less, about 10 minutes or less, about 8 minutes or less, about 7 minutes or less, about 6 minutes or less, about 5 minutes or less, about 4 minutes or less, about 3 minutes or less, about 2 minutes or less, about 1 minute or less, about 50 seconds or less, about 40 seconds or less, about 30 seconds or less, about 20 seconds or less, about 10 seconds or less, or about 5 seconds or less). In some aspects, the connection time is from about 1 second to about 2 minutes or from about 5 seconds to about 1 minute. The two electrodes are then disconnected from the circuit to allow charge to accumulate. After sufficient charge has accumulated, the two electrodes can be reconnected to the circuit to measure the ketone signal (i.e., the measurement of the ketone signal plus the background signal) and the background signal (i.e., the measurement of the background signal only). The ketone concentration can be correlated with the measured ketone signal minus the measured background signal.

[0147] In one aspect, this disclosure relates to a method for sensing ketones, the method comprising:

[0148] The biofluid containing ketones is brought into contact with the following electrodes: a first sensing electrode containing a ketone-responsive enzyme and a redox medium, and a second sensing electrode containing a redox medium but not a ketone-responsive enzyme.

[0149] The first and second sensing electrodes are connected to the circuit and a potential of less than +40 mV is applied to the two electrodes to provide a steady state.

[0150] Disconnect the first and second sensing electrodes from the circuit;

[0151] Accumulate the charge generated by the biofluid reacting with the first and second sensing electrodes for a set time period (e.g., a set accumulation time period);

[0152] After a set time period, the first and second sensing electrodes are connected (e.g., reconnected) to the circuit; and

[0153] The ketone signal is measured by subtracting the signal from the second sensing electrode from the signal obtained from the first sensing electrode.

[0154] In some aspects of the ketone sensing method, the connection step and the disconnection / accumulation step are performed alternately between the ketone sensing electrode and the background sensing electrode. Specifically, this disclosure relates to a method for sensing ketones, the method comprising:

[0155] (a) Contacting the first and second sensing electrodes with a biological fluid containing ketones, wherein the first sensing electrode contains a ketone-responsive enzyme and a redox medium and the second sensing electrode contains a redox medium and does not contain a ketone-responsive enzyme.

[0156] (b) Connect the first sensing electrode to the circuit and apply a potential of less than +40 mV to provide a steady state;

[0157] (c) Disconnect the first sensing electrode from the circuit and connect the second sensing electrode to the circuit and apply a potential of less than +40 mV to provide a steady state;

[0158] (d) Accumulate the charge generated by the biofluid reacting with the first sensing electrode for a first set time period;

[0159] (e) After the first set time period, connect (e.g., reconnect) the first sensing electrode to the circuit and disconnect the second sensing electrode from the circuit;

[0160] (f) Accumulate the charge generated by the biofluid reacting with the second sensing electrode for a second set time period;

[0161] (g) After the second predetermined time period, connect (e.g., reconnect) the second sensing electrode to the circuit; and

[0162] (h) The ketone signal is measured by subtracting the signal obtained from the second sensing electrode from the signal obtained from the first sensing electrode.

[0163] In some aspects of ketone sensing methods, both the ketone sensing electrode and the background sensing electrode are in contact with a biological fluid containing ketones. The duration of contact between the two sensing electrodes and the fluid can be any suitable time period that allows for the detection of ketones and / or background signals. Typically, the duration of contact between the two sensing electrodes and the biological fluid (e.g., a contact-defined time period) can be about 1 second or longer (e.g., about 2 seconds or longer, about 3 seconds or longer, about 4 seconds or longer, about 5 seconds or longer, about 10 seconds or longer, about 20 seconds or longer, about 30 seconds or longer, about 40 seconds or longer, about 50 seconds or longer, about 1 minute or longer, about 2 minutes or longer, about 3 minutes or longer, about 4 minutes or longer, about 5 minutes or longer, about 6 minutes or longer, about 7 minutes or longer, about 8 minutes). (or longer or about 10 minutes or longer) to about 30 minutes or less (e.g., about 25 minutes or less, about 20 minutes or less, about 15 minutes or less, about 10 minutes or less, about 8 minutes or less, about 7 minutes or less, about 6 minutes or less, about 5 minutes or less, about 4 minutes or less, about 3 minutes or less, about 2 minutes or less, about 1 minute or less, about 50 seconds or less, about 40 seconds or less, about 30 seconds or less, about 20 seconds or less, about 10 seconds or less, or about 5 seconds or less). In some aspects, the contact time period is from about 1 second to about 2 minutes or from about 5 seconds to about 1 minute.

[0164] In some aspects, the measurement step may include determining the concentration of a ketone in a fluid (e.g., a biological fluid), which can be correlated by subtracting a signal from a second sensing electrode (a second signal) from a signal from a first sensing electrode (a first signal). In some aspects, the ketone concentration (mM) can be determined by the following formula:

[0165] (Ketone signal (nA) - Blank signal (nA)) / Ketone response (nA / mM).

[0166] A known concentration of ketone can be added to a control sample to determine the ketone response value (nA / mM) of the sensor.

[0167] The disconnection and connection in steps (b) and (c) can be simultaneous or sequential. In some aspects of this alternation method, the disconnection and connection in steps (b) and (c) are simultaneous.

[0168] The accumulated time period (which includes the first and second set time periods) can be any suitable time period that allows sufficient charge to accumulate after the circuit is reconnected to detect ketones and / or background. Typically, the set time period 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 set time period can 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 all ketones present at the ketone sensing electrode to react completely, interferences at the background sensing electrode to react, or both. For example, the set time period can be about 30 seconds or more and about 30 minutes or less, about 1 to about 20 minutes, about 1 to about 15 minutes, about 1 to about 10 minutes, about 1 to about 8 minutes, about 1 to about 5 minutes, about 2 to about 8 minutes, about 2 to about 5 minutes, or about 30 seconds or more. The first set time period can be the same as or different from the second set time period. In some respects, the first set time period can be the same as the second set time period. In other respects, the first set time period can be different from the second set time period. In any of these aspects, the set time period, the first set time period, the second set time period, or any combination thereof may be 30 seconds or longer.

[0169] Surprisingly, it was found that lowering the sensing potential reduced the interaction between the sensing electrode and background interference (e.g., electroactive interference), which in turn reduced the interference signal. In some respects, the potential applied to the ketone sensor can be less than +40 mV to about -250 mV, including less than +40 mV to about -225 mV, less than +40 mV to about -200 mV, less than +40 mV to about -175 mV, less than +40 mV to about -150 mV, less than +40 mV to about -125 mV, about +30 mV to about -250 mV, about +30 mV to about -225 mV, about +30 mV to about -200 mV, about +30 mV to about -175 mV, about +30 mV to about -150 mV, about +30 mV to about -125 mV, about +20 mV to about -250 mV, about +20 mV to about -225 mV, about +20 mV to about -200 mV, about +20 mV to about -175 mV, about +20 mV to about -20 ... The ranges of approximately 10 mV to -150 mV, approximately +20 mV to -125 mV, approximately +10 mV to -250 mV, approximately +10 mV to -225 mV, approximately +10 mV to -200 mV, approximately +10 mV to -175 mV, approximately +10 mV to -150 mV, approximately +10 mV to -125 mV, approximately +5 mV to -250 mV, approximately +5 mV to -225 mV, approximately +5 mV to -200 mV, approximately +5 mV to -175 mV, approximately +5 mV to -150 mV, and approximately +5 mV to -125 mV are each relative to the Ag / AgCl reference electrode. In some respects, the applied potential may be about +5 mV to about -125 mV, about -5 mV to about -100 mV, about -10 mV to about -90 mV, or about -20 mV to about -80 mV, each relative to the Ag / AgCl reference electrode.In some respects, the applied potential may be approximately +35 mV, approximately +30 mV, approximately +25 mV, approximately +20 mV, approximately +15 mV, approximately +10 mV, approximately +5 mV, approximately -5 mV, approximately -10 mV, approximately -15 mV, approximately -20 mV, approximately -25 mV, approximately -30 mV, approximately -40 mV, approximately -50 mV, approximately -60 mV, approximately -70 mV, approximately -80 mV, approximately -90 mV, approximately -100 mV, approximately -110 mV, approximately -120 mV, approximately -130 mV, approximately -140 mV, approximately -150 mV, approximately -160 mV, approximately -170 mV, approximately -180 mV, approximately -190 mV, approximately -200 mV, approximately -210 mV, approximately -220 mV, etc. mV, approximately -230 mV, approximately -240 mV, or approximately -250 mV, each relative to the Ag / AgCl reference electrode. In certain specific aspects, the applied potential may be approximately -80 mV, relative to Ag / AgCl.

[0170] In any of these aspects, the sensing electrode may comprise a working electrode and a ketone sensing layer or 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) can 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 an 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 metal compounds of these elements. In some aspects, the working electrode (e.g., the working electrode in the first sensing electrode, the working electrode in the second sensing electrode) may comprise carbon.

[0171] In the first sensing electrode, a ketone-sensing layer disposed on at least a portion of the working electrode senses ketones and includes a ketone-responsive enzyme and a redox mediator. In some aspects, the ketone to be sensed can be an endogenous ketone. For example, the sensed ketone can be acetone, acetoacetic acid, acetoacetate, β-hydroxybutyrate (β-HBA), or any combination thereof. β-hydroxybutyrate (β-HBA) is strictly a carboxylic acid rather than a ketone, but is commonly referred to in the industry as a blood ketone. In some aspects, the sensed ketone can be acetoacetate, β-HBA, β-hydroxybutyrate (β-HB), or any combination thereof. In some aspects, the sensed ketone can be β-hydroxybutyrate (β-HB).

[0172] The ketone-sensing layer contains a ketone-responsive enzyme that acts as a catalyst for electron transfer. In some aspects, the ketone-responsive enzyme may be 3-hydroxybutyrate dehydrogenase (3-HBDH), glucose dehydrogenase, alcohol dehydrogenase, or a combination thereof.

[0173] In some aspects, the first sensing electrode may contain an NAD(P)H oxidoreductase, such as a flavotransferase. If necessary, one or more cofactors may be added to the ketone-responsive enzyme or the NAD(P)H oxidoreductase. Suitable cofactors include, for example, oxidized nicotinamide adenine dinucleotide (NAD) or reduced nicotinamide adenine dinucleotide (NADH), or derivatives thereof. Not wishing to be theoretically constrained, it is believed that ketone-responsive enzymes (such as 3-HBDH) do not readily transfer electrons directly to the redox medium. Therefore, an NAD(P)H oxidoreductase (e.g., a flavotransferase) and optionally a cofactor (e.g., NAD) may be added to transfer electrons from the ketone-responsive enzyme to the redox medium. In any of these aspects, the first sensing electrode may contain a ketone-responsive enzyme (e.g., 3-HBDH), an NAD(P)H oxidoreductase, and nicotinamide adenine dinucleotide phosphate (NAD(P)+) or a derivative thereof.

[0174] In some aspects, the ketone sensing layer may further comprise albumin, which can act as an enzyme stabilizer. In one aspect, the albumin may be serum albumin, such as bovine serum albumin (BSA) or human serum albumin (HSA). In some aspects, the sensing layer may comprise human serum albumin.

[0175] In some aspects, the sensing layer may include an albumin stabilizer to an enzyme (e.g., 3-HBDH) ratio of about 40:1 to about 1:40, such as about 35:1 to about 1:35, about 30:1 to about 1:30, about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1. In some aspects, the sensing layer may include an albumin stabilizer to enzyme ratio of about 1:1 to about 1:10, such as about 1:1 to about 1:9, about 1:1 to about 1:8, about 1:1 to about 1:7, about 1:1 to about 1:6, about 1:1 to about 1:5, about 1:2 to about 1:9, about 1:3 to about 1:8, about 1:3 to about 1:7, or about 1:4 to about 1:6.

[0176] In one specific embodiment, β-hydroxybutyrate dehydrogenase can convert β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD+) into acetoacetate and reduced nicotinamide adenine dinucleotide (NADH), respectively. The enzyme cofactors NAD and NADH can help facilitate the synergistic enzymatic reaction disclosed herein. NADH can then be reduced under the mediation of a flavotransferase, and the electrons transferred in this process provide the basis for ketone detection at the working electrode. The obtained electrochemical signal can then be correlated with the amount of ketone initially present in the sample at the time of measurement. Thus, a 1:1 molar correspondence exists between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted, providing a basis for the detection and quantification of ketones based on the current measured at the working electrode. As described herein, the transfer of electrons from the reduction of NADH to the working electrode can occur using electron transfer reagents such as osmium (Os) compounds. Albumin can optionally be present as a stabilizer for the enzyme.

[0177] Ketone-responsive enzymes may be present in any suitable amount, including from about 1% to about 50% by weight relative to redox media (e.g., from about 1% to about 40% by weight, from about 1% to about 30% by weight, from about 1% to about 20% by weight, from about 1% to about 15% by weight, from about 1% to about 10% by weight, or from about 1% to about 5% by weight).

[0178] NAD(P)H oxidoreductase (e.g., flavoxin) may be present in the ketone sensing 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).

[0179] In any respect, the ketone sensing layer may contain a pH buffer. The buffer may be any suitable composition that is water-soluble and controls (i.e., maintains) the pH of the sensing 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 respects, the pH may be controlled within a range of about 6 to about 8. For example, the buffer may contain phosphates (e.g., sodium dihydrogen phosphate and disodium hydrogen phosphate), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 2-( N -morpholino)ethanesulfonic acid (MES), 3-( N2-Morpholino)propanesulfonic acid (MOPS), 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS), carbonates (e.g., carbonic acid and carbonates, such as sodium carbonate; sodium carbonate and sodium bicarbonate) or citrates (e.g., citric acid 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, such as sodium chloride, potassium chloride, and magnesium chloride). In one aspect, the buffer may be phosphate-buffered saline (PBS) containing disodium hydrogen phosphate, sodium chloride, and optionally potassium chloride and potassium dihydrogen phosphate. In another aspect, the buffer may be MES or phosphate buffer, which may contain phosphate, sodium chloride, potassium chloride, and / or magnesium chloride.

[0180] In some respects, the buffer solution may typically be an aqueous buffer solution. In other respects, a non-aqueous solvent, such as an alcohol (e.g., ethanol), may be present. In some respects, the buffer solution may contain water as the sole solvent. In other respects, the buffer solution may contain water in any suitable ratio and at least one (e.g., 1, 2, or 3) non-aqueous solvent, such as a non-aqueous solvent to water volume ratio of 99.9:0.1 to 0.1:99.9. In some aspects, the volume ratio of the non-aqueous solvent to water can 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 one specific embodiment, ethanol (EtOH) and water in a volume ratio ranging from 50:50 to 90:10 EtOH:H2O (e.g., 70:30, about 75:25, about 80:20, about 85:15, or about 90:10, etc.) can be used.

[0181] In several aspects, the second sensing electrode is a background sensing electrode comprising a working electrode and a background sensing layer located on a portion of the working electrode. As described herein, the background sensing layer senses background interference and contains a redox mediator but not a ketone-responsive enzyme. The redox materials in the first and second sensing electrodes may be the same or different. In one aspect, the redox mediators in the first and second sensing electrodes are the same material. In one aspect, the background sensing layer does not contain 3-HBDH. Optional components, such as NAD(P)H oxidoreductase (e.g., flavoprotein), one or more cofactors, albumin (e.g., human serum albumin (HSA)), and pH buffers (each as described herein), may be present in the background sensing layer. In some aspects, the background sensing layer may contain albumin (e.g., HSA), NAD(P)H oxidoreductase (e.g., flavoprotein), and oxidized nicotinamide adenine dinucleotide (NAD) or reduced nicotinamide adenine dinucleotide (NADH) or derivatives thereof. In some respects, the background sensing layer may contain human serum albumin, flavoprotein, and oxidized nicotinamide adenine dinucleotide (NAD).

[0182] In some aspects, the background sensing layer contains a ketone-responsive enzyme (e.g., the only ketone-responsive enzyme), but does not contain any other sensing components such as albumin (e.g., HSA), NAD(P)H oxidoreductase (e.g., xanthocarboxylase), oxidized nicotinamide adenine dinucleotide (NAD) or reduced nicotinamide adenine dinucleotide (NADH) or derivatives thereof, or redox mediators. In some aspects, the background sensing layer is composed of a ketone-responsive enzyme.

[0183] In some aspects, the background sensing layer does not contain albumin (e.g., HSA). In some aspects, the background sensing layer does not contain flavoprotein transfection enzymes. In some aspects, the background sensing layer does not contain added NAD or NADH. In some aspects, the background sensing layer contains environmental NAD and NADH, but contains no added NAD and / or NADH at all or substantially. In some aspects, the background sensing layer does not contain redox mediators.

[0184] In some aspects, the background sensing layer does not contain ketone-responsive enzymes or albumins (e.g., HSA). In some aspects, the background sensing layer does not contain ketone-responsive enzymes or flavoprotein transducers. In some aspects, the background sensing layer does not contain ketone-responsive enzymes or added NAD or NADH. In some aspects, the background sensing layer does not contain ketone-responsive enzymes or redox mediators.

[0185] In some aspects, the background sensing layer does not contain ketone-responsive enzymes, albumins (e.g., HSA), or flavoprotein transducers. In some aspects, the background sensing layer does not contain ketone-responsive enzymes, flavoprotein transducers, or added NAD. In some aspects, the background sensing layer does not contain ketone-responsive enzymes, albumins (e.g., HSA), or flavoprotein transducers. In some aspects, the background sensing layer does not contain ketone-responsive enzymes, flavoprotein transducers, or added NAD.

[0186] The ketone sensing layer and / or the background sensing layer may be disposed continuously or discontinuously on at least a portion of the respective working electrode. A discontinuous application means that the sensing layer may be formed in a discrete shape on the working electrode, such as dots, lines, or multiple (e.g., arrays) dots and / or lines. The number of dots or lines is not particularly limited, but may range from 2 to approximately 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, including approximately 3 to approximately 8 or approximately 4 to approximately 6). In any aspect herein, the ketone sensing layer, the background sensing layer, or both may be continuous on the respective working electrode. In other aspects, the ketone sensing layer, the background sensing layer, or both may be discontinuous on the respective working electrode.

[0187] The total size of one or more creatine sensing layers (e.g., the combined area of ​​all points, layers, or active regions) can be at least about 0.05 mm. 2 But it can be up to about 100 mm 2 In some respects, the overall size can be approximately 0.05 mm. 2 approximately 100 mm 2 Approximately 0.05 mm 2 Approximately 75 mm 2 Approximately 0.05 mm 2 Approximately 50 mm 2 Approximately 0.05 mm 2 Approximately 40 mm 2 Approximately 0.05 mm 2 Approximately 30 mm 2 Approximately 0.05 mm 2 approximately 25 mm 2 Approximately 0.05 mm 2 approximately 15 mm 2 Approximately 0.05 mm 2 approximately 10mm 2 Approximately 0.05 mm 2 Approximately 5 mm 2 Approximately 0.05 mm 2 approximately 1 mm 2 or approximately 0.05 mm 2 To approximately 0.1 mm 2In one particular aspect, the overall size of the one or more sensing layers can be from about 0.05 to about 0.1 mm. 2 Approximately 0.05 to approximately 100 mm 2 Approximately 0.1 to approximately 50 mm 2 Approximately 0.5 to approximately 30 mm 2 Approximately 1 to approximately 20 mm 2 or about 1 to about 15 mm 2 Within the range.

[0188] Each or multiple sensing layers (including a ketone sensing layer, a background sensing layer, or both) typically have a thickness in the range of about 0.1-10 μm. For example, each sensing layer may be 0.1 μm thick or greater (e.g., 0.2 μm or greater, 0.3 μm or greater, 0.5 μm or greater, 0.8 μm or greater, 1 μm or greater, 2 μm or greater, 3 μm or greater, 5 μm or greater, or 8 μm or greater) and typically have 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 embodiment, 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.

[0189] In some aspects, conductive materials (such as, for example, carbon nanotubes, graphene, or metal nanoparticles) can be incorporated into one or more sensing layers (including a ketone sensing layer, a background sensing layer, or both) to facilitate the rapid attainment of steady-state current. The conductive material may be included in the range of about 0.1% to about 50% (pbw) by weight in each sensing layer (e.g., about 1 to about 50 pbw, about 1 to about 10 pbw, or about 0.1 to about 10 pbw).

[0190] The first sensing electrode (e.g., a ketone sensing layer) and the second sensing electrode (e.g., a background sensing layer) contain a redox medium. Each redox medium may be the same or different. In some respects, the redox medium of the first and second sensing electrodes may be the same.

[0191] In one aspect, the redox medium may comprise a polymer and an electron transfer reagent.

[0192] In some respects, the polymer in the redox medium can be any suitable polymer capable of transferring electrons between the electron transfer reagent and the working electrode. For example, the polymer can 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 a carboxypentyl group, or poly(sodium 4-styrene sulfonate). These polymers can be considered precursor polymers because they are further modified to immobilize (e.g., link) the electron transfer complex. In some aspects, the polymer may comprise 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 aspects, the polymer may comprise repeating polymer or copolymer units, which may contain at least one (e.g., 1, 2, 3, 4, 5, or 6) side-chain pyridyl groups, imidazole groups, or both pyridyl and imidazole groups. For example, suitable polymers may comprise partially or completely quaternized poly(4-vinylpyridine) and poly(1-vinylimidazole), wherein the quaternized pyridyl and imidazole groups can be used to form spacers by reacting with an electron transfer agent (e.g., complexation).

[0193] In some aspects, the electron transfer agent in the redox medium may comprise a transition metal complex. The transition metal in the transition metal complex can be any suitable transition metal that can be efficiently reduced and oxidized in the methods described herein. For example, the transition metal complex may comprise osmium, ruthenium, iron, cobalt, vanadium, or combinations thereof. In some aspects, the transition metal may be ruthenium or osmium, particularly osmium. According to some aspects, suitable electron transfer agents may comprise low-potential osmium complexes, such as those described in U.S. Patent Nos. 6,134,461, 6,605,200, 6,736,957, 7,501,053, and 7,754,093, the disclosure of each of which is incorporated herein by reference in its entirety. Other suitable examples of electron transfer media combined with polymers may include those described in U.S. Patent Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosure of which is incorporated herein by reference in its entirety.

[0194] The transition metal complex may further comprise at least one ligand, which may be monodentate or polydentate (e.g., bidentate, tridentate, tetradentate). Typically, the complex will comprise enough ligands to provide a complete coordination sphere. In some aspects, at least one ligand (e.g., 1, 2, 3, 4, 5, or 6) may comprise a nitrogen-containing heterocycle.

[0195] Monodentate ligands include, for example, -F, -Cl, -Br, -I, -CN, -SCN, -OH, NH3, alkylamines, dialkylamines, trialkylamines, alkoxy groups, heterocyclic compounds, compounds containing such groups, solvent molecules (e.g., H2O, EtOH), or reactive groups. For example, alkyl groups of the ligand (e.g., C...) 1-12 C 1-6 C 1-4 C 1-3 The aryl (e.g., phenyl, benzyl, naphthyl) moiety may optionally be substituted with F, Cl, Br, I, alkylamino, dialkylamino, trialkylammonium (except at the aryl moiety), alkoxy, alkylthio, or aryl. Examples of suitable heterocyclic monodentate ligands include imidazole, pyrazole, oxazole, thiazole, pyridine, and pyrazine, 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).

[0196] Suitable examples of bidentate ligands include, for example, 1,10-phenanthroline, amino acids, oxalic acid, acetylacetone, diaminoalkanes, o-diaminoaromatics, and 2,2-diaminoalkanes. - Bimidazole, 2,2 - Bisoxazole, 2,2 -Bithiazole, 2-(2-pyridyl)imidazol and 2,2 -Bipyridines, each of which can 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). Bidetent ligands particularly suitable for electron transfer complexes include substituted and unsubstituted 2,2-dimethylpyridines. -Bimidazole, 2-(2-pyridyl)imidazole and 2,2 -Bipyridine. Examples of suitable tripentate ligands include, for example, diethylenetriamine, 2,2-diphenyltriamine, etc. ,2 - Tripyridine, 2,6-bis( N -pyrazolyl)pyridines, each of which may be substituted or unsubstituted (e.g., substituted by one or more alkyl groups (such as methyl) or one or more reactive groups).

[0197] A suitable 2,2 - Bimidazole ligands can be ligands according to formula (I):

[0198]

[0199] (I).

[0200] In equation (I), R 1 and R 2 They may be the same or different, and each may be a substituted or unsubstituted alkyl, alkenyl, or aryl group. Typically, R... 1 and R 2 The same or different and each of them is an unsubstituted C 1-12 Alkyl (e.g., C10) 1-4 Alkyl group). In some respects, R 1 and R 2 They are all methyl groups.

[0201] In equation (I), R 3 R 4 R 5 and R 6 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, alkylacylamino, arylcarboxylamino, hydrazyl, alkylhydrazyl, hydroxyamino, alkoxyamino, alkylthio, alkyl, alkenyl, or aryl. Alternatively, R 3 and R 4 Combined land or R 5 and R 6 They can be combined independently to form saturated or unsaturated 5- or 6-membered rings (e.g., benzo[a] rings). Typically, the alkyl and alkoxy moieties are C12-24-32-36 ... 1-12 The alkyl or aryl moiety of any substituent may optionally be substituted by one or more substituents (e.g., 1, 2, 3, 4, 5, or 6), such as F, Cl, Br, I, amino, alkylamino, dialkylamino, trialkylammonium (except on the aryl moiety), alkoxy, alkylthio, aryl, or reactive groups (e.g., CO₂H). Typically, R 3 R 4 R 5 and R 6 The same or different and each is H or unsubstituted C 1-12 Alkyl (e.g., C10) 1-4 Alkyl group). In some respects, R 3 R 4 R 5 and R 6 Both are H.

[0202] A suitable 2-(2-pyridyl)imidazolium ligand can be a ligand according to formula (II):

[0203]

[0204] (II).

[0205] In equation (II), R 1 It can be a substituted or unsubstituted alkyl, alkenyl, or aryl group. Typically, R... 1 It is the unreplaced C 1-12 Alkyl (e.g., C10) 1-4 Alkyl groups or C groups optionally substituted with reactive groups 1-12 Alkyl group. In some respects, R 1 It is a methyl group.

[0206] In equation (II), R 3 R 4 R a R b R c and R d 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, alkylacylamino, arylcarboxylamino, hydrazyl, alkylhydrazyl, hydroxyamino, alkoxyamino, alkylthio, alkyl, alkenyl, or aryl. Alternatively, R 3 and R 4 Combined land or R a R b R c and R d Two adjacent substituents (e.g., R) a and R b R b and R c , or R c and R d These can be combined to independently form saturated or unsaturated 5- or 6-membered rings (e.g., benzo[a] ring). Typically, the alkyl and alkoxy moieties are C12-24-24-36 ... 1-12 The alkyl or aryl moiety of any substituent may optionally be substituted by one or more (e.g., 1, 2, 3, 4, 5, or 6) substituents such as F, Cl, Br, I, amino, alkylamino, dialkylamino, trialkylammonium (except on the aryl moiety), alkoxy, alkylthio, aryl, or reactive groups (e.g., CO₂H). Typically, R 3 R 4 R a Rb R c and R d The same or different and each is H or unsubstituted C 1-12 Alkyl (e.g., C10) 1-4 Alkyl group). In some respects, R 3 R 4 R a R b R c and R d Both are H.

[0207] A suitable 2,2 - The bipyridine ligand can be a ligand according to formula (III):

[0208]

[0209] (III).

[0210] In equation (III), R 16 R 17 R 18 R 19 R 20 R 21 R 22 and R 23 The groups may be 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, alkylacylamino, arylcarboxylamino, hydrazyl, alkylhydrazyl, hydroxyamino, alkoxyamino, alkylthio, alkyl, alkenyl, or aryl. Typically, the alkyl and alkoxy moieties are C10 and C20, respectively. 1-12 The alkyl or aryl moiety of any substituent may optionally be replaced by one or more substituents (e.g., 1, 2, 3, 4, 5, or 6), such as F, Cl, Br, I, amino, alkylamino, dialkylamino, trialkylammonium (except on the aryl moiety), alkoxy, alkylthio, aryl, or reactive groups (e.g., CO2H).

[0211] Specific examples of suitable combinations include R 16 and R 23 All are H or all are methyl and / or R 17 and R 23 All are H or all are methyl and / or R 18 and R 21 All are H or all are methyl and / or R 19 and R 20All are H or all are methyl. An alternative combination is one in which 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 They can be combined to form saturated or unsaturated 5- or 6-membered rings (e.g., benzo[a] ring).

[0212] In one aspect, the one or more ligands are 4,4 -Dimethyl-2,2- β-Bipyridine, mono-, di-, or poly-alkoxy-2,2- -Bipyridine (e.g., 4,4) -dimethoxy-2,2 1,7-Bipyridine), 4,7-dimethyl-1,10-phenanthroline, mono-, di-, or poly-alkoxy-1,10-phenanthroline (e.g., 4,7-dimethoxy-1,10-phenanthroline), or any combination thereof.

[0213] In some aspects, the transition metal complex may include counter ions (X) to balance the charge of the transition metal. Typically, there may be 1 to 5 (i.e., 1, 2, 3, 4, or 5) counter ions. The multiple counter ions in the complex are not necessarily all the same. Examples of suitable counter ions include anions such as halide ions (e.g., fluoride, chloride, bromide, or iodide ions), sulfate, phosphate, hexafluorophosphate, and tetrafluoroborate, and cations (e.g., monovalent cations) such as lithium, sodium, potassium, tetraalkylammonium, and ammonium. In some aspects, the counter ion is a halide ion, such as chloride.

[0214] In one aspect, the transition metal complex may be an osmium transition metal complex, which may contain one or more ligands, wherein at least one (e.g., 1, 2, 3, 4, 5, or 6) of the ligands may contain a nitrogen-containing heterocycle (e.g., imidazole, pyrazole, oxazole, thiazole, pyridine, and pyrazine). In other aspects, the osmium transition metal complex may contain one or more ligands selected from 4,4 -Dimethyl-2,2- β-Bipyridine, mono-, di-, or poly-alkoxy-2,2- -Bipyridine (e.g., 4,4) -dimethoxy-2,2 -Bipyridine), 4,7-dimethyl-1,10-phenanthroline, mono-, di-, or poly-alkoxy-1,10-phenanthroline (e.g., 4,7-dimethoxy-1,10-phenanthroline).

[0215] In one aspect, the redox medium may comprise an osmium complex bonded to a polymer or copolymer of poly(1-vinylimidazole) or poly(4-vinylpyridine). The poly(4-vinylpyridine)-based polymer is a prepolymer, as shown in the following structure, which has been modified to link the osmium complex (e.g., a poly(biimidizyl)osmium complex):

[0216] ,

[0217] Where n can be 2, n It can be 17, and n It can be 1. Other reactive groups and / or spacer groups can be used.

[0218] In one aspect, the electronic redox medium may comprise an osmium-containing poly(4-vinylpyridine)-based polymer, as shown below.

[0219]

[0220] Where n is 2, n It is 17, and n It is 1.

[0221] In some respects, the electron transfer agent can be attached (e.g., non-leaching and / or covalently bonded) to a polymer in a redox medium. For example, covalent bonding of the electron transfer agent to the polymer can be achieved by polymerizing monomer units with covalently bonded electron transfer agents, or the electron transfer agent can be reacted with the polymer separately after the polymer has been synthesized.

[0222] According to certain aspects, bifunctional spacers can be used to link (e.g., covalently bond) an electron transfer reagent to a polymer in a redox medium, wherein 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 reagent (e.g., a functional group capable of reacting with a ligand of a coordinating metal ion). Typically, a covalent bond is formed between the two reactive groups to create the linkage. Suitable reactive groups include, for example, activated esters (e.g., succinimide, benzotriazolyl, or aryl groups substituted with one or more electron-withdrawing groups (such as sulfonyl, nitro, cyano, or halogen), acryloylamino, acyl azide, acyl halide, carboxyl (-COO- or -CO2H), aldehyde, ketone, alkyl halide, alkyl sulfonic acid, acid anhydride, aziridine, epoxy, triazine halide, imide ester, isocyanate, isothiocyanate, maleimide, sulfonyl halide, amino, thiol (-SH), hydroxyl, pyridyl, imidazole, and hydroxyamino. The reaction between two reactive groups can form a covalent bond between the transition metal complex and the polymer, which is a carboxylamino, thioether, hydrazone, oxime, alkylamino, ester, carboxylic acid ester, imidazolium, pyridinium, ether, thioether, aminotriazinyl, triazinyl ether, amidine, urea, urethanyl, thiourea, thioether, sulfonamide, or any combination thereof. In addition to the reactive groups, the bifunctional spacer may further contain an alkylene group (i.e., -(CH2)). n -) and / or ethoxy groups (i.e., -(CH2CH2O) m - where n and m are each an independent integer from 1 to 12 (e.g., 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).

[0223] In some aspects, the redox medium may further comprise a crosslinking agent. Generally, the crosslinking agent is any suitable multifunctional (e.g., bifunctional) short-chain molecule capable of attaching (e.g., covalently bonding) an electron transfer agent to the polymer of the redox medium. For example, the crosslinking agent may comprise polyepoxides (e.g., polyethylene glycol diglycidyl ether (PEGDGE), ethylene glycol diglycidyl ether (EGDGE), resorcinol diglycidyl ether, 1,2,7,8-diepoxyoctane, Gly3), cyanuric chloride, etc. N -Hydroxysuccinimide, imide ester, epichlorohydrin, or combinations thereof. In one aspect, the crosslinking agent is polyethylene glycol diglycidyl ether (PEGDGE) of the following formula:

[0224] ,

[0225] Where n is an integer from 1 to 50 (e.g., 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, about 5 to 50, about 5 to 45, about 5 to 40, about 5 to 35, or about 5 to 30).

[0226] In one specific embodiment, PEGDGE can be PEGDGE200, PEGDGE400 (n is 10), PEGDGE500, PEGDGE600, PEGDGE1000, or PEGDGE2000, where the numbers represent the average molecular weight (M). n In one aspect, the crosslinking agent may be PEGDGE400.

[0227] The redox medium can be applied to the working electrode using any suitable technique, such as spraying, painting, inkjet printing, stencil printing, roll coating, dip coating, or any combination thereof. In some aspects, the redox medium can be applied by immersing at least a portion of the working electrode in a redox medium solution. It can be applied in one or multiple applications (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 applications). In some aspects, the redox medium can be applied in 1, 2, 3, or 4 applications (e.g., coating). In some aspects, the redox medium can be applied in 1 or 2 applications (e.g., coating).

[0228] In some aspects, one or more enzymes in the ketone sensing layer or background sensing layer can be linked (e.g., covalently linked or non-leachingly bound) to a polymeric portion of the redox medium in the first sensing electrode. In some aspects, one or more enzymes can be covalently bonded to the polymeric portion of the redox medium. The covalent bonding of one or more enzymes to the redox medium (e.g., polymer) can occur via a crosslinking agent (as described herein) and the reaction sites on the enzyme. Thus, in such a case, the enzyme can be electronically “linked” to the working electrode via the redox medium. In one aspect, a hydrogel can be formed after crosslinking the enzyme and its wires on the electrode. In another aspect, at least a portion of the enzyme can diffuse into the polymer and / or hydrogel and attach (but not necessarily covalently bonded) to the polymer.

[0229] In one aspect, the first sensing electrode, the second sensing electrode, or both may comprise a membrane covering at least the ketone sensing layer and / or the background sensing layer, and optional other components. The covering layer forms an outer membrane that provides stability to the sensing reagent (e.g., ketone-responsive enzymes, redox mediators), limits mass transfer, improves biocompatibility, and / or prevents electrode contamination. The membrane may optionally cover all or part of the working electrode and optionally cover any counter electrode or reference electrode that may be present. In one aspect, the membrane covers (e.g., encapsulates) the entire sensing system (e.g., the sensor tail), including the first and second sensing electrodes and their respective sensing layers, and any counter electrode, reference electrode, and / or substrate that may be present.

[0230] The membrane may comprise one or more polymer membrane materials whose physical structure allows for analyte flux to the sensing layer (i.e., the membrane is a mass-contained membrane). The composition of the membrane may be varied (e.g., degree of hydrophobicity and / or degree of crosslinking) to facilitate the desired flux of ketones to the sensing electrode, thereby providing the desired signal strength and stability, as further described below. In one aspect, the membrane may be permeated with at least one analyte ketone.

[0231] A film can be coated on at least the ketone sensing layer, at least the background sensing layer, or both using any suitable technique. Typically, the film can be coated by spraying, painting, inkjet printing, roller coating, dip coating, or any combination thereof. The coating steps can be performed once or multiple times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times), which will affect the thickness of the film coating. In one aspect, the coating steps can be performed twice to form a bilayer.

[0232] Generally, if multiple coatings are applied, subsequent coatings must be applied only after the first coating has dried. The amount of time between coating steps varies depending on the type of film, the working electrode and sensing layer, and atmospheric conditions. Generally, drying time can be 1 minute or longer (e.g., 2 min or longer, 3 min or longer, 5 min or longer, 10 min or longer, 15 min or longer, or 20 min or longer). Once the film coating has been applied, it can be cured. In one aspect, the coating can be cured for 12 hours or longer (e.g., 18 hours or longer, 24 hours or longer, 30 hours or longer, 36 hours or longer, 42 hours or longer, or 48 hours or longer). Curing can be carried out at room temperature (i.e., about 20°C) or at slightly elevated temperatures (e.g., 100°C or lower, 80°C or lower, 70°C or lower, 60°C or lower, 50°C or lower, 40°C or lower, 30°C or lower, or 25°C or lower). Generally, the curing will not occur at temperatures below approximately 20°C.

[0233] The membrane typically has a thickness ranging from about 1 μm to about 100 μm. For example, in some aspects, the membrane may have a thickness of about 1 μm or greater (e.g., about 5 μm or greater, about 10 μm or greater, about 15 μm or greater, about 20 μm or greater, about 25 μm or greater, about 30 μm or greater, about 35 μm or greater, about 40 μm or greater, about 50 μm or greater, about 60 μm or greater, about 70 μm or greater, about 80 μm or greater, or about 90 μm or greater) 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 less, about 50 μm or less, 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). The film may have a thickness of about 5 to about 85 μm, about 10 to about 65 μm, about 20 to about 50 μm, about 20 to about 40 μm, about 25 to about 35 μm, or about 30 μm.

[0234] In one aspect, the membrane may comprise optionally crosslinked poly(4-vinylpyridine), poly(vinyl alcohol), poly(acrylic acid), poly(methacrylic acid), a membrane based on a tetrafluoroethylene-perfluoro-3,6-dioxa-4-methyl-7-octenesulfonic acid copolymer (e.g., NAFION™ membrane), polyurethane, or combinations thereof. In some aspects, the mass transfer confinement membrane may comprise at least a poly(4-vinylpyridine) homopolymer or copolymer (e.g., poly(4-vinylpyridine)-co-polystyrene sulfonate (PVP-co-PSS)), wherein the poly(4-vinylpyridine) may optionally be crosslinked.

[0235] Suitable poly(4-vinylpyridine) copolymers contained in mass transfer limiting membranes may contain up to about 25% comonomer (based on the total amount of monomers in the copolymer), such as about 0.1% to about 5% comonomer, or about 5% to about 15% comonomer, or about 15% to about 25% comonomer, or about 1% to about 10% comonomer. There are no particular limitations on suitable comonomers, provided that the mass transfer limiting membrane provides sufficient ketone permeability such that the analyte sensitivity is about 1 nA / mM or greater when exposed to ketones. In some aspects, the mass transfer limiting membrane may contain optionally crosslinked poly(4-vinylpyridine)-co-polystyrene sulfonate (PVP-co-PSS).

[0236] In some aspects, the membrane may comprise multiple layers, each with a different composition and / or degree of crosslinking. In one embodiment, the membrane coating may be a bilayer membrane comprising a first layer and a second layer, the first layer comprising a poly(4-vinylpyridine) homopolymer or copolymer, and the second layer comprising a crosslinked poly(4-vinylpyridine) homopolymer or copolymer (e.g., crosslinked with PEGDGE). In some aspects, the membrane may comprise a bilayer consisting of: (i) a poly(4-vinylpyridine) homopolymer crosslinked with a crosslinking agent; and (ii) optionally a copolymer of poly(4-vinylpyridine) crosslinked with a crosslinking agent, as described herein (e.g., high molecular weight (such as 400 g / mol) poly(ethylene glycol) diglycidyl ether). In some aspects, the second layer may comprise a crosslinked polyvinylpyridine-co-styrene polymer. In some respects, the second layer may comprise a polyvinylpyridine-co-styrene polymer, wherein a portion of the pyridine nitrogen atoms are functionalized with a non-crosslinked poly(ethylene glycol) tail and a portion of the pyridine nitrogen atoms are functionalized with an alkyl sulfonic acid group.

[0237] The film can be coated on at least the ketone sensing layer and / or the background sensing layer using any suitable technique. In some aspects, the film can be coated by spraying, painting, inkjet printing, roll coating, dip coating, or any combination thereof. In one aspect, the coating includes immersing a ketone sensor (e.g., the sensor tail) comprising the ketone sensing layer and the background sensing layer into a solution comprising a polymer and a solvent to provide an immersed ketone sensor. The coating step can be performed once or multiple times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times), which will affect the thickness of the film coating. In one aspect, the coating step can be performed twice to form a bilayer. In one aspect, the coating step can be dip coating. In one aspect, the coating step can be dip coating performed 2 to 6 times (i.e., 2, 3, 4, 5, or 6 times).

[0238] The membrane typically has a thickness ranging from about 1 μm to about 100 μm. For example, in some aspects, the membrane may have a thickness of about 1 μm or greater (e.g., about 5 μm or greater, about 10 μm or greater, about 15 μm or greater, about 20 μm or greater, about 25 μm or greater, about 30 μm or greater, about 35 μm or greater, about 40 μm or greater, about 50 μm or greater, about 60 μm or greater, about 70 μm or greater, about 80 μm or greater, or about 90 μm or greater), and may typically have 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 less, about 50 μm or less, 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). The film may have a thickness of about 5 to about 80 μm, about 10 to about 60 μm, about 20 to about 50 μm, about 20 to about 40 μm, about 25 to about 35 μm, or about 30 μm.

[0239] In some aspects, the first and second electrodes are components of a sensor (e.g., a ketone sensor), which may be contained within a sensor housing designed for adhesion to tissue (e.g., skin). If necessary, the sensor housing may include an adhesive layer capable of adhering to the desired tissue. The sensor housing may house all necessary components of the sensor, such as circuitry and the power supply required for sensor operation. In some aspects, the power supply (e.g., a coin cell battery) and / or active circuitry are not contained within the sensor housing. A processor may be communicatively coupled to the sensor, wherein the processor is physically located within the sensor housing or readout device. The power supply may include one or more batteries, which may be rechargeable or disposable. Power management circuitry may regulate battery charging and power monitoring, boost power, or perform direct current (DC) conversion.

[0240] In some aspects, the sensor may include a sensor tail (e.g., an insertion tip) for penetrating (e.g., implanting) tissue. The sensor tail may include at least a first sensing electrode and a second sensing electrode. A counter electrode may be present in combination with one or two working electrodes. The respective electrodes may be at least partially stacked (layered) and / or laterally spaced apart on the sensor tail. Generally, the sensor tail may have sufficient size and shape to be positioned below the tissue surface (e.g., penetrating the skin (dermis)) and into the subcutaneous space, and to contact the wearer's biofluids (such as interstitial fluid). Suitable sensor configurations may be substantially flat, substantially cylindrical, or any other suitable shape. In one embodiment, the sensor tail may be about 5 mm in length, about 0.6 mm in width, and about 0.25 mm in thickness. Suitable tissues include, for example, skin, including the dermis, interstitial layer, and / or subcutaneous layer of the skin. In any sensor configuration disclosed herein, the respective electrodes may be electrically isolated from each other by a dielectric material or similar insulator.

[0241] In some aspects, the sensor may include a reference electrode, a counter electrode, or both a reference electrode and a counter electrode as part of a first sensing electrode, a second sensing electrode, or both. In one aspect, the counter electrode may be carbon (e.g., screen-printed carbon), and the reference electrode may be Ag / AgCl. In some aspects, a working electrode and a second electrode that simultaneously serves as both a counter electrode and a reference electrode (i.e., a counter electrode / reference electrode) may be used as part of a first sensing electrode, a second sensing electrode, or both.

[0242] In one embodiment, electrode contacts may be located on a first portion of the sensor, which lies above the skin surface and extends into the tail of the sensor. A first working electrode, a reference electrode, and a counter electrode may be located in a second portion of the sensor, and a second working electrode, a reference electrode, and a counter electrode may be located in a third portion of the sensor, wherein the second and third portions may typically be located at the bottom of the tail of the sensor. The first working electrode may comprise a ketone sensing layer, and the second working electrode may comprise a background sensing layer, each as described herein.

[0243] In some aspects, the sensor may include at least one insulating layer (e.g., a dielectric layer) as part of a first sensing electrode, a second sensing electrode, or both. In some aspects, the insulating layer may be made of a suitable dielectric material, which may be solid. In one embodiment, the insulating layer may be formed of ceramic, mica, glass, barium strontium titanate, plastic (e.g., polystyrene, polytetrafluoroethylene, polyethylene terephthalate, polyethylene, polypropylene, polymethyl methacrylate, polysulfone, polydimethylsiloxane, polyvinyl chloride, or combinations thereof) or metal oxide (e.g., silicon dioxide, alumina, titanium dioxide, zirconium oxide, tantalum oxide, etc.).

[0244] In some aspects, the sensor may include a substrate, on which the first and second sensing electrodes may be disposed. The substrate may be formed of any suitable inert material. In some aspects, 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 aspects, the substrate may be disposed 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.

[0245] The sensor may be part of a system that includes a first sensing electrode (e.g., a ketone sensing electrode), a second sensing electrode (e.g., a background sensing electrode), and circuitry configured to connect and disconnect from the first and second sensing electrodes. In one aspect, the system may be a ketone sensor comprising: a first sensing electrode including a first working electrode, a ketone sensing layer, and a redox medium; a second sensing electrode serving as a background sensing layer, including a second working electrode, a redox medium, and free of a ketone-responsive enzyme; and a membrane comprising a PVP covering at least the ketone sensing layer and the background sensing layer. The ketone sensing layer may contain 3-HBDH, a yellow transducer, nicotinamide adenine dinucleotide phosphate (NAD(P)+) or a derivative thereof, optionally albumin, and an osmium-containing poly(4-vinylpyridine)-based polymer as the redox medium. The background sensing layer may contain a yellow transducer, nicotinamide adenine dinucleotide phosphate (NAD(P)+) or a derivative thereof, albumin, and an osmium-containing poly(4-vinylpyridine)-based polymer as the redox medium.

[0246] In the presence of a biofluid containing the analyte, a first sensing electrode (ketone sensing electrode) oxidizes the analyte, and the amount of oxidation is measured as the amount of electron charge generated by the reaction. A second sensing electrode (background sensing electrode) oxidizes background interferences (e.g., electroactive interferences) present in the biofluid, and the amount of oxidation is measured as the amount of electron charge generated by the reaction. As long as the first and second sensing electrodes are not connected to other electrodes, the charge generated by the redox reaction continues to accumulate on the respective electrodes. The accumulation of charge (electrons) over a set time period results in a low concentration of ketone and background that is easily measured and quantified compared to other known methods. After the set time period of charge accumulation, the first and / or second sensing electrodes can be connected to at least one (e.g., 1, 2, 3, or 4) other electrodes (such as counter electrodes and / or reference electrodes) to form a circuit. After the circuit is connected, the electrons accumulated on the first and / or second sensing electrodes can be released in the form of an electrical signal, the amplitude of which can be measured and correlated with the amount of ketone present at the sensing electrode. Subtracting the measured background signal (i.e., the measured value of ketone signal + background signal) from the measured ketone signal (i.e., the measured value of background signal only) provides the ketone-only signal, which is proportional to the ketone concentration.

[0247] The sensing of analyte (A) relies on an oxidoreductase (AOx) that is electrically "connected" to the working electrode of the sensor via a redox medium. In normal amperometric sensing, the electrode is set at a potential (voltage) so that the analyte reacts at a constant rate, proportional to the analyte concentration. For the analyte oxidation reaction (A to A... + Electrons will flow from the analyte (A) to the analyte-specific enzyme (AOx), and then to the redox medium (e.g., Os). 3+ The electrons eventually flow to the working electrode at a constant rate, generating a steady-state current. If the working electrode is disconnected from the circuit, the electron flow from the redox polymer to the working electrode will stop, resulting in no current flow in the circuit. However, the analyte will still undergo enzymatic oxidation, which in turn leads to the reduction of the redox medium (e.g., Os). 3+ Restore to Os 2+ This leads to the reduced form of the redox medium (e.g., Os). 2+ It accumulates over time because of the electrons (e) from the analyte. -The analyte is stored in a redox medium. When the working electrode is reconnected to the circuit and kept at its original potential (voltage), the accumulated reduced form of the redox medium is oxidized, resulting in a large current spike. As the redox system reaches steady state again, the current decays back to its original ampere current. This two-step process forms the basis of accumulation-mode sensing: in the first step, the sensor's working electrode is disconnected from the circuit for a set period of time (also known as the accumulation time), allowing the charge from the analyte to accumulate in the redox polymer; and in the second step, after the accumulation time, the sensor's working electrode is reconnected to the circuit, releasing the accumulated charge and measuring it as a spike.

[0248] In some respects, in the ketone sensor used in this paper, the potential (voltage) sufficient to drive the redox reaction and reduce background interference can be less than +40 mV (e.g., less than +30 mV, less than +20 mV, less than +10 mV, less than +5 mV, less than 0 mV, less than -5 mV, less than -10 mV, less than -20 mV, less than -30 mV, less than -40 mV, less than -50 mV, less than -60 mV, less than -70 mV, less than -80 mV, less than -90 mV, less than -100 mV, less than -110 mV, less than -120 mV, less than -130 mV, less than -140 mV, less than -150 mV, less than -160 mV, less than -170 mV, less than -180 mV, less than -190 mV, less than -200 mV, less than -210 mV, less than -220 mV, less than -230 mV). mV or less than -240 mV), relative to Ag / AgCl.

[0249] In some respects, ketone signals and background signals can be measured at different times. In other respects, ketone signals and background signals can be obtained simultaneously through the first and second channels.

[0250] Since ketones are present in relatively low concentrations in biological fluids (e.g., serum), this system is designed to detect low concentrations of analytes by allowing the analytes to accumulate on the enzyme biosensor. In the context of ketone detection, low concentrations can be about 1 mM or less (e.g., about 900 μM or less, about 800 μM or less, about 700 μM or less, about 600 μM or less, about 500 μM or less, about 400 μM or less, about 300 μM or less, about 200 μM or less, or about 100 μM or less) to about 10 μM or greater (e.g., about 20 μM or greater, about 30 μM or greater, about 40 μM or greater, about 50 μM or greater, about 60 μM or greater, about 70 μM or greater, about 80 μM or greater, about 90 μM or greater, about 100 μM or greater, about 120 μM or greater, about 140 μM or greater, or about 150 μM or greater). For example, the concentration of ketone in the analyte can be about 10 μM to about 1 mM, about 50 to about 400 μM, about 60 to about 300 μM, or about 70 to about 200 μM.

[0251] In some aspects, the ketone sensor is exposed to a biological fluid in vivo. Generally, this method uses the systems disclosed herein (e.g., ketone sensors) to measure ketone concentrations and can be used in in vivo monitoring systems that, when placed in the body of a user (e.g., a patient, such as a human), come into contact with the user's biological fluid and sense the ketones contained therein. The in vivo monitoring system may include one or more readout devices that receive sensed analyte data from a sensor control device. The readout device may process and / or display the sensed analyte data or sensor data to the user in any number of formats. In some aspects, the readout device may be a mobile communication device, such as a dedicated readout device (configured to communicate with a sensor control device), optionally used in conjunction with a computer system, mobile phone (e.g., a smartphone with WiFi or Internet support), tablet computer, personal digital assistant (PDA), or mobile smart wearable electronic device (e.g., smart glass, smart glasses, watch, bracelet, or necklace). For example, U.S. Patent 11,371,957 describes how a readout device can be configured as an in vivo monitoring system, the disclosure of which is incorporated herein by reference in its entirety.

[0252] The readout device typically includes an input component, a display, and processing circuitry, which may include one or more processors, microprocessors, controllers, and / or microcontrollers, each of which may be a separate chip or distributed across multiple different chips (and as part of multiple different chips). The processing circuitry may include a communication processor with onboard memory and an application processor with onboard memory. The readout device may further include radio frequency (RF) communication circuitry (coupled to an RF antenna), memory, multifunction circuitry with one or more associated antennas, a power supply, power management circuitry, and / or a clock. It should be recognized that other hardware and functions may be included in the readout device.

[0253] In addition to the detection methods using sensors (e.g., ketone sensors) described above, this disclosure relates to a ketone sensor comprising:

[0254] A first sensing electrode includes a first working electrode and a ketone sensing layer located on a portion of the first working electrode, wherein the ketone sensing layer includes a ketone-responsive enzyme and a redox mediator; and

[0255] The second sensing electrode includes a second working electrode and a background sensing layer located on a portion of the second working electrode, wherein the background sensing layer contains a redox medium and does not contain a ketone-responsive enzyme.

[0256] The ketone-responsive enzyme is as described herein. In some aspects, the ketone-responsive enzyme may be 3-hydroxybutyrate dehydrogenase. In some aspects, the first sensing electrode may comprise NAD(P)H oxidoreductase and nicotinamide adenine dinucleotide phosphate (NAD(P)+) or a derivative thereof. In some aspects, the first sensing electrode may comprise albumin, as described herein. In some aspects, the first sensing electrode may comprise a pH buffer, as described herein.

[0257] In some respects, the ketone-responsive enzyme can be linked to a redox medium, as described herein.

[0258] In some respects, the redox medium may comprise polymers and electron transfer reagents, each as described herein.

[0259] In some aspects, the polymer may comprise poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene). In some aspects, the polymer comprises repeating polymer or copolymer units, said repeating unit comprising at least one side-chain pyridyl group, imidazole group, or both pyridyl and imidazole groups. In some aspects, the polymer may be crosslinked with a crosslinking agent, as described herein. In some aspects, the crosslinking agent may be a polyepoxide, cyanuric chloride, N-hydroxysuccinimide, imine ester, epichlorohydrin, or a combination thereof. In some aspects, the crosslinking agent may be polyethylene glycol diglycidyl ether (PEGDGE), as described herein.

[0260] In some aspects, the electron transfer reagent may comprise a transition metal complex, as described herein. In some aspects, the transition metal complex may comprise osmium, ruthenium, iron, cobalt, or combinations thereof. In some aspects, the transition metal complex may be an osmium transition metal complex comprising one or more ligands, wherein at least one ligand comprises a nitrogen-containing heterocycle. In some aspects, the redox medium may comprise an osmium complex bonded to a poly(vinylpyridine)-based polymer.

[0261] In some respects, the ketone sensing layer or the background sensing layer may be continuous on the working electrode, or both sensing layers may be continuous, as described herein. In other respects, the ketone sensing layer or the background sensing layer may be discontinuous on the working electrode, or both sensing layers may be discontinuous, as described herein.

[0262] In some aspects, the sensor may comprise a membrane, as described herein, which covers at least a ketone sensing layer, at least a background sensing layer, or both. In some aspects, the membrane comprises poly(4-vinylpyridine), which is optionally crosslinked.

[0263] In some aspects, the sensor may include a housing, as described herein. In some aspects, the sensor may include a sensor tail, as described herein, configured for implantation in tissue, wherein the first and second sensing electrodes may be disposed on the sensor tail. In some aspects, the sensor may include a reference electrode, a counter electrode, or both a reference electrode and a counter electrode, each as described herein. In some aspects, the sensor may include at least one insulating layer, as described herein. In some aspects, the sensor may include at least one substrate, as described herein, wherein the first sensing electrode or the second sensing electrode is disposed on the substrate, or both sensing electrodes are disposed on the substrate.

[0264] In some respects, the ketone sensor or its method of use may provide about 3 times or more (e.g., about 4 times or more, about 5 times or more, about 6 times or more, about 7 times or more, about 8 times or more, about 9 times or more, about 10 times or more, about 11 times or more, about 12 times or more, about 13 times or more, about 14 times or more, or about 15 times or more) sensor accuracy relative to Ag / AgCl sensing potential less than +40 mV (e.g., sensing potential at -80 mV), background subtraction, and accumulation mode sensing.

[0265] In some respects, compared to ketone sensors that do not include one, two, or all three of the following features, the ketone sensor or its method of use can provide a signal that is about 3 times or more (e.g., about 4 times or more, about 5 times or more, about 6 times or more, about 7 times or more, about 8 times or more, about 9 times or more, about 10 times or more, about 11 times or more, about 12 times or more, about 13 times or more, about 14 times or more, or about 15 times or more) more: a sensing potential of less than +40 mV relative to Ag / AgCl (e.g., a sensing potential of -80 mV), background subtraction, and accumulation mode sensing.

[0266] In some aspects, the ketone sensor can provide accurate ketone measurements (e.g., within about 20%, about 18%, about 15%, about 12%, about 10%, about 8%, about 5%, about 4%, about 3%, about 2%, or about 1% of the actual value). In some aspects, the ketone sensor can provide ketone measurements within about 10% of the actual value (e.g., relative to a control value). In any of these aspects, the ketone sensor can provide accurate ketone measurements over a period of 1 day or longer (e.g., 2 days or longer, 3 days or longer, 4 days or longer, 5 days or longer, 6 days or longer, 7 days or longer, 8 days or longer, 9 days or longer, 10 days or longer, 11 days or longer, 12 days or longer, 13 days or longer, 14 days or longer, 15 days or longer, 16 days or longer, 17 days or longer, 18 days or longer, 19 days or longer, 20 days or longer, or 21 days or longer). In some aspects, the ketone sensor can provide accurate ketone measurements over a period of 7 days or longer. In some aspects, the ketone sensor can provide accurate ketone measurements over a period of 14 days or longer. In some aspects, the ketone sensor can provide accurate ketone measurements over a period of 21 days or longer.

[0267] Example

[0268] The embodiments provided below are for illustrative purposes only, and the aspects described herein should in no way be construed as being limited to these embodiments. Rather, these aspects should be construed as encompassing any and all variations that may arise as a result of the teachings provided herein.

[0269] Example 1

[0270] To determine the relationship between the sensing potential and background interference, a ketone sensor with a first sensing electrode and a second sensing electrode was prepared according to Tables 1 and 2. A membrane solution was prepared according to Table 3.

[0271] Table 1

[0272]

[0273] HBDH: Hydroxybutyrate dehydrogenase

[0274] HSA: Human serum albumin

[0275] NAD: Nicotinamide adenine dinucleotide

[0276] Os-PVP: Osmium-containing poly(4-vinylpyridine)-based polymer

[0277] PEGDGE400: Polyethylene glycol diglycidyl ether with an average molecular weight (Mn) of approximately 400 g / mol.

[0278] MES: 2-(N-morpholino)ethanesulfonic acid

[0279] Table 2

[0280]

[0281] PVP: Poly(4-vinylpyridine) (PVP)

[0282] PVPS: Crosslinked polyvinylpyridine-co-styrene polymer

[0283] Table 3

[0284]

[0285] PVP: Poly(4-vinylpyridine) (PVP)

[0286] PEGDGE400: Polyethylene glycol diglycidyl ether with an average molecular weight (Mn) of approximately 400 g / mol.

[0287] PDMS: Poly(dimethylsiloxane)

[0288] EtOH: Ethanol

[0289] PVPS: Crosslinked polyvinylpyridine-co-styrene polymer

[0290] Gly-3: Gly-Gly-Gly, where Gly is glycine.

[0291] Using standard amperometry, the sensor was tested in single-donor serum at +40 mV or -80 mV relative to Ag / AgCl, and the concentration of ketone (D-β-hydroxybutyrate) was measured to be 190 μM. Aliquots of ketone (+100 μM, +100 μM, +300 μM, and +500 μM D-β-hydroxybutyrate) were cumulatively added to the serum every 15 minutes. After the addition of the first aliquot of 100 μM, the total ketone (D-β-hydroxybutyrate) concentration was 290 μM; after the addition of the second aliquot of 100 μM, the total ketone (D-β-hydroxybutyrate) concentration was 390 μM; after the addition of the third aliquot of 300 μM, the total ketone (D-β-hydroxybutyrate) concentration was 690 μM; and after the addition of the fourth aliquot of 500 μM, the total ketone (D-β-hydroxybutyrate) concentration was 1,190 μM. Time (hours) and current ( ) were recorded using a multichannel potentiostat at a 5-minute data acquisition (DAQ) rate. Figure 6 ).

[0292] As in Figure 6 As observed, the standard sensing chemistry showed a higher signal than the blank sensing chemistry due to the signal from ketones in the serum. However, the blank sensing chemistry showed a much lower background at -80 mV than at +40 mV, indicating that the reduced sensing potential significantly reduced the background signal. In turn, this made ketone measurements more accurate.

[0293] Figure 7 Showing Figure 6 A focused view of the aliquot of the added ketone. (As shown in...) Figure 7 As seen, 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 the background interference from the serum alone. The blank signal can be subtracted from the standard signal to determine the signal from ketones alone. However, because the ketone concentration is very low, this difference cannot be accurately determined due to the noise associated with the electronic device. This background noise is even more pronounced for wearable sensors.

[0294] Example 2

[0295] To determine the effectiveness of the cumulative mode sensing, the same sensor as in Example 1 was used. In cumulative detection mode, the blank electrode and ketone electrode were kept unconnected / open-circuited for 2.5 minutes, and then connected at -80 mV for 2.5 minutes. Every 30 minutes, aliquots of ketone (+100 μM, +100 μM, +300 μM, and +500 μM D-β-hydroxybutyrate, respectively) were cumulatively added to serum, thus continuously repeating this cycle. After the addition of the first aliquot of 100 μM, the total ketone (D-β-hydroxybutyrate) concentration was 290 μM; after the addition of the second aliquot of 100 μM, the total ketone (D-β-hydroxybutyrate) concentration was 390 μM; after the addition of the third aliquot of 300 μM, the total ketone (D-β-hydroxybutyrate) concentration was 690 μM; and after the addition of the fourth aliquot of 500 μM, the total ketone (D-β-hydroxybutyrate) concentration was 1,190 μM.

[0296] Figure 8A The relationship between current (nA) and time (hours) is shown. Figure 8B The integrated charge of each peak is displayed. (As shown in...) Figure 8A As observed, the peak height is >5 nA, which is significantly higher than the 0.1 to 0.3 nA observed in previous experiments using the standard amperometric method. Figure 6 This indicates that, by providing an acceptable signal-to-noise ratio, cumulative detection can improve the mM ketone signal to a level similar to that of other analytes, such as glucose sensors.

[0297] Based on these measurements, the ketone concentrations were measured, as shown in Table 4.

[0298] Table 4

[0299]

[0300] As seen in Table 4, the predicted ketones without background subtraction significantly overestimated serum ketone concentrations (by approximately 232%), while the values ​​using background were significantly more accurate (by approximately 16%).

[0301] Example 3

[0302] According to Tables 5 and 6, two ketone sensors (“Sensor 1” and “Sensor 2”) were prepared, each with a first sensing electrode and a second sensing electrode, to test whether reduced sensing potential, background subtraction, and accumulation patterns would enhance the monitoring of low concentrations of ketones in vivo. As shown in Tables 5 and 6, the sensing chemical was deposited on the sensing electrode using a non-contact piezoelectric dispensing system in a pattern of 5 drops wide and 10 drops high (first coating). Each drop was approximately 0.6 nL, and the drops were connected together to form a connected “groove” pattern. The sensing chemical was dried and then redispension in a 5 x 10 pattern (second coating).

[0303] The membrane solution was prepared according to Table 7. A membrane was deposited on the working electrode by dipping the electrode five times in the prepared membrane solution at a speed of 5 mm / s. The electrode was then baked at 56°C for 3 days to form the ketone sensor.

[0304] Table 5

[0305]

[0306] Table 6

[0307]

[0308] Table 7

[0309]

[0310] Sensors 1 and 2 were placed on both arms of the subject, and ketone concentrations were continuously monitored over a 16-hour period starting at 4:00 PM. Subjects consumed incremental increments (2 mL, 4 mL, 8 mL, 16 mL, and 32 mL) of a ketone beverage (KETONE-IQ) hourly. Sensor 1 measured ketone concentrations using a cumulative detection method (the blank and ketone electrodes were left unconnected / open for 2.5 minutes, and then connected at -80 mV for 2.5 minutes). Sensor 2 measured ketone concentrations at -80 mV (relative to Ag / AgCl) using a standard amperometric method.

[0311] As in Figures 9A-9B As observed, cumulative detection (i.e., sensor 1) enhances the signal by approximately 10 times and reduces noise compared to the standard amperometric method (sensor 2). The signal-to-noise ratio of sensor 1 is also significantly higher than that of sensor 2. For example, in... Figure 10 As observed, background subtraction stabilizes the accumulated detection signal, especially in noisy regions. The reduced sensing potential (-80 mV) also reduces background interference, as can be seen from the relatively low signal in the background channel.

[0312] Example 4

[0313] Measurements using blood ketone test strips confirmed the consistency and sensitivity of ketone readings for ≤1 mM ketone concentrations. Sensors 1 and 2 were placed on the subject's opposite arms, and ketone concentrations were continuously monitored. Sensors 1 and 2 were used to measure ketone concentrations using a cumulative detection method (the blank electrode and ketone electrode were left unconnected / open for 2.5 minutes, and then connected at -80 mV for 2.5 minutes).

[0314] Vitamin C (2g) was administered to subjects at 8:30 AM, followed by a ketone beverage (100mL) at 10:00 AM. Ketone concentrations were continuously monitored throughout the day using sensors 1 and 2. Reference measurements were also performed using blood ketone test strips. Figures 11A-11B As shown, sensors 1 and 2 exhibit high signal and low noise at ≤1 mM ketone concentrations. In sensors 1 and 2, the background signal at -80 mV is less significant than that at +40 mV, but vitamin C is indeed detected in the background channel (as a background interfering agent). Plotting the signals of sensors 1 and 2 against blood ketone test strip measurements after background subtraction confirms that sensors 1 and 2 provide consistent and sensitive ketone readings at ≤1 mM ketone concentrations. Figure 12 ).

[0315] It should be understood that the detailed description section, rather than the summary and abstract section, is intended to interpret the claims. The summary and abstract section may set forth one or more (but not all) exemplary aspects of the invention as conceived by the inventors, and is therefore not intended to limit the invention and the appended claims in any way.

[0316] The present disclosure has been described above using functional structural units to illustrate the implementation of specific functions and their relationships. For ease of description, the boundaries of these functional structural units have been arbitrarily defined. Alternative boundaries can be defined as long as the specific functions and their relationships are properly executed.

[0317] The foregoing description of specific aspects will fully reveal the overall nature of the invention, enabling others to easily modify and / or adapt these specific aspects for various applications without extensive experimentation by applying knowledge within the scope of the art, without departing from the general conception of the invention. Therefore, such modifications and alterations are intended to fall within the meaning and scope of equivalents to the aspects disclosed herein, based on the teachings and guidance presented herein. It should be understood that the wording or terminology used herein is for descriptive rather than limiting purposes, and that the terminology or terminology in this specification will be interpreted by a person skilled in the art in light of the teachings and guidance.

[0318] The breadth and scope of this invention should not be limited by any of the foregoing exemplary aspects, but should be defined only by the following claims and their equivalents.

[0319] The claims in this application differ from those in the parent application or other related applications. Therefore, the applicant withdraws any abandonment of the scope of the claims made in the parent application or any prior application related to this application. Consequently, it is suggested that the examiner may need to reconsider any such prior abandonment and the relevant information cited to avoid such abandonment. Furthermore, the examiner is reminded that any abandonment made in this application should not be construed as applicable to or contrary to the parent application.

Claims

1. A method for sensing a ketone, the method comprising: contacting a biological fluid comprising a ketone with i) a first sensing electrode comprising a ketone-responsive enzyme and a redox mediator; and ii) a second sensing electrode comprising a redox mediator and not comprising a ketone-responsive enzyme; connecting the first and second sensing electrodes to an electrical circuit and applying a potential of less than +40 mV to both electrodes to provide a steady state; disconnecting the first and second sensing electrodes from the electrical circuit; accumulating a charge generated by the biological fluid reacting with the first and second sensing electrodes for a set period of time; connecting the first and second sensing electrodes to the electrical circuit after the set period of time; and measuring a ketone signal by subtracting the signal from the second sensing electrode from the signal from the first sensing electrode.

2. A method for sensing a ketone, the method comprising: (a) contacting first and second sensing electrodes with a biological fluid comprising a ketone, wherein the first sensing electrode comprises a ketone-responsive enzyme and a redox mediator and the second sensing electrode comprises a redox mediator and not comprising a ketone-responsive enzyme; (b) connecting the first sensing electrode to an electrical circuit and applying a potential of less than +40 mV to provide a steady state; (c) disconnecting the first sensing electrode from the electrical circuit and connecting the second sensing electrode to an electrical circuit and applying a potential of less than +40 mV to provide a steady state; (d) accumulating a charge generated by the biological fluid reacting with the first sensing electrode for a first set period of time; (e) connecting the first sensing electrode to the electrical circuit and disconnecting the second sensing electrode from the electrical circuit after the first set period of time; (f) accumulating a charge generated by the biological fluid reacting with the second sensing electrode for a second set period of time; (g) connecting the second sensing electrode to the electrical circuit after the second set period of time; and (h) measuring a ketone signal by subtracting the signal from the second sensing electrode from the signal from the first sensing electrode.

3. The method of claim 2, wherein the disconnecting and connecting of step (b) and step (c) are simultaneous.

4. The method of any one of claims 1-3, wherein the set period of time, the first set period of time, the second set period of time, or any combination thereof is 30 seconds or more.

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

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

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

8. The method of claim 7, wherein the ketone sensing layer or the background sensing layer is continuous, or both sensing layers are continuous. ​ 9. The method of claim 7 or 8, wherein the ketone sensing layer or the background sensing layer is discontinuous, or both sensing layers are discontinuous.

10. The method of claim 7, 8, or 9, further comprising a membrane covering at least the ketone sensing layer, at least the background sensing layer, or both.

11. The method of claim 10, wherein the membrane comprises poly(4-vinylpyridine).

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

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

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

15. The method of any one of claims 1-14, wherein the first sensing electrode further comprises albumin.

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

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

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

19. The method of claim 18, wherein the polymer comprises poly(vinylpyridine), poly(thiophene), poly(aniline), poly(pyrole), or poly(acetylene).

20. The method of claim 19, wherein the polymer comprises a polymer or copolymer repeat unit comprising at least one pendant pyridyl group, imidazolyl group, or both a pyridyl and imidazolyl group.

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

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

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

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

25. The method of claim 24, wherein the crosslinking agent is a polyepoxide, cyanuric chloride, N-hydroxysuccinimide, imido ester, epichlorohydrin, or a combination thereof.

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

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

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

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

30. The method of claim 28 or 29, wherein the sensor further comprises a reference electrode, a counter electrode, or both a reference electrode and a counter electrode.

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

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

33. A ketone sensor comprising a first sensing electrode comprising a first working electrode and a ketone sensing layer disposed on a portion of the first working electrode, wherein the ketone sensing layer comprises a ketone-responsive enzyme and a redox mediator; and a second sensing electrode comprising a second working electrode and a background sensing layer disposed on a portion of the second working electrode, wherein the background sensing layer comprises a redox mediator and does not comprise a ketone-responsive enzyme.

34. The ketone sensor of claim 33, wherein the ketone-responsive enzyme is 3-hydroxybutyric acid dehydrogenase.

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

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

37. The ketone sensor of any one of claims 33-36, wherein the first sensing electrode further comprises albumin.

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

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

40. The ketone sensor of claim 39, wherein the polymer comprises a poly(vinylpyridine), a poly(thiophene), a poly(aniline), a poly(pyrole), or a poly(acetylene).

41. The ketone sensor of claim 40, wherein the polymer comprises a polymeric or copolymeric repeat unit comprising at least one pendant pyridyl group, imidazolyl group, or both a pyridyl and imidazolyl group.

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

43. The ketone sensor of claim 42, wherein the crosslinking agent is a polyepoxide, a cyanuric chloride, an N-hydroxysuccinimide, an imidate, an epichlorohydrin, or a combination thereof.

44. The ketone sensor of claim 42 or 43, wherein the crosslinking agent is polyethylene glycol diglycidyl ether (PEG DGE).

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

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

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

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

49. The ketone sensor of any one of claims 33-48, wherein the ketone sensing layer or the background sensing layer is continuous over the working electrode, or both sensing layers are continuous.

50. The ketone sensor of any one of claims 33-48, wherein the ketone sensing layer or the background sensing layer is discontinuous over the working electrode, or both sensing layers are discontinuous.

51. The ketone sensor of any one of claims 33-50, further comprising a membrane covering at least the ketone sensing layer, at least the background sensing layer, or both.

52. The ketone sensor of claim 51, wherein the membrane comprises poly(4-vinylpyridine), which is optionally crosslinked.

53. The ketone sensor of any one of claims 33-52, further comprising a housing.

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

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

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

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