Analyte sensor and sensing method for detecting alcohol

Abstract

Devices, methods and systems for detecting an alcohol concentration in an individual, in particular in an individual. An alcohol sensing composition includes at least one alcohol responsive active region comprising a synergistic enzyme system having at least a first enzyme and a second enzyme capable of synergistic action to facilitate alcohol detection. At least one enzyme in the synergistic enzyme system is a ketoreductase.

Description

[0001] This application is a divisional application of CN202080070792.3.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 894,306, filed on August 30, 2019, entitled “ANALYTE SENSOR AND SENSING METHOD FOR DETECTING ALCOHOL,” the entire contents of which are incorporated herein by reference.

[0004] background

[0005] The detection of various analytes in an individual can sometimes be crucial to 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.

[0006] Analyte monitoring in an individual can be performed periodically or continuously over a period of time. Periodic analyte monitoring can be performed by extracting a body fluid, such as a blood sample, at one or more time intervals and analyzing it in vitro. Continuous analyte monitoring can be performed using one or more sensors that are at least partially implanted in an individual's tissue, such as the skin, subcutaneously, or intravenously, so that analysis can be performed in vivo. For example, an implantable sensor can collect analyte data at any specified rate based on the individual's specific health needs and / or previously measured analyte levels.

[0007] Any analyte may be suitable for in vivo analysis, provided that the appropriate chemical can be identified to sense the analyte. Indeed, in vivo amperometric sensors configured for measuring glucose have been developed and improved in recent years. It may also be desirable to monitor other analytes that are commonly affected by physiological disorders, including but not limited to lactate, oxygen, pH, A1c, ketones, drug levels, etc.

[0008] Another analyte that may be particularly important to the medical welfare of an individual is alcohol level. In fact, information related to alcohol levels in an individual can be used to predict or monitor the level of another analyte of interest. For example, alcohol may alter the glycemic control of an individual, whose blood sugar levels are naturally dysregulated or lack homeostasis without intervention, which may be harmful to the individual. Other analytes that may be dysregulated by alcohol include, among others, triglycerides (e.g., related to heart disease, stroke, blood pressure, obesity), gamma-glutamyl transferase (GGT) (e.g., related to cancer, hepatitis, bone disease), and cortisol (e.g., related to stress, inflammation). Therefore, it is necessary to monitor the alcohol level of an individual.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The following drawings are included to illustrate certain aspects of the present disclosure and should not be considered to be exclusive embodiments. The disclosed subject matter is capable of considerable modification, alteration, combination, and equivalents in form and function without departing from the scope of the present disclosure.

[0011] Figure 1 A diagram of an exemplary sensing system that may incorporate an analyte sensor of the present disclosure is shown.

[0012] Figure 2A A diagram showing an exemplary two-electrode analyte sensor configuration with a single working electrode that is compatible for use in some embodiments of the present disclosure is shown. Figure 2B and 2C A diagram showing an exemplary three-electrode analyte sensor configuration with a single working electrode compatible for use in some embodiments of the present disclosure is shown.

[0013] Figure 3 A diagram of an exemplary analyte sensor configuration having two working electrodes, one reference electrode, and one counter electrode compatible for use in some embodiments of the present disclosure is shown.

[0014] Figure 4A Shown is a synergistic enzyme system related to alcohol detection using ketoreductase, nicotinamide adenine dinucleotide, and diaphorase located on a working electrode according to various embodiments of the present disclosure.

[0015] Figure 4B Shown is a coordinated enzyme system associated with alcohol detection using ketoreductase, nicotinamide adenine dinucleotide phosphate, and diaphorase located on a working electrode according to various embodiments of the present disclosure.

[0016] Figures 5A-5D Shown are graphical representations of the responses of various KRT-containing sensors to various ethanol concentrations. Figure 5E Shows the Figures 5A-5C Graph of the response of four KRT-containing sensors at 4 mM ethanol, showing the change (Δ) in the current response from baseline (no EtOH exposure) to 4 mM ethanol.

[0017] Fig. 6A Shown are graphical representations of the responses of various KRT-containing sensors to various ethanol concentrations. Figure 6B Shows Fig. 6A Linear sensitivity response to the ethanol concentration measured in . Figure 6C Shows Fig. 6A Figure 2 is a graphical representation of the stability response of various sensors containing KRT.

[0018] Figure 7Shown is a graphical representation of the stability responses of various KRT-containing sensors and an ADH control sensor to aldehyde exposure.

[0019] Figure 8 Shown are graphical representations of the results of kinetic assays of various KRTs compared to an ADH control.

[0020] Fig.9A and 9B Shown is a graphical representation of the results of kinetic assays of two KRTs in different alcohol types.

[0021] Fig. 10A and 10B A graphical representation of the response of two KRT-containing sensors of various compositions to IPA is shown.

[0022] Fig.11A and 11B Shown is a graphical representation of the stability response of various KRT-containing sensors with different additional polymer and cross-linker compositions.

[0023] Fig. 12A Shown is a graphical representation of the response of a particular KRT-containing sensor comprising a particular polymer composition to various ethanol concentrations. Fig. 12B Shows Fig. 12A The linear sensitivity response to the ethanol concentration measured in . ADH control refers to the alcohol dehydrogenase (ADH) from the signal; Figures 12A-12B The linearity of ADH and KRT A15 was compared.

[0024] Fig.13 Shown is a graphical representation of the stability response of a particular KRT-containing sensor comprising a particular polymer composition. Fig.13 The stability of ADH and KRT A15 beakers was compared.

[0025] Fig.14 Shown is a graphical representation of the stability response of a particular KRT-containing sensor with an immersed membrane in two different solvents.

[0026] Fig.15 Shown is a graphical representation of the stability response of various KRT-containing sensors with different membrane compositions.

[0027] Fig.16 Shown is a graphical representation of the stability response of various KRT-containing sensors with different membrane and cross-linker compositions.

[0028] Details

[0029] The present disclosure generally describes analyte sensors and methods for detecting alcohol levels in vivo using enzymes.

[0030] Analyte sensors are commonly used to detect a variety of analytes, often using enzymes with a particular specificity for a particular substrate. For example, glucose-responsive analyte sensors represent a well-studied and still developing field to help diabetic individuals better manage their health. However, other in vivo analytes are also important and may have significant value in determining and / or monitoring an individual's health, including determining and / or monitoring the effects of an analyte of interest on dysregulation of other analytes.

[0031] The alcohol concentration in the body of an individual can vary significantly based on the amount of alcohol consumed, alcohol intoxication, and / or based on various physiological factors. For example, the ability of an individual to metabolize alcohol varies, resulting in different alcohol levels between individuals who consume the same amount of alcohol per body weight. In fact, the equilibrium concentration of alcohol in a tissue depends at least on the water content, blood flow rate, and the mass of the tissue. Alcohol can pass through biological membranes, so alcohol can easily flow from the bloodstream to all tissues and body fluids, and the flow is proportional to the water content of the tissues and body fluids. In addition, in rare cases, an individual may produce a large amount of alcohol by endogenous fermentation in the digestive system without any drinking. As described above, the presence of certain concentrations of alcohol may additionally affect the function of one or more other analytes of an individual, thereby further affecting the health of the individual.

[0032] Current alcohol measurements are performed by collecting physical blood samples, urine samples, saliva samples, or using breath tests. However, such measurements are static in time and, in some cases, may be plagued by false positive or false negative results and, therefore, are at least occasionally inaccurate. In contrast, the present disclosure provides an analyte sensor that responds to alcohol over time to facilitate health management. As used herein, the term "alcohol" and its grammatical variants refer to any primary, secondary, or tertiary alcohol. For example, the alcohol sensor of the present disclosure can detect ethanol, methanol, butanol, propanol, isopropanol, and the like, and any combination thereof.

[0033] The alcohol sensors described herein are responsive to alcohol levels in the body and are capable of providing a "continuous" measurement of the alcohol levels. That is, the alcohol sensors described herein are operable to provide multiple alcohol concentration measurements over an extended (continuous) period of time (e.g., seconds, minutes, or hours to days, weeks, or months).

[0034] Alcohol sensors can provide many advantages for monitoring the dynamic levels of alcohol associated with the resulting physiological conditions and the effects on other analytes of interest. For example, the sensors of the present disclosure utilize specialized enzymatic reactions, including one or more ketoreductases (referred to herein as "KRTs"). The sensing chemistry comprising KRTs described herein advantageously overcomes various obstacles associated with typical detection of alcohol levels in individuals, including overcoming enzyme inhibition caused by one or more enzymatic reaction products of the sensing chemistry as discussed in more detail below. By overcoming enzyme inhibition, the alcohol sensors of the present disclosure can, in particular, allow the use of a single mass limiting membrane and the manufacture of more cost-effective sensors that require less complex sensing area configurations without affecting effectiveness. The chemistry comprising KRTs further advantageously accepts primary and secondary alcohols, including a particularly unexpected affinity for secondary alcohol detection.

[0035] Individuals wearing the continuous alcohol sensors described herein can access real-time alcohol level information to make various decisions based on the information, such as whether it is safe to drive, whether their glucose or other analyte levels may be disordered based on alcohol levels and therefore require action, etc., and any combination thereof. In addition, the alcohol sensors of the present disclosure can be used to monitor, test and / or assess the alcohol levels of individuals suffering from alcohol abuse, alcoholism or addiction. In doing so, the health of these individuals can be monitored by the individuals themselves or by health practitioners or law enforcement professionals. Sensor electronics and processing algorithms associated with operating the sensors and sensing systems described herein (including their display units or their devices) can also provide guidance, instructions, recommendations and / or outputs about alcohol concentrations and recommended actions based thereon. Suitable processing algorithms, processors, memories, electronic components, etc. can reside in any trusted computer system, remote terminal, cloud server, reader device and / or the housing of the sensor itself. Instructions, recommendations, outputs, etc. can be displayed on a suitable display unit or device that electronically communicates with one or more of these components. The display unit or device can be a dedicated reader device or a personal communication device, such as a mobile phone (e.g., or Android device). Alternatively, the display unit or device may be a third-party server, cloud server, or remote terminal that communicates with various software-related and healthcare-related applications (e.g., personal health monitors). Suitable remote terminals, cloud servers, etc. may further relay the output to related auxiliary devices, such as smart home devices, wearable technology, personal health monitors, etc., or a combination thereof.

[0036] The present disclosure further describes a sensing system comprising one or more alcohol sensors. The system may include various sensing components, such as a processor and / or coded instructions (algorithms) therein, which are suitable for processing sensor data received from an alcohol sensor and determining one or more alcohol concentrations therefrom. In some embodiments, the processor and / or coded instructions may then analyze alcohol concentration to determine one or more recommendations based on a specific alcohol concentration. For example, alcohol concentration or other amounts that may be derived from alcohol concentration at a specific evaluation time may be used to suggest whether another health analyte may be negatively impacted. In this case, the processor and / or coded instructions may suggest various ways in which an individual may react based on detected alcohol concentration, including immediate and future action suggestions. For example, according to the specific alcohol concentration of an individual, the recommendation may be whether to drive a motor vehicle, whether to perform an action to compensate for another analyte affected (e.g., if the analyte is glucose, whether to consume carbohydrates or inject insulin), whether to seek medical attention, etc., and any combination thereof.

[0037] Before describing the alcohol sensor of the present disclosure in more detail, a brief overview of suitable in vivo analyte sensor configurations and sensor systems employing analyte sensors will first be provided so that the embodiments of the present disclosure can be better understood. It should be understood that, according to various embodiments of the present disclosure, any sensor system and analyte sensor configuration described below can feature one or more enzymes for detecting alcohol and generally for detecting alcohol in vivo.

[0038] Figure 1Shown is a diagram of an exemplary sensing system of an alcohol sensor that can be combined with the present disclosure. As shown, sensing system 100 includes sensor control device 102 and reader device 120, which are configured to communicate with each other through local communication paths or links, and local communication paths or links can be wired or wireless, unidirectional or bidirectional and encrypted or non-encrypted. According to some embodiments, reader device 120 can be configured to view the analyte concentration determined by sensor 104 or a processor associated therewith and an output medium for alarm or notification and allowing one or more user inputs. Reader device 120 can be a multi-function smart phone or a dedicated electronic reader instrument. Although only one reader device 120 is shown, multiple reader devices 120 can be present in some cases. Reader device 120 can also communicate with remote terminal 170 and / or trusted computer system 180 through communication path / link 141 and / or 142 respectively, and communication path / link 141 and / or 142 can also be wired or wireless, unidirectional or bidirectional and encrypted or non-encrypted. Reader device 120 may also or alternatively communicate with network 150 (e.g., a mobile phone network, the Internet, or a cloud server) via communication path / link 151. Network 150 may further be communicatively coupled to remote terminal 170 via communication path / link 152 and / or to trusted computer system 180 via communication path / link 153. Alternatively or additionally, sensor 104 may communicate directly with remote terminal 170 and / or trusted computer system 180 without an intervening reader device 120. For example, according to some embodiments, sensor 104 may communicate with remote terminal 170 and / or trusted computer system 180 via a direct communication link to network 150, as described in U.S. Patent Application Publication 2011 / 0213225 and incorporated herein by reference in its entirety.

[0039] Any suitable electronic communication protocol may be used for each communication path or link, such as near field communication (NFC), radio frequency identification (RFID), or low energy consumption Protocol, WiFi, etc. According to some embodiments, the remote terminal 170 and / or the trusted computer system 180 may be accessed by individuals other than the primary user who are interested in the user's alcohol level. The reader device 120 may include a display 122 and an optional input component 121. The display 122 may include, for example, a touch screen interface for outputting information related to the sensor control device and inputting information by a user.

[0040] The sensor control device 102 includes a sensor housing 103, which can house circuitry and a power source for operating the sensor 104. Optionally, the power source and / or active circuitry can be omitted. A processor (not shown) can be communicatively coupled to the sensor 104, wherein the processor is physically located within the sensor housing 103 or the reader device 120. According to some embodiments, the sensor 104 protrudes from the underside of the sensor housing 103 and extends through an adhesive layer 105, which is suitable for adhering the sensor housing 103 to a tissue surface, such as skin.

[0041] Sensor 104 is suitable for at least partially inserting into the tissue of interest, such as in the dermis or subcutaneous layer of the skin. Sensor 104 may include a sensor tail of sufficient length to be inserted into the desired depth in a given tissue. The sensor tail may include at least one working electrode and one or more active areas (sensing areas / sensing points or sensing layers) located on at least one working electrode, which are active for sensing alcohol (or in some cases one or more additional analytes). In some embodiments, the active area is in the form of one or more discrete points (e.g., one point to about 10 points or more points), and its size range may be from about 0.01mm 2 To about 1mm 2 , including any values ​​and subsets therebetween, although larger or smaller individual active region points are also contemplated herein.

[0042] According to one or more embodiments of the present disclosure, one or more active regions may include one or more enzymes to facilitate at least alcohol detection. The active region may include a polymeric material to which, in some embodiments, one or more (or all) enzymes are chemically bonded (e.g., covalently bonded, ionically bonded, etc.) or otherwise fixed (e.g., not bound to a matrix) according to some embodiments. In some embodiments, each active region may be coated with a mass-restricted or biocompatible membrane and / or further include an electron transfer agent to facilitate at least alcohol detection.

[0043] In various embodiments of the present disclosure, at least the alcohol level in any biological fluid of interest can be monitored, such as dermal fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, etc. In specific embodiments, the analyte sensor of the present disclosure can be adapted to measure dermal fluid or interstitial fluid to determine alcohol concentration in vivo.

[0044] Continue to refer Figure 1In some embodiments, the sensor control unit 102 can automatically forward the data obtained by the sensor 104 to the reader device 120. For example, the alcohol concentration data can be automatically and periodically transmitted, such as at a certain frequency when the data is obtained or after a certain time period has passed, and the data is stored in the memory until the transmission (for example, every few seconds, every minute, five minutes or other predetermined time period). In other embodiments, the sensor control unit 102 can communicate with the reader device 120 in a non-automatic manner and not according to a set schedule. For example, when the sensor electronic device is brought into the communication range of the reader device 120, NFC or RFID technology can be used to communicate data from the sensor control unit 102. Before communicating to the reader device 120, the data can remain stored in the memory of the sensor control device 102. Therefore, the patient does not have to keep in close proximity with the reader device 120 all the time, but can upload data at a convenient time. In other embodiments, a combination of automatic and non-automatic data transmission can be achieved. For example, data transmission can continue on an automatic basis until the reader device 120 is no longer in the communication range of the sensor control unit 102. Although automatic and non-automatic data transmission from the sensor control unit 102 has been described with reference to the reader device 120, such transmission mechanisms are equally applicable to the remote terminal 170 and / or the trusted computer system 180 without departing from the scope of the present disclosure.

[0045] The introducer may exist briefly to facilitate the introduction of sensor 104 into the tissue. In an exemplary embodiment, the introducer may include a needle or similar sharp. It should be appreciated that other types of introducers, such as a sheath or a blade, may be present in alternative embodiments. More specifically, before tissue insertion, a needle or other introducer may reside briefly near sensor 104 or exist simultaneously with sensor 104, and then be withdrawn subsequently. When present, a needle or other introducer may facilitate the insertion of sensor 104 into the tissue by opening an entry path for sensor 104 to follow. For example, according to one or more embodiments, as a path into the dermis, a needle may facilitate penetration of the epidermis to allow the implantation of sensor 104 to occur. After opening the entry path, the needle or other introducer may be withdrawn so that there is no risk of sharps. In an exemplary embodiment, the cross-section of a suitable needle may be solid or hollow, beveled or non-beveled, and / or circular or non-circular. In more specific embodiments, suitable needles can be comparable to acupuncture needles in cross-sectional diameter and / or tip design, which can have a cross-sectional diameter of about 150 microns to about 300 microns (e.g., about 250 microns). However, it should be appreciated that suitable needles can have larger or smaller cross-sectional diameters if desired for a particular application.

[0046] In some embodiments, the tip of the needle or introducer (when present) may be angled on the end of the sensor 104 so that the needle or introducer first penetrates the tissue and opens an access path for the sensor 104. In other exemplary embodiments, the sensor 104 may reside within a lumen or groove of the needle or inserter, wherein the needle similarly opens an access path for the sensor 104. In either case, the needle is subsequently withdrawn after facilitating sensor insertion.

[0047] The alcohol sensors disclosed herein can have an active area located on a single working electrode (e.g., located on one or both sides of a single working electrode) or on two or more separate working electrodes (e.g., located on one or both sides of two or more separate working electrodes). According to various embodiments of the present disclosure and as further described herein, a single working electrode sensor configuration can employ a two-electrode or three-electrode detection pattern. Figures 2A-2C Sensor configurations featuring a single working electrode are described. Each of these sensor configurations can be suitably combined with one or more alcohol-responsive active regions according to various embodiments of the present disclosure. Figure 3 4 and 5 describe sensor configurations featuring multiple working electrodes. When multiple working electrodes are present, one or more alcohol responsive active regions, one or more (or all) of the multiple working electrodes, or one of the working electrodes can be used to simultaneously detect another analyte of interest while detecting alcohol levels.

[0048] When there is a single working electrode in the alcohol sensor of the present disclosure, the three-electrode detection pattern may include a working electrode, a counter electrode and a reference electrode. The related two-electrode detection pattern may include a working electrode and a second electrode, wherein the second electrode is used as one of the counter electrode or the reference electrode, or is used as the counter electrode and the reference electrode (i.e., the counter electrode / reference electrode) at the same time. In the two-electrode and three-electrode detection patterns, one or more active areas of the alcohol sensor may be in contact with the working electrode. One or more active areas may contain one or more enzymes according to the embodiments of the present disclosure. In some embodiments, various electrodes may be at least partially stacked (layered) on top of each other. In some or other embodiments, various electrodes may be laterally spaced apart from each other on the tail of the sensor. Similarly, the related active areas on each electrode may be vertically stacked on top of each other or laterally spaced apart. In either case, the various electrodes may be electrically insulated from each other by dielectric materials or similar insulators.

[0049] Figure 2AFigures of exemplary dual-electrode sensor configurations with a single working electrode compatible with alcohol detection according to embodiments of the present disclosure are shown. As shown, sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter electrode / reference electrode 216. Alternatively, working electrode 214 and counter electrode / reference electrode 216 may be located on the same side of substrate 212, with dielectric material inserted therebetween (configuration not shown). Active region 218 is disposed on at least a portion of working electrode 214 as at least one layer. In various embodiments, active region 218 may include multiple points or a single point configured to detect one or more analytes of interest. In general, one or more enzymes may be present in active region 218 (i.e., in a single point or multiple points).

[0050] Still reference Figure 2A According to some embodiments, the membrane 220 covers at least the active area 218 and may optionally cover some or all of the working electrode 214 and / or the counter / reference electrode 216, or the entire analyte sensor 200. One or both faces of the analyte sensor 200 may be covered with the membrane 220. The membrane 220 may include one or more polymeric membrane materials having the ability to restrict analyte flux to the active area 218 and / or biocompatibility capabilities. The sensor 200 may be used to determine at least alcohol by any of coulometric, amperometric, voltammetric, potentiometric electrochemical, or iontophoretic (including reverse iontophoretic) detection techniques.

[0051] Figure 2B and 2C A diagram showing an exemplary three-electrode analyte sensor configuration having a single working electrode compatible for use in embodiments of the present disclosure. In addition to including an additional electrode (represented as electrode 217 in sensors 201 and 202 ( Figure 2B and 2C )) In addition, a three-electrode analyte sensor configuration using a single working electrode can be similar to Figure 2A The configuration shown in sensor 200 in FIG. 2 is shown. Using additional electrode 217, counter electrode / reference electrode 216 can then be used as a counter electrode or reference electrode, and additional electrode 217 performs another electrode function that is not otherwise considered. Working electrode 214 continues to perform its original function. Additional electrode 217 can be disposed on working electrode 214 or electrode 216 with a separator layer of dielectric material therebetween. For example, as Figure 2B As shown, dielectric layers 219a, 219b, and 219c separate electrodes 214, 216, and 217 from each other to provide electrical insulation. Figure 2CAs shown, at least one of the electrodes 214, 216, and 217 may be located on opposite sides of the substrate 212. Thus, in some embodiments, the electrode 214 (working electrode) and the electrode 216 (counter electrode) may be located on opposite sides of the substrate 212, wherein the electrode 217 (reference electrode) is located on one of the electrodes 214 or 216 and is separated therefrom by a dielectric material. Alternatively, in some embodiments, the electrode 214 (working electrode) and the electrode 216 (reference electrode) may be located on opposite sides of the substrate 212, wherein the electrode 217 (counter electrode) is located on one of the electrodes 214 or 216 and is separated therefrom by a dielectric material. In yet other embodiments, the reference electrode and the counter electrode may be located on one side of the substrate 212, while the working electrode is located on the opposite side. Optionally, a reference material layer 230 (e.g., Ag / AgCl) may be present on the electrode 217, wherein the location of the reference material layer 230 is not limited Figure 2B and 2C The location depicted in .

[0052] and Figure 2A As with the sensor 200 shown in FIG. 1 , the active area 218 in the analyte sensors 201 and 202 may include multiple spots or a single spot configured to detect at least alcohol. In general, one or more enzymes may be present in the active area 218 of the sensors 201 and 202. In addition, the analyte sensors 201 and 202 may be used to analyze at least alcohol by any of coulometric, amperometric, voltammetric, potentiometric electrochemical, or iontophoretic detection techniques.

[0053] Continue to refer Figure 2A and 2B , and like sensor 200, membrane 220 may also cover active area 218 and other sensor components in sensors 201 and 202. In some embodiments, additional electrode 217 may be covered with membrane 220. Although Figure 2B and 2C All electrodes 214, 216, and 217 have been depicted as being covered with a film 220, but it should be understood that in some embodiments only the working electrode 214 may be covered or in other embodiments only the working electrode 214 and another electrode may be covered. In addition, the thickness of the film 220 at each of the electrodes 214, 216, and / or 217 may be the same or different, and the amount of surface area covered by the film 220 at each of the electrodes 214, 216, and / or 217 may be the same or different. As in a dual electrode analyte sensor configuration ( Figure 2A ), one or both faces of the analyte sensors 201 and 202 may be Figure 2B and 2C In the sensor configuration, the membrane 220 may be covered or the entire analyte sensor 201 and 202 may be covered. Figure 2B and 2C The three-electrode sensor configuration shown in should be understood to be non-limiting to the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of the present disclosure.

[0054] Figure 3 A schematic diagram of an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode compatible for use with the alcohol sensor described in the present disclosure is shown. Figure 3 As shown, the sensor 300 includes working electrodes 304 and 306 disposed on opposite surfaces of a substrate 302. An active region 310 is disposed on the surface of the working electrode 304, and an active region 312 is disposed on the surface of the working electrode 306. In general, one or more enzymes may be present in active regions 310 and 312 configured to detect at least alcohol. For example, both active regions 310 and 312 may be configured to detect alcohol concentration. Alternatively, one of the active regions 310 or 312 may be configured to detect alcohol concentration, while the other active region is configured to detect another analyte of interest (e.g., glucose, lactic acid, etc.). The counter electrode 320 is electrically insulated from the working electrode 304 by a dielectric layer 322, while the reference electrode 321 is electrically insulated from the working electrode 306 by a dielectric layer 323. Outer dielectric layers 330 and 332 are located on the reference electrode 321 and the counter electrode 320, respectively. According to various embodiments, the film 340 may cover at least the active regions 310 and 312. Other components of analyte sensor 300 may also be covered with membrane 340, and as described above, one or both faces of analyte sensor 300, or a portion thereof, may be covered with membrane 340. As with analyte sensors 200, 201, and 202, sensor 300 may be used to measure at least alcohol by any of coulometric, amperometric, voltammetric, potentiometric electrochemical, or iontophoretic detection techniques.

[0055] Having multiple working electrodes and being different from Figure 3 The features of the alternative sensor configurations shown may have counter electrodes / reference electrodes that are not separate counter and reference electrodes 320 and 321, and / or feature layer and / or membrane arrangements that are different from those explicitly depicted. For example, the positioning of the counter electrode 320 and the reference electrode 321 may be different from Figure 3 In addition, working electrodes 304 and 306 do not have to be Figure 3 The illustrated approach resides on opposite sides of substrate 302 .

[0056] Although the above description related to Figures 2 and 3 is mainly directed to an analyte sensor configuration with two working electrodes, it should be understood that more than two working electrodes can be successfully included through the expansion of the disclosure herein. Additional working electrodes can allow additional active areas and corresponding sensing capabilities to be imparted to such sensors having such features.

[0057] In addition, although Fig. 2 and 3 show a planar substrate (e.g., relatively flat) with a conductive structure (e.g., electrode) and an active area disposed thereon, it should be understood that the analyte sensor used in the embodiments of the present disclosure can have various other configurations without departing from the scope of the present disclosure. For example, the substrate can be substantially non-planar (e.g., relatively curved, semi-hemispherical or spherical), cylindrical, spiral, other irregular shapes, and any combination thereof. Similarly, two or more electrodes can be substantially non-planar (e.g., relatively curved, semi-hemispherical or spherical), cylindrical, spiral, other irregular shapes, and any combination thereof. The electrodes can be layered, concentric or otherwise arranged, and are generally insulated by one or more insulating regions. The sensing region disposed on at least one working electrode can further cover at least a portion (or all) of the working electrode as a single layer or as a discrete region of various shapes, such as square, circular, semi-circular, arcuate, rectangular, polygonal or other irregular shapes.

[0058] According to various embodiments of the present disclosure, an electron transfer agent may be present in one or more active regions of any alcohol sensor or alcohol sensor configuration disclosed herein. When the alcohol analyte (enzyme substrate) undergoes a redox reaction, a suitable electron transfer agent may facilitate the transfer of electrons to the working electrode. The selection of an electron transfer agent in each active region may determine the observed redox potential of the alcohol analyte.

[0059] Suitable electron transfer agents may include electroreducible and electrooxidizable ions, complexes or molecules (e.g., quinones) having a redox potential that is higher or lower than the redox potential of a standard calomel electrode (SCE) by a few hundred millivolts. According to some embodiments, suitable electron transfer agents may include low potential osmium complexes, such as those described in U.S. Patents 6,134,461 and 6,605,200, the entire contents of which are incorporated herein by reference. Other examples include those described in U.S. Patents 6,736,957, 7,501,053, and 7,754,093; the disclosures of each of which are incorporated herein by reference in their entirety. Other suitable electron transfer agents may include metal compounds or complexes such as ruthenium, osmium, iron (e.g., polyethylene ferrocene or hexacyanoferrate), cobalt, metallocene compounds thereof, and the like. Suitable ligands for metal complexes may also include, for example, bidentate or higher-dentate ligands, such as bipyridine, biimidazole, phenanthroline, or pyridyl (imidazole). Other suitable bidentate ligands may include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes or ortho-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate or higher dentate ligands may be present in the metal complex to achieve a complete coordination sphere. In some embodiments, the selected electron transfer agent for the alcohol responsive active region described herein is an osmium complex.

[0060] According to various embodiments of the present disclosure, one or more polymers may be present in each of the one or more active regions of any alcohol sensor or alcohol sensor configuration disclosed herein. For example, the enzymes may each be chemically bonded or otherwise immobilized in a single polymer. In other embodiments, such as when multiple enzymes are used, one or more enzymes may be polymerized into a first polymer and one or more other enzymes may be polymerized into a second polymer, with both the polymers and the enzymes forming the active region of the alcohol sensor.

[0061] Suitable polymers for inclusion in the active region may include, but are not limited to, polyvinylpyridine (e.g., poly(4-vinylpyridine)), polyvinylimidazole (e.g., poly(1-vinylimidazole)), any copolymers thereof, and any combination thereof. For example, exemplary copolymers that may be suitable for inclusion in the active region include those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile. When multiple active regions are present, the one or more polymers in each active region may be the same or different. Any combination of the above polymers may also be used without departing from the scope of the present disclosure.

[0062] According to various embodiments of the present disclosure, the electron transfer agent can be covalently bonded to one or more polymers in one or more active regions. The mode of covalent bonding is not considered to be particularly limited. The covalent bonding of the electron transfer agent to one or more polymers can occur by polymerizing monomer units with covalently bound electron transfer agents, or the electron transfer agent can react with one or more polymers respectively after it has been synthesized. According to some embodiments, a bifunctional spacer can covalently bond the electron transfer agent to one or more polymers in the active region, wherein the first functional group reacts with one or more polymers (e.g., a functional group capable of quaternizing a pyridine nitrogen atom or an imidazole nitrogen atom) and the second functional group reacts with the electron transfer agent (e.g., a functional group reacting with a ligand of a coordinated metal ion).

[0063] In an exemplary embodiment, one or more polymers in the active region of the alcohol sensor disclosed herein can be poly (4-vinyl pyridine), wherein a portion of the monomer units are functionalized with alkyl carboxylate side chains, a portion of the monomer units are linked to an osmium electron transfer agent having an amino spacer group, and a portion of the monomer units are not functionalized. That is, the polymer can be a redox polymer of an osmium-modified poly (vinyl pyridine)-based polymer, referred to herein as "X7".

[0064] Similarly, according to some or other various embodiments of the present disclosure, one or more enzymes in one or more active regions can be covalently bonded to one or more polymers. When multiple enzymes are present in a single active region, all multiple enzymes can be covalently bonded to a single polymer or two or more separate polymers, which can be the same or different. For example, if two or more enzymes are covalently bonded to separate different polymers, then one of the polymers can be coated on another to form an active region. In other embodiments, only a part can be covalently bonded to one or more polymers in the multiple enzymes. For example, a first enzyme can be covalently bonded to a first polymer, and a second enzyme can be non-covalently associated with a first polymer or a second polymer.

[0065] The covalent bonding of enzyme to polymer can occur by introducing a cross-linking agent of a suitable cross-linking agent. Suitable cross-linking agents for reacting with the free amino groups in the enzyme can include such cross-linking agents, such as polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin or its derivative variants. Suitable cross-linking agents for reacting with the free carboxylic acid groups in the enzyme can include, for example, carbodiimide. Cross-linking is usually intermolecular, but can be intramolecular in some embodiments.

[0066] As described above, the electron transfer agent and / or one or more enzymes may also be associated with one or more polymers in the active region by means other than covalent bonding. In some embodiments, the electron transfer agent and / or one or more enzymes may be ionically or coordinately associated with one or more polymers. For example, a charged polymer may be ionically associated with an electron transfer agent or enzyme of opposite charge. In other embodiments, the electron transfer agent and / or one or more enzymes may be physically entrained within one or more polymers without being bonded thereto.

[0067] In some embodiments, stabilizers can be incorporated into the active region of the alcohol sensor described herein to improve the functionality of the sensor and achieve the desired sensitivity and stability. For example, such stabilizers can include antioxidants and / or chaperones to stabilize one or more enzymes. Examples of suitable stabilizers can include, but are not limited to, serum albumin (e.g., human or bovine serum albumin or other compatible albumins), glutaraldehyde cross-linked albumin, catalase, glutaraldehyde cross-linked catalase, other enzyme antioxidants, and the like, and any combination thereof. Stabilizers can be conjugated or non-conjugated, and can be chemically bound or unbound to the active region. The amount of stabilizer can vary depending on the type of stabilizer selected, but can be about 1% to about 50% of the weight of the total active region (i.e., the weight of the combined components of the active region), including any value and subset therebetween, such as about 1% to about 25% by weight of the total active region, or about 10% to about 25% by weight.

[0068] In certain embodiments, a mass limiting membrane or a biocompatible membrane may be disposed over at least a portion of the active region. Suitable membranes may be composed of cross-linked polymers containing heterocyclic nitrogen groups, such as polymers of polyvinyl pyridine and / or polyvinyl imidazole. Embodiments also include membranes made of polyurethane, polyether urethane or chemically related materials, membranes made of polysiloxanes, and the like.

[0069] In some embodiments, the membrane can be formed by in situ cross-linked polymers, including those discussed above, with zwitterionic moieties, non-pyridine copolymer components and optionally hydrophilic or hydrophobic polymers modified in another part, and / or in a buffer solution (e.g., an alcohol buffer solution or other biological buffer solution, such as HEPES). The modified polymer can be made from a precursor polymer containing a heterocyclic nitrogen group. For example, the precursor polymer can be polyvinyl pyridine and / or polyvinyl imidazole. Optionally, hydrophilic or hydrophobic modifiers can be used to "fine tune" the permeability of the resulting membrane to alcohol or even a specific type of alcohol of interest. Optional hydrophilic modifiers, such as poly (ethylene glycol), hydroxyl or polyhydroxy modifiers, etc., and any combination thereof, can be used to enhance the biocompatibility of the resulting membrane.

[0070] The membrane can be applied to the active area by placing one or more droplets of the membrane solution on at least one or more working electrodes of the alcohol sensor, for example by immersing the sensor tail in the membrane solution, by spraying the membrane solution on the sensor tail, by hot pressing or melting any size layer (e.g., discrete or fully enclosed), vapor deposition of the membrane solution, powder coating of the membrane solution, etc., and any combination thereof.

[0071] In general, the thickness of the membrane is controlled by the concentration of the membrane solution, the number of droplets of the membrane solution applied, the number of times the sensor tail is immersed in the membrane solution, the volume of the membrane solution sprayed on the sensor tail, and the like, as well as any combination of these factors. In some embodiments, the membrane described herein may have a thickness ranging from about 0.1 micrometers (μm) to about 1000 μm, including any values ​​and subsets therebetween, such as from about 1 μm to about 500 μm, or from about 10 μm to about 100 μm. As described above, the membrane may cover one or more active areas, and in some embodiments, the active area may have a thickness of about 0.1 μm to about 10 μm, including any values ​​and subsets therebetween. In addition, the size of the active area may range from about 0.001 mm 2 To about 1mm 2 , including any values ​​and subsets therebetween. However, it should be understood that thicker or thinner films, thicker and thinner active areas, and larger or smaller active areas are also contemplated herein.

[0072] In some embodiments, the membrane may include compounds including, but not limited to, poly(styrene-co-maleic anhydride), dodecylamine, and poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol) (2-aminopropyl ether) crosslinked with poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol) bis(2-aminopropyl ether); poly(N-isopropylacrylamide); copolymers of poly(ethylene oxide) and poly(propylene oxide); polyvinyl pyridine; derivatives of polyvinyl pyridine; polyvinylimidazole; derivatives of polyvinylimidazole; etc.; and any combination thereof. In some embodiments, the membrane may be composed of polyvinyl pyridine-co-polystyrene polymers in which a portion of the pyridinic nitrogen atoms are functionalized with non-crosslinked poly(ethylene glycol) tails and a portion of the pyridinic nitrogen atoms are functionalized with alkyl sulfonic acid groups. Other membrane compounds may include suitable copolymers of 4-vinyl pyridine and styrene and polyether arms that do not contain amines, either alone or in combination with any of the above membrane compounds. Any combination of the above membrane polymers may also be used without departing from the scope of the present disclosure.

[0073] The membrane compounds described herein can be further cross-linked with one or more cross-linking agents, including those cross-linking agents listed above for enzymes. For example, suitable cross-linking agents may include, but are not limited to, polyethylene glycol diglycidyl ether (PEGDGE), glycerol triglycidyl ether (Gly3), polydimethylsiloxane diglycidyl ether (PDMS-DGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin or its derivative variants, and any combination thereof. Branched forms with similar terminal chemicals (terminal chemistry) are also applicable to the present disclosure. For example, in some embodiments, Formula 1 can be cross-linked with triglycidyl glyceryl ether and / or PEDGE and / or polydimethylsiloxane diglycidyl ether (PDMS-DGE).

[0074] In some embodiments, the membrane composition used as the mass transfer limiting layer of the present disclosure may include polydimethylsiloxane (PDMS), polydimethylsiloxane diglycidyl ether (PDMS-DGE), aminopropyl-terminated polydimethylsiloxane, and the like, and any combination thereof, which is used as a leveling agent (e.g., for reducing the contact angle of the membrane composition or the sensing element composition). Branched forms with similar terminal chemistry are also suitable for the present disclosure. Certain leveling agents may be additionally included, such as those found in U.S. Pat. No. 8,983,568, the disclosure of which is incorporated herein by reference in its entirety.

[0075] In some cases, the membrane can form one or more bonds with one or more elements of the active region. As used herein, the term "bond" and its grammatical variants refer to any type of interaction between atoms or molecules that allows chemical compounds to form associations with each other, such as but not limited to covalent bonds, ionic bonds, dipole-dipole interactions, hydrogen bonds, London dispersion forces, etc., and any combination thereof. For example, in-situ polymerization of the membrane can form crosslinks between one or more polymers of the membrane and one or more polymers in the active region. In some embodiments, crosslinking of the membrane to the active region helps reduce delamination of the membrane from the sensor.

[0076] As previously described, the present disclosure provides various methods, systems and devices for detecting at least one alcohol (e.g., alcohol concentration or level) in a bodily fluid of an individual. Detection can be in vitro or in vivo using the active area sensing chemistry described herein to overcome one or more obstacles in manufacturing desired alcohol sensors.

[0077] One or more enzymes, such as alcohol oxidase, can be used to detect alcohol. As a representative example, alcohol oxidase interacts with alcohol (generally primary alcohol) to form acetaldehyde (C2H4O) and hydrogen peroxide (H2O2). Other primary and secondary alcohols react to form aldehydes with correspondingly higher or lower carbon contents. Alcohol oxidase only catalyzes the forward conversion of alcohol to acetaldehyde and contains a strongly bound flavin cofactor. Therefore, in theory, alcohol oxidase can be used in analyte sensors for detecting alcohol by measuring the acetaldehyde or hydrogen peroxide products produced in the enzymatic reaction. However, there are two problems with this approach. First, both acetaldehyde and hydrogen peroxide have an inhibitory effect on alcohol oxidase. Therefore, if these compounds are not removed from the sensor environment, the alcohol oxidase becomes inactive for promoting alcohol oxidation, making the sensor unusable for measuring alcohol. In addition, if acetaldehyde and hydrogen peroxide are isolated or quenched with other reagents (further complicating the sensing composition of the active area), there is no longer material or sufficient material available for electrochemical detection. Second, alcohol oxidase does not freely exchange electrons with redox mediators other than molecular oxygen. Therefore, electron transfer agents associated with the polymer in the active region of the sensor, such as osmium and other transition metal complexes discussed herein, are ineffective in cycling the alcohol oxidase from the inactive reduced state to the oxidized state that can react with alcohols.

[0078] Alcohol dehydrogenases are another group of enzymes that promote the conversion of alcohols (generally primary alcohols) to aldehydes or ketones. As a representative example, in the oxidation of nicotinamide adenine dinucleotide (NAD + ), alcohol dehydrogenase interacts with ethanol to form acetaldehyde (C2H4O) and reduced nicotinamide adenine dinucleotide (NADH) in the presence of NADH. Other primary and secondary alcohols react to form aldehydes with correspondingly higher or lower carbon contents. Unlike alcohol oxidases, alcohol dehydrogenases not only catalyze the forward conversion of alcohols to aldehydes (generally referred to herein as acetaldehyde, but other aldehydes with higher or lower carbon contents may be produced), but also react reversibly. Further unlike alcohol oxidases, alcohol dehydrogenases do not contain bound cofactors, and therefore require exogenous cofactors to make the enzyme active to promote alcohol oxidation. In addition, the activity of alcohol dehydrogenases is controlled, at least in part, based on the product inhibition of NADH and acetaldehyde (see Figure 4). These characteristics generally make alcohol dehydrogenases unsuitable for sensing chemicals for designing alcohol sensors.

[0079] Embodiments of the present disclosure utilize an oxidoreductase approach to detect alcohol levels using one or more analyte sensor embodiments of the present disclosure. More specifically, the sensing chemistry described in the present disclosure utilizes two enzymes to interact synergistically in at least one active region on a working electrode, one of which is KRT. As used herein, the term "synergistic enzyme" or "synergistic enzyme system" and grammatical variants thereof refer to at least two enzymes that can synergistically (combine) interact with each other. KRT is an oxidoreductase with NADH-dependent or NAD(P)(H)-dependent catalytic activity, primarily known to be able to reduce aldehydes and ketones. Typically, KRTs are relatively inefficient as alcohol dehydrogenases because they lack metal cofactors, which are used to maintain and position alcohol groups by alcohol dehydrogenases. KRTs typically prefer NAD(P)H rather than NADH. Therefore, their effective use in detecting individual alcohol levels is considered unfavorable.

[0080] Embodiments of the present disclosure provide a synergistic enzyme system suitable for detecting alcohol, wherein the synergistic enzyme system utilizes KRT. KRTs described for use in sensing chemicals of the present disclosure for detecting alcohol include wild-type (naturally occurring) and non-natural engineered peptide chains that exhibit KRT functionality. For example, the aldehyde KRT (aldo-KRT) family may be suitable for embodiments of the present disclosure. Without being bound by theory, it is further believed that changes in one or more residues in the active site of a KRT may alter its reaction mechanism, thereby altering its affinity for detecting alcohol. In other words, certain KRTs may be more effective in alcohol detection compared to other KRTs, depending on their chemical composition.

[0081] More specifically, the synergistic enzyme system described herein utilizes KRT and diaphorase. In the sensor configuration of the present disclosure, KRT can convert alcohol and oxidized NAD + or oxidized nicotinamide adenine dinucleotide phosphate (NAD(P) + ) are converted into aldehydes (e.g., acetaldehyde) and reduced NADH or reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H), respectively. Aldehydes are used as representative molecules indicating the presence of alcohols. NADH or NAD(P)H can be reduced under the mediation of diaphorase, wherein the electrons transferred in this process provide the basis for alcohol detection at one or more working electrodes.

[0082] exist Figure 4A The NAD + Cofactor-mediated synergistic reaction between KRT and diaphorase; Figure 4B The NAD(P) for alcohol detection is shown in + Cofactor-mediated synergistic reaction between KRT and diaphorase. Figure 4A and4B In the embodiment, diaphorase is chemically bonded (e.g., covalently) to a polymer disposed on a working electrode (i.e., a working electrode in the active region of the alcohol sensor), and KRT is chemically bonded (e.g., covalently) to the same polymer (e.g., via a cross-linking agent) or to a separate polymer. For example, in one embodiment, diaphorase can be chemically bonded to a first polymer disposed on the working electrode, and KRT can be chemically bonded to a second polymer disposed on the first polymer. The membrane is disposed over the entire active region, and a specific cofactor (e.g., NAD + or NAD(P) + ) can be non-covalently associated with the polymer in the active region, but in other cases the membrane is limited. In addition to diaphorase and / or KRT, an osmium complex or other transition metal complex capable of exchanging electrons with the enzyme is also chemically (e.g., covalently) bonded to a polymer disposed on the working electrode. For example, as described above, X7 comprises a covalently bound polymer and an electron transfer agent. In some embodiments, the active region comprises an X7 polymer, diaphorase and KRT are chemically bound to the X7 polymer, and diaphorase and KRT are covered with a membrane.

[0083] Each of KRT and diaphorase can be present in an amount from about 1% to about 50% by weight of the total active area (i.e., the weight of the combined components of the active area), including any values ​​and subsets therebetween, such as from about 1% to about 40%, or from about 10% to about 40% by weight of the total active area.

[0084] If you can Figure 4A and 4B As understood in the KRT oxidation of alcohol, the amount of aldehyde (e.g., acetaldehyde) formed enzymatically is proportional to the amount of alcohol. Therefore, the current generated at the working electrode during KRT oxidation of alcohol can be proportional to the amount of acetaldehyde present, and by extension, the amount of alcohol present (i.e., alcohol concentration or level). The working electrode current and alcohol concentration can be related by reference to a lookup table of currents at known alcohol concentrations or by using a calibration curve.

[0085] Therefore, in some embodiments, the present disclosure provides an alcohol-responsive active region based on a synergistic enzyme system of ketoreductase and diaphorase. More specifically, the present disclosure provides an analyte sensor and a system, which includes a sensor tail, the sensor tail including at least one working electrode and at least one alcohol-responsive active region disposed on the surface of the working electrode, the alcohol-responsive active region comprising an enzyme system, the enzyme system comprising at least two enzymes that can act synergistically to promote alcohol detection, wherein one of the at least two enzymes is a ketoreductase. The membrane may further cover at least one alcohol-responsive active region. In addition, at least the sensor tail of the analyte sensor may be configured to be at least partially inserted into a tissue, such as skin, subcutaneous or intravenous, so that analysis can be performed in vivo. In other embodiments, the analyte sensor (including the sensor tail) may be completely implanted in the tissue. Therefore, the present disclosure provides a method for sensing alcohol using the above-mentioned analyte sensor. Specifically, at least one alcohol-responsive active region is exposed to body fluids, and during the duration (e.g., one day or longer, such as up to about one month or longer) of the user wearing the analyte sensor, the active region can continuously measure alcohol. A signal may be detected (eg, by electrochemical detection) from the alcohol-responsive active region of the analyte sensor that is proportional to the alcohol concentration.

[0086] Embodiments disclosed herein include:

[0087] Embodiment A: An analyte sensor comprising: a sensor tail comprising at least one working electrode; and at least one alcohol-responsive active area disposed on a surface of the working electrode, the at least one alcohol-responsive active area comprising an enzyme system comprising at least a first enzyme and a second enzyme capable of acting synergistically to facilitate alcohol detection, wherein the first enzyme is a ketone reductase.

[0088] Embodiment B: A method comprising: detecting a signal proportional to an alcohol concentration using an analyte sensor, the analyte sensor comprising: a sensor tail comprising at least one working electrode, the sensor tail being configured for implantation in tissue; and at least one alcohol-responsive active region disposed on a surface of the working electrode, the at least one alcohol-responsive active region comprising an enzyme system comprising at least a first enzyme and a second enzyme capable of acting synergistically to facilitate alcohol detection, wherein the first enzyme is a ketoreductase.

[0089] Embodiment C: An electrode assembly comprising: at least one working electrode; and at least one alcohol-responsive active area disposed on a surface of the working electrode, the at least one alcohol-responsive active area comprising an enzyme system comprising at least a first enzyme and a second enzyme capable of acting synergistically to facilitate alcohol detection, wherein the first enzyme is a ketone reductase.

[0090] Embodiment D: An alcohol sensing composition comprising: at least one alcohol responsive active region comprising an enzyme system comprising at least a first enzyme and a second enzyme capable of acting synergistically to facilitate alcohol detection, wherein the first enzyme is a ketoreductase.

[0091] Embodiment E: A system comprising: an analyte sensor comprising: a sensor tail comprising at least one working electrode; and at least one alcohol-responsive active area disposed on a surface of the working electrode, the at least one alcohol-responsive active area comprising an enzyme system comprising at least a first enzyme and a second enzyme capable of acting synergistically to facilitate alcohol detection, wherein the first enzyme is a ketoreductase; and a receiver configured to receive a signal from the analyte sensor that is proportional to the alcohol concentration.

[0092] Each of embodiments A, B, C, D and E may have one or more additional elements in any combination of the following:

[0093] Element 1: wherein the second enzyme is diaphorase.

[0094] Element 2: wherein the ketoreductase is an aldo-ketoreductase.

[0095] Element 3: Ketoreductase is composed of Manufactured KRED-P1-A04, KRED-P2-C11, KRED-P2-G03, or KRED-P2-H07.

[0096] Element 4: wherein a membrane is disposed on at least one alcohol responsive active region.

[0097] Element 5: wherein a membrane is disposed on at least one alcohol responsive active region, and the membrane is one of polyvinylpyridine, polyvinylimidazone or any copolymer thereof.

[0098] Element 6: wherein at least one alcohol responsive active region comprises a polymer.

[0099] Element 7: wherein at least one alcohol-responsive active region comprises a polymer, and the ketoreductase of the first enzyme and the second enzyme are chemically bonded to the polymer.

[0100] Element 8: wherein at least one alcohol responsive active region comprises an electron transfer agent.

[0101] Element 9: wherein at least one alcohol responsive active region comprises a stabilizer.

[0102] As non-limiting examples, exemplary combinations suitable for A, B, C, D, and E include:

[0103] Elements 1 and 2; 1 and 3; 1 and 4; 1 and 5; 1 and 6; 1 and 7; 1 and 8; 2 and 3; 2 and 4; 2 and 5; 2 and 6; 2 and 7; 2 and 8; 3 and 4; 3 and 5; 3 and 6; 3 and 7; 3 and 8; 4 and 5; 4 and 6; 4 and 7; 4 and 8; 5 and 6; 5 and 7; 5 and 8; 6 and 7; 6 and 8; 7 and 8; and any non-limiting combination of one, more than one, or all of 1, 2, 3, 4, 5, 6, 7, and 8.

[0104] To facilitate a better understanding of the embodiments described herein, including the advantages of the alcohol sensor as described above and compared to other alcohol detection chemistries, the following examples of various representative embodiments are given. The following examples should in no way be construed as limiting or defining the scope of the invention.

[0105] Example 1: Example 1 evaluated the functionality of an alcohol sensor comprising KRT using a dual-layer active area system. Example 1 was used to screen 24 candidate KRTs for alcohol sensors.

[0106] Alcohol sensor preparation . Alcohol responsive active regions containing various experimental KRTs were prepared using a dual layer active region system. The active region was coated onto a carbon working electrode (note that other electrode surface types may be used according to embodiments of the present disclosure), wherein a first layer composition (Table 1 below) was initially coated directly onto the carbon working electrode, and a second layer composition (Table 2 below) was coated onto the first layer composition, the first and second layers comprising the active region. After the first layer composition was deposited, the first layer composition was cured overnight at room temperature (RT) (approximately 25° C.); after the first layer composition was cured, the second layer composition was deposited and allowed to cure overnight at RT. Thereafter, a PVP film was applied to the working electrode using a coating solution formulated with 40:1 polyvinylpyridine-co-polystyrene (15) (i.e., with 15% styrene) and PEGDGE 400. The film was deposited on the active region (3x5 mm / second dipping) and allowed to cure overnight at RT, and then cured for 48 hours at 56° C. in a dried vial. It should be understood that the components of Tables 1 and 2 can be applied as a monolayer (i.e., a mixture of all components) with or without the additional crosslinker (PEGDGE 400) in Table 2 without departing from the scope of the present disclosure. The layered active regions used in this example are to facilitate testing and comparison of a large number of KRT sensor samples.

[0107] Table 1

[0108]

[0109] Table 2

[0110]

[0111] Using the Redwood City, CA The obtained KRTs were used to prepare various sensors using the method provided in Example 1 above. Table 3 provides the performance of each of the 24 KRTs tested. Enzyme code (" Ref."), and their respective cofactors (NAD+ or NADP+), and are identified below as samples A1-A24 ("ID").

[0112] Table 3

[0113]

[0114] Beaker Calibration for A1-A24 Sensors The sensor comprising A1-A24 KRT prepared according to Example 1 was subjected to alcohol sensing analysis by immersing the electrode in 100 mM PBS buffer solution at 33°C and different concentrations of ethanol (2, 4, 6, 10, 15, 20, 30 and 40 mM ethanol). Figures 5A-5D The responses of each of A1-A24 are shown. As shown, various KRT sensors had no response, only minimal response, or inconsistent response to ethanol. Figure 5E is a static view of the beaker calibration at 4 mM ethanol, showing the change (Δ) in the current response from baseline (no EtOH exposure) to 4 mM ethanol. As shown in the figure, at 4 mM ethanol, the Δ in the sensor response prepared using KRT IDs A1, A11, A15, and A16 showed a significant response to ethanol, which is also reflected in Figures 5A-5C Various other ethanol concentrations in the range of 400 Å and 800 Å could therefore be candidate KRTs for alcohol sensing. Therefore, KRT IDs A1, A11, A15, and A16 were further evaluated.

[0115] Example 2: Example 2 evaluated the functionality of alcohol sensors comprising KRT IDs A1, A11, A15, and A16 compared to a control alcohol dehydrogenase active region system.

[0116] Alcohol sensor preparation Experimental A1, A11, A15, and A16 KRT alcohol-responsive active regions were prepared using a single-layer active region system and compared to control sensors containing ADH. ADH was obtained from Sigma-Aldrich Corp., St. Louis, MO, under the product number A3263. The ADC control used NAD +Cofactor. The active area was coated on the carbon working electrode in the form of a monolayer composition (Table 4 below). After deposition of the active area, the active area was cured overnight at RT. Thereafter, the PVP film was applied to the working electrode using a film coating solution containing 4 mL 100 mg / mL polyvinyl pyridine and 100 μl 100 mg / mL PEGDGE 400. The film was deposited on the active area (3x5 mm / second dip) and cured overnight at RT, then cured for 48 hours at 56°C in a dried vial.

[0117] Table 4

[0118]

[0119] Beaker Calibration for A1-A24 Sensors The sensors comprising A1, A11, A15 and A16 KRT prepared according to Example 2 were subjected to alcohol sensing analysis by immersing the electrodes in 100 mM PBS buffer solution at RT and different concentrations of ethanol (2, 4, 6, 10, 15, 20, 30 and 40 mM ethanol). Fig. 6A The response of each of A1, A11, A15 and A16 is shown. As shown, each KRT exhibited a measurable response to increasing ethanol concentrations. Figure 6B The linear sensitivity response of the A1, A11, A25, and A16 KRT sensors based on beaker calibration is shown, demonstrating positive driving forces, particularly for A1, A11, and A15.

[0120] Beaker stability The beaker stability (long term stability) of the A1, A11, A15 and A16 sensors of Example 2 was evaluated in 30 mM EtOH in 100 mM PBS at 52°C. Figure 6C The results shown indicate that after 4 days, each signal experienced a different rate of sensor decline (reduced stability in detecting alcohol), as provided in Table 5. Notably, the signal decline for the A15 sensor was significantly lower than that for the other KRT sensors tested.

[0121] Table 5

[0122]

[0123] Aldehyde inhibition The effect of aldehyde inhibition (e.g., acetaldehyde inhibition discussed above) on the performance of the A1, A11, A15, and A16 sensors of Example 2 was compared to the ADH control sensor of Example 2. The sensors were incubated over time in 30 mM EtOH in 100 mM PBS at 33°C and then spiked with 1 mM acetaldehyde (at approximately 19.3 hours). The normalized signal results are shown in Figure 7As shown, the A15 and A16 sensors had no or negligible effects of aldehyde inhibition up to about 20 hours; the A1 and A11 sensors showed slightly greater inhibition; and the ADH control sensor showed significant inhibition. The percentage of signal reduction due to aldehyde inhibition for the tested sensors is provided in Table 6.

[0124] Table 6

[0125]

[0126] Example 3. Standard kinetic assays were performed at RT for KRT IDs A15 and A16 and compared to an ADH control (ADH described in Example 2 above). + (or NADP + ) and measure the optical absorption ("OD") of reduced NADH (or NADPH) at 340 nm. The concentration of A15 and A16 used in the kinetic assay was 0.167 mg / mL, and the concentration of ADH control used in the kinetic assay was 0.0167 μg / mL (orders lower than A15 and A16), both in PBS buffer. The results of the kinetic assay are shown graphically in Figure 8 The numerical results are provided in Table 7. Notably, the amount of ADH control enzyme was significantly reduced compared to the A15 and A16 KRT enzymes, but still resulted in approximately 260% greater kinetic activity, but A15 and A16 showed significantly greater resistance to aldehyde inhibition (see Figure 7 ).

[0127] Table 7

[0128] ADH A15 A16 OD / min 0.0132 0.0051 0.0043 (OD / min) / (mg / mL) 790 0.031 0.026 Unit / mg activity 288 0.011 0.009

[0129] Example 4. Standard kinetic assays were performed at RT for KRT IDs A15 and A16, each in various types of alcohols, and compared to an ADH control (ADH described in Example 2 above). Cofactor NAD(P) was used in the reaction + , and the optical absorption ("OD") of reduced NAD(P)H at 340 nm was measured. Testing of A15 and A16 in the kinetic assay is shown in Fig.9A and 9B The assay was performed in ethanol (EtOH), isopropanol (IPA), propanol (nPA), butanol (1-BuA), and methanol (MeOH). The "blank" excludes the KRT enzyme. The results of the KRT kinetic assay are shown graphically in Fig.9A and 9B9A relates to A15 and 9B relates to A16. Numerical results are provided in Table 8, including results measured against an ADH control (not otherwise represented in graphical form). Notably, KRT exhibited significantly higher enzyme activity in IPA compared to the ADH control, further demonstrating that the KRT sensor described herein can be used for alcohols other than primary alcohols.

[0130] Table 8

[0131] Unit / mg activity ADH A15 A16 MeOH 0.000 0.001 0.000 EtOH 288.000 0.012 0.009 IPA 0.709 2.553 7.091 nPA 4.211 0.032 0.042 1-BuA 1.309 0.047 0.131

[0132] Example 5. In this example, a sensor comprising KRT having additional polymers and / or cross-linking agents in addition to the constituent elements of Example 2 was evaluated.

[0133] IPA Response . The effects of IPA on A15 and A16 are further illustrated with respect to the results of Example 4. A15 and A16 sensors were prepared according to Example 2, wherein in some cases one or more additional polymers or cross-linkers were added to the active region composition. The additional polymer was poly(1-vinyl imidazole) ("PVI") and the additional cross-linker was glutaraldehyde ("Glut"). The current response of the sensor was initially measured in 30 mM EtOH in 100 mM PBS buffer at 33°C for approximately 3.5 hours, and then increasing concentrations of IPA (1, 2, and 3 mM IPA) were spiked (buffer exchange and IPA addition). The results are shown in Fig. 10A and 10B , 10A involves A15, and 10B involves A16. Fig. 10A and 10B The additional polymers and / or cross-linkers used for each sensor tested are shown in Table 9 as mg / mL of total active area with a “-” symbol indicating that the component was not added.

[0134] Table 9

[0135] Fig. 10A / 10B Legend Reference PVI (mg / mL) Glut(mg / mL) A15 ─ ─ A15PVI4 4 ─ A15Glut05 ─ 0.5 A15PVI4Glut05 4 0.5 A15PVI4Glut1 4 1 A16PVI4 4 ─ A16Glut05 ─ 0.5 A16PVI4Glut05 4 0.5 A16Glut1 ─ 1 A16PVI4Glut1 4 1

[0136] like Fig. 10A and 10B As shown in Figure 2, the response to 1 mM IPA was about 12 times greater than that to 30 mM EtOH. Fig. 10A As shown in Figure 2, the A15 sensor containing glutaraldehyde crosslinker generally showed higher sensitivity to IPA, among which the A15 sensor containing only glutaraldehyde (and no PVI) showed the highest sensitivity. Similar results are shown in Figure 2 for the reference A16 sensor. Fig. 10B However, a lower concentration of glutaraldehyde (0.5 mg / mL) showed higher sensitivity to IPA compared with a higher concentration of glutaraldehyde (1 mg / mL).

[0137] Beaker stability The beaker stability (long term stability) of the A15 and A16 sensors of Example 5 was evaluated in 30 mM EtOH in 100 mM PBS at 33°C. The results are shown in Fig.11A and 11B , where 11A relates to A15 and 11B relates to A16. As shown, the stability of the A15 sensor is greater than the stability of the A16 sensor. In addition, the stability of the A15 sensor containing the additional glutaraldehyde crosslinker is better than the stability of the other A15 compositions. However, precipitation of the active zone was observed in the A15PVI4Glut05 and A15PVI4Glut1 samples; more specifically, turbidity appeared in the active zone when both PVI and glutaraldehyde were included. Table 10 provides the sensor value drop experienced by the A15 sensor over a 5-day period.

[0138] Table 10

[0139]

[0140] Example 6. In this example, a sensor comprising KRT with an additional polymer was compared to an ADH control sensor.

[0141] Beaker Calibration The A15 sensor prepared according to Example 2 and also containing 8 mg / mL of PVI in the active region was tested for alcohol sensing analysis to determine its response to various concentrations of EtOH (1, 2, 3, 5, 7, 10, 15, 20, 25, and 30 mM EtOH) in 100 mM PBS buffer at 33°C. The KRT A15 sensor was compared to an ADH control sensor prepared according to Example 2. Fig. 12A The normalized signal response of each of the A15 sensor and the ADH control sensor containing the additional PVI polymer (labeled as A15+8PVI) is shown. As shown, the A15 sensor exhibits higher ethanol sensitivity (i.e., the signal loss in the ADH control is significantly greater after each higher concentration of EtOH), as shown in FIG. Fig. 12B As further shown in , the linear sensitivity response of the two tested sensors is shown.

[0142] Beaker stability The beaker stability (long term stability) of the A15 and ADH control sensors of Example 6 were evaluated in 30 mM EtOH in 100 mM PBS at 33°C. Fig.13 The results shown in demonstrate that the stability of the KRT A15 sensor is significantly better than that of the ADH control sensor.

[0143] Example 7. The beaker stability (long-term stability) of the A15 sensor prepared according to Example 2 was evaluated in two different membrane solvents, one membrane solvent comprising EtOH and the second membrane solvent comprising EtOH and HEPES buffer in a ratio of 80:20. The results are shown in Fig.14 It was demonstrated that the stability of the sensor in EtOH solvent alone exhibited greater stability compared to diluted EtOH solvent.

[0144] Example 8. Beaker stability (long term stability) of analyte sensors comprising active regions prepared according to Example 2 with different membrane compositions (and different from Example 2) to which 1 mg / mL glutaraldehyde was added was evaluated in 30 mM EtOH in 100 mM PBS buffer at 33°C. Three separate membrane compositions were prepared and coated on A15 active regions (further comprising glutaraldehyde) comprising 1%, 2% or 4% PEGDGE 400 in PVP. The results of signal stability are shown in Fig.15 The short-term stability of the tested films based on the composition of the present invention is greater than the long-term stability. The numerical signal decreases within 5 and 10 days are shown in Table 11.

[0145] Table 11

[0146]

[0147] Example 9. The beaker stability (long term stability) of an analyte sensor comprising an active region prepared according to Example 2 with different membrane compositions (and different from Example 2) to which 1 mg / mL glutaraldehyde was added was evaluated in 30 mM EtOH in 100 mM PBS buffer at 33°C. Three separate membrane compositions were prepared and coated on an A15 active region (further comprising glutaraldehyde) comprising PVP without any crosslinker, polyvinylpyridine-co-polystyrene (30) without any crosslinker (i.e., with 30% styrene) ("PVPSty30"), or PVPSty30 with 4% PEGDGE 400 crosslinker. The results of the signal stability are shown in Fig.16 , indicating that the addition of cross-linkers does not seem to have a significant effect on the stability of the KRT sensor.

[0148] In general, embodiments of the present disclosure demonstrate that a synergistic enzyme system comprising KRT enzymes is feasible as an alcohol sensor for detecting alcohol levels in an individual, particularly in vivo (but can also be used for in vivo measurements). Of the KRTs tested in the present disclosure, the KRT sensor comprising A15 exhibited the greatest stability (beaker stability) and A16 exhibited the least product inhibition; and both had much higher specific activity for IPA than for EtOH (e.g., 200-fold for A15 and 700-fold for A16).

[0149] Unless otherwise indicated, all numbers expressing quantities and the like in this specification and the associated claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and the attached claims are approximations that may vary depending on the desired properties sought to be obtained by embodiments of the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0150] One or more exemplary embodiments in combination with various features are presented herein. For the sake of clarity, all features of physical implementation are not described or shown in this application. It is to be understood that when developing a physical implementation comprising an embodiment of the present invention, many decisions specific to implementation must be made to achieve the developer's goal, such as meeting system-related, business-related, government-related and other constraints, which vary from time to time due to implementation. Although the labor of developers may be time-consuming, such labor will be routine work for those skilled in the art and for those who benefit from the present disclosure.

[0151] Although various systems, tools, and methods are described herein as "comprising" various components or steps, the systems, tools, and methods may also "consist essentially of" or "consist of" the various components and steps.

[0152] As used herein, the phrase "at least one" preceding a series of items, with the terms "and" or "or" separating any items, modifies the list as a whole, rather than each member (i.e., each item) of the list. The phrase "at least one" is permitted to have a meaning that includes at least one of any item, and / or at least one of any combination of these items, and / or at least one of each item. For example, the phrases "at least one of A, B, and C" or "at least one of A, B, or C" each refer to only A, only B, or only C; any combination of A, B, and C; and / or at least one of each of A, B, and C.

[0153] Therefore, the disclosed systems, tools and methods are well suited for achieving the mentioned purposes and advantages and those inherent therein. The specific embodiments disclosed above are exemplary only, because the teachings of the present disclosure can be modified and practiced in different but equivalent ways that are obvious to those skilled in the art who benefit from the teachings herein. In addition, except as described in the appended claims, it is not intended to limit the details of the construction or design shown herein. It is therefore obvious that the specific exemplary embodiments disclosed above can be changed, combined or modified, and all these changes are considered to be within the scope of the present disclosure. The systems, tools and methods exemplarily disclosed herein can be appropriately practiced in the absence of any elements not specifically disclosed herein and / or any optional elements disclosed herein. Although systems, tools and methods are described with "comprising", "containing" or "including" various components or steps, systems, tools and methods can also be "essentially composed of" or "composed of" various components and steps. All numbers and ranges disclosed above may vary. Whenever a numerical range with a lower limit and an upper limit is disclosed, any number and any included range falling within the range are specifically disclosed. Specifically, each value range disclosed herein (in the form of "from about a to about b," or equivalently, "from approximately a to b," or equivalently, "from approximately ab") should be understood to list every number and range contained within the broader value range. In addition, unless otherwise clearly and unambiguously defined by the patentee, the terms in the claims have their plain, ordinary meaning. In addition, the indefinite articles "a" or "an" used in the claims are defined herein to mean the element or elements into which they are introduced. If there is a conflict in the use of a word or term in this specification with one or more patents or other documents that may be incorporated herein by reference, the definition consistent with this specification shall be adopted.

Claims

1. An analyte sensor comprising: a sensor tail comprising at least one working electrode; and At least one alcohol-responsive active area is disposed on the surface of the working electrode, the at least one alcohol-responsive active area comprising an enzyme system comprising at least a first enzyme and a second enzyme that can act synergistically to facilitate alcohol detection, wherein the first enzyme is a ketoreductase.

2. The analyte sensor of claim 1, wherein the second enzyme is diaphorase. The analyte sensor of claim 1 , wherein the ketoreductase is an aldo-ketoreductase.

4. The analyte sensor according to claim 1, wherein the ketoreductase is Manufactured KRED-P1-A04, KRED-P2-C11, KRED-P2-G03, or KRED-P2-H07. The analyte sensor of claim 1 , wherein a membrane is disposed over the at least one alcohol responsive active region.

6. The analyte sensor of claim 5, wherein the membrane is one of polyvinylpyridine, polyvinylimidazole, or any copolymer thereof. The analyte sensor of claim 1 , wherein the at least one alcohol-responsive active region comprises a polymer.

8. The analyte sensor of claim 7, wherein the ketoreductase of the first enzyme and the second enzyme are chemically bound to the polymer.

9. The analyte sensor of claim 1, wherein the at least one alcohol-responsive active region comprises an electron transfer agent.

10. The analyte sensor of claim 1, wherein the at least one alcohol-responsive active region comprises a stabilizer.

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