Pyruvic acid sensors and related methods

Abstract

The present disclosure relates to a sensing composition comprising a pyruvate responsive enzyme and a thiamine pyrophosphate (TPP). In particular, the sensing composition may comprise a pyruvate responsive enzyme (e.g., pyruvate oxidase), a TPP, an optional flavin adenine dinucleotide (FAD), a stabilizer (e.g., albumin), and a redox mediator. The sensing composition forms a sensing layer on a working electrode to form a sensing electrode. The sensing electrode, together with an electrical circuit, may form part of a system for sensing pyruvic acid. The present disclosure further relates to a method for sensing pyruvic acid comprising accumulating a charge derived from the reaction of pyruvic acid with the sensing composition for a set period of time and then measuring a signal from the accumulated charge.

Description

Background Art

[0001] Detecting various analytes within an individual is crucial for monitoring their health and well-being. Deviations from normal analyte levels can often indicate an underlying physiological condition, such as a metabolic condition or disease or exposure to a specific environmental factor or stimulus. For example, glucose levels can be particularly important to detect and monitor in individuals with diabetes.

[0002] Pyruvate is crucial for many aspects of eukaryotic and human metabolism. Pyruvate is the end product of glycolysis and is ultimately transported to mitochondria as the main fuel input supporting the citric acid cycle carbon flux. The proper regulation of pyruvate flux is crucial for maintaining cellular function under various circumstances. Due to the pyruvate metabolism dysfunction caused by the excessive inhibition of pyruvate dehydrogenase (PDH) by pyruvate dehydrogenase kinase (PDK), it is present in chronic progressive diseases, such as chronic obstructive pulmonary disease (COPD), obesity, diabetes and aging. For example, in patients with type 2 diabetes, increased serum and intramuscular lipids increase PDK activity, which then reduces PDH activity and the pyruvate flux entering the citric acid cycle. It is believed that restoring normal pyruvate metabolism can alleviate the main aspects of the metabolic pathology present in type 2 diabetes. In addition, healthy subjects with a high lipid, low carbohydrate diet may have impaired PDH activity by PDK upregulation, so understanding pyruvate levels may be important for monitoring patient health.

[0003] Therefore, there is a need for a biosensor for sensing pyruvate to monitor the health of patients, including those with diabetes. Brief Overview

[0004] Some previous methods for sensing pyruvate rely on amperometric detection of the catalytic conversion of pyruvate using pyruvate-responsive enzymes such as pyruvate oxidase. Such sensors allow for rough detection of elevated pyruvate concentrations. However, such pyruvate concentrations may not reflect physiological samples. Changes in pyruvate concentrations caused by changes in underlying biochemical processes may be very small and may not be easily detected using such sensors. Therefore, there is a need for improved sensors and related methods that can reliably detect low (e.g., micromolar) pyruvate concentrations.

[0005] The present disclosure surprisingly discloses that the sensitivity of pyruvate sensors can be improved by controlling the concentration of a cofactor of a pyruvate-responsive enzyme. The present disclosure also discloses electrochemical methods that surprisingly improve the sensitivity of pyruvate sensors (such as those disclosed herein) to low pyruvate concentrations (such as may be present in physiological samples).

[0006] Compared to those known in the art, the disclosed compositions, sensors and methods provide significant advantages. For example, sensitivity can be improved, which means that lower concentrations of pyruvic acid can be detected. Because the sensitivity of the response can be improved when sensing pyruvic acid, it is possible to design sensor elements that are less invasive, more sensitive, more reliable and / or more cost-effective than known sensors. Because lower concentrations of pyruvic acid can be detected, more accurate information about health and health care conditions can be obtained, and changes that may not have been detected previously can be monitored. The use of electrochemical technology provided herein can further improve the sensitivity of pyruvic acid sensing that can be achieved. In addition, such technology can allow electronic devices with improved properties (such as increased lifespan in actual use, such as when used for continuous or semi-continuous pyruvic acid monitoring, reduced complexity, reduced size and / or more cost-effective construction).

[0007] The present disclosure relates to sensing compositions for detecting pyruvic acid. In some embodiments, the sensing compositions can be used as part of a sensing electrode in a sensor. In some embodiments, the sensing compositions can produce a sensor for sensing pyruvic acid. Thus, the present disclosure also relates to sensors comprising the sensing compositions. The present disclosure also relates to sensors obtainable from (e.g., formed from) the sensing compositions. Methods of sensing pyruvic acid using the sensing compositions and / or sensors are also provided.

[0008] In one embodiment, the sensing composition comprises a pyruvate responsive enzyme and thiamine pyrophosphate (TPP). In one embodiment, the TPP is a cofactor for the pyruvate responsive enzyme. In some embodiments, the pyruvate responsive enzyme comprises pyruvate oxidase.

[0009] In some embodiments, the sensing composition further comprises one or more additional cofactors of the pyruvate-responsive enzyme. In some embodiments, the one or more additional cofactors comprise flavin adenine dinucleotide (FAD).

[0010] In some embodiments, the sensing composition further comprises a stabilizer (e.g., an enzyme stabilizer). In some embodiments, the stabilizer is or comprises a protein. In some embodiments, the stabilizer is or comprises albumin. In some embodiments, the albumin is bovine serum albumin (BSA) or human serum albumin (HSA).

[0011] In some embodiments, the pyruvate-responsive enzyme (e.g., pyruvate oxidase) and TPP are present in the composition, for example, and in certain embodiments, in a weight ratio of about 500:1 to about 1:1, e.g., about 100:1 to about 1:1, or about 50:1 to about 1:1, or about 10:1 to about 1:1.

[0012] In some embodiments, the sensing composition may further comprise a pH buffer.

[0013] In some embodiments, the redox mediator may include a polymer and an electron transfer agent. The polymer may include poly (4-vinyl pyridine), poly (1-vinyl imidazole), poly (thiophene), poly (aniline), poly (pyrrole) or poly (acetylene). In other embodiments, the polymer may include a polymer or copolymer repeat unit, and the polymer or copolymer repeat unit may include at least one (e.g., 1, 2, 3, 4, 5 or 6) pendant pyridyl, imidazole or both pyridyl and imidazole. In some embodiments, the electron transfer agent may include a transition metal complex, such as a transition metal complex that may include osmium, ruthenium, iron, cobalt, vanadium or a combination thereof. In one embodiment, the transition metal complex may be an osmium transition metal complex, which may include one or more ligands, wherein at least one (e.g., 1, 2, 3, 4, 5 or 6) ligand includes a nitrogen-containing heterocycle. In one embodiment, the redox mediator may include an osmium complex bonded to a poly (4-vinyl pyridine) -based polymer.

[0014] In some embodiments, the sensing composition may further comprise a cross-linking agent. The cross-linking agent may be polyepoxide, cyanuric chloride, N-hydroxysuccinimide, imidate, epichlorohydrin, or a combination thereof. In one embodiment, the cross-linking agent is polyethylene glycol diglycidyl ether (PEGDGE).

[0015] In some embodiments, the sensing composition comprises an electron transfer agent. In some embodiments, the electron transfer agent can transfer electrons from the pyruvate response enzyme to a substrate, such as an organic or inorganic surface, such as an electrode surface. In some embodiments, the electron transfer agent is a redox mediator. In some embodiments, the redox mediator is a redox polymer. In one embodiment, the pyruvate response enzyme (e.g., pyruvate oxidase) can be fixed (e.g., attached) to the redox mediator in the sensing composition. In some embodiments, the redox polymer is attached to the pyruvate response enzyme. In some embodiments, the redox polymer is attached to the pyruvate response enzyme and a substrate such as an electrode surface.

[0016] In another embodiment, the present disclosure provides a sensing electrode comprising a working electrode, which may include a pyruvic acid sensing layer formed from the sensing composition described herein. The pyruvic acid sensing layer may be continuous or discontinuous. In one embodiment, the sensing electrode may further include a membrane covering at least the pyruvic acid sensing layer. In some embodiments, the membrane is permeable to pyruvic acid and / or has a reduced permeability to TPP relative to pyruvic acid. In some embodiments, the membrane may comprise poly (4-vinyl pyridine).

[0017] In another embodiment, the present invention provides a system for sensing pyruvate, comprising a working electrode, a sensing element disposed on at least a portion of the working electrode, and a circuit configured to connect and disconnect with the working electrode, wherein the sensing element is configured to accumulate charge derived from the reaction of pyruvate with a pyruvate-responsive enzyme (e.g., pyruvate oxidase) over a set time period. The sensing element is formed from a sensing composition as described herein. In one embodiment, the system may further comprise a sensor tail configured for insertion into tissue, wherein the working electrode is disposed on the sensor tail.

[0018] The present disclosure also provides a method for sensing pyruvate, comprising providing a sensing electrode as described herein, connecting the sensing electrode to a circuit to provide a steady-state current, disconnecting the sensing electrode from the circuit, contacting the sensing electrode with an analyte that may contain pyruvate, accumulating charge derived from the reaction of pyruvate with a pyruvate-responsive enzyme (e.g., pyruvate oxidase) and a redox mediator over a set time period, connecting the sensing electrode to the circuit after the set time period, and measuring a signal from the accumulated charge. In some embodiments, the set time period is 30 seconds or longer.

[0019] Additional embodiments and advantages of the disclosure will be set forth in part in the description which follows and will be derived from the description, or may be learned by practice of the disclosure.

[0020] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the claims. Description of attached figures / figures

[0021] Figure 1 is a schematic diagram illustrating the pyruvate sensor chemistry reacting with pyruvate oxidase in the presence of the cofactor thiamine pyrophosphate (TPP) and an osmium-based redox material designated "X7" attached ("wired") to the working electrode.

[0022] Figure 2is a graph of current (nA) versus time (hours) at various pyruvate concentrations, tested for a sensing electrode prepared from a sensing composition comprising a pyruvate oxidase to TPP weight ratio of 200:1 ("1xTPP").

[0023] Figure 3 is a graph of current (nA) versus time (hours) at various pyruvate concentrations, tested for a sensing electrode prepared from a sensing composition comprising a pyruvate oxidase to TPP weight ratio of 1.73:1 ("100xTPP").

[0024] Figure 4 is a plot of accumulated charge (nC) versus pyruvate concentration (μM) for the 1×TPP sensor (solid line) and the 100×TPP sensor (dashed line). Detailed description

[0025] The headings provided herein are not limitations of the various embodiments of the present disclosure, which can be defined by reference to the entire specification. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present disclosure will be limited only by the appended claims. definition

[0026] For convenience, the meanings of some terms and phrases used in the specification, examples and appended claims are provided below. Unless otherwise specified or implied from the context, the following terms and phrases include the meanings provided below. Definitions are provided to help describe specific embodiments and are not intended to limit the claimed technology, as the scope of the technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the technology belongs. If there is a clear difference between the use of a term in the art and its definition provided herein, the definition provided in the specification shall prevail.

[0027] The articles "a," "an," and "the" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0028] As used herein, unless otherwise indicated, the term "about" refers to ±10% of the specified value.

[0029] The term "at least" preceding a number or series of numbers is understood to include the number associated with the term "at least," as well as all subsequent numbers or integers that can logically be included, as is clear from the context. When at least precedes a series of numbers or a range, it should be understood that "at least" can modify each number in the 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 in a method step, that component is included in that step, and additional components are optional.

[0030] As used herein, the terms "comprising," "including," "having," "including," "containing," and the like are open-ended terms meaning "including, but not limited to." To the extent that a given embodiment disclosed herein "comprising" certain elements, it should be understood that this disclosure also specifically contemplates and discloses embodiments that "consist essentially of" and "consist of" those elements.

[0031] As used herein, the terms "consisting essentially of," "consisting essentially of," and the like should be interpreted as semi-closed terms, meaning that other ingredients that materially affect the basic and novel characteristics of the embodiment are not included.

[0032] As used herein, the terms "consisting of," "composed of," and the like should be interpreted as closed terms such that an embodiment "consisting of a particular set of elements" excludes any elements, steps, or ingredients not specified in that embodiment.

[0033] The terms "measure," "measured," and "measured" as used herein may encompass one or more of the corresponding terms "determine," "determine," "determined," "calculate," "calculate," and "calculated."

[0034] As used herein, an "analyte" is an enzyme substrate to be measured or detected. The analyte can be derived from, for example, a biological fluid and can be tested in vivo, ex vivo, or in vitro. Pyruvate is an exemplary analyte in the present disclosure.

[0035] As used herein, a "sensor" is a device configured to detect the presence and / or absence of an analyte in a sample and / or measure the level (e.g., concentration) of an analyte in a sample through electrochemical oxidation and reduction reactions on the sensor. These reactions are converted into electrical signals that can be correlated with the amount, concentration, or level of the analyte in the sample.

[0036] As used herein, a "working electrode" is an electrode at which the analyte (or a second compound whose level depends on the level of the analyte) is electrooxidized or electroreduced with or without the action of an electron transfer agent.

[0037] As used herein, "counter electrode" refers to an electrode paired with a working electrode, through which a current equal in magnitude and opposite in sign to the current through the working electrode is passed. In the context of the embodiments of the present disclosure, unless otherwise indicated, the term "counter electrode" includes a) a counter electrode and b) a counter electrode that also serves as a reference electrode (i.e., a counter / reference electrode).

[0038] As used herein, "reference electrode" includes a) a reference electrode and b) a reference electrode that also functions as a counter electrode (ie, a counter / reference electrode), unless otherwise indicated.

[0039] As used herein, "electrolysis" is the electrooxidation or electroreduction of a compound directly at an electrode or via one or more electron transfer agents.

[0040] As used herein, a component is "immobilized" or "attached" to a polymer and / or sensor when, for example, the component is entrapped on, embedded within, covalently bound, ionically bound, electrostatically bound, or coordinately bound thereto, which reduces or eliminates mobility.

[0041] As used herein, a "non-leachable" or "non-releasable" compound, or a compound that is "non-leachably disposed," is intended to define a compound that is immobilized on a sensor such that it does not substantially diffuse out of the sensing layer of the working electrode during use of the sensor (e.g., while the sensor is implanted in a patient or while a sample is being measured).

[0042] As used herein, an "electron transfer agent" is a compound that carries electrons between the analyte and the working electrode, either directly or in conjunction with other electron transfer agents. An example of an electron transfer agent is a redox mediator.

[0043] As used herein, a "redox mediator" is an electron transfer agent that is used to carry electrons between an analyte, an analyte-reduced or analyte-oxidized enzyme and an electrode, either directly or through one or more additional electron transfer agents. A redox mediator comprising a polymer backbone may also be referred to as a "redox polymer."

[0044] As used herein, the term "precursor polymer" refers to the starting polymer before the various modifier groups are attached to form the modified polymer.

[0045] "Substituted" functional groups (e.g., substituted alkyl, alkenyl, alkoxy, aryl) include at least one substituent (e.g., 1, 2, 3, 4, or 5), which can be, for example, halogen, alkoxy, thiol, aryl, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxyl, amino, alkylamino, dialkylamino, trialkylammonium, alkanoylamino, arylcarboxamido, hydrazine, alkylthio, alkenyl, and reactive groups.

[0046] " reactive group " is the functional group of molecule (such as polymer, cross-linking agent, enzyme), and it can react with another compound to couple at least a portion (such as another reactive group) of the another compound to the molecule.Reactive groups include carboxyl, activated ester, sulfonyl halide, sulfonate, isocyanate, isothiocyanate, epoxide, aziridine, halide, aldehyde, ketone, amine, acrylamide, thiol, acyl azide, acyl halide, hydrazine, hydroxylamine, alkyl halide, imidazole, pyridine, phenol, alkyl sulfonate, halotriazine, imidoester, maleimide, hydrazide, hydroxyl and photoreactive aryl azido groups.As understood in the art, activated ester generally includes esters of succinimidyl, benzotriazolyl or aryl substituted by electron-withdrawing groups such as sulfo, nitro, cyano or halogen groups; Or carboxylic acids activated by carbodiimide.

[0047] As used herein, a "sensing layer" is a component of a sensor that includes ingredients that promote electrolysis of an analyte. The sensing layer may include ingredients such as a redox mediator (e.g., an electron transfer agent or a redox polymer), a catalyst (e.g., an analyte-catalyst) that catalyzes a reaction of the analyte to produce a response at the working electrode, or both an electron transfer agent and a catalyst. In some embodiments of the present disclosure, the sensor includes a sensing layer that is non-leachably disposed near or on the working electrode.

[0048] As used herein, a "sensing element" is an application or area of ​​an analyte-specific enzyme arranged together with a sensing layer. Thus, the sensing element is capable of interacting with the analyte. The sensing layer may have more than one sensing element, which constitutes an analyte detection area disposed on the working electrode. In some embodiments, the sensing element comprises an analyte-specific enzyme and an electron transfer agent (e.g., an electron transfer agent). In some embodiments, the sensing element comprises an analyte-specific enzyme, a redox mediator, and a cross-linking agent.

[0049] As used herein, a "cross-linking agent" or "cross-linker" is 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 cross-linking) or at least two parts of the same molecule together (intramolecular cross-linking). Cross-linkers with more than two reactive groups are capable of both intermolecular and intramolecular cross-linking.

[0050] A "membrane solution" is a solution containing components for crosslinking and membrane formation, including, for example, a polymer (eg, a modified polymer containing a heterocyclic nitrogen group), a crosslinker, and a solvent (eg, a buffer or an alcohol-buffer mixed solvent).

[0051] As used herein, "configured to accumulate charge" is an arrangement of a working electrode and circuitry that allows for the accumulation of electrons generated by the oxidation of an analyte (e.g., pyruvate). The oxidation that occurs at or on the sensing element of the working electrode is not connected to the circuitry, thereby generating the accumulation of electrons.

[0052] As used herein, a "set time period" is the amount of time required to accumulate charge (electrons) to provide sufficient signal output that can measure and quantify a given analyte (e.g., pyruvate). Typically, and as discussed in detail elsewhere herein, the set time period is 30 seconds or longer and 30 minutes or shorter.

[0053] As used herein, "biological fluid" is any body fluid or body fluid derivative in which an analyte can be measured. Examples of biological fluids include, for example, dermal fluid, subcutaneous fluid, interstitial fluid, plasma, blood (e.g., from a vein or blood vessel), lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, sweat, or tears.

[0054] The term "patient" refers to a living animal and thus encompasses, for example, living mammals and living humans. The term "user" may be used herein as a term encompassing the term "patient."

[0055] As used herein, "accumulation mode sensing" refers to the accumulation of electrons resulting from the oxidation of an analyte at or on a sensing element that is not connected to a working electrode of an electrical circuit, thereby generating an accumulation of electrons. Compositions and methods of the present disclosure

[0056] In one embodiment, the present invention relates to a sensor composition for detecting pyruvate. In some embodiments, the sensor composition comprises a pyruvate-responsive enzyme and thiamine pyrophosphate (TPP). In some embodiments, the pyruvate-responsive enzyme is pyruvate oxidase.

[0057] In some embodiments, the sensing composition comprises additional components. In some embodiments, the sensing composition comprises one or more cofactors of a pyruvate-responsive enzyme (e.g., FAD); and / or one or more enzyme stabilizers (e.g., albumin). In some embodiments, the sensing composition comprises an electron transfer agent, such as a redox mediator.

[0058] In one embodiment, the present disclosure relates to a sensing composition comprising a pyruvate-responsive enzyme (eg, pyruvate oxidase), thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), albumin, and a redox mediator.

[0059] In some embodiments, the composition comprises about 1 to about 100 mg / mL of a pyruvate-responsive enzyme (e.g., pyruvate oxidase), e.g., about 5 to about 50 mg / mL, e.g., about 10 to about 30 mg / mL or about 20 mg / mL.

[0060] In some embodiments, the composition comprises about 0.04 mg / mL to about 40 mg / mL TPP, e.g., about 0.1 to about 20 mg / mL, e.g., about 0.5 to about 18 mg / mL, e.g., about 1 mg / mL to about 16 mg / mL, e.g., about 5 mg / mL to about 15 mg / mL, e.g., about 10 to about 12 mg / mL TPP.

[0061] In some embodiments, the relative amount of pyruvate response enzyme and TPP is expressed with the weight ratio of pyruvate response enzyme (e.g., pyruvate oxidase) to TPP. In some embodiments, pyruvate response enzyme (e.g., pyruvate oxidase) and TPP are present in the composition with a weight ratio of about 500:1 to about 1:1. For example, TPP is used at a concentration within the same concentration range of 1 / 500 of the concentration of pyruvate response enzyme (e.g., pyruvate oxidase) to pyruvate response enzyme (e.g., pyruvate oxidase). TPP and FAD are cofactors of pyruvate response enzyme (e.g., pyruvate oxidase). Surprisingly, it was found that the amount of TPP in the composition affected the ability of the sensor to fully detect pyruvate. It was further found that, by increasing the ratio of TPP to pyruvate response enzyme, the sensitivity of pyruvate sensing using the composition can be increased. Advantageously, the minimum concentration of TPP in the sensing composition can be used to affect detection. In particular, it has been surprisingly discovered that an amount of TPP that is about five percent or more by weight of the amount of pyruvate-responsive enzyme (e.g., pyruvate oxidase) should be used in the compositions and their uses in the sensing methods for detecting pyruvate. Thus, in some embodiments, the pyruvate-responsive enzyme (e.g., pyruvate oxidase) and TPP are present in the sensing compositions described herein such that the weight ratio of the pyruvate-responsive enzyme (e.g., pyruvate oxidase) to TPP is at least 500: 1. In some embodiments, the weight ratio of the pyruvate-responsive enzyme (e.g., pyruvate oxidase) to TPP can be at least about 500: 1, at least about 400: 1, at least about 300: 1, at least about 200: 1, at least about 100: 1, at least about 50: 1, at least about 20: 1, at least about 10: 1, at least about 5: 1, at least about 4: 1, at least about 3: 1, or at least about 2: 1.

[0062] In some embodiments, the weight ratio of the pyruvate-responsive enzyme (e.g., pyruvate oxidase) to TPP can range from about 500:1 to about 1:1 (e.g., about 450:1 to about 1:1, about 400:1 to about 1:1, about 350:1 to about 1:1, about 300:1 to about 1:1, about 250:1 to about 1:1, about 200:1 to about 1:1, about 150:1 to about 1:1, about 100:1 to about 1:1, about 90:1 to about 1:1, about 80:1 to about 1:1, about 70:1 to about 1:1, about 60:1 to about 1:1). 1 to about 1:1, about 50:1 to about 1:1, about 45:1 to about 1:1, about 40 to about 1:1, about 35:1 to about 1:1, about 30:1 to about 1:1, about 25:1 to about 1:1, about 20:1 to about 1:1, about 15:1 to about 1:1, about 10:1 to about 1:1, about 9:1 to about 1:1, about 8:1 to about 1:1, about 7:1 to about 1:1, about 6:1 to about 1:1, about 5:1 to about 1:1, about 4:1 to about 1:1, about 3:1 to about 1:1, or about 2:1 to about 1:1). For example, the weight ratio of the pyruvate responsive enzyme (e.g., pyruvate oxidase) to TPP can be about 500: 1, about 475: 1, about 450: 1, about 425: 1, about 400: 1, about 375: 1, about 350: 1, about 325: 1, about 300: 1, about 275: 1, about 250: 1, about 225: 1, about 200: 1, about 175: 1, about 150: 1, about 125: 1, about 100: 1, about 95: 1, about 90: 1, about 85: 1, about 80: 1, about 75: 1, about 70: 1, about 65: 1, about 85: 1, about 80 ... 1, about 5:1, about 60: 1, about 55: 1, about 50: 1, about 45: 1, about 50: 1, about 45: 1, about 40: 1, about 35: 1, about 30: 1, about 25: 1, about 20: 1, about 15: 1, about 10: 1, about 9.5: 1, about 9: 1, about 8.5: 1, about 8: 1, about 7.5: 1, about 7: 1, about 6.5: 1, about 6: 1, about 5.5: 1, about 5: 1, about 4: 1, about 3.5: 1, about 3: 1, about 2.5: 1, about 2: 1, about 1.5: 1, or about 1 : 1. In one embodiment, the weight ratio of the pyruvate responsive enzyme (e.g., pyruvate oxidase) to TPP can be less than about 200: 1 to about 1 : 1. In one embodiment, the weight ratio of the pyruvate-responsive enzyme (e.g., pyruvate oxidase) to TPP can be about 150: 1 to about 1: 1. In one embodiment, the weight ratio of the pyruvate-responsive enzyme (e.g., pyruvate oxidase) to TPP can be about 100: 1 to about 1: 1. In one embodiment, the weight ratio of the pyruvate-responsive enzyme (e.g., pyruvate oxidase) to TPP can be about 50: 1 to about 1: 1. In one embodiment, the weight ratio of the pyruvate-responsive enzyme (e.g., pyruvate oxidase) to TPP can be about 25: 1 to about 1: 1.In one embodiment, the weight ratio of the pyruvate-responsive enzyme (e.g., pyruvate oxidase) to TPP can be about 10:1 to about 1:1. In one embodiment, the weight ratio of the pyruvate-responsive enzyme (e.g., pyruvate oxidase) to TPP can be about 5:1 to about 1:1. In one embodiment, the weight ratio of the pyruvate-responsive enzyme (e.g., pyruvate oxidase) to TPP can be about 1:1 to about 3:1, or about 1:1 to about 2.5:1, or about 2:1 to about 1:1. In certain embodiments, the weight ratio of the pyruvate-responsive enzyme (e.g., pyruvate oxidase) to TPP can be about 2:1.

[0063] Those skilled in the art will appreciate that the weight ratio of pyruvic acid responsive enzyme (e.g., pyruvic acid oxidase) to TPP can be calculated based on the concentration of, for example, reagent in the composition. As a non-limiting illustration, a composition comprising 20 mg / mL pyruvic acid responsive enzyme (e.g., pyruvic acid oxidase) and 0.1 mg / mL has a weight ratio of pyruvic acid responsive enzyme (e.g., pyruvic acid oxidase) to TPP of 200:1; and a composition comprising 20 mg / mL pyruvic acid responsive enzyme (e.g., pyruvic acid oxidase) and 11.5 mg / mL has a weight ratio of pyruvic acid responsive enzyme (e.g., pyruvic acid oxidase) to TPP of 1.7:1 (to one decimal place).

[0064] The amount of the pyruvic acid response enzyme (e.g., pyruvic acid oxidase) and TPP present in the sensing composition can also be represented by the weight ratio of TPP to the pyruvic acid response enzyme (e.g., pyruvic acid oxidase). The weight ratio of the pyruvic acid response enzyme (e.g., pyruvic acid oxidase) to TPP is 500:1, and the weight ratio of the corresponding TPP to the pyruvic acid response enzyme (e.g., pyruvic acid oxidase) is 0.002:1. Therefore, the composition comprising 20mg / mL pyruvic acid response enzyme (e.g., pyruvic acid oxidase) and 0.1mg / mL has a weight ratio of TPP to the pyruvic acid response enzyme (e.g., pyruvic acid oxidase) of 0.005:1; and the composition comprising 20mg / mL pyruvic acid response enzyme (e.g., pyruvic acid oxidase) and 11.5mg / mL has a weight ratio of TPP to the pyruvic acid response enzyme (e.g., pyruvic acid oxidase) of 0.575:1.

[0065] Thus, in some embodiments, the weight ratio of TPP to the pyruvate-responsive enzyme (e.g., pyruvate oxidase) is at least 0.002: 1. In some embodiments, the weight ratio of TPP to the pyruvate-responsive enzyme (e.g., pyruvate oxidase) is at least about 0.005: 1, such as at least about 0.01: 1, such as about 0.02: 1, such as at least about 0.05: 1, such as at least about 0.1: 1, such as at least about 0.2: 1, such as at least about 0.25: 1, such as at least about 0.33: 1, such as at least about 0.5: 1.

[0066] In some embodiments, the weight ratio of TPP to the pyruvate-responsive enzyme (e.g., pyruvate oxidase) ranges from about 0.002:1 to about 1:1, e.g., about 0.005:1 to 1:1, e.g., about 0.01:1 to 1:1, e.g., 0.02:1 to 1:1, e.g., about 0.05:1 to 1:1, e.g., about 0.1:1 to 1:1, e.g., about 0.2:1 to 1:1, e.g., about 0.25:1 to 1:1, e.g., about 0.33:1 to 1:1, e.g., about 0.5:1 to 1:1.

[0067] In some embodiments, the relative amounts of the pyruvate-responsive enzyme and TPP are expressed as a molar ratio of TPP to pyruvate-responsive enzyme (eg, pyruvate oxidase).

[0068] In some embodiments, TPP is in molar excess relative to the pyruvate-responsive enzyme (e.g., pyruvate oxidase). In some embodiments, the molar ratio of TPP to the pyruvate-responsive enzyme (e.g., pyruvate oxidase) can be at least about 1:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 10:1, at least about 20:1, at least about 50:1, at least about 100:1, at least about 200:1, at least about 300:1, or more.

[0069] In some embodiments, the molar ratio is from about 1.2:1 to about 611.3:1, for example, from about 1.3:1 to about 611.3:1, for example, from about 1.4:1 to about 611.3:1, for example, from about 1.4:1 to about 611.3:1, for example, from about 1.5:1 to about 611.3:1, for example, from about 1.6:1 to about 611.3:1, for example, from about 1.7:1 to about 611.3:1, for example, from about 1.9:1 to about 611.3:1, for example, from about 2.0:1 to about 611.3:1, for example, from about 2.2:1 to about 611.3 :1, for example, about 2.4:1 to about 611.3:1, for example, about 2.7:1 to about 611.3:1, for example, about 3.1:1 to about 611.3:1, for example, about 3.5:1 to about 611.3:1, for example, about 4.1:1 to about 611.3:1, for example, about 4.9:1 to about 611.3:1, for example, about 6.1:1 to about 611.3:1, for example, about 6.8:1 to about 611.3:1, for example, about 7.6:1 to about 611.3:1, for example, about 8.7:1 to about 611.3:1, for example, about 10.2 :1 to about 611.3:1, for example, about 12.2:1 to about 611.3:1, for example, about 13.6:1 to about 611.3:1, for example, about 15.3:1 to about 611.3:1, for example, about 17.5:1 to about 611.3:1, for example, about 20.4:1 to about 611.3:1, for example, about 24.5:1 to about 611.3:1, for example, about 30.6:1 to about 611.3:1, for example, about 40.8:1 to about 611.3:1, for example, about 61.1:1 to about 611.3:1, for example, about 67.9 :1 to about 611.3:1, for example, about 76.4:1 to about 611.3:1, for example, about 87.3:1 to about 611.3:1, for example, about 101.9:1 to about 611.3:1, for example, about 122.3:1 to about 611.3:1, for example, about 152.8:1 to about 611.3:1, for example, about 174.7:1 to about 611.3:1, for example, about 203.8:1 to about 611.3:1, for example, about 244.5:1 to about 611.3:1, for example, about 305.7:1 to about 611.3:1.

[0070] Pyruvate responsive enzyme is any enzyme or combination of enzymes that converts pyruvate (or pyruvate-TPP adduct) into acetyl phosphate. In some embodiments, pyruvate responsive enzyme can include pyruvate oxidase, pyruvate dehydrogenase, pyruvate decarboxylase or its combination. In some embodiments, pyruvate responsive enzyme is pyruvate oxidase. In certain embodiments, one or more cofactors (such as coenzyme) can be used in combination with pyruvate responsive enzyme. In some embodiments, the cofactor can be thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD) or its combination. In some embodiments, TPP is the cofactor of pyruvate responsive enzyme. In some embodiments, pyruvate responsive enzyme is TPP dependent enzyme. In some embodiments, pyruvate responsive enzyme is pyruvate oxidase. In some embodiments, pyruvate responsive enzyme is phosphate-dependent pyruvate oxidase.

[0071] When the pyruvate responsive enzyme is or includes pyruvate oxidase, any suitable pyruvate oxidase can be used. Pyruvate oxidase is commercially available from suppliers such as Toyobo USA (New York). In some embodiments, pyruvate oxidase is derived from Pseudomonas species (Pseudomonas sp.). In some embodiments, pyruvate oxidase has Enzyme Commission numbering EC 1.2.3.3 and / or Chemical Abstracts Service (CAS) registration number 9001-96-1. In some embodiments, pyruvate oxidase is an enzyme.

[0072] Typically, the molecular weight of the pyruvate responsive enzyme is from about 100 to about 400 kDa, such as from about 200 to about 300 kDa, such as from about 220 to about 280 kDa, such as from about 250 to about 270 kDa, such as 260 kDa. An exemplary pyruvate oxidase is commercially available from Toyoba USA as product PYO-311 and has a molecular weight of about 260 kDa.

[0073] The sensing composition may further comprise a stabilizer (e.g., an enzyme stabilizer). In some embodiments, the stabilizer is or comprises a protein, such as albumin or casein, catalase, or a small organic molecule, such as a polyol, a carboxylic acid, a carboxylate, a carboxylate, and a sugar, or any combination thereof. In some embodiments, the stabilizer is albumin. In one embodiment, the albumin may be a serum albumin, such as bovine serum albumin or human serum albumin. In certain embodiments, the sensing composition may further comprise bovine serum albumin.

[0074] In some embodiments, the albumin is present in the composition in an amount of about 1 to about 50 mg / mL, such as about 2 to about 20 mg / mL, such as about 5 to about 15 mg / mL, such as about 10 mg / mL.

[0075] In some embodiments, the sensing composition comprises a pyruvate-responsive enzyme, thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), albumin, and a redox mediator.

[0076] In some embodiments, the sensing composition comprises FAD. In some embodiments, FAD is present in the composition in an amount of about 0.01 to about 1 mg / mL, such as about 0.05 to about 0.5 mg / mL, such as about 0.1 mg / mL.

[0077] In any embodiment, the sensing composition may include a pH buffer. The buffer may be any suitable composition that is water-soluble and controls (i.e., maintains) the pH of the sensing composition within a pH range of about 5 to about 8 (e.g., maintaining a pH of about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, or about 8). In some embodiments, the pH may be controlled within a range of about 6 to about 8. For example, the buffer may include phosphates (e.g., sodium dihydrogen phosphate and disodium hydrogen phosphate), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 3-(N-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 include one or more (e.g., 1, 2, 3, or 4) additional salts (e.g., halide salts of Group I or Group II, such as sodium chloride, potassium chloride, magnesium chloride). In one embodiment, the buffer may be phosphate-buffered saline (PBS) comprising sodium hydrogen phosphate, sodium chloride, and optionally potassium chloride and potassium dihydrogen phosphate. In another embodiment, the buffer may be HEPES or a phosphate buffer that may comprise phosphate, sodium chloride, and magnesium chloride. In some embodiments, the sensing composition comprises a buffer salt at a concentration of about 10 mM to about 500 mM, such as about 20 mM to about 100 mM, such as 50 mM. For example, in some embodiments, the sensing composition comprises a phosphate (e.g., sodium phosphate) at about 10 mM to about 500 mM, such as about 20 mM to about 100 mM, such as 50 mM. In some embodiments, the sensing composition comprises one or more Group I or Group II halide salts at a concentration of about 10 mM to about 500 mM, such as about 20 mM to about 100 mM, such as 50 mM to about 60 mM. In some embodiments, the sensing composition comprises about 10 mM to about 500 mM, such as about 20 mM to about 100 mM, such as 50 mM to about 60 mM NaCl and / or MgCl2, such as about 50 mM NaCl and about 10 mM MgCl2. In some embodiments, the sensing composition comprises about 50 mM phosphate, about 50 mM NaCl, and about 10 mM MgCl2, and is buffered to about pH 6.

[0078] The buffer is typically an aqueous buffer, but other non-aqueous solvents may also be present, such as alcohols (e.g., ethanol). In some embodiments, the buffer comprises water as the sole solvent. In other embodiments, the buffer may comprise water and at least one (e.g., 1, 2, or 3) non-aqueous solvent in any suitable ratio, such as a volume ratio of 99.9:0.1 to 0.1:99.9 of non-aqueous solvent to water. In some embodiments, the volume ratio of the non-aqueous solvent to water is 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 a specific example, ethanol (EtOH) and water are used in a volume ratio ranging from 50:50 to 90:10 EtOH:HO (e.g., 70:30, about 75:25, about 80:20, about 85:15, or about 90:10, etc.).

[0079] In one embodiment, the redox mediator may comprise a polymer and an electron transfer agent.

[0080] The polymer in the redox mediator can be any suitable polymer that allows electrons to transfer between the electron transfer agent and the working electrode. For example, the polymer can be poly (4-vinyl pyridine), poly (1-vinylimidazole), poly (thiophene), 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-vinyl pyridine) and poly (sodium 4-styrene sulfonate) quaternized with carboxypentyl groups. These polymers can be considered as precursor polymers because the polymer is further modified to fix (e.g., attach) the electron transfer complex. In some embodiments, the polymer can include poly (4-vinyl pyridine), poly (1-vinylimidazole), poly (thiophene), poly (aniline), poly (pyrrole) or poly (acetylene). In other embodiments, the polymer may comprise polymer or copolymer repeat units that may comprise at least one (e.g., 1, 2, 3, 4, 5, or 6) pendant pyridyl, imidazole, or both pyridyl and imidazole groups. For example, suitable polymers include partially or fully quaternized poly(4-vinylpyridine) and poly(1-vinylimidazole), wherein the quaternized pyridine and imidazole groups, respectively, are available to form spacers by reaction (e.g., complexation) with an electron transfer agent.

[0081] The electron transfer agent in the redox mediator generally includes a transition metal complex. The transition metal in the transition metal complex can be any suitable transition metal that can be effectively reduced and oxidized in the method described herein. For example, the transition metal complex can include osmium, ruthenium, iron, cobalt, vanadium or a combination thereof. In some embodiments, the transition metal can be ruthenium or osmium, particularly osmium. According to some embodiments, suitable electron transfer agents can include 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 disclosures of which are incorporated herein by reference in their entirety. Other suitable examples of electron transfer mediators and polymer-bound electron transfer mediators can include those described in U.S. Patent Nos. 8,444,834, 8,268,143 and 6,605,201, the disclosures of which are incorporated herein by reference in their entirety.

[0082] 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 include enough ligands to provide a complete coordination sphere. In some embodiments, at least one ligand (e.g., 1, 2, 3, 4, 5, or 6) may comprise a nitrogen-containing heterocycle.

[0083] Monodentate ligands include, for example, -F, -Cl, -Br, -I, -CN, -SCN, -OH, NH3, alkylamines, dialkylamines, trialkylamines, alkoxy groups, heterocyclic compounds, compounds containing these groups, solvent molecules (e.g., H2O, EtOH), or reactive groups. For example, the alkyl group of the ligand (e.g., C 1-12 , C 1-6 , C 1-4 , C 1-3 ) or aryl (e.g., phenyl, benzyl, naphthyl) moieties can be optionally substituted with, for example, F, Cl, Br, I, alkylamino, dialkylamino, trialkylammonium (in addition to the aryl moiety), alkoxy, alkylthio, and aryl groups. Examples of suitable heterocyclic monodentate ligands include imidazole, pyrazole, oxazole, thiazole, pyridine, and pyrazine, each of which can be unsubstituted or substituted as described herein (e.g., substituted with at least one reactive group, e.g., 1, 2, 3, or 4 reactive groups).

[0084] Examples of suitable bidentate ligands include, for example, 1,10-phenanthroline, amino acids, oxalic acid, acetylacetone, diaminoalkanes, o-diaminoarenes, 2,2'-biimidazole, 2,2'-bioxazole, 2,2'-bithiazole, 2-(2-pyridyl)imidazole, and 2,2'-bipyridine, each of which may be unsubstituted or substituted as described herein (e.g., substituted with at least one reactive group, such as 1, 2, 3, or 4 reactive groups). Particularly suitable bidentate ligands for electron transfer complexes include substituted and unsubstituted 2,2'-biimidazole, 2-(2-pyridyl)imidazole, and 2,2'-bipyridine. Examples of suitable tridentate ligands include, for example, diethylenetriamine, 2,2',2"-terpyridine, 2,6-bis(N-pyrazolyl)pyridine, each of which may be substituted or unsubstituted (e.g., substituted with one or more alkyl groups such as methyl or one or more reactive groups).

[0085] Suitable 2,2'-biimidazole ligands may be ligands according to formula (I):

[0086] In formula (I), R 1 and R 2 are the same or different and are each a substituted or unsubstituted alkyl, alkenyl or aryl group. 1 and R 2 are the same or different and are each unsubstituted C 1-12 Alkyl (e.g. C 1-4 In some embodiments, R 1 and R 2 All are methyl.

[0087] In formula (I), R 3 、R 4 、R 5 and R 6 are the same or different and are each H, F, Cl, Br, I, NO2, CN, CO2H, SO3H, SH, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazine, alkylhydrazine, hydroxyamino, alkoxyamino, alkylthio, alkyl, alkenyl or aryl. Alternatively, R 3 and R 4 Combination or R 5 and R 6 The alkyl and alkoxy moieties are C 1-12The alkyl or aryl portion of any substituent may be optionally substituted with one or more substituents (e.g., 1, 2, 3, 4, 5, or 6) such as F, Cl, Br, I, amino, alkylamino, dialkylamino, trialkylammonium (except on the aryl portion), alkoxy, alkylthio, aryl, or a reactive group (e.g., CO2H). Typically, R 3 、R 4 、R 5 and R 6 are the same or different and are each H or unsubstituted C 1-12 Alkyl (e.g. C 1-4 In some embodiments, R 3 、R 4 、R 5 and R 6 All are H.

[0088] Suitable 2-(2-pyridyl)imidazole ligands may be ligands according to formula (II):

[0089] In formula (II), R 1 is a substituted or unsubstituted alkyl, alkenyl or aryl group. 1 For unsubstituted C 1-12 Alkyl (e.g. C 1-4 alkyl) or C optionally substituted by a reactive group 1-12 In some embodiments, R 1 It's methyl.

[0090] In formula (II), R 3’ 、R 4’ 、R a 、R b 、R c and R d are the same or different and are each H, F, Cl, Br, I, NO2, CN, CO2H, SO3H, SH, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazine, alkylhydrazine, hydroxyamino, alkoxyamino, alkylthio, alkyl, alkenyl or aryl. Alternatively, R 3’ and R 4’ combination, or R a 、R b 、R c and R d Two adjacent substituents (such as R a and R b 、R b and R c or R c and Rd ) are combined to independently form a saturated or unsaturated 5- or 6-membered ring (e.g., benzo). Typically, the alkyl and alkoxy moieties are C 1-12 The alkyl or aryl portion of any substituent may be optionally substituted with one or more substituents (e.g., 1, 2, 3, 4, 5, or 6) such as F, Cl, Br, I, amino, alkylamino, dialkylamino, trialkylammonium (except on the aryl portion), alkoxy, alkylthio, aryl, or a reactive group (e.g., CO2H). Typically, R 3’ 、R 4’ 、R a 、R b 、R c and R d are the same or different and are each H or unsubstituted C 1-12 Alkyl (e.g. C 1-4 In some embodiments, R 3’ 、R 4’ 、R a 、R b 、R c and R d All are H.

[0091] Suitable 2,2'-bipyridine ligands may be ligands according to formula (III):

[0092] In formula (III), R 16 、R 17 、R 18 、R 19 、R 20 、R 21 、R 22 and R 23 The same or different and each is H, F, Cl, Br, I, NO2, CN, CO2H, SO3H, SH, alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, alkanoylamino, arylcarboxamido, hydrazine, alkylhydrazine, hydroxyamino, alkoxyamino, alkylthio, alkyl, alkenyl or aryl. Typically, the alkyl and alkoxy moieties are C 1-12 The alkyl or aryl portion of any substituent may be optionally substituted with one or more substituents (e.g., 1, 2, 3, 4, 5, or 6) such as F, Cl, Br, I, amino, alkylamino, dialkylamino, trialkylammonium (except on the aryl portion), alkoxy, alkylthio, aryl, or a reactive group (e.g., CO2H).

[0093] Specific examples of suitable combinations include R 16 and R 23are all H or are all methyl, and / or R 17 and R 23 are all H or are all methyl, and / or R 18 and R 21 are all H or are all methyl, and / or R 19 and R 20 are all H or are all methyl. An alternative combination is one or more pairs of adjacent 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 ) are combined to form a saturated or unsaturated 5- or 6-membered ring (e.g., benzo).

[0094] In one embodiment, the one or more ligands are 4,4'-dimethyl-2,2'-bipyridine, mono-, di- or polyalkoxy-2,2'-bipyridine (e.g., 4,4'-dimethoxy-2,2'-bipyridine), 4,7-dimethyl-1,10-phenanthroline, mono-, di- or polyalkoxy-1,10-phenanthroline (e.g., 4,7-dimethoxy-1,10-phenanthroline), or a combination of any of these.

[0095] In some embodiments, the transition metal complex will include a counterion (X) that balances the charge of the transition metal. Typically, there will be 1 to 5 (i.e., 1, 2, 3, 4, or 5) counterions. The multiple counterions in the complex are not necessarily all the same. Examples of suitable counterions include anions such as halides (e.g., fluoride, chloride, bromide, or iodide), sulfate, phosphate, hexafluorophosphate, and tetrafluoroborate, and cations (e.g., monovalent cations), such as lithium, sodium, potassium, tetraalkylammonium, and ammonium. In some embodiments, the counterion is a halides, such as chloride.

[0096] In one embodiment, the transition metal complex can be an osmium transition metal complex, which can include one or more ligands, wherein at least one (e.g., 1, 2, 3, 4, 5, or 6) ligand can include a nitrogen-containing heterocycle (e.g., imidazole, pyrazole, oxazole, thiazole, pyridine, and pyrazine). In some embodiments, the osmium transition metal complex can include one or more ligands selected from 4,4'-dimethyl-2,2'-bipyridine, monoalkoxy-2,2'-bipyridine, dialkoxy-2,2'-bipyridine, or polyalkoxy-2,2'-bipyridine (e.g., 4,4'-dimethoxy-2,2'-bipyridine), 4,7-dimethyl-1,10-phenanthroline, and monoalkoxy-1,10-phenanthroline, dialkoxy-1,10-phenanthroline, or polyalkoxy-1,10-phenanthroline (e.g., 4,7-dimethoxy-1,10-phenanthroline).

[0097] In one embodiment, the redox mediator 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 may be a prepolymer modified to attach an osmium complex (e.g., a poly(biimidazole-based)osmium complex) as shown in the following structure. Wherein n can be 2, n' can be 17, and n" can be 1. Other reactive groups and / or spacer groups can be used.

[0098] In one embodiment, the electronic redox mediator may include an osmium-containing poly(4-vinylpyridine)-based polymer, referred to herein as "X7," as shown below. Where n is 2, n' is 17, and n" is 1.

[0099] The electron transfer agent is typically attached (e.g., non-leaching and / or covalently bonded) to the polymer in the redox material. For example, the covalent bonding of the electron transfer agent to the polymer can be performed by polymerizing monomer units with the covalently bound electron transfer agent, or the electron transfer agent can be reacted separately with the polymer after the polymer has been synthesized.

[0100] According to some embodiments, a bifunctional spacer can be used to link (e.g., covalently bond) an electron transfer agent to a polymer in a redox material, wherein a first reactive group reacts with the polymer (e.g., a functional group capable of quaternizing a pyridinium nitrogen atom or an imidazole nitrogen atom), and a second reactive group reacts with the electron transfer agent (e.g., a functional group that reacts with a ligand that coordinates a metal ion). Typically, a covalent bond is formed between the two reactive groups to create the link. Suitable reactive groups include, for example, activated esters (e.g., succinimidyl, benzotriazolyl, or aryl substituted with one or more electron withdrawing groups such as sulfo, nitro, cyano, or halogen), acrylamido, acyl azide, acyl halide, carboxyl (-COO- or -CO2H), aldehyde, ketone, alkyl halide, alkyl sulfonate, anhydride, aziridine, epoxy, halotriazine, imidoester, isocyanato, isothiocyanato, maleimido, sulfonyl halide, amino, thiol (-SH), hydroxyl, pyridyl, imidazolyl, and hydroxyamino groups. The reaction between the two reactive groups can form a covalent bond between the transition metal complex and the polymer, wherein the covalent bond is a formamido, thioether, hydrazone, oxime, alkylamino, ester, carboxylate, imidazolium, pyridinium, ether, thioether, aminotriazinyl, triazinyl ether, amidine, urea, urethane, thiourea, thioether, sulfonamide, or any combination thereof. In addition to the reactive groups, the bifunctional spacer can generally further comprise an alkylene group (i.e., -(CH2) n -) and / or ethyleneoxy (i.e. -(CH2CH2O) m -, wherein n and m are each independently 1 to 12 (eg, 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).

[0101] In some embodiments, the sensing composition, particularly the redox mediator may further include a cross-linking agent. Typically, a cross-linking agent is any suitable multifunctional (e.g., bifunctional) short-chain molecule that enables an electron transfer agent to be connected (e.g., covalently bonded) to the polymer of the redox mediator. For example, a cross-linking agent may include polyepoxides (e.g., polyethylene glycol diglycidyl ether (PEGDGE), ethylene glycol diglycidyl ether (EGDGE), resorcinol diglycidyl ether, 1,2,7,8-diepoxyoctane, Gly3), cyanuric chloride, N-hydroxysuccinimide, imidoester, epichlorohydrin, or a combination thereof. In one embodiment, a cross-linking agent is polyethylene glycol diglycidyl ether (PEGDGE) of the formula below.

[0102] In one embodiment, PEGDGE is PEGDGE200, PEGDGE400 (n is 10), PEGDGE500, PEGDGE600, PEGDGE1000 or PEGDGE2000, wherein the number represents the average molecular weight (M n In one embodiment, the cross-linking agent is PEGDGE400.

[0103] In one embodiment, at least a portion of the pyruvate responsive enzyme (e.g., pyruvate oxidase) present in the sensing composition is non-leachingly attached to a redox mediator. In some embodiments, the enzyme is covalently linked to the polymer portion of the redox mediator. The covalent bonding of the enzyme to the redox material (e.g., polymer) can occur through a cross-linking agent as described herein and a reactive site on the enzyme. Therefore, the pyruvate responsive enzyme (e.g., pyruvate oxidase) is electrically "wired" to the working electrode through the redox material. In one embodiment, a hydrogel is formed when the enzyme and its wire are cross-linked on the electrode. In another embodiment, at least a portion of the pyruvate responsive enzyme (e.g., pyruvate oxidase) can diffuse into the hydrogel and become attached but not necessarily covalently bonded to the polymer.

[0104] In some embodiments, when the pyruvic acid responsive enzyme is entrained in a polymer and / or is connected (for example, covalently bound) to a polymer, the pyruvic acid responsive enzyme retains enzymatic (for example, pyruvic acid oxidation) activity. In some embodiments, compared with the enzymatic (for example, pyruvic acid oxidation) activity of the pyruvic acid responsive enzyme in solution, when connected to a polymer, the pyruvic acid responsive enzyme retains at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or more enzymatic (for example, pyruvic acid oxidation) activity of POX activity. Can use known in the art, for example, as the conventional method used in the examples, measure enzyme (for example, pyruvic acid oxidation) activity.

[0105] In one embodiment, an enzyme sensor composition is provided comprising (i) a pyruvate-responsive enzyme, typically pyruvate oxidase; optionally wherein the composition comprises about 5 to about 50 mg / mL of the pyruvate-responsive enzyme and (ii) TPP, optionally wherein the composition comprises about 0.1 to about 20 mg / mL of TPP; wherein: In the composition, (a) the weight ratio of the pyruvate-responsive enzyme to TPP is at least 500:1, typically from about 500:1 to about 1:1, more typically from about 200:1 to about 1:1; and / or (b) the molar ratio of the TPP to the pyruvate-responsive enzyme is at least 1:1, typically from about 1.2:1 to about 611.3:1, more typically from about 3.1:1 to about 611.3:1; The composition typically comprises from about 0.01 to about 1 mg / mL of FAD; and / or from about 1 to about 50 mg / mL of an enzyme stabilizer, typically albumin, more typically BSA or HSA; and / or from about 10 mM to about 500 mM of a buffer salt and / or from about 10 mM to about 500 mM of one or more Group I or Group II halide salts and / or has a pH of from about 5 to about 8; Optionally, wherein at least a portion of the pyruvate-responsive enzyme present in the sensing composition is attached to or entrained in a redox mediator comprising a polymer and a transition metal complex, typically an osmium complex.

[0106] In one embodiment, an enzyme sensing composition is provided, comprising: about 10 to about 30 mg / mL, e.g., about 20 mg / mL, of pyruvate oxidase, optionally wherein the pyruvate oxidase is derived from Pseudomonas sp.; about 0.1 to about 20 mg / mL, e.g., about 5 mg / mL to about 15 mg / mL, of TPP; about 0.05 to about 0.5 mg / mL FAD; about 5 to about 15 mg / mL of albumin, typically BSA or HSA; From about 20 mM to about 100 mM buffer salt; typically phosphate; and From about 20 mM to about 100 mM of one or more Group I or Group II halide salts, typically NaCl and / or MgCl2; wherein (i) the pH of the composition is from about 6 to about 8, typically about pH 6; and (ii) (a) the weight ratio of the pyruvate oxidase to TPP is from about 10:1 to about 1:1, typically from about 2:1 to about 1:1; and / or (b) the molar ratio of the TPP to pyruvate oxidase is from about 61.1:1 to about 611.3:1, more typically from about 305.7:1 to about 611.3:1; Optionally, wherein at least a portion of the pyruvate-responsive enzyme present in the sensing composition is non-leachably attached to a redox mediator comprising an osmium complex bonded to a polymer or copolymer of poly(1-vinylimidazole) or poly(4-vinylpyridine).

[0107] As described in more detail herein, pyruvic acid can be sensed by electrochemical sensing using the methods and compositions disclosed herein. Therefore, in one embodiment, an electrode comprising a pyruvic acid responsive enzyme (e.g., pyruvic acid oxidase) and TPP as described herein is provided. The ratio of the pyruvic acid responsive enzyme (e.g., pyruvic acid oxidase) to TPP can be as described herein. The electrode can also include other components of the sensing composition as described herein. Therefore, in one embodiment, an electrode comprising an enzyme sensing composition as described herein is provided, typically wherein the composition is typically included in a sensing layer on a working electrode.

[0108] Also provided herein is a method of preparing an electrode for sensing pyruvate, comprising disposing a sensing composition as described in more detail herein on the surface of the electrode. In some embodiments, the method further comprises incorporating the electrode into a sensor or other device as described herein.

[0109] The present disclosure further relates to a sensing electrode, which may include a working electrode comprising a pyruvic acid sensing layer formed from (or comprising) a sensing composition as described herein. The working electrode may be any suitable conductive material, such as carbon, gold, palladium, or platinum. The pyruvic acid sensing layer may be continuous or discontinuous (e.g., forming a point, a line, or a plurality of points and / or lines (i.e., an array)). The number of points is not considered to be particularly limited, but may be in the range of 2 to about 10 (e.g., about 3 to about 8, or about 4 to about 6). In one embodiment, the pyruvic acid sensing layer may be continuous. In other embodiments, the pyruvic acid sensing layer may be discontinuous.

[0110] The total size (combined area of ​​all points or layers) of the one or more sensing layers may be at least about 0.05 mm 2 and can be up to about 100mm 2 In some embodiments, the overall size may be approximately 100 mm 2 or smaller, about 75mm 2 or smaller, about 50mm 2 or smaller, about 40mm 2 or smaller, about 30mm 2 or smaller, about 25mm 2 or smaller, about 15mm 2 or smaller, about 10mm 2 or smaller, about 5mm 2 or smaller, about 1mm 2 or smaller, or about 0.1mm 2 In one embodiment, the total size of the one or more sensing layers ranges from about 0.05 to about 0.1 mm. 2 , about 0.05 to about 100mm 2, about 0.1 to about 50mm 2 , about 0.5 to about 30mm 2 , about 1 to about 20mm 2 , or from about 1 to about 15 mm 2 .

[0111] The one or more sensing layers typically have a thickness in the range of about 0.1-10 μm. For example, the thickness of each sensing layer should be 0.1 μm 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 will 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 example, each sensing layer present has 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.

[0112] In some embodiments, conductive materials such as carbon nanotubes, graphene, or metal nanoparticles can be incorporated into one or more sensing layers to facilitate rapid acquisition of steady-state current. The conductive materials can be included in the range of about 0.1% to about 50% by weight (pbw) of the sensing layer (e.g., about 1 to about 50 pbw, about 1 to about 10 pbw, or about 0.1 to about 10 pbw).

[0113] In one embodiment, the sensing electrode may further include a film covering at least the pyruvic acid sensing layer and optionally other components. The outer coating forms an outer film that provides stability, mass transfer restriction, biocompatibility, and / or prevents electrode scaling for sensing reagents (e.g., analyte-specific enzymes and redox mediators). The film may optionally coat all or part of the working electrode and optionally coat any counter electrode or reference electrode that may be present. In one embodiment, the film coats (e.g., encapsulates) the entire system, including the sensing electrode with its sensing layer, and any counter electrode, reference electrode, and / or substrate that may be present.

[0114] Membrane can comprise one or more polymeric membrane materials, and it has the physical structure (that is, film is mass transfer limiting membrane) that allows analyte flux to sensing layer.The composition of membrane can change (such as hydrophobicity degree and / or cross-linking degree) to promote the desired flux of pyruvic acid (and any other other analyte) to sensing electrode, thereby provide desired signal intensity and stability, as further described herein.In one embodiment, membrane is permeable to pyruvic acid.Generally, serum contains low concentration of pyruvic acid (such as about 80 to about 160 μ M), so that permeable membrane can detect pyruvic acid.In one embodiment, described membrane has enough pyruvic acid permeability, provides about 1nA / mM or higher analyte sensitivity when being exposed to pyruvic acid.In this embodiment or other embodiments, the membrane has the permeability of reduction to TPP relative to pyruvic acid.The permeability of this reduction minimizes the amount of TPP leached from pyruvic acid sensing layer by membrane.In one embodiment, described membrane is hydrophobic.

[0115] The membrane can be coated onto at least the pyruvic acid sensing layer by any suitable technique. Typically, the membrane will be coated by spraying, painting, inkjet printing, roller coating, dip coating, or any combination thereof. The coating step can be performed one or more times (e.g., 2, 3, 4, or 5 times), which will affect the thickness of the membrane coating. In one embodiment, the coating step can be performed twice to form a double layer.

[0116] Typically, if multiple coatings are applied, the first coating is dried before subsequent coatings are applied. The amount of time between coating steps will vary according to the type of film, working electrode and sensing layer and atmospheric conditions. Typically, the drying time will 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 is applied, the coating can be cured. In one embodiment, 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 a slightly elevated temperature (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). Typically, curing will not occur below about 20 ° C.

[0117] The thickness of the film typically ranges from about 1 μm to about 100 μm. For example, in some embodiments, the thickness of the film can be 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 or less). In one example, the film can have a thickness of about 5 to about 80 μm, about 10 to about 80 μm, about 10 to about 60 μm, about 15 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. In one example, the film can have a thickness of about 15 to about 35 μm. In one example, the film can have a thickness of about 20 to about 35 μm. In one example, the thickness of the film may be 20 μm to about 30 μm. In one example, the thickness of the film may be about 25 μm.

[0118] In one embodiment, the membrane may comprise optionally cross-linked poly(4-vinylpyridine), poly(vinyl alcohol), poly(acrylic acid), poly(methacrylic acid), or a combination thereof. In one example, the mass transfer limiting membrane may comprise at least a poly(4-vinylpyridine) homopolymer or copolymer, wherein the poly(4-vinylpyridine) may be optionally cross-linked. In particular, the membrane may comprise poly(4-vinylpyridine) cross-linked with a cross-linking agent as described herein, such as high molecular weight (e.g., molecular weight 400 g / mol) poly(ethylene glycol) diglycidyl ether.

[0119] Suitable poly(4-vinylpyridine) copolymers for inclusion in the mass transfer limiting membrane can contain up to about 25% comonomer (based on the total amount of monomers in the copolymer), such as from about 0.1% to about 5% comonomer, or from about 5% to about 15% comonomer, or from about 15% to about 25% comonomer, or from about 1% to about 10% comonomer. Suitable comonomers are not particularly limited, provided that the mass transfer limiting membrane provides sufficient pyruvate permeability to provide an analyte sensitivity of about 1 nA / mM or greater upon exposure to pyruvate.

[0120] In some embodiments, the membrane can comprise multiple layers, each layer having a different composition and / or degree of cross-linking. In one example, the membrane coating can be a bilayer membrane comprising a first layer formed from a poly(4-vinylpyridine) homopolymer or copolymer and a second layer formed from a cross-linked (e.g., cross-linked with PEGDGE) poly(4-vinylpyridine) homopolymer or copolymer. Variations in composition between the layers allow for tuning of the membrane's permeability to both pyruvic acid and TPP.

[0121] The present disclosure also relates to a system for sensing pyruvic acid. The system may include a working electrode, a sensing element disposed on at least a portion of the working electrode, and a circuit configured to connect and disconnect with the working electrode. The sensing element may include a sensing composition as described herein, and the sensing element is configured to accumulate charge derived from pyruvic acid reacting with a pyruvic acid responsive enzyme (e.g., pyruvic acid oxidase) over a set time period. In one embodiment, the system may be a sensor (e.g., an enzyme biosensor). In one example, the sensor chemistry is Figure 1 Described in, wherein X7 is a redox material.

[0122] In some embodiments, the disclosed systems and methods are used to sense pyruvic acid in biological samples. The samples include both biological samples and environmental samples. The samples can be detected in a laboratory setting, on-site, or at any other suitable location. The samples can be brought to a sensor as described herein for testing, or the sensor can be applied at the source of the sample.

[0123] Biological sample can be obtained from any source, including animal, plant and microorganism, and contains fluid, solid, tissue and gas.The material containing interested analyte obtained from clinical or forensic environment is also within the expected meaning of term sample.Biological sample includes but is not limited to whole blood, serum, blood plasma, saliva, eye lens fluid, amniotic fluid, synovial fluid, cerebrospinal fluid, tears, lymph, tissue fluid, peritoneal fluid, bronchial lavage fluid, ascites, bone marrow aspirate, pleural effusion, urine, milk, sweat, sputum, semen, mucus, feces, tissue (skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, myocardial tissue, brain tissue, bone marrow, cervical tissue, skin etc.), organ (such as biopsy sample), vaginal fluid, aqueous humor, cerumen, gastric juice, gastrointestinal fluid, nasal washing liquid, liposuction, sebum, tears, breathing and vitreous humor.This type of sample can be assessed in vitro, in vitro or in vivo.

[0124] In some embodiments, the sample can be in a processed form, including dried (eg, dried blood spots) and fixed (eg, formalin-fixed paraffin-embedded (FFPE)) samples. In some embodiments, the sample is located in an animal.

[0125] Since pyruvic acid is present in biological fluids (e.g., serum) at relatively low concentrations, the system is designed to detect low concentrations of analyte by allowing the accumulation of the analyte on the enzyme biosensor. In the context of detecting pyruvic acid, low concentrations can be about 1 mM or less (e.g., 900 μm or less, 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 400 μm or less, 300 μm or less, 200 μm or less, or 100 μm or less) and 10 μM or more (e.g., 20 μm or more, 30 μm or more, 40 μm or more, 50 μm or more, 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, 100 μm or more, 120 μm or more, 140 μm or more, or 150 μm or more). For example, the concentration of pyruvate in a sample or 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. In some embodiments, the methods disclosed herein are methods for detecting a pyruvate concentration of 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, such as about 80 to about 160 μM.

[0126] Thus, in some embodiments, provided herein is a system comprising a pyruvate-responsive enzyme, such as pyruvate oxidase, as disclosed herein; TPP; and a sample comprising pyruvate, wherein the ratio (e.g., weight ratio) of the pyruvate-responsive enzyme to the TPP is as described herein (e.g., a weight ratio of about 500:1 to about 1:1). In some embodiments, pyruvate is present in a biological sample as described herein. In some embodiments, pyruvate is present in the sample at a concentration of about 1 μM to about 1 mM.

[0127] In one embodiment, the sensor can include a working electrode and another electrode (e.g., a counter electrode and / or a reference electrode), wherein the working electrode is provided with a sensing element arranged on at least a portion of the working electrode. If necessary, other components, such as substrates, can be present. A substrate (e.g., a carbon-based substrate, a plastic substrate) can be arranged between the working electrode and the counter electrode and / or the reference electrode. The sensing element can be formed by a sensing composition as described herein, for example, it can include at least a pyruvate-responsive enzyme (e.g., pyruvate oxidase), TPP, FAD, albumin, and a redox mediator. The working electrode comprising the sensing element forms the sensing electrode. In the presence of an analyte, the sensing electrode oxidizes the analyte, and the amount of oxidation is measured as the amount of electronic charge generated by the reaction. As long as the sensing electrode is not connected to another electrode, the charge from the redox reaction will continue to accumulate on the sensing electrode. The charge (electrons) accumulated within a set time period allows low concentrations of pyruvic acid to produce a signal output that is easy to measure and quantify compared to other known methods. After a set period of time for charge accumulation, the sensing electrode is connected to at least one (e.g., 1, 2, 3, or 4) other electrodes (such as a counter electrode and / or a reference electrode) to complete an electrical circuit. When the circuit is connected, the electrons accumulated on the sensing electrode are discharged as an electrical signal, the amplitude of which is measured and correlated to the amount of pyruvate present at the sensing electrode.

[0128] The detection of the analyte (A) relies on electrically "wiring" the oxidoreductase (AOx) to the working electrode of the sensor via the redox material. During normal amperometric detection, the electrode is held at a potential (voltage) that allows the analyte to react at a constant rate that is proportional to the analyte concentration. For the analyte oxidation reaction (A to A+), electrons will flow at a constant rate from the analyte (A) to the analyte-specific enzyme (AOx), to the redox material (e.g., Os 3+ ), flows to the working electrode, thereby generating a steady-state current. If the working electrode is disconnected from the circuit, the flow of electrons from the redox polymer to the working electrode will cease, resulting in no current flowing through the circuit. However, the analyte will still undergo enzymatic oxidation, which in turn results in the reduction of the redox material (e.g., Os 3+ Reduced to Os 2+ This results in the redox material in reduced form (e.g. Os 2+ ) accumulates over time because electrons from the analyte (e -) is stored in the redox material. When the working electrode is reconnected to the circuit so that it remains at its original potential (voltage), the accumulation of reduced redox material will be oxidized, resulting in a large current spike. Then, when the redox system reaches steady state again, the current will decay back to the original amperometric current. This two-step process forms the basis of accumulation mode sensing: a first step, in which the working electrode of the sensor is disconnected or not connected to the circuit for a set period of time (also called the accumulation time), allowing the charge from the analyte to "accumulate" in the redox polymer, and a second step, in which the working electrode of the sensor is connected to the circuit after the accumulation time, allowing the accumulated charge to discharge and be measured as a spike.

[0129] In the example of three-electrode arrangement, there may be a sensing electrode (i.e., a working electrode with a pyruvic acid sensing layer), a reference electrode, and a counter electrode for accumulation mode sensing, wherein when the circuit is connected, the sensing electrode is maintained at a potential (voltage) sufficient to drive the redox reaction of the analyte under steady-state conditions. For example, for the pyruvic acid sensor described herein, the potential (voltage) sufficient to drive the redox reaction is +40mV relative to Ag / AgCl. When the circuit is not connected, the working electrode is electrically disconnected from the circuit so that the charge (e.g., electron) from the analyte can be stored in the redox material until the sensing electrode is reconnected to the circuit and the stored charge is measured. In one aspect, the counter electrode can be carbon (e.g., screen-printed carbon), and the reference electrode can be Ag / AgCl. In a two-electrode example, a sensing electrode and a second electrode (i.e., pair / reference electrode) serving as both the counter electrode and the reference electrode are used.

[0130] In some embodiments, the time period of described setting can be any suitable time period, and it allows to just accumulate enough electric charges to detect pyruvic acid once circuit reconnects.Conventionally, the time period of described setting can be about 30 seconds or longer (such as 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).Conventionally, the time period of described setting can be about 30 minutes or shorter (such as 25 minutes or shorter, 20 minutes or shorter, 15 minutes or shorter, 10 minutes or shorter, 8 minutes or shorter, 7 minutes or shorter, 6 minutes or shorter, 5 minutes or shorter, 4 minutes or shorter, 3 minutes or shorter, 2 minutes or shorter or 1 minute or shorter), to allow all pyruvic acids present at sensing electrode place to react completely. For example, the time period of the setting can be about 30 seconds or longer and about 30 minutes or shorter, about 1 to about 20 minutes, about 1 to about 15 minutes, about 1 to about 10 minutes, about 2 to about 8 minutes, about 2 to about 5 minutes or about 30 seconds or longer. In some embodiments, the time period of the setting can be about 30 seconds to about 1 hour, such as about 1 minute to about 45 minutes, such as about 2 minutes to about 30 minutes, such as about 5 minutes to about 15 minutes.

[0131] Thus, in some embodiments, a method of sensing pyruvate is provided, comprising: contacting a sample comprising pyruvate with a sensing electrode described herein; and detecting accumulated charge resulting from the reaction of pyruvate with pyruvate oxidase and a redox mediator.

[0132] In some embodiments, the sensors disclosed herein are configured to penetrate the skin of a subject. The sensor may include a component capable of penetrating the skin of a subject. For example, the component may be an insertable tip, tail, probe, or needle capable of penetrating the skin of a subject. In some embodiments, the methods disclosed herein are used to detect subcutaneous and / or interstitial pyruvate concentrations.

[0133] In one embodiment, the system (e.g., sensor) can further include a sensor tail (e.g., insertion tip, implantable portion) configured to penetrate (e.g., be implanted) into tissue, wherein the working electrode is disposed on the sensor tail (e.g., insertion tip, implantable portion). Typically, the sensor tail (e.g., insertion tip, implantable portion) can be of sufficient size and shape to be positioned below the surface of the tissue (e.g., penetrate the skin (dermis)) and enter the subcutaneous space and come into contact with the wearer's biological fluid (e.g., tissue fluid). In one example, the sensor tail (e.g., insertion tip, implantable portion) can have a length of about 5 mm, a width of about 0.6 mm, and a thickness of about 0.25 mm. Suitable tissues include, for example, skin, including the dermis, interstitial layer, and / or subcutaneous layer of the skin.

[0134] In one example, the electrode contacts are positioned on a first portion of the sensor that is located above the skin surface and extend to a location in the tail of the sensor (e.g., insertion tip, implantable portion). The working electrode, reference electrode, and counter electrode are located in a second portion of the sensor, typically at the bottom of the tail of the sensor (e.g., insertion tip, implantable portion). As described herein, the working electrode will include a pyruvate sensing layer and may optionally include a sensing layer for an analyte other than pyruvate.

[0135] The system (e.g., a sensor) may further be contained in a sensor housing that is configured to adhere 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 accommodate all necessary components of the sensor, such as circuits and power supplies for operating the sensor. In some embodiments, a power supply (e.g., a button cell) and / or active circuitry is not contained in the sensor housing. The processor may be communicatively coupled to the sensor, wherein the processor is physically located within the sensor housing or reader device. The power supply may include one or more batteries, which may be rechargeable or single-use disposable batteries. The power management circuit may regulate battery charging and power monitoring, boost power, or perform direct current (DC) conversion.

[0136] The present invention also relates to a method for detecting pyruvate. In one embodiment, the method may include contacting a sample containing pyruvate with a sensing electrode as described herein; and detecting the accumulated charge derived from the reaction of pyruvate with pyruvate oxidase and a redox mediator. In some embodiments, the method may include providing a sensing electrode as described herein, connecting the sensing electrode to a circuit to provide a steady state, disconnecting the sensing electrode from the circuit, contacting the sensing electrode with an analyte that may contain pyruvate, accumulating the charge derived from the reaction of pyruvate with a pyruvate-responsive enzyme (e.g., pyruvate oxidase) and a redox mediator over a set time period, connecting the sensing electrode to the circuit after the set time period; and measuring a signal from the accumulated charge.

[0137] As described herein, the set time period of the method can be any suitable time period that allows sufficient charge to build up to detect pyruvic acid once the circuit is reconnected. In one embodiment, the set time period can be 30 seconds or longer.

[0138] Typically, the method uses a system (such as a sensor) for measuring low concentrations of pyruvic acid as disclosed herein, and can be used for an in vivo monitoring system, wherein the system is positioned in a user (such as a patient, such as a human) in vivo, contacts the user's biological fluid and senses one or more analyte levels contained therein. The in vivo monitoring system may include one or more reader devices receiving the sensed analyte data from a sensor control device. The reader device can process and / or display analyte data or sensor data sensed in any quantity form to the user. In some embodiments, the reader device can be a mobile communication device, such as a dedicated reader device (configured to communicate with a sensor control device), which is optionally combined with a computer system, a mobile phone (such as WiFi or an internet-enabled smart phone), a tablet computer, a personal digital assistant (PDA), or a mobile intelligent wearable electronic component (such as smart glasses, smart glasses, watches, bracelets, or necklaces). For example, in U.S. Patent No. 11,371,957, a reader device is described to be configured to an in vivo monitoring system, and its disclosure is incorporated herein by reference in its entirety.

[0139] The reader 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 discrete chip or distributed across multiple different chips (as well as portions of multiple different chips). The processing circuitry may include a communications processor with onboard memory and an application processor with onboard memory. The reader device may further include RF communications 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 will be appreciated that other hardware and functionality may be included in the reader device.

[0140] Due to the association between sleep and physical and mental health and well-being, sleep monitoring is increasingly gaining attention. Pyruvic acid is related to sleep quality, wherein pyruvic acid levels increase during REM sleep cycles and decrease during wakefulness. Therefore, monitoring the pyruvic acid levels in a subject or a sample obtained from a subject can allow tracking or monitoring sleep (e.g., both sleep quality and sleep quantity). Information from sleep monitoring is useful for many individuals, such as those seeking to improve their sleep quality (e.g., individuals suffering from sleep disorders such as insomnia) and / or individuals suffering from mental or physical disorders characterized by poor sleep, wherein monitoring sleep quality can inform individual potential illnesses and efficacy or other treatments thereof.

[0141] Weight loss is another area where pyruvate monitoring is valuable. Pyruvate is involved in lipogenesis and fat metabolism in the body. Therefore, monitoring pyruvate levels in a subject or a sample obtained from a subject can provide information about the subject's health status. Monitoring pyruvate levels can inform dietary and lifestyle choices made by an individual.

[0142] Pyruvate and lactate levels also fluctuate during exercise. Therefore, monitoring pyruvate in a subject or a sample obtained from a subject can provide information about the subject's health and wellness, including recovery rate after exercise.

[0143] As described above, pyruvate sensing can be used to determine and monitor a variety of health and wellness conditions, including chronic, progressive diseases such as chronic obstructive pulmonary disease (COPD), obesity, diabetes, and aging; sleep quality and quantity; weight loss and diet; and fitness and recovery from exercise. Information about this can be provided by detecting and / or monitoring the presence, absence, or concentration of pyruvate in a sample from a subject.

[0144] Thus, in some embodiments, there is provided a method for detecting the presence, absence, or concentration of pyruvate in a sample, the method comprising: contacting the sample with an electrode as described herein; and One or more measurements characterized by pyruvate oxidation are made by the pyruvate-responsive enzyme in the sensing composition of the electrode.

[0145] In some embodiments, electrodes are present in a system and / or device as described herein.

[0146] Also provided is a use of a pyruvate-responsive enzyme (optionally pyruvate oxidase) and TPP for detecting the presence, absence, or concentration of pyruvate in a sample, wherein the weight ratio of the pyruvate-responsive enzyme to the TPP is from about 500:1 to about 1:1. The use can be further as described herein.

[0147] Also provided is a method for determining the health and / or well-being of a subject, the method comprising contacting a biological sample from the subject with a sensor as described herein; and determining the health and / or well-being of the subject. In some embodiments, determining the health and / or well-being of the subject comprises monitoring and / or detecting one or more chronic progressive diseases, such as chronic obstructive pulmonary disease (COPD), obesity, diabetes, and aging; sleep quality and quantity; weight loss and diet; and fitness and recovery from exercise.

[0148] The present disclosure is further illustrated by the following embodiments.

[0149] (1) A sensing composition comprising a pyruvate-responsive enzyme, thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD), albumin, and a redox mediator.

[0150] (2) The sensing composition according to embodiment 1, wherein the pyruvate-responsive enzyme comprises pyruvate oxidase.

[0151] (3) The sensing composition according to embodiment 1 or 2, wherein the pyruvate oxidase and TPP are present in the composition in a weight ratio of about 500:1 to about 1:1.

[0152] (4) The sensing composition according to any one of embodiments 1 to 3, wherein the albumin is bovine serum albumin.

[0153] (5) The sensing composition according to any one of embodiments 1 to 4, further comprising a pH buffer.

[0154] (6) The sensing composition according to any one of embodiments 1 to 5, wherein the redox mediator comprises a polymer and an electron transfer agent.

[0155] (7) The sensing composition according to embodiment 6, wherein the polymer comprises poly(4-vinylpyridine), poly(1-vinylimidazole), poly(thiophene), poly(aniline), poly(pyrrole) or poly(acetylene).

[0156] (8) The sensing composition according to embodiment 6, wherein the polymer comprises a polymer or copolymer repeating unit, wherein the polymer or copolymer repeating unit comprises at least one pendant pyridyl group, imidazole group, or both a pyridyl group and an imidazole group.

[0157] (9) The sensing composition according to any one of embodiments 6 to 8, wherein the electron transfer agent comprises a transition metal complex.

[0158] (10) The sensing composition according to embodiment 9, wherein the transition metal complex comprises osmium, ruthenium, iron, cobalt, vanadium or a combination thereof.

[0159] (11) The sensing composition according to embodiment 9 or 10, 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.

[0160] (12) The sensing composition according to any one of embodiments 1 to 11, wherein the redox mediator comprises an osmium complex bonded to a poly(4-vinylpyridine)-based polymer.

[0161] (13) The sensing composition according to any one of embodiments 1 to 12, further comprising a cross-linking agent.

[0162] (14) The sensing composition according to embodiment 13, wherein the cross-linking agent is polyepoxide, cyanuric chloride, N-hydroxysuccinimide, imidate, epichlorohydrin or a combination thereof.

[0163] (15) The sensing composition according to embodiment 13 or 14, wherein the cross-linking agent is polyethylene glycol diglycidyl ether (PEGDGE).

[0164] (16) The sensing composition according to any one of embodiments 1-15, wherein the pyruvate oxidase is attached to the redox mediator.

[0165] (17) A sensing electrode comprising a working electrode, wherein the working electrode comprises a pyruvic acid sensing layer formed from the sensing composition according to any one of embodiments 1 to 16.

[0166] (18) The sensing electrode according to embodiment 17, wherein the pyruvate sensing layer is continuous.

[0167] (19) The sensing electrode according to embodiment 17, wherein the pyruvate sensing layer is discontinuous.

[0168] (20) The sensing electrode according to any one of embodiments 17 to 19 further includes a film covering at least the pyruvic acid sensing layer.

[0169] (21) The sensing electrode according to embodiment 20, wherein the membrane is permeable to pyruvate.

[0170] (22) The sensing electrode according to embodiment 20 or 21, wherein the membrane has reduced permeability to TPP relative to pyruvate.

[0171] (23) A sensing electrode according to any one of embodiments 20-22, wherein the membrane comprises poly(4-vinylpyridine).

[0172] (24) A system for sensing pyruvate, comprising a working electrode, a sensing element disposed on at least a portion of the working electrode, and a circuit configured to connect to and disconnect from the working electrode, wherein the sensing element comprises the sensing composition according to any one of embodiments 1-15, and the sensing element is configured to accumulate charge derived from the reaction of pyruvate with pyruvate oxidase over a set time period.

[0173] (25) The system according to embodiment 24 further includes a sensor tail configured for implantation into tissue, wherein the working electrode is disposed on the sensor tail.

[0174] (26) A method for sensing pyruvate, the method comprising: providing a sensing electrode according to any one of embodiments 17-23; connecting the sensing electrode to a circuit to provide a stable state; disconnecting the sensing electrode from the circuit; contacting the sensing electrode with an analyte containing pyruvate; accumulating charge derived from the reaction of pyruvate with pyruvate oxidase and a redox mediator over a set time period; connecting the sensing electrode to a circuit after the set time period; and measuring a signal from the accumulated charge.

[0175] (27) The method according to embodiment 26, wherein the set time period is 30 seconds or longer.

[0176] (28) A method for preparing an electrode for sensing pyruvate, comprising disposing the sensing composition described herein (e.g., according to any one of Embodiments 1-16) on the surface of the electrode.

[0177] (29) A system comprising: a pyruvate-responsive enzyme, optionally pyruvate oxidase; TPP; and A sample comprising pyruvate; optionally wherein the pyruvate is present in the sample at a concentration of about 1 μM to about 1 mM; wherein the weight ratio of the pyruvate-responsive enzyme to TPP is about 500:1 to about 1:1.

[0178] (30) A method for detecting the presence, absence or concentration of pyruvic acid in a sample, comprising: contacting the sample with a sensing electrode as described herein (e.g., according to any one of Embodiments 17-23), optionally wherein the sensing electrode is comprised in a system according to Embodiment 29; and One or more measurements characterized by pyruvate oxidation are made by the pyruvate-responsive enzyme in the sensing composition of the electrode.

[0179] (31) A method for determining a subject's health and / or well-being, comprising: contacting a biological sample from the subject with a sensor described herein (e.g., according to any one of embodiments 1-16); and Determine the subject's health and / or well-being.

[0180] (32) Use of a pyruvate-responsive enzyme (optionally pyruvate oxidase) and TPP for detecting the presence, absence, or concentration of pyruvate in a sample, wherein the weight ratio of the pyruvate-responsive enzyme to TPP is from about 500:1 to about 1:1. Example

[0181] These examples are for illustrative purposes only, and the embodiments described herein should in no way be construed as being limited to these examples. Rather, the embodiments should be construed to encompass any and all variations that become evident as a result of the teachings provided herein. Example 1

[0182] Using a solution prepared as described in Table 1 below, a sensing layer containing pyruvate oxidase (POX) was deposited. FAD and TPP were present as cofactors together with phosphate buffer, bovine serum albumin (BSA), an osmium-based redox material called X7, and a crosslinker PEGDGE400. Two different concentrations of TPP were tested. One sample contained pyruvate oxidase (from Pseudomonas sp., Toyobo USA) and TPP ("1×TPP", corresponding to a molar ratio of TPP:POX of 3.1:1) at a weight ratio of 200:1, and the second sample contained 100 times more TPP than the first sample to provide a pyruvate oxidase and TPP at a weight ratio of 1.73:1 ("100×TPP", corresponding to a molar ratio of TPP:POX of 353.4:1). Each solution of the sensing composition was applied to a carbon electrode to provide a continuous pyruvate sensing layer. Each sensing layer was then cured overnight at 25°C.

[0183] A mass transfer limiting membrane was coated onto the electrode with the pyruvic acid sensing layer using an alcohol buffer solution of the materials specified in Table 2. The membrane was deposited onto the pyruvic acid sensing layer prepared above using dip coating. The membrane solution was deposited using two dips, with a wait time of approximately 10 minutes between dips. After dip coating, the membrane was cured at 25°C for 24 hours and then at 56°C for 48 hours in a dried vial to provide the sensing electrode. Spray coating, screen printing, or similar processes can also be used to deposit the mass transfer limiting membrane onto at least the sensing layer. The membrane thickness was measured to be approximately 18 μm. Table 1 *50 mM phosphate, 50 mM NaCl, 10 nM MgCl2, pH 6.0 Table 2 Reagents final EtOH / HEPES buffer (80 / 20) mg / mL Poly(4-vinylpyridine) (PVP) 96 PEGDGE400 4 Example 2

[0184] The sensing electrode prepared in Example 1 was tested in the system to measure the detection signal (nA) at various pyruvic acid concentrations over time. The system was tested using an accumulated charge method. In particular, the sensing electrode was connected to a circuit to provide a stable state. The sensing electrode was then disconnected from the circuit. The sensing electrode was contacted with various samples containing pyruvic acid (0 μM, 50 μM, 100 μM, 150 μM, and 200 μM) at different concentrations. While the sensing electrode was still disconnected from the circuit, the charge generated by the reaction of pyruvic acid with pyruvic oxidase and redox mediator (X7) accumulated for a set 30-minute period. The sensing electrode was then reconnected to the circuit after the set period of time; and the signal from the accumulated charge was measured.

[0185] Figure 2 and 3 The results for the 1xTPP composition and the 100xTPP composition are shown separately. Using these data, the accumulated charge (measured in nanocoulombs) was plotted against the pyruvate concentration (μM), as shown in FIG. Figure 4 shown. Figure 4 It was shown that when the amount of TPP was increased relative to pyruvate oxidase, the pyruvate signal was improved using the accumulation detection technique.

[0186] It should be understood that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more, but not all, exemplary embodiments of the present invention as contemplated by the inventor(s), and thus, are not intended to limit the present invention and the appended claims in any way.

[0187] The present disclosure has been described above with reference to functional building blocks that illustrate the implementation of specific functions and their relationships. For ease of description, the boundaries of these functional building blocks have been arbitrarily defined herein. Alternative boundaries may be defined as long as the specified functions and their relationships are appropriately performed.

[0188] The foregoing description of the specific embodiments will fully reveal the general nature of the invention so that others can, by applying knowledge within the technical scope of the art, easily modify and / or adapt these specific embodiments for various applications without departing from the general concept of the invention, without undue experimentation. Therefore, based on the teachings and guidance given herein, these adjustments and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments. It should be understood that the wording or terminology herein is for descriptive and not limiting purposes, so that the terms or wording of this specification will be interpreted by those skilled in the art based on the teachings and guidance.

[0189] The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0190] The claims in this application differ from those in the parent application or other related applications. Therefore, the applicant revokes any waiver of claim scope in the parent application or any predecessor application related to this application. Therefore, the examiner is advised that any such prior disclaimers and cited references (which were raised to avoid) may need to be re-examined. Furthermore, the examiner is reminded that any disclaimers made in this application should not be read into or against the parent application.

Claims

1. A sensing composition comprising a pyruvate-responsive enzyme, thiamine pyrophosphate (TPP), optionally flavin adenine dinucleotide (FAD), a stabilizer, and a redox mediator.

2. The sensing composition of claim 1, wherein the pyruvate-responsive enzyme comprises pyruvate oxidase.

3. The sensing composition of claim 1 or 2, wherein the pyruvate oxidase and TPP are present in the composition in a weight ratio of about 500:1 to about 1:

1.

4. The sensing composition of claim 3, wherein the pyruvate oxidase and TPP are present in the composition in a weight ratio of about 100:1 to about 1:1, or about 50:1 to about 1:1, or about 10:1 to about 1:

1.

5. The sensing composition according to any one of claims 1 to 4, wherein the stabilizer is albumin. The sensing composition according to any one of claims 1 to 5 , further comprising a pH buffer.

7. The sensing composition of any one of claims 1-6, wherein the redox mediator comprises a polymer and an electron transfer agent.

8. The sensing composition of claim 7, wherein the polymer comprises poly(4-vinylpyridine), poly(1-vinylimidazole), poly(thiophene), poly(aniline), poly(pyrrole), or poly(acetylene).

9. The sensing composition of claim 7, wherein the polymer comprises a polymer or copolymer repeat unit comprising at least one pendant pyridyl group, imidazole group, or both a pyridyl group and an imidazole group.

10. The sensing composition of any one of claims 7 to 9, wherein the electron transfer agent comprises a transition metal complex.

11. The sensing composition of claim 10, wherein the transition metal complex comprises osmium, ruthenium, iron, cobalt, vanadium, or a combination thereof.

12. The sensing composition of claim 10 or 11, 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.

13. The sensing composition of any one of claims 1 to 12, wherein the redox mediator comprises an osmium complex bonded to a poly(4-vinylpyridine)-based polymer.

14. The sensing composition according to any one of claims 1 to 13, further comprising a cross-linking agent.

15. The sensing composition of claim 14, wherein the cross-linking agent is polyepoxide, cyanuric chloride, N-hydroxysuccinimide, imidate, epichlorohydrin, or a combination thereof.

16. The sensing composition according to claim 14 or 15, wherein the cross-linking agent is polyethylene glycol diglycidyl ether (PEGDGE).

17. The sensing composition of any one of claims 1 to 16, wherein the pyruvate oxidase is attached to the redox mediator.

18. A sensing electrode comprising a working electrode, wherein the working electrode comprises a pyruvate sensing layer, wherein the pyruvate sensing layer comprises the sensing composition according to any one of claims 1 to 17. The sensing electrode according to claim 18 , wherein the pyruvate sensing layer is continuous.

20. The sensing electrode of claim 18, wherein the pyruvate sensing layer is discontinuous.

21. The sensing electrode according to any one of claims 18 to 20, further comprising a film covering at least the pyruvic acid sensing layer.

22. The sensing electrode of claim 21, wherein the membrane is permeable to pyruvate.

23. The sensing electrode of claim 21 or 22, wherein the membrane has a reduced permeability to TPP relative to pyruvate.

24. The sensing electrode of any one of claims 21-23, wherein the membrane comprises poly(4-vinylpyridine).

25. A system for sensing pyruvate, comprising: An electrode comprising the sensing composition according to any one of claims 1 to 17; and A circuit is configured for electrochemically detecting pyruvate at the electrode.

26. A system for sensing pyruvate, comprising: Working electrode, a sensing element disposed on at least a portion of the working electrode, and a circuit configured to connect and disconnect with the working electrode, in The sensing element comprises the sensing composition according to any one of claims 1 to 17, and The sensing element is configured to accumulate charge resulting from the reaction of pyruvate with pyruvate oxidase over a set period of time.

27. The system of claim 25 or 26, further comprising an implantable portion configured for insertion into tissue, wherein the working electrode is disposed on the implantable portion.

28. A method for sensing pyruvate, comprising: contacting a sample comprising pyruvate with a sensing electrode according to any one of claims 18 to 24; and The accumulated charge originating from the reaction of pyruvate with pyruvate oxidase and a redox mediator is detected.

29. A method for sensing pyruvate, comprising: Providing a sensing electrode according to any one of claims 18 to 24; connecting the sensing electrode to a circuit to provide a stable state; disconnecting the sensing electrode from the circuit; contacting the sensing electrode with an analyte comprising pyruvate; accumulating charge derived from the reaction of pyruvate with pyruvate oxidase and a redox mediator over a set period of time; After the set time period, connecting the sensing electrodes to a circuit, and A signal from the accumulated charge is measured.

30. The method of claim 29, wherein the set time period is 30 seconds or longer.

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