Alcohol-sensing composition, alcohol sensor, and method for monitoring alcohol levels

Abstract

To provide a device, a method, and a system for detecting an alcohol concentration of an individual, particularly, an alcohol concentration in vivo of an individual.SOLUTION: An alcohol sensing composition includes at least one alcohol responsive active region containing a coordinated enzyme system containing at least a first enzyme and a second enzyme capable of coordinative acting in order to facilitate alcohol detection. At least one of the enzymes of the coordinated enzyme system is keto-reductase.SELECTED DRAWING: Figure 4A

Description

[Background technology]

[0001] The detection of various analytes in an individual can sometimes be essential for monitoring the state of health and well-being: deviations from normal analyte levels are often indicative of an underlying physiological condition, such as a metabolic state or disease, or exposure to a particular environmental factor or stimuli.

[0002] Analyte monitoring of an individual may occur periodically or continuously over a period of time. Periodic analyte monitoring may be performed by withdrawing samples of a bodily fluid, such as blood, at one or more time intervals and analyzing them ex vivo. Continuous analyte monitoring may be performed using one or more sensors that remain at least partially implanted within the individual's tissue, such as intradermally, subcutaneously, or intravenously, so that analysis may be performed in vivo. The implanted sensors may collect analyte data at any prescribed rate, depending, for example, on the individual's particular health needs and / or previously measured analyte levels.

[0003] Any analyte may be suitable for in vivo analysis if the appropriate chemistry for detecting the analyte can be identified. Indeed, in vivo amperometric sensors configured for analyzing glucose have been developed and improved in recent years. Other analytes commonly subject to physiological dysregulation that may also be desirable to monitor include, but are not limited to, lactate, oxygen, pH, A1c, ketones, drug levels, etc.

[0004] Another analyte that may be particularly important to an individual's health is alcohol level. Indeed, information related to an individual's in vivo alcohol level can be used to predict or monitor the level of another analyte of interest. For example, alcohol can alter glycemic control in individuals whose blood glucose levels are naturally dysregulated or who lack homeostasis without intervention, which can be harmful to the individual. Other analytes that may be dysregulated by alcohol include triglycerides (e.g., associated with heart disease, stroke, blood pressure, and obesity), gamma-glutamyltransferase (GGT) (e.g., associated with cancer, hepatitis, and bone disease), and cortisol (e.g., associated with stress and inflammation), among others. Therefore, monitoring an individual's alcohol level is desirable. [Brief explanation of the drawings]

[0005] The accompanying drawings are included to illustrate certain aspects of the present disclosure and should not be considered as 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. [Figure 1] 1 shows a diagram of an exemplary sensing system that can incorporate an analyte sensor of the present disclosure. [Figure 2A] FIG. 1 shows a diagram of an exemplary two-electrode analyte sensor configuration having a single working electrode suitable for use in some embodiments disclosed herein. [Figure 2B] FIG. 1 shows a diagram of an exemplary three-electrode analyte sensor configuration with a single working electrode suitable for use in some embodiments disclosed herein. [Figure 2C] Same as above. [Figure 3] FIG. 1 shows a diagram of an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode suitable for use in some embodiments disclosed herein. [Figure 4A]1 illustrates a cooperative enzyme system associated with alcohol detection using a ketoreductase, nicotinamide adenine dinucleotide, and diaphorase disposed on a working electrode, according to various embodiments of the present disclosure. [Figure 4B] 1 illustrates a cooperative enzyme system associated with alcohol detection using a ketoreductase, nicotinamide adenine dinucleotide phosphate, and diaphorase disposed on a working electrode, according to various embodiments of the present disclosure. [Figure 5A] 1 shows a graphical representation of the response of various KRT-containing sensors to various ethanol concentrations. [Figure 5B] 1 shows a graphical representation of the response of various KRT-containing sensors to various ethanol concentrations. [Figure 5C] 1 shows a graphical representation of the response of various KRT-containing sensors to various ethanol concentrations. [Figure 5D] 1 shows a graphical representation of the response of various KRT-containing sensors to various ethanol concentrations. [Figure 5E] 5A-5C show graphical representations of the response of four of the KRT-containing sensors from FIGS. 5A-5C to 4 mM ethanol. The change (Δ) in current response from baseline (no EtOH exposure) to 4 mM ethanol is shown. [Figure 6A] 1 shows a graphical representation of the response of various KRT-containing sensors to various ethanol concentrations. [Figure 6B] Figure 6A shows the linear sensitivity response of the measured ethanol concentration. [Figure 6C] FIG. 6B shows a graphical representation of the stability response of the various KRT-containing sensors of FIG. 6A. [Figure 7] 1 shows a graphical representation of the stability response of various KRT-containing sensors and an ADH control sensor to aldehyde exposure. [Figure 8] 1 shows a graphical representation of the results of a kinetic assay of various KRTs compared to an ADH control. [Figure 9A] 9A and 9B show graphical representations of the results of kinetic assays of two KRTs in different alcohol types. [Figure 9B] Same as above. [Figure 10A] 10A and 10B show graphical representations of the response of two KRT-containing sensors of various compositions to IPA. [Figure 10B] Same as above. [Figure 11A] 11A and 11B show a graphical representation of the stability response of various KRT-containing sensors with different additive polymer and crosslinker compositions. [Figure 11B] Same as above. [Figure 12A] 1 shows a graphical representation of the response of a particular KRT-containing sensor with a particular polymer composition to the inclusion of various ethanol concentrations. [Figure 12B] Figure 12A shows the linear sensitivity response of the measured ethanol concentration. The ADH control refers to the signal from alcohol dehydrogenase (ADH). Figures 12A-12B compare the linearity of ADH and KRT A15. [Figure 13] Figure 13 shows a graphical representation of the stability response of specific KRT-containing sensors with specific polymer compositions. Figure 13 compares the beaker stability of ADH and KRT A15. [Figure 14] 1 shows a graphical representation of the stability response of a particular KRT-containing sensor with a membrane immersed in two different solvents. [Figure 15] 1 shows a graphical representation of the stability response of various KRT-containing sensors with different membrane compositions. [Figure 16] 1 shows a graphical representation of the stability response of various KRT-containing sensors with different membrane and cross-linker compositions. DETAILED DESCRIPTION OF THE INVENTION

[0006] Detailed Description The present disclosure generally describes enzyme-based analyte sensors and methods for the detection of in vivo alcohol levels.

[0007] Analyte sensors are commonly used to detect various analytes and typically employ enzymes with specific specificity for particular substrates. For example, glucose-responsive analyte sensors represent a well-studied and still evolving field for helping diabetic patients better manage their health. However, other in vivo analytes are also important and may be of great value in determining and / or monitoring an individual's health. This includes determining and / or monitoring the effect of an analyte of interest on the dysregulation of other analytes.

[0008] An individual's in vivo alcohol concentration can vary dramatically based on alcohol consumption, intoxication, and / or various physiological factors. For example, individuals differ in their ability to metabolize alcohol, and therefore, even when consuming the same amount of alcohol per body weight, alcohol levels will vary between individuals. In fact, the equilibrium concentration of alcohol in tissues depends at least on water content, blood flow rate, and tissue mass. Because alcohol can cross biological membranes, it can easily flow from the bloodstream to all tissues and body fluids, and this flow is proportional to the water content of the tissues and body fluids. Furthermore, in rare cases, an individual may produce large amounts of alcohol through endogenous fermentation within the digestive system without consuming alcohol. As noted above, the presence of certain concentrations of alcohol may further affect the function of one or more other analytes in an individual, further impacting the individual's health.

[0009] Current alcohol measurements are performed by taking physical blood, urine, or saliva samples or using breath tests. However, such measurements are static in time and, in some cases, can suffer from false positive or false negative results, and therefore can be inaccurate at least occasionally. 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 grammatical variations thereof refer to any primary, secondary, or tertiary alcohol. For example, the alcohol sensor of the present disclosure can detect ethanol, methanol, butanol, propanol, isopropyl alcohol, etc., and any combination thereof.

[0010] The alcohol sensors described herein are responsive to in vivo alcohol levels and can provide "continuous" measurements of said alcohol levels, i.e., the alcohol sensors described herein can be operable to provide multiple alcohol concentration measurements over an extended (continuous) period, such as from a few seconds, minutes, or hours to days, weeks, or months.

[0011] Alcohol sensors may offer many advantages for monitoring dynamic alcohol levels related to the resulting physiological state and effects on other analytes of interest. For example, the disclosed sensors utilize a specialized enzymatic reaction involving one or more ketoreductases (referred to herein as "KRTs"). The KRT-containing sensing chemistries described herein advantageously overcome various hurdles associated with typical detection of an individual's alcohol level. These include overcoming enzyme inhibition by one or more products of the sensing chemistry's enzymatic reaction, as discussed in more detail below. By overcoming enzyme inhibition, the disclosed alcohol sensors may, among other things, enable the use of a single mass-limiting membrane and the fabrication of more cost-effective sensors while reducing the complexity of the required sensing region configuration, without compromising efficacy. KRT-containing chemistries further advantageously enable sensing of both primary and secondary alcohols, with the affinity for secondary alcohol detection being particularly unexpected.

[0012] An individual wearing a continuous alcohol sensor described herein can access real-time alcohol level information and make various decisions based thereon, such as whether it is safe to drive, whether glucose or other analyte levels are likely to become dysregulated based on the alcohol level and therefore whether action is necessary, and any combination thereof. Additionally, the alcohol sensors of the present disclosure can be used to monitor, test, and / or assess the alcohol levels of individuals suffering from alcohol misuse, abuse, or dependence. In doing so, the health of such individuals can be monitored by the individuals themselves or by medical or law enforcement professionals. The sensor electronics and processing algorithms associated with the operation of the sensors and detection systems described herein, including display units and devices thereof, can also provide instructions, guidance, recommendations, and / or output regarding alcohol concentration and suggested actions based thereon. Suitable processing algorithms, processors, memory, electronic components, etc., can reside in a trusted computer system, a remote terminal, a cloud server, a reader device, and / or the housing of the sensor itself. Guidance, recommendations, output, etc., can be displayed on a suitable display unit or device in electronic communication with one or more of these components. The display unit or device may be a dedicated reader device or a personal communication device such as a mobile phone (e.g., an iPhone® or ANDROID® device). Alternatively, the display unit or device may be a remote terminal that communicates with various software and healthcare-related applications, such as a third-party server, a cloud server, or a personal health monitor. A suitable remote terminal, cloud server, or the like may further relay the output to associated secondary devices, such as smart home devices, wearable technology, personal health monitors, or the like, or combinations thereof.

[0013] The present disclosure further describes a detection system incorporating one or more alcohol sensors. The system may include various detection components, such as a processor and / or coding instructions (algorithms), adapted to process sensor data received from the alcohol sensor and determine one or more alcohol concentrations therefrom. The processor and / or coding instructions, in some embodiments, can then analyze the alcohol concentration and determine one or more recommendations based on the particular alcohol concentration. For example, the alcohol concentration at a particular evaluation time or other quantities derivable from the alcohol concentration can be used to suggest whether another health analyte may be adversely affected. In such cases, the processor and / or coding instructions can suggest various ways in which an individual can respond based on the detected alcohol concentration, including suggesting immediate and future actions. For example, depending on the individual's particular alcohol concentration, recommendations may include whether to drive a motor vehicle, whether to take action to compensate for another affected analyte (e.g., whether to ingest carbohydrates or inject insulin if the analyte is glucose), whether to seek medical attention, etc., and any combination thereof.

[0014] Before describing the alcohol sensors of the present disclosure in more detail, a brief overview of suitable in-vivo analyte sensor configurations and sensor systems using analyte sensors will first be provided so that embodiments of the present disclosure may be better understood. It should be understood that any of the sensor systems and analyte sensor configurations described below may feature one or more enzymes used to detect alcohol, and generally for detecting alcohol in vivo, in accordance with various embodiments of the present disclosure.

[0015] FIG. 1 illustrates a diagram of an exemplary detection system that can incorporate an alcohol sensor of the present disclosure. As shown, detection system 100 includes a sensor control device 102 and a reader device 120 configured to communicate with each other via a local communication path or link that may be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. Reader device 120, according to some embodiments, may provide an output medium for displaying analyte concentrations and alerts or notifications determined by sensor 104 or its associated processor, as well as allowing for one or more user inputs. Reader device 120 may be a general-purpose smartphone or a dedicated electronic reader device. While only one reader device 120 is shown, multiple reader devices 120 may be present in certain cases. Reader device 120 may also communicate with a remote terminal 170 and / or a trusted computer system 180, respectively, via communication paths / links 141 and / or 142, which may also be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. Additionally or alternatively, reader device 120 may communicate with network 150 (e.g., a cellular network, the Internet, or a cloud server) via communication path / link 151. Network 150 may be further communicatively connected to remote terminal 170 via communication path / link 152 and / or to trusted computer system 180 via communication path / link 153. Alternatively, or in addition, sensor 104 may communicate directly with remote terminal 170 and / or trusted computer system 180 without the presence of an intervening reader device 120. For example, sensor 104 may communicate with remote terminal 170 and / or trusted computer system 180 via a direct communication link to network 150 according to some embodiments, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is incorporated herein by reference in its entirety.

[0016] Any suitable electronic communication protocol, such as near field communication (NFC), radio frequency identification (RFID), BLUETOOTH® or BLUETOOTH® low energy protocol, WiFi®, or the like, may be used for each of the communication paths or links. The remote terminal 170 and / or trusted computer system 180, according to some embodiments, are accessible 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 touchscreen interface for outputting information related to the sensor control device and user input information.

[0017] The sensor control device 102 includes a sensor housing 103 that can house circuitry and a power source for operating the sensor 104. Optionally, the power source and / or active circuitry may be omitted. A processor (not shown) may be communicatively connected to the sensor 104, and the processor may be physically located within the sensor housing 103 or the reader device 120. The sensor 104 protrudes from an underside of the sensor housing 103 and extends through an adhesive layer 105. The adhesive layer 105, according to some embodiments, is adapted to adhere the sensor housing 103 to a tissue surface, such as skin.

[0018] The sensor 104 is adapted to be at least partially inserted into a tissue of interest, such as into the dermis or subcutaneous layer of the skin. The sensor 104 may include a sensor tail of sufficient length to insert to a desired depth into a given tissue. The sensor tail may include at least one working electrode and one or more active areas (sensing areas / spots or sensing layers) disposed on the at least one working electrode and active for sensing alcohol (or, optionally, one or more additional analytes). In some embodiments, the active areas are in the form of one or more discrete spots (e.g., 1 to about 10 spots, or more) and are about 0.01 mm in size. 2 ~about 1mm 2and includes any value and subset therebetween, although larger or smaller individual active area spots are also contemplated herein.

[0019] 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 the detection of alcohol. According to some embodiments, the active region may include a polymeric material to which one or more (or all) enzymes are chemically bound (e.g., covalently, ionically, etc.) or otherwise immobilized (e.g., unbound in a matrix). In some embodiments, each active region may be covered with a mass-limiting or biocompatible membrane and / or may further include an electron transfer agent to facilitate at least the detection of alcohol.

[0020] In various embodiments of the present disclosure, at least the alcohol level may be monitored in any biological fluid of a subject, such as dermal fluid, interstitial fluid, plasma, blood, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, etc. In certain embodiments, the analyte sensor of the present disclosure may be adapted to assay dermal fluid or interstitial fluid to determine the concentration of alcohol in vivo.

[0021] Continuing with reference to FIG. 1 , in some embodiments, the sensor control unit 102 can automatically transfer data obtained by the sensor 104 to the reader device 120. For example, alcohol concentration data may be stored in memory as the data is acquired until it is transmitted (e.g., every few seconds, every minute, every five minutes, or at other predetermined intervals), and may be communicated automatically and periodically, such as at a specific frequency or after a specific period of time has elapsed. In other embodiments, the sensor control unit 102 can communicate with the reader device 120 in a non-automatic manner rather than according to a set schedule. For example, data may be communicated from the sensor control unit 102 using NFC or RFID technology when the sensor electronics come within communication range of the reader device 120. The data may remain stored in the memory of the sensor control unit 102 until communicated to the reader device 120. Thus, the patient need not be constantly in proximity to the reader device 120 but can instead upload data at a convenient time. In still other embodiments, a combination of automatic and non-automatic data transfer may be implemented. For example, data transfer may continue automatically until the reader device 120 is no longer within communication range of the sensor control unit 102. Although automatic and non-automatic data transfer from the sensor control unit 102 has been described with reference to the reader device 120, such transfer mechanisms are equally applicable to the remote terminal 170 and / or the trusted computer system 180 without departing from the scope of this disclosure.

[0022] An introducer may be temporarily present to facilitate the introduction of the sensor 104 into the tissue. In an exemplary embodiment, the introducer may include a needle or similar sharp. It should be appreciated that in alternative embodiments, other types of introducers, such as a sheath or blade, may be present. More specifically, the needle or other introducer may be temporarily present near or concurrent with the sensor 104 prior to tissue insertion and then withdrawn. While present, the needle or other introducer may facilitate the insertion of the sensor 104 into the tissue by opening an access path for the sensor 104 to follow. For example, according to one or more embodiments, the needle may facilitate penetration of the epidermis as an access path to the dermis, allowing implantation of the sensor 104 to occur. After opening the access path, the needle or other introducer may be withdrawn to avoid presenting a sharps hazard. In an exemplary embodiment, a suitable needle may be solid or hollow, beveled or non-beveled, and / or circular or non-circular in cross section. In more specific embodiments, suitable needles may be comparable in cross-sectional diameter and / or tip design to acupuncture needles, which may have a cross-sectional diameter of about 150 micrometers to about 300 micrometers (e.g., about 250 micrometers). However, it should be recognized that suitable needles may have larger or smaller cross-sectional diameters as needed for a particular application.

[0023] In some embodiments, the tip of the needle or introducer (while present) may be angled above the end of the sensor 104 so that the needle or introducer penetrates the tissue first, opening an access path for the sensor 104. In other exemplary embodiments, the sensor 104 may reside within a lumen or channel of the needle or inserter, the needle similarly opening an access path for the sensor 104. In either case, the needle facilitates insertion of the sensor and is then withdrawn.

[0024] Alcohol sensors disclosed herein may feature active regions located on a single working electrode (e.g., on one or both sides of a single working electrode) or on two or more separate working electrodes (e.g., on one or both sides of two working electrodes). According to various embodiments of the present disclosure and as further described herein, single-working electrode sensor configurations can use two-electrode or three-electrode detection motifs. Sensor configurations featuring a single working electrode are described below with reference to FIGS. 2A-2C. Each of these sensor configurations can suitably incorporate one or more alcohol-responsive active regions according to various embodiments of the present disclosure. Thereafter, sensor configurations featuring multiple working electrodes are described with reference to FIGS. 3 and 4. 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 detect another analyte of interest in coordination with the detection of alcohol level.

[0025] When a single working electrode is present in the alcohol sensor of the present disclosure, a three-electrode detection motif may include a working electrode, a counter electrode, and a reference electrode. A related two-electrode detection motif may include a working electrode and a second electrode, with the second electrode functioning as either a counter electrode or a reference electrode, or both a counter electrode and a reference electrode (i.e., a counter / reference electrode). In both two-electrode and three-electrode detection motifs, one or more active regions of the alcohol sensor may be in contact with the working electrode. According to embodiments of the present disclosure, one or more active regions may include one or more enzymes. In some embodiments, the various electrodes may be at least partially stacked (layered) on top of each other. In some or other embodiments, the various electrodes may be laterally spaced apart from each other on the sensor tail. Similarly, the associated active regions on each electrode may be stacked vertically on top of each other or laterally spaced apart. In either case, the various electrodes may be electrically insulated from each other by a dielectric or similar insulator.

[0026] FIG. 2A shows a diagram of an exemplary two-electrode sensor configuration having a single working electrode suitable for use in detecting alcohol according to embodiments of the present disclosure. As shown, the sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter / reference electrode 216. Alternatively, the working electrode 214 and the counter / reference electrode 216 may be disposed on the same side of the substrate 212 with a dielectric material interposed therebetween (configuration not shown). An active region 218 is disposed as at least one layer over at least a portion of the working electrode 214. In various embodiments, the active region 218 may include multiple spots or a single spot configured for the detection of one or more analytes of interest. Collectively, one or more enzymes may be present in the active region 218 (i.e., as a single spot or multiple spots).

[0027] 2A , membrane 220 covers at least active area 218 and, optionally, according to some embodiments, can cover part or all of working electrode 210 and / or counter / reference electrode 216, or the entire analyte sensor 200. One or both sides of analyte sensor 200 can be covered with membrane 220. Membrane 220 can include one or more polymeric membrane materials capable of restricting analyte flow to active area 218 and / or having biocompatibility capabilities. Sensor 200 can be operable to assay at least alcohol by any of the following detection techniques: coulometric, amperometric, voltammetric, potentiometric electrochemical, or iontophoretic (including reverse iontophoresis).

[0028] 2B and 2C show diagrams of exemplary three-electrode analyte sensor configurations with a single working electrode suitable for use in embodiments of the present disclosure. Three-electrode analyte sensor configurations utilizing a single working electrode can be similar to that shown for sensor 200 in FIG. 2A , except that sensors 201 and 202 ( FIGS. 2B and 2C ) include an additional electrode, represented as electrode 217. With additional electrode 217, counter / reference electrode 216 can function as either a counter electrode or a reference electrode, while additional electrode 217 performs the other electrode function. Working electrode 214 continues to perform its original function. Additional electrode 217 can be disposed on either working electrode 210 or electrode 216, with a dielectric separation layer between them. For example, as shown in FIG. 2B , dielectric layers 219 a, 219 b, and 219 c separate electrodes 214, 216, and 217 from one another, providing electrical insulation. Alternatively, at least one of electrodes 214, 216, and 217 may be disposed on opposite sides of substrate 212, as shown in FIG. 2C . Thus, in some embodiments, electrode 214 (working electrode) and electrode 216 (counter electrode) may be disposed on opposite sides of substrate 212, and electrode 217 (reference electrode) may be disposed on one of electrodes 214 or 216 and separated therefrom by a dielectric material. Alternatively, in some embodiments, electrode 214 (working electrode) and electrode 216 (reference electrode) may be disposed on opposite sides of substrate 212, and electrode 217 (counter electrode) may be disposed on one of electrodes 214 or 216 and separated therefrom by a dielectric material. In yet other embodiments, the reference electrode and counter electrode may be disposed on one side of substrate 212, and the working electrode may be disposed on the opposite side. Optionally, a reference material layer 230 (eg, Ag / AgCl) may be present on the electrode 217, and the location of the reference material layer 230 is not limited to that shown in FIGS. 2B and 2C.

[0029] 2A, the active areas 218 of the analyte sensors 201 and 202 may include multiple spots or a single spot configured for the detection of at least alcohol. Collectively, one or more enzymes may be present in the active areas 218 of the sensors 201 and 202. Additionally, the analyte sensors 201 and 202 may be operable to assay at least alcohol by any of the following detection techniques: coulometric, amperometric, voltammetric, potentiometric electrochemical, or iontophoretic.

[0030] 2A and 2B , similar to analyte sensor 200, membrane 220 can also cover active area 218, as well as other sensor components, in sensors 201 and 202. In some embodiments, additional electrode 217 can be covered by membrane 220. While FIGS. 2B and 2C depict all of electrodes 214, 216, and 217 as being covered by membrane 220, it should be understood that in some embodiments, only working electrode 214 can be covered, or in some embodiments, only working electrode 214 and one other electrode can be covered. Furthermore, the thickness of membrane 220 on each of electrodes 214, 216, and / or 217 can be the same or different, and the amount of surface area of ​​each of electrodes 214, 216, and / or 217 that membrane 220 covers can also be the same or different. As in the two-electrode analyte sensor configuration (FIG. 2A), one or both sides of analyte sensors 201 and 202 may be covered with membrane 220 in the sensor configurations of FIGS. 2B and 2C, or the entire analyte sensors 201 and 202 may be covered. Thus, the three-electrode sensor configurations shown in FIGS. 2B and 2C should be understood as non-limiting examples of embodiments disclosed herein, with alternative electrode and / or layer configurations falling within the scope of the present disclosure.

[0031] FIG. 3 shows a diagram of an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode suitable for use in the alcohol sensor described in this disclosure. As shown in FIG. 3, sensor 300 includes working electrodes 304 and 306, each disposed on opposing sides of substrate 302. Active area 310 is disposed on the surface of working electrode 304, and active area 312 is disposed on the surface of working electrode 306. Collectively, one or more enzymes may be present in active areas 310 and 312 configured for the detection of at least alcohol. For example, both active areas 310 and 312 may be configured to detect alcohol concentration. Alternatively, one of active areas 310 or 312 may be configured to detect alcohol concentration, while the other active area may be configured to detect another analyte of interest (e.g., glucose, lactate, etc.). Counter electrode 320 is electrically insulated from working electrode 304 by dielectric layer 322, and reference electrode 321 is electrically insulated from working electrode 306 by dielectric layer 323. Outer dielectric layers 330 and 332 are disposed on reference electrode 321 and counter electrode 320, respectively. A membrane 340 can cover at least active areas 310 and 312, according to various embodiments. Other components of analyte sensor 300 may also optionally be covered with membrane 340, and as noted above, one or both sides of analyte sensor 300, or portions thereof, may be covered with membrane 340. Similar to analyte sensors 200, 201, and 202, sensor 300 can be operable to assay at least alcohol by any of a coulometric, amperometric, voltammetric, potentiometric electrochemical, or iontophoretic detection technique.

[0032] Alternative sensor configurations having multiple working electrodes and different from that shown in Figure 3 may feature counter / reference electrodes instead of separate counter and reference electrodes 320 and 321, and / or different layer and / or film arrangements than those explicitly shown. For example, the arrangement of counter electrode 320 and reference electrode 321 may be reversed from that shown in Figure 3. Furthermore, working electrodes 304 and 306 do not necessarily have to be on opposite sides of substrate 302 in the manner shown in Figure 3.

[0033] 2 and 3 are primarily directed to analyte sensor configurations having two working electrodes, it should be understood that more than two working electrodes may be successfully incorporated through extensions of the disclosure herein. Additional working electrodes allow for additional active areas and corresponding sensing capabilities to be imparted to such sensors having such features.

[0034] 2 and 3 illustrate a planar substrate (e.g., relatively flat) having conductive structures (e.g., electrodes) and active regions disposed thereon. It should be understood that analyte sensors for use in embodiments of the present invention can have a variety of other configurations without departing from the scope of the present disclosure. For example, the substrate can be substantially non-planar (e.g., relatively curved, hemispherical, or spherical), cylindrical, spiral, other irregular, and any combination thereof. Similarly, two or more electrodes can be substantially non-planar (e.g., relatively curved, hemispherical, or spherical), cylindrical, spiral, other irregular, and any combination thereof. The electrodes can be arranged concentrically in layers or otherwise, typically separated by one or more insulating regions. A sensing region disposed on at least the working electrode can further cover at least a portion (or all) of the working electrode, either as a single layer or as discrete regions of various shapes, such as square, circular, semicircular, arcuate, rectangular, polygonal, or other irregular shapes.

[0035] According to various embodiments of the present disclosure, an electron transfer agent can be present in one or more of the active areas of any of the alcohol sensors or alcohol sensor configurations disclosed herein. A suitable electron transfer agent can facilitate the transport of electrons to the working electrode when the alcohol analyte (enzyme substrate) undergoes a redox reaction. The selection of the electron transfer agent within each active area can determine the redox potential observed for the alcohol analyte.

[0036] Suitable electron transfer agents may include electroreducible and electrooxidizable ions, complexes, or molecules (e.g., quinones) with redox potentials several hundred millivolts above or below the redox potential of a standard calomel electrode (SCE). According to some embodiments, suitable electron transfer agents may include low-potential osmium complexes, such as those described in U.S. Pat. Nos. 6,134,461 and 6,605,200, the disclosures of which are incorporated herein by reference in their entireties. Additional examples include those described in U.S. Pat. Nos. 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are incorporated herein by reference in their entireties. Other suitable electron transfer agents may include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), cobalt, metallocene compounds thereof, and the like. Suitable ligands for metal complexes can also include bidentate or higher dentate ligands, such as, for example, bipyridine, biimidazole, phenanthroline, or pyridyl (imidazole). Other suitable bidentate ligands can include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher dentate ligands can be present in the metal complex to achieve a complete coordination sphere. In some embodiments, the electron transfer agent selected for use in the alcohol-responsive active regions described herein is an osmium complex.

[0037] According to various embodiments of the present disclosure, one or more polymers may be present in each of one or more active regions of any of the alcohol sensors or alcohol sensor configurations disclosed herein. For example, each enzyme may be chemically bonded or otherwise immobilized to a single polymer. In other embodiments, such as when multiple enzymes are used, one or more enzymes may be polymerized to a first polymer and one or more other enzymes may be polymerized to a second polymer, with both the polymer and the enzymes forming the active region of the alcohol sensor.

[0038] 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 combinations thereof. Exemplary copolymers that may be suitable for inclusion in the active region include those containing monomer units such as, for example, 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 foregoing polymers may also be used without departing from the scope of the present disclosure.

[0039] According to various embodiments of the present disclosure, an electron transfer agent can be covalently bonded to one or more polymers in one or more active regions. The method of covalent attachment is not believed to be particularly limiting. Covalent attachment of the electron transfer agent to one or more polymers can occur by polymerizing a monomer unit having a covalently attached electron transfer agent, or the electron transfer agent can be reacted separately with one or more polymers after they have already 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 a first functional group is reactive with one or more polymers (e.g., a functional group capable of quaternizing a pyridine nitrogen atom or an imidazole nitrogen atom) and a second functional group is reactive with the electron transfer agent (e.g., a functional group that reacts with a ligand that coordinates a metal ion).

[0040] In an exemplary embodiment, one or more polymers in the active region of the alcohol sensor disclosed herein may be poly(4-vinylpyridine), in which some of the monomer units are functionalized with alkyl carboxylate side chains, some of the monomer units are attached to an osmium electron transfer agent with an amide spacer group, and some of the monomer units are unfunctionalized. That is, the polymer may be an osmium-decorated poly(vinylpyridine)-based polymer redox polymer, referred to herein as "X7."

[0041] Similarly, according to some or various other embodiments of the present disclosure, one or more enzymes in one or more active regions can be covalently bound to one or more polymers. When multiple enzymes are present in a single active region, all of the multiple enzymes can be covalently bound to a single polymer or to two or more separate polymers, which can be the same or different. For example, when two or more enzymes are covalently bound to separate, distinct polymers, one polymer can be coated over the other to form the active region. In other embodiments, only a portion of the multiple enzymes can be covalently bound to one or more polymers. For example, a first enzyme can be covalently bound to a first polymer, and a second enzyme can be non-covalently bound to either the first polymer or the second polymer.

[0042] Covalent attachment of the enzyme to the polymer can occur via a cross-linking agent introduced with an appropriate cross-linking agent. Suitable cross-linking agents for reaction with free amino groups in the enzyme can include, for example, polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanogen chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatized variants thereof. Suitable cross-linking agents for reaction with free carboxylic acid groups in the enzyme can include, for example, carbodiimides. Cross-linking is generally intermolecular, but in some embodiments can be intramolecular.

[0043] As noted above, the electron transfer agent and / or one or more enzymes can be bound to 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 can be ionically or coordinatively bound to one or more polymers. For example, a charged polymer can be ionically bound to an oppositely charged electron transfer agent or enzyme. In yet other embodiments, the electron transfer agent and / or one or more enzymes can be physically entrained within one or more polymers without being bound to the polymer.

[0044] In some embodiments, stabilizers can be incorporated into the active region of the alcohol sensors described herein to improve sensor function and achieve desired sensitivity and stability. Such stabilizers can include, for example, antioxidants and / or companion proteins to stabilize one or more enzymes. Examples of suitable stabilizers include, but are not limited to, serum albumin (e.g., human or bovine serum albumin or other compatible albumin), glutaraldehyde-crosslinked albumin, catalase, glutaraldehyde-crosslinked catalase, other enzyme antioxidants, and the like, and any combination thereof. The stabilizer can be conjugated or unconjugated and may or may not be chemically bound to the active region. The amount of stabilizer can vary depending on the type of stabilizer selected, but can be in an amount of about 1% to about 50% by weight of the total active region (i.e., the combined weight of the active region components). This includes 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.

[0045] In certain embodiments, a mass-limiting or biocompatible membrane can be disposed over at least a portion of the active area. Suitable membranes can be composed of cross-linked polymers containing heterocyclic nitrogen groups, such as polymers of polyvinylpyridine and / or polyvinylimidazole. Embodiments also include membranes made of polyurethane, polyetherurethane, or chemically related materials, membranes made of silicone, and the like.

[0046] In some embodiments, membranes can be formed by in situ crosslinking of polymers containing zwitterionic moieties, non-pyridine copolymer components, and optionally modified with additional moieties that are either hydrophilic or hydrophobic and / or have other desirable properties in a buffer solution (e.g., an alcohol buffer or other biological buffer such as HEPES). The modified polymer can be made from a precursor polymer containing heterocyclic nitrogen groups. For example, the precursor polymer can be polyvinylpyridine and / or polyvinylimidazole. Optionally, hydrophilic or hydrophobic modifiers can be used to "fine-tune" the permeability of the resulting membrane to alcohols or to specific types of alcohols of interest. Optional hydrophilic modifiers, such as poly(ethylene glycol), hydroxyl or polyhydroxyl modifiers, and any combination thereof, can be used to enhance the biocompatibility of the resulting membrane.

[0047] The film may be applied onto the active area by placing one or more droplets of the film solution on at least one or more working electrodes of the alcohol sensor, for example, by dipping the sensor tail into the film solution, by spraying the film solution onto the sensor tail, by heat pressing or melting the film in any sized layer (discrete or all-inclusive, etc.), by vapor deposition of the film solution, powder coating of the film solution, etc., and any combination thereof.

[0048] In general, the thickness of the membrane is controlled by the concentration of the membrane solution, the number of droplets of membrane solution applied, the number of times the sensor tail is dipped into the membrane solution, the amount of membrane solution sprayed onto the sensor tail, and the like, and any combination of these factors. In some embodiments, the membranes described herein can have a thickness ranging from about 0.1 micrometers (μm) to about 1000 μm, including any value and subset therebetween, such as about 1 μm to about 500 μm, or about 10 μm to about 100 μm. As noted above, the membrane can cover one or more active regions, and in some embodiments, the active regions can have a thickness ranging from about 0.1 μm to about 10 μm, including any value and subset therebetween. Additionally, the active regions can have a thickness ranging from about 0.001 mm in size. 2 ~about 1mm 2 and includes any value and subset 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.

[0049] In some embodiments, the membrane may contain 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); polyvinylpyridine; derivatives of polyvinylpyridine; polyvinylimidazole; derivatives of polyvinylimidazole, etc.; and any combination thereof. In some embodiments, the membrane may be composed of a polyvinylpyridine-co-styrene polymer in which a portion of the pyridine nitrogen atoms are functionalized with uncrosslinked poly(ethylene glycol) tails and a portion of the pyridine nitrogen atoms are functionalized with alkylsulfonic acid groups. Other membrane compounds may include suitable copolymers of 4-vinylpyridine and styrene and non-amine-containing polyether arms, either alone or in combination with the aforementioned membrane compounds. Any combination of the aforementioned membrane polymers may also be used without departing from the scope of the present disclosure.

[0050] The membrane compounds described herein can be further crosslinked with one or more crosslinkers, including those listed above for enzymes. For example, suitable crosslinkers include, but are not limited to, polyethylene glycol diglycidyl ether (PEG-DGE), glycerol triglycidyl ether (Gly3), polydimethylsiloxane diglycidyl ether (PDMS-DGE), or other polyepoxides, cyanogen chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatized variants thereof, and any combination thereof. Branched versions with similar terminal chemical structures are also suitable for the present disclosure. For example, in some embodiments, Formula 1 may be crosslinked with triglycidyl glycerol ether and / or PEG-DGE and / or polydimethylsiloxane diglycidyl ether (PDMS-DGE).

[0051] In some embodiments, the film composition for use as a mass transport 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, for use as a leveling agent (e.g., to reduce the contact angle of the film composition or sensing element composition). Branched versions with similar terminal chemical structures are also suitable for the present disclosure. Specific leveling agents may also be 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.

[0052] In some cases, the membrane may form one or more bonds with one or more elements of the active region. As used herein, the term "bond" and grammatical variations thereof refer to any type of interaction, atomic or molecular, that allows compounds to form bonds with one another, such as, but not limited to, covalent bonds, ionic bonds, dipole-dipole interactions, hydrogen bonds, London dispersion forces, and the like, 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 the membrane to the active region facilitates reducing the occurrence of delamination of the membrane from the sensor.

[0053] As previously mentioned, the present disclosure provides various methods, systems, and devices for detecting at least alcohol (e.g., alcohol concentration or level) in an individual's bodily fluid. Detection can be in vitro or in vivo utilizing the active area sensing chemistries described herein to overcome one or more hurdles in the fabrication of desirable alcohol sensors.

[0054] Alcohols can be detected using one or more enzymes, such as alcohol oxidase. As a representative example, alcohol oxidase interacts with alcohols, mostly primary alcohols, to form acetaldehyde (CHO) and hydrogen peroxide (HO). Other primary and secondary alcohols react to form corresponding aldehydes with higher or lower carbon content. Alcohol oxidase catalyzes only the forward conversion of alcohols to acetaldehyde and contains a tightly bound flavin cofactor. Therefore, in theory, alcohol oxidase could be used in analyte sensors to detect alcohols by analyzing either the acetaldehyde or hydrogen peroxide products produced in the enzymatic reaction. However, this approach presents two problems. First, both acetaldehyde and hydrogen peroxide are inhibitory to alcohol oxidase. Therefore, if these compounds are not removed from the sensor environment, alcohol oxidase becomes inactive with respect to promoting alcohol oxidation, thereby rendering the sensor nonfunctional with respect to analyzing alcohols. Furthermore, if acetaldehyde and hydrogen peroxide are sequestered or quenched with other agents (which further complicate the sensing composition of the active region), there will be no species or enough species available for electrochemical detection. Second, alcohol oxidase does not freely exchange electrons with redox mediators other than molecular oxygen. Therefore, electron transfer agents bound to polymers in the active region of the sensor, such as osmium and other transition metal complexes described herein, are ineffective at cycling alcohol oxidase from an inactive reduced state to an oxidized state that reacts with alcohol.

[0055] Alcohol dehydrogenases are another group of enzymes that catalyze the conversion of alcohols (usually primary alcohols) to aldehydes or ketones. As a typical example, alcohol dehydrogenases oxidize nicotinamide adenine dinucleotide (NAD +In the presence of acetaldehyde, alcohol dehydrogenase interacts with ethanol to form acetaldehyde (CHO) and reduced nicotinamide adenine dinucleotide (NADH). Other primary and secondary alcohols react to form corresponding aldehydes of higher or lower carbon content. Unlike alcohol oxidase, alcohol dehydrogenase not only catalyzes the forward conversion of alcohols to aldehydes (generically referred to herein as acetaldehyde, although other aldehydes with higher or lower carbon content may be produced), but also performs the reverse reaction. Further unlike alcohol oxidase, alcohol dehydrogenase does not contain a bound cofactor and therefore requires exogenous cofactors to activate the enzyme for promoting alcohol oxidation. Furthermore, the activity of alcohol dehydrogenase is controlled, at least in part, by product inhibition by NADH and acetaldehyde (see Figure 4). These properties generally make alcohol dehydrogenase unsuitable for use in designing sensing chemistries for alcohol sensors.

[0056] Embodiments of the present disclosure utilize an oxidoreductase scheme for detecting 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 that interact cooperatively with each other in at least one active area on a working electrode, where one of the enzymes is KRT. As used herein, the terms "cooperative enzyme" or "cooperative enzyme system," and grammatical variations thereof, refer to at least two enzymes that can interact cooperatively with each other. KRT is a type of oxidoreductase known to have NADH- or NAD(P)(H)-dependent catalytic activity and to be capable of reducing primarily aldehydes and ketones. Typically, KRT lacks the metal cofactor used by alcohol dehydrogenases to retain and position the alcohol group, making it a relatively inefficient alcohol dehydrogenase. Furthermore, KRT typically prefers NAD(P)H over NADH. Therefore, their effective use in detecting alcohol levels in individuals has not been considered beneficial.

[0057] Embodiments of the present disclosure provide cooperative enzyme systems suitable for use in the detection of alcohol, which utilize KRTs. KRTs described for use in the disclosed sensing chemistries for alcohol detection include wild-type (naturally occurring) and non-naturally engineered peptide chains that exhibit KRT functionality. For example, the aldo-KRT family may be suitable for use in embodiments of the present disclosure. Without being bound by theory, it is further believed that altering one or more residues in the active site of a KRT may alter its reaction mechanism and therefore its affinity for alcohol detection. Thus, certain KRTs may be more effective at alcohol detection than other KRTs, depending on their chemical makeup.

[0058] More specifically, the cooperative enzyme system described herein utilizes KRT and diphorase. In the sensor configuration of the present disclosure, KRT reacts with alcohol and oxidized NAD. + or oxidized nicotinamide adenine dinucleotide phosphate (NAD(P) + ) can be converted to an aldehyde (e.g., acetaldehyde) and reduced NADH or reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H), respectively. The aldehyde serves as a representative molecule for the presence of alcohol. NADH or NAD(P)H can undergo diaphorase-mediated reduction, and the electrons transferred during this process provide the basis for alcohol detection at one or more working electrodes.

[0059] NAD for alcohol detection + The cooperative reaction between KRT and diaphorase mediated by the cofactors is shown in Figure 4A. +A cooperative reaction between KRT and diaphorase mediated by a cofactor is shown in Figure 4B. In Figures 4A and 4B, diaphorase is chemically bound (e.g., covalently) to a polymer disposed on the working electrode (e.g., in the active area of ​​an alcohol sensor), and KRT is chemically bound (e.g., covalently) to the same (e.g., via a crosslinker) or to a different polymer. For example, in one embodiment, diaphorase may be chemically bound to a first polymer disposed on the working electrode, and KRT may be chemically bound to a second polymer disposed on the first polymer. A membrane is placed over the entire active area, and a specific cofactor (e.g., NAD) is introduced into the membrane. + or NAD(P) + The enzyme (either of these) may be non-covalently bound to the polymer in the active region, but the membrane may be otherwise confined. 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) bound to a polymer disposed on the working electrode. For example, X7, as described above, includes both a covalently bound polymer and an electron transfer agent. In some embodiments, the active region includes an X7 polymer to which diaphorase and KRT are chemically bound, and which is then covered by a membrane.

[0060] KRT and diaphorase can each be present in an amount ranging from about 1% to about 50% by weight of the total active area (i.e., the combined weight of the active area components), including any value and subset therebetween, such as about 1% to about 40% by weight, or about 10% to about 40% by weight of the total active area.

[0061] 4A and 4B, the amount of aldehyde (e.g., acetaldehyde) formed by the enzyme 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, which in turn can be proportional to the amount of alcohol present (i.e., alcohol concentration or level). Correlation of working electrode current to alcohol concentration can be performed by referencing a lookup table of currents at known alcohol concentrations or by utilizing a calibration curve.

[0062] Thus, in some embodiments, the present disclosure provides an alcohol-responsive active region based on a cooperative enzyme system of ketoreductase and diaphorase. More specifically, the present disclosure provides analyte sensors and systems including at least a working electrode and a sensor tail including at least one alcohol-responsive active region disposed on the surface of the working electrode, the alcohol-responsive active region including an enzyme system comprising at least two enzymes capable of acting cooperatively to facilitate alcohol detection, one of the at least two enzymes being a ketoreductase. A membrane may further cover the at least one alcohol-responsive active region. Furthermore, at least the sensor tail of the analyte sensor may be configured for at least partial insertion into tissue, such as transdermal, subcutaneous, or intravenous, so that analysis may be performed in vivo. In other embodiments, the analyte sensor (including the sensor tail) may be completely implanted within tissue. Thus, the present disclosure provides methods for detecting alcohol using the analyte sensor described above. In particular, at least one alcohol-responsive active region is exposed to bodily fluids, and the active region can continuously analyze alcohol while the user wears the analyte sensor (e.g., for one day or more, e.g., up to about one month or more). A signal can be detected (e.g., by electrochemical detection) from the alcohol-responsive active region of the analyte sensor that is proportional to the concentration of alcohol.

[0063] Embodiments disclosed herein include the following: Embodiment A: An analyte sensor comprising: a sensor tail including at least a working electrode; and at least one alcohol-responsive active region disposed on a surface of the working electrode, wherein the at least one alcohol-responsive active region comprises an enzyme system including at least a first enzyme and a second enzyme capable of acting in concert to facilitate the detection of alcohol, wherein the first enzyme is a ketoreductase.

[0064] Embodiment B: A method comprising detecting a signal proportional to a concentration of alcohol using an analyte sensor, the analyte sensor comprising: a sensor tail including at least a working electrode, the sensor tail 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 including at least a first enzyme and a second enzyme capable of acting in concert to facilitate detection of alcohol, the first enzyme being a ketoreductase.

[0065] Embodiment C: An electrode assembly comprising: at least one working electrode; and at least one alcohol-responsive active region disposed on a surface of the at least one working electrode, wherein the alcohol-responsive active region comprises an enzyme system comprising at least a first enzyme and a second enzyme capable of acting in concert to facilitate the detection of alcohol, wherein the first enzyme is a ketoreductase.

[0066] 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 in concert to facilitate the detection of alcohol, wherein the first enzyme is a ketoreductase.

[0067] Embodiment E: A system comprising: a sensor tail including at least a working electrode; and an analyte sensor including at least one alcohol-responsive active region disposed on a surface of the working electrode; and a receiver configured to receive a signal proportional to a concentration of alcohol from the analyte sensor, wherein the at least one alcohol-responsive active region comprises an enzyme system including at least a first enzyme and a second enzyme capable of acting in concert to facilitate detection of alcohol, wherein the first enzyme is a ketoreductase.

[0068] Each of embodiments A, B, C, D, and E can have one or more of the following additional elements in any combination. Element 1: The second enzyme is diaphorase.

[0069] Element 2: The ketoreductase is an aldo-ketoreductase. Element 3: The ketoreductase is KRED-P1-A04, KRED-P2-C11, KRED-P2-G03, or KRED-P2-H07 manufactured by Codexis.

[0070] Element 4: A membrane is disposed over the at least one alcohol-responsive active area. Element 5: A membrane is disposed over the at least one alcohol-responsive active area, the membrane being one of polyvinylpyridine, polyvinylimidazone, or any copolymer thereof.

[0071] Element 6: The at least one alcohol-responsive active region comprises a polymer. Element 7: The at least one alcohol-responsive active region comprises a polymer, and the ketoreductase of the first enzyme and the second enzyme are chemically bound to the polymer.

[0072] Element 8: The at least one alcohol-responsive active region comprises an electron transfer agent. Element 9: The at least one alcohol-responsive active region comprises a stabilizer. By way of non-limiting example, exemplary combinations applicable to A, B, C, D, and E include the following:

[0073] Non-limiting combinations of one, more, or all of 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 combination of one, more, or all of 1, 2, 3, 4, 5, 6, 7, and 8.

[0074] To facilitate a better understanding of the embodiments described herein, including the advantages of the alcohol sensor as described above, compared to other alcohol detection chemistries, the following examples of various representative embodiments are provided and should not be construed as limiting or defining the scope of the present invention.

[0075] Example 1: Example 1 evaluates the function of an alcohol sensor containing a KRT using a two-layer active area system. Example 1 was used to screen 24 KRT candidates for use in an alcohol sensor.

[0076] Alcohol Sensor Preparation. Various experimental KRT-containing alcohol-responsive active regions were prepared using a two-layer active region system. The active region was coated onto a carbon working electrode (note that other types of electrode surfaces can be used according to embodiments of the present disclosure). Here, the first layer composition (Table 1 below) was first coated directly onto the working electrode, and the second layer composition (Table 2 below) was coated onto the first layer composition. The first and second layers comprise the active region. Following deposition of the first layer composition, the first layer composition was cured overnight at room temperature (RT) (approximately 25°C). After the first layer composition cured, the second layer composition was deposited and cured overnight at RT. A PVP film was then applied to the working electrode using a coating solution formulated with 40:1 polyvinylpyridine-co-styrene (15) (i.e., containing 15% styrene) and PEG-DGE400. The film was deposited (3 x 5 mm / sec dip) onto the active area and cured overnight at RT, then at 56°C for 48 hours in a dry vial. It should be understood that the components in Tables 1 and 2 can be applied in a single layer (i.e., a mixture of all components) with or without the additional crosslinker (PEGDGE400) in Table 2 without departing from the scope of this disclosure. The layered active area used in this example is to facilitate testing and comparison of multiple KRT sensor samples.

[0077] [Table 1]

[0078] [Table 2]

[0079] Various sensors were prepared as provided in Example 1 above using KRTs obtained from Codexis, headquartered in Redwood City, California. The Codexis® enzyme code ("Codexis® Ref.") and respective cofactor (NAD+ or NADP+) for each of the 24 KRTs tested are listed in Table 3 and are hereafter referred to as Samples A1 through A24 ("ID").

[0080] [Table 3]

[0081] Beaker Calibration of Sensors A1-A24. Alcohol-sensing analysis of sensors containing KRT IDs A1-A24 prepared according to Example 1 was performed by immersing the electrodes in 100 mM PBS buffer and various concentrations of ethanol (2, 4, 6, 10, 15, 20, 30, and 40 mM ethanol) at 33 °C. Figures 5A-5D show the responses of each of A1-A24. As shown, the various KRT sensors had no, minimal, or inconsistent response to ethanol. Figure 5E is a static view of the beaker calibration at 4 mM ethanol, showing the change in current response (Δ) from baseline (no EtOH exposure) to 4 mM ethanol. As shown, the Δ of the sensor responses prepared using KRT IDs A1, A11, A15, and A16 at 4 mM ethanol showed substantial responses to ethanol. These are reflected in the various other ethanol concentrations in Figures 5A-5C, making them potential candidates for use in alcohol sensing. Therefore, KRT IDs A1, A11, A15, and A16 were further evaluated.

[0082] Example 2: Example 2 evaluates the function of alcohol sensors containing KRT IDs A1, A11, A15, and A16 in comparison to a control alcohol dehydrogenase active site system.

[0083] Alcohol Sensor Preparation. Experimental A1, A11, A15, and A16 KRT alcohol-responsive active regions were prepared using a monolayer active region system and compared to a control ADH-containing sensor. ADH was obtained from Sigma-Aldrich Corp., headquartered in St. Louis, Missouri, product number A3263. The ADC control was NAD. + The active area was coated onto a carbon working electrode with a monolayer composition (Table 4 below). Following active area deposition, the active area was cured overnight at room temperature. A PVP film was then applied to the working electrode using a film coating solution containing 4 mL of 100 mg / mL polyvinylpyridine and 100 μL of 100 mg / mL PEG-DGE400. The film was deposited onto the active area (3 × 5 mm / s immersion) and cured overnight at room temperature, then at 56 °C for 48 hours in a dry vial.

[0084] [Table 4]

[0085] Beaker calibration of A1-A24 sensors. Alcohol-sensing analysis of sensors containing A1, A11, A15, and A16 KRTs prepared according to Example 2 was performed by immersing the electrodes in 100 mM PBS buffer at room temperature and various concentrations of ethanol (2, 4, 6, 10, 15, 20, 30, and 40 mM ethanol). Figure 6A shows the response of each of A1, A11, A15, and A16. As shown, each KRT exhibits a measurable response to increasing ethanol concentrations. Figure 6B shows the linear sensitivity response based on beaker calibration of the A1, A11, A25, and A16 KRT sensors. A positive driving force is observed, particularly for A1, A11, and A15.

[0086] Beaker Stability. The beaker stability (long-term stability) of the A1, A11, A15, and A16 sensors from Example 2 was evaluated in 30 mM EtOH in 100 mM PBS at 52°C. The results, shown in Figure 6C, indicate that after 4 days, each signal underwent a different rate of sensor drop (decrease in stability for alcohol detection), as shown in Table 5. However, the signal drop for the A15 sensor was significantly less than that of the other KRT sensors tested.

[0087] [Table 5]

[0088] Aldehyde Inhibition. The performance of the A1, A11, A15, and A16 sensors from Example 2 was compared to the ADH control sensor from Example 2 for aldehyde inhibition (e.g., acetaldehyde inhibition as described above). The sensors were incubated in 30 mM EtOH in 100 mM PBS at 33°C over time and then spiked with 1 mM acetaldehyde (approximately 19.3 hours). The normalized signal results are shown in Figure 7. As shown, the effect of aldehyde inhibition was nonexistent or negligible for the A15 and A16 sensors up to approximately 20 hours. The A1 and A11 sensors showed slightly greater inhibition. The ADH control sensor showed substantial inhibition. The percent signal loss due to aldehyde inhibition for the tested sensors is shown in Table 6.

[0089] [Table 6]

[0090] Example 3. A standard kinetic assay performed at RT was performed on KRT IDs A15 and A16 and compared to an ADH control (ADH as described in Example 2 above). + (or NADP +) was used in the reaction, and the absorbance ("OD") of reduced NADH (or NADPH) at 340 nm was measured. The concentrations of A15 and A16 used in the kinetic assay were 0.167 mg / mL, and the concentration of the ADH control used in the kinetic assay was 0.0167 μg / mL (an order of magnitude lower than A15 and A16) (both concentrations in PBS buffer). The results of the kinetic assay are shown graphically in Figure 8. Numerical results are shown in Table 7. Notably, a substantially lower amount of the ADH control enzyme compared to the A15 and A16 KRT enzymes resulted in approximately 260% higher kinetic activity, while A15 and A16 exhibit significantly higher resistance to aldehyde inhibition (see Figure 7).

[0091] [Table 7]

[0092] Example 4. Standard kinetic assays performed at RT were performed on KRT IDs A15 and A16, each in a variety of alcohols, and compared to an ADH control (ADH as described in Example 2 above). + The absorbance (OD) of reduced NAD(P)H at 340 nm was measured. Testing of A15 and A16 in kinetic assays is shown in Figures 9A and 9B. Tests were performed in ethanol (EtOH), isopropyl alcohol (IPA), propanol (nPA), butanol (1-BuA), and methanol (MeOH). The "blank" omitted the KRT enzyme. The results of the KRT kinetic assay are shown graphically in Figures 9A and 9B. Figure 9A is for A15, and Figure 9B is for A16. Numerical results are shown in Table 8, which also includes results measured with an ADH control (not presented in graphical format). Notably, KRT showed substantially greater enzymatic activity in IPA compared to the ADH control, further demonstrating that the KRT sensors described herein can be used with alcohols other than primary alcohols.

[0093] [Table 8]

[0094] Example 5. In this example, KRT-containing sensors with the components of Example 2 plus comparative additional polymers and / or crosslinkers were evaluated. IPA Response. Building on the results of Example 4, the effect of IPA on A15 and A16 was further illustrated. A15 and A16 sensors were prepared according to Example 2, and in some cases, one or more additional polymers or crosslinkers were added to the active region composition. The additional polymer was poly(1-vinylimidazole) (“PVI”), and the additional crosslinker was glutaraldehyde (“Glut”). The current response of the sensors was first measured in 30 mM EtOH in 100 mM PBS buffer at 33°C for approximately 3.5 hours, and then spiked with increasing concentrations of IPA (1, 2, and 3 mM IPA). The results are shown in Figures 10A and 10B. Figure 10A is for A15, and Figure 10B is for A16. The additional polymer and / or crosslinker for each sensor tested is listed in Table 9, based on the references in the legends of Figures 10A and 10B. Here, total active area is expressed as mg / mL, and a "-" sign indicates that no component was added.

[0095] [Table 9]

[0096] As shown in Figures 10A and 10B, the response to 1 mM IPA was approximately 12-fold greater than the response to 30 mM EtOH. Furthermore, as shown in Figure 10A, the A15 sensors containing glutaraldehyde crosslinker generally exhibited higher sensitivity to IPA, with the A15 sensor containing only glutaraldehyde (and no PVI) exhibiting the highest sensitivity. Similar results are shown in Figure 10B for the A16 sensor. However, a lower concentration of glutaraldehyde (0.5 mg / mL) exhibited higher sensitivity to IPA compared to a higher concentration of glutaraldehyde (1 mg / mL).

[0097] Beaker Stability. The beaker stability (long-term stability) of the A15 and A16 sensors from Example 5 was evaluated in 30 mM EtOH in 100 mM PBS at 33°C. The results are shown in Figures 11A and 11B. Figure 11A is for A15, and Figure 11B is for A16. As shown, the stability of the A15 sensor is superior to that of the A16 sensor. Furthermore, the stability of the A15 sensor containing the additional glutaraldehyde crosslinker is superior to that of the other A15 compositions. However, precipitation of the active region was observed in the A15PVI4Glut05 and A15PVI4Glutl samples. More specifically, when both PVI and glutaraldehyde were present, the active region appeared cloudy. Table 10 shows the sensor dropout values ​​experienced by the A15 sensor over a 5-day period.

[0098] [Table 10]

[0099] Example 6. In this example, a KRT-containing sensor with additional polymer was compared to an ADH control sensor. Beaker Calibration. The alcohol-sensing assay of an A15 sensor prepared according to Example 2 and further containing 8 mg / mL of PVI in the active area was tested 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. Figure 12A shows the normalized signal response of each A15 sensor containing additional PVI polymer (labeled A15 + 8 PVI) and the ADH control sensor. As shown, the A15 sensor exhibits higher ethanol sensitivity (i.e., the signal loss is significantly greater in the ADH control after each higher concentration of EtOH). This is further illustrated in Figure 12B, which shows the linear sensitivity response of the two sensors tested.

[0100] Beaker Stability. The beaker stability (long-term stability) of the A15 and ADH control sensors of Example 6 was evaluated in 30 mM EtOH in 100 mM PBS at 33° C. The results, shown in FIG. 13, indicate that the stability of the KRT A15 sensor is significantly superior to that of the ADH control sensor.

[0101] 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 of the membrane solvents contained EtOH, and the other contained EtOH and HEPES buffer in an 80:20 ratio. The results are shown in Figure 14 and indicate that the stability of the sensor in EtOH solvent alone was higher than that in diluted EtOH solvent.

[0102] Example 8. Beaker stability (long-term stability) of analyte sensors with different membrane compositions (different from that of Example 2) containing active regions prepared according to Example 2 with the addition of 1 mg / mL glutaraldehyde was investigated using 30 mM PBS in 100 mM PBS buffer at 33°C. EtOH was evaluated. Three separate membrane compositions were prepared and coated onto A15 active areas (additionally containing glutaraldehyde) containing either 1%, 2%, or 4% PEG-EtOH in PVP. Signal stability results are shown in Figure 15 and followed very similar profiles. The short-term stability of the tested membranes was greater than the long-term stability based on this composition. Numerical signal degradation over 5 and 10 days is shown in Table 11.

[0103] [Table 11]

[0104] Example 9. Beaker stability (long-term stability) of analyte sensors with different membrane compositions (different from that of Example 2) containing active regions prepared according to Example 2 with the addition of 1 mg / mL glutaraldehyde was investigated using 30 mM PBS in 100 mM PBS buffer at 33° C. Three separate membrane compositions were prepared and coated onto A15 active areas (additionally containing glutaraldehyde) containing either PVP without crosslinker, polyvinylpyridine-co-styrene (30) without crosslinker (i.e., with 30% styrene) ("PVPSty30"), or PVPSty30 with a 4% PEG-DGE400 crosslinker. Signal stability results are shown in Figure 16. The addition of the crosslinker does not appear to significantly affect the stability of the KRT sensor.

[0105] Overall, embodiments of the present disclosure demonstrate that cooperative enzyme systems including KRT enzymes may be feasible as alcohol sensors for detecting an individual's alcohol level, particularly in vivo (although they may also be used for in vivo measurements). Of the KRTs tested in this disclosure, KRT sensors including A15 exhibited the greatest stability (beaker stability), while A16 exhibited the least product inhibition. Both have much higher specific activity toward IPA than EtOH (e.g., 200-fold for A15 and 700-fold for A16).

[0106] Unless otherwise indicated, all numbers expressing quantities and the like in this specification and the related claims should be understood to be modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon 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 application of 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.

[0107] One or more exemplary embodiments incorporating various features are presented herein. For clarity, not all features of a physical implementation are described or shown herein. It is understood that in developing a physical embodiment incorporating embodiments of the present invention, numerous implementation-specific decisions must be made to achieve the developer's goals, such as compliance with system-related, business-related, government-related, and other constraints. These will vary from implementation to implementation and from case to case. While the developer's efforts may be time-consuming, such efforts would nevertheless be routine for one of ordinary skill in the art and would have the benefit of this disclosure.

[0108] Although various systems, tools, and methods are described herein in terms of "including" various components or steps, the systems, tools, and methods may also "consist essentially of" or "consist of" various components and steps.

[0109] As used herein, the phrase "at least one of" followed by the word "and" or "or" preceding a list of items and separating any of the items modifies the list as a whole, rather than each member (i.e., each item) of the list. The phrase "at least one of" allows for the meaning of including at least one of any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. By way of example, the phrase "at least one of A, B, and C" or "at least one of A, B, or C" refers, respectively, to A only, B only, or C only; any combination of A, B, and C; and / or at least one of each of A, B, and C.

[0110] Thus, the disclosed systems, tools, and methods are well adapted to achieve the objects and advantages mentioned, as well as those inherent therein. The specific embodiments disclosed above are illustrative only, as the teachings of the present disclosure may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Moreover, no limitations are intended to the details of construction or design shown herein, except as described in the appended claims. Accordingly, it will be apparent that the specific exemplary embodiments disclosed above may be altered, combined, or modified, and all such variations are deemed to be within the scope of the present disclosure. The systems, tools, and methods illustratively disclosed herein may suitably be practiced in the absence of elements not specifically disclosed herein and / or optional elements disclosed herein. While systems, tools, and methods are described in terms of "including" various components or steps, the systems, tools, and methods may also "consist essentially of" or "consist of" the various components and steps. All of the numbers and ranges disclosed above may be subject to some variation. Whenever a numerical range with a lower and upper limit is disclosed, any number within that range and any included range is specifically disclosed. Specifically, all range values ​​disclosed herein (in the form "about a to about b," or, equivalently, "about a to b," or, equivalently, "about a to b") should be understood to describe all numbers and ranges within that broader range. Additionally, unless expressly and unambiguously defined by the patent owner, claim terms have their ordinary meanings. If there is a discrepancy in the usage of a word or term between this specification and one or more patents or other documents incorporated herein by reference, the definition in this specification shall control.

Claims

1. An alcohol-sensing composition comprising: ketoreductases, diaphorase, oxidized nicotinamide adenine dinucleotide (NAD+) or oxidized nicotinamide adenine dinucleotide phosphate (NAD(P)+) as a cofactor; an electron transfer agent, and polymer wherein one or more of the ketoreductase, the diaphorase, and the oxidized nicotinamide adenine dinucleotide (NAD+) or oxidized nicotinamide adenine dinucleotide phosphate (NAD(P)+) are covalently attached to the polymer.

2. The composition of claim 1 , wherein the ketoreductase is an aldo-ketoreductase.

3. 2. The composition of claim 1, wherein the ketoreductase is KRED-P1-A04, KRED-P2-C11, KRED-P2-G03, or KRED-P2-H07.

4. The composition of claim 1 , wherein the ketoreductase and the diaphorase are covalently attached to the polymer.

5. 2. The composition of claim 1, wherein the oxidized nicotinamide adenine dinucleotide (NAD+) or oxidized nicotinamide adenine dinucleotide phosphate (NAD(P)+) is covalently attached to the polymer.

6. The composition of claim 1 , wherein the polymer is polyvinylpyridine, polyvinylimidazole, a copolymer thereof, or a combination thereof.

7. The composition of claim 1 , wherein the electron transfer agent comprises a transition metal complex.

8. 8. The composition of claim 7, wherein the transition metal complex is a ruthenium-containing complex or an osmium-containing complex.

9. The composition of claim 1 , wherein the electron transfer agent comprises an osmium complex covalently bound to a polymer, the polymer being a poly(vinylpyridine)-based polymer.

10. The composition of claim 1 further comprising a stabilizer.

11. An alcohol sensor comprising the alcohol-sensing composition of claim 1.

12. 12. The alcohol sensor of claim 11, wherein the reaction between the ketoreductase and diaphorase mediated by the cofactor produces a signal proportional to alcohol concentration.

13. 1. A method for monitoring an alcohol level in a subject using an alcohol sensor, comprising: a) applying a potential to a working electrode of the alcohol sensor, the alcohol sensor having been inserted into the subject's tissue, the alcohol sensor comprising: The working electrode; at least one alcohol-responsive active region disposed on a surface of the working electrode, the at least one alcohol-responsive active region comprising at least (i) a ketoreductase, (ii) a diaphorase, (iii) an oxidized form of nicotinamide adenine dinucleotide (NAD+) or an oxidized form of nicotinamide adenine dinucleotide phosphate (NAD(P)+) as a cofactor, (iv) an electron transfer agent, and (v) a polymer, wherein one or more of the ketoreductase, the diaphorase, and the oxidized form of nicotinamide adenine dinucleotide (NAD+) or an oxidized form of nicotinamide adenine dinucleotide phosphate (NAD(P)+) are covalently bound to the polymer; sensor electronics coupled to the alcohol sensor and including a processor and a memory, the memory storing a baseline signal for comparison with a signal generated by the cofactor-mediated reaction of the ketoreductase with diaphorase, and the processor configured to monitor an alcohol level based on a comparison of the generated signal with the baseline signal; b) determining an alcohol level by comparing the generated signal to a baseline signal over a period of time; and c) transmitting said alcohol level to a reader device or a personal communication device; A method comprising:

14. The method of claim 13, wherein the period of time is one minute.

15. 14. The method of claim 13, wherein the period of time is five minutes.

16. The method of claim 13, wherein the alcohol level is transferred to a reader device.

17. The method of claim 13 , wherein the alcohol level is transferred to a personal communication device.

18. The method of claim 17 , wherein the personal communication device is a mobile phone.

19. 14. The method of claim 13, wherein the ketoreductase is KRED-P1-A04, KRED-P2-C11, KRED-P2-G03, or KRED-P2-H07.

20. 14. The method of claim 13, wherein the electron transfer agent comprises an osmium complex covalently bound to a polymer, the polymer being a poly(vinylpyridine)-based polymer.

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