Analytical sensor and detection method for alcohol detection

Abstract

The present invention provides an apparatus, method, and system for detecting an individual's alcohol concentration, particularly an individual's internal alcohol concentration. [Solution] The alcohol-sensing composition includes at least one alcohol-responsive active region comprising a cooperative enzyme system having at least a first enzyme and a second enzyme that can work together to facilitate the detection of alcohol. At least one of the enzymes in the cooperative enzyme system is ketoreductase.

Description

[Background technology]

[0001] The detection of various analytes within an individual can sometimes be essential for monitoring their health and well-being. Deviations from normal analyte levels often indicate underlying physiological conditions such as metabolic state or disease, or exposure to specific environmental factors or stimuli.

[0002] Individual analyte monitoring may be performed periodically or continuously over a set period. Periodic analyte monitoring may be performed by taking samples of bodily fluids, such as blood, at one or more time intervals and analyzing them in vitro. Continuous analyte monitoring may be performed using one or more sensors that remain at least partially implanted in the individual's tissue, such as intracutaneously, subcutaneously, or intravenously, so that analysis can be performed in vivo. The implanted sensors can collect analyte data at any indicated rate, for example, depending on the individual's specific health needs and / or previously measured analyte levels.

[0003] Any analyte may be suitable for in vivo analysis if the appropriate chemical properties for detecting it can be identified. In fact, in vivo amperometry sensors configured to analyze glucose have been developed and improved in recent years. Other analytes commonly exposed to physiological dysregulations that would be similarly desirable to monitor include, but are not limited to, levels of lactate, oxygen, pH, A1c, ketones, and drugs.

[0004] Another analyte that may be particularly important to an individual's health is their alcohol level. In fact, information related to an individual's in vivo alcohol level can be used to predict or monitor levels of other analytes of interest. For example, alcohol can alter the blood glucose control of individuals whose blood glucose levels are naturally dysregulated or lack homeostasis without intervention, which can be harmful to the individual. Other analytes that can be dysregulated by alcohol include, among others, 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). Therefore, it is desirable to monitor an individual's alcohol level. [Brief explanation of the drawing]

[0005] The accompanying drawings are included to illustrate specific aspects of the disclosure and should not be considered exclusive embodiments. The disclosed subject matter can be substantially modified, altered, combined, and replaced with equivalents in form and function without departing from the scope of the disclosure. [Figure 1] An example of a detection system that may incorporate the analyte sensor of this disclosure is shown in the diagram. [Figure 2A] A diagram shows an exemplary two-electrode analyte sensor configuration having a single working electrode, suitable for use in some embodiments disclosed herein. [Figure 2B] A diagram shows an exemplary three-electrode analyte sensor configuration having a single working electrode, suitable for use in some embodiments disclosed herein. [Figure 2C] Same as above. [Figure 3] A diagram shows 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]This disclosure presents various embodiments of a cooperative enzyme system related to alcohol detection using ketoreductase, nicotinamide adenine dinucleotide, and diaphorase placed on a working electrode. [Figure 4B] Various embodiments of this disclosure illustrate a coordinated enzyme system related to alcohol detection using ketoreductase, nicotinamide adenine dinucleotide phosphate, and diaphorase placed on a working electrode. [Figure 5A] This graph shows the response of various KRT-containing sensors to various ethanol concentrations. [Figure 5B] This graph shows the response of various KRT-containing sensors to various ethanol concentrations. [Figure 5C] This graph shows the response of various KRT-containing sensors to various ethanol concentrations. [Figure 5D] This graph shows the response of various KRT-containing sensors to various ethanol concentrations. [Figure 5E] Figures 5A-5C show graphs of the responses of four of the KRT-containing sensors at 4 mM ethanol. The graphs show the change (Δ) in current response from baseline (no EtOH exposure) to 4 mM ethanol. [Figure 6A] This graph shows the response of various KRT-containing sensors to various ethanol concentrations. [Figure 6B] Figure 6A shows the linear sensitivity response of ethanol concentrations measured. [Figure 6C] Figure 6A shows a graphical representation of the stability response of various KRT-containing sensors. [Figure 7] This graph shows the stability response of various KRT-containing sensors and ADH-controlled sensors to aldehyde exposure. [Figure 8] This graph shows the results of various KRT kinetic assays compared to ADH controls. [Figure 9A] Figures 9A and 9B show graphical representations of the results of kinetic assays of two KRTs with different alcohol types. [Figure 9B] Same as above. [Figure 10A] Figures 10A and 10B show graphical representations of the responses of two KRT-containing sensors of various compositions to IPA. [Figure 10B] Same as above. [Figure 11A] Figures 11A and 11B show graphical representations of the stability responses of various KRT-containing sensors having different additional polymer and crosslinker compositions. [Figure 11B] Same as above. [Figure 12A] A graphical representation of the response of a specific KRT-containing sensor containing a specific polymer composition to containing various ethanol concentrations is shown. [Figure 12B] Figure 12A shows the linear sensitivity response of the ethanol concentration measured. ADH control means alcohol dehydrogenase (ADH) from the signal. Figures 12A - 12B compare the linearity of ADH and KRT A15. [Figure 13] A graphical representation of the stability response of a specific KRT-containing sensor containing a specific polymer composition is shown. Figure 13 compares the beaker stability of ADH and KRT A15. [Figure 14] A graphical representation of the stability response of a specific KRT-containing sensor having a membrane immersed in two different solvents is shown. [Figure 15] Graphical representations of the stability responses of various KRT-containing sensors having different membrane compositions are shown. [Figure 16] Graphical representations of the stability responses of various KRT-containing sensors having different membrane and crosslinker compositions are shown. **DETAILED DESCRIPTION**

[0006] Detailed Description The present disclosure generally describes analyte sensors and methods using enzymes 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 that helps people with diabetes 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 effects of the analyte of interest on the dysregulation of other analytes.

[0008] An individual's internal alcohol concentration can vary dramatically based on alcohol consumption, addiction, and / or various physiological factors. For example, the ability to metabolize alcohol differs from person to person, and therefore, even with the same dose of alcohol per unit of body weight, alcohol levels will differ between individuals. In fact, the equilibrium concentration of alcohol in tissues depends at least on water content, blood flow velocity, and tissue mass. Since alcohol can pass through biological membranes, it can easily flow from the bloodstream into 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 in the digestive system even without consuming alcohol. As described above, the presence of a certain concentration of alcohol can further affect the function of one or more other analytes in an individual, and may further affect 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 can sometimes suffer from false positive or false negative results, and therefore can be inaccurate at least occasionally. In contrast, this disclosure provides an analyte sensor that responds to alcohol over time to facilitate health management. As used herein, the term “alcohol,” and its grammatical variations thereof, refers to any primary, secondary, or tertiary alcohol. For example, the alcohol sensors of this disclosure can detect ethanol, methanol, butanol, propanol, isopropyl alcohol, and any combination thereof.

[0010] The alcohol sensors described herein can respond to in vivo alcohol levels and provide "continuous" measurements of said alcohol levels. That is, the alcohol sensors described herein may be capable of providing multiple alcohol concentration measurements over extended (continuous) periods such as seconds, minutes, or hours, or days, weeks, or months.

[0011] Alcohol sensors can offer numerous advantages for monitoring the dynamic levels of alcohol, relating to the resulting physiological state and its impact on other analytes of interest. For example, the sensors of this disclosure utilize a specific enzymatic reaction involving one or more ketoreductases (hereinafter referred to as "KRT"). The detection chemistry involving KRT described herein advantageously overcomes various hurdles associated with the typical detection of individual alcohol levels. These include overcoming enzymatic inhibition by one or more products of the enzymatic reaction of the detection chemistry, as will be discussed in more detail below. By overcoming enzymatic inhibition, the alcohol sensors of this disclosure can, among other things, enable the manufacture of more cost-effective sensors without compromising effectiveness, while minimizing the use of a single mass-limiting membrane and the complexity of the required detection area configuration. The chemical properties involving KRT are even more advantageous, as they can sense both primary and secondary alcohols, with a particularly unexpected affinity for detecting secondary alcohols.

[0012] Individuals wearing the continuous alcohol sensor described herein can access real-time alcohol level information and, based on that, make various decisions, 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 required, and any combination thereof. Furthermore, the alcohol sensor of this disclosure can be used to monitor, test, and / or evaluate the alcohol levels of individuals suffering from alcohol misuse, abuse, or addiction. In doing so, the health of such individuals can be monitored by the individuals themselves or by healthcare professionals or law enforcement experts. Sensor electronics and processing algorithms related to the operation of the sensor and detection system described herein, including display units and their devices, may also provide instructions, guidance, recommendations, and / or outputs regarding alcohol concentration and suggested actions thereunder. Appropriate processing algorithms, processors, memory, electronic components, etc., may reside in any of the following: a reliable computer system, a remote terminal, a cloud server, a reader device, and / or the housing of the sensor itself. Guidance, recommendations, outputs, etc., may be displayed on an appropriate display unit or device electronically communicating 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 a third-party server, cloud server, or various software and healthcare-related applications such as a personal health monitor. A suitable remote terminal, cloud server, etc., can further relay its output to related secondary devices such as smart home devices, wearable technology, personal health monitors, or a combination thereof.

[0013] This 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 sensors and determine one or more alcohol concentrations therefrom. The processor and / or coding instructions can then, in some embodiments, analyze the alcohol concentration to determine one or more recommendations based on a particular alcohol concentration. For example, it may suggest whether another health analyte may be adversely affected using the alcohol concentration at a particular evaluation time or other quantities that can be derived from the alcohol concentration. In such cases, the processor and / or coding instructions may suggest a variety of ways in which an individual can respond based on the detected alcohol concentration, including suggestions for immediate and future actions. For example, depending on an individual's particular alcohol concentration, recommendations could be whether to drive a car, whether to take action to compensate for another affected analyte (e.g., if the analyte is glucose, whether to take carbohydrates or inject insulin), whether to see a doctor, and any combination thereof.

[0014] Before describing the alcohol sensors of this disclosure in more detail, a brief overview of appropriate in vivo analyte sensor configurations and sensor systems using analyte sensors is first provided to better understand the embodiments of this disclosure. 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, in general, to detect alcohol in vivo, according to various embodiments of this disclosure.

[0015] Figure 1 shows a diagram of an exemplary detection system in which the alcohol sensor of this disclosure may be incorporated. As shown, the detection system 100 includes a sensor control device 102 and a reader device 120 configured to communicate with each other via a wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted local communication path or link. The reader device 120 may, according to some embodiments, constitute an output medium for displaying analyte concentrations and warnings or notifications determined by the sensor 104 or an associated processor, and may allow one or more user inputs. The reader device 120 may be a multipurpose smartphone or a dedicated electronic reader device. Although only one reader device 120 is shown, in certain cases, there may be multiple reader devices 120. The reader device 120 may also communicate with remote terminals 170 and / or trusted computer systems 180, respectively, via a communication path / link 141 and / or 142, which is also wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. In addition to or instead of the above, the reader device 120 may communicate with a network 150 (e.g., a cellular network, the Internet, or a cloud server) via a communication path / link 151. The network 150 may be further communicated to a remote terminal 170 via a communication path / link 152 and / or to a reliable computer system 180 via a communication path / link 153. Alternatively, or in addition to the above, the sensor 104 may communicate directly with the remote terminal 170 and / or the reliable computer system 180 without the presence of the intervening reader device 120. For example, according to some embodiments, the sensor 104 may communicate with the remote terminal 170 and / or the reliable computer system 180 via a direct communication link to the network 150, 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, or Wi-Fi®, can be used for each of the communication paths or links. The remote terminal 170 and / or reliable computer system 180 are accessible, according to some embodiments, to 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 capable of housing circuits and a power supply for operating the sensor 104. Optionally, the power supply and / or active circuits may be omitted. A processor (not shown) may be communicatively connected to the sensor 104, and the processor is physically located within the sensor housing 103 or the reader device 120. The sensor 104 protrudes from the underside of the sensor housing 103 and extends through an adhesive layer 105. According to some embodiments, the adhesive layer 105 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 the target tissue, such as within the dermis or subcutaneous layer of the skin. The sensor 104 may include a sensor tail of sufficient length to be inserted to a desired depth in a given tissue. The sensor tail may include at least one working electrode and one or more active regions (detection regions / spots or detection layers) positioned on at least one working electrode and active for detecting alcohol (or optionally, one or more additional analytes). In some embodiments, the active regions are in the form of one or more individual spots (e.g., 1 to about 10 spots, or more) and are about 0.01 mm in size. 2 ~about 1mm 2This range includes any values ​​and subsets between them, but larger or smaller individual active region spots are also intended in this application.

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

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

[0021] Continuing to refer to Figure 1, in some embodiments, the sensor control unit 102 can automatically transfer data obtained from the sensor 104 to the reader device 120. For example, once alcohol concentration data is acquired, it is stored in memory until it is transmitted (e.g., every few seconds, every minute, every five minutes, or at other predetermined intervals) and can be communicated automatically and periodically 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 enter the communication range of the reader device 120. The data may remain stored in the memory of the sensor control unit 102 until it is communicated to the reader device 120. Thus, the patient does not need to be in constant proximity to the reader device 120, but can instead upload data at a convenient time. In yet another embodiment, a combination of automatic and non-automatic data transfer can be implemented. For example, data transfer can be automatically continued until the reader device 120 is out of the communication range of the sensor control unit 102. While 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 a reliable computer system 180 without departing from the scope of this disclosure.

[0022] To facilitate the introduction of the sensor 104 into the tissue, an introducer may be temporarily present. In exemplary embodiments, the introducer may include a needle or a similar sharp object. In alternative embodiments, it should be recognized that 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 simultaneously with the sensor 104 before 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, thereby enabling the implantation of the sensor 104. After opening the access path, the needle or other introducer may be withdrawn to avoid posing a sharp hazard. In exemplary embodiments, a suitable needle may be solid or hollow, beveled or non-beveled, and / or have a circular or non-circular cross-section. In more specific embodiments, a suitable needle may be comparable to an acupuncture needle having a cross-sectional diameter of about 150 to about 300 micrometers (e.g., about 250 micrometers) in terms of cross-sectional diameter and / or tip design. However, it should be recognized that a suitable needle may have a larger or smaller cross-sectional diameter if required for a particular application.

[0023] In some embodiments, the tip of the needle or introducer (while present) may be angled over 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 be located within the lumen or groove of the needle or inserter, and the needle similarly opens an access path for the sensor 104. In either case, the needle is withdrawn after facilitating the insertion of the sensor.

[0024] The alcohol sensors disclosed herein may feature an active region located on a single working electrode (e.g., one or both sides of a single working electrode) or on two or more separate working electrodes (e.g., one or both sides of two working electrodes). According to various embodiments of this disclosure and as further described herein, the single working electrode sensor configuration can utilize a two-electrode or three-electrode detection motif. Sensor configurations featuring a single working electrode are described below with reference to Figures 2A-2C. Each of these sensor configurations can appropriately incorporate one or more alcohol-responsive active regions according to various embodiments of this disclosure. Sensor configurations featuring multiple working electrodes are then described with reference to Figures 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 conjunction with the detection of the alcohol level.

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

[0026] Figure 2A shows an exemplary two-electrode sensor configuration having a single working electrode, suitable for use in alcohol detection according to embodiments of the present disclosure. As shown, the sensor 200 includes a substrate 212 positioned between a working electrode 214 and a counter / reference electrode 216. Alternatively, the working electrode 214 and the counter / reference electrode 216 may be positioned on the same side of the substrate 212 with a dielectric material inserted between them (configuration not shown). The active region 218 is positioned as at least one layer on 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] Continuing to refer to Figure 2A, the membrane 220 covers at least the active region 218 and, optionally according to some embodiments, can cover part or all of the working electrode 210 and / or the counter / reference electrode 216, or the entire analyte sensor 200. One or both sides of the analyte sensor 200 may be covered by the membrane 220. The membrane 220 may comprise one or more polymer membrane materials having the ability to restrict the flow of analyte to the active region 218 and / or biocompatible. The sensor 200 may be operable to assay at least alcohol by any of the detection techniques by coulometry, amperometry, voltammetry, potentiometry, or iontophoresis (including reverse iontophoresis).

[0028] Figures 2B and 2C illustrate exemplary three-electrode analyte sensor configurations having 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 those shown for sensor 200 in Figure 2A, except that sensors 201 and 202 (Figures 2B and 2C) include an additional electrode, represented as electrode 217. With the additional electrode 217, the counter / reference electrode 216 can function as either the counter electrode or the reference electrode, while the additional electrode 217 performs the other electrode function. The working electrode 214 continues to perform its original function. The additional electrode 217 can be positioned on either the working electrode 210 or electrode 216, with a dielectric isolation layer between them. For example, as shown in Figure 2B, dielectric layers 219a, 219b, and 219c provide electrical isolation by separating electrodes 214, 216, and 217 from each other. Alternatively, at least one of electrodes 214, 216, and 217 may be located on the opposite side of the substrate 212, as shown in Figure 2C. Thus, in some embodiments, electrodes 214 (working electrode) and 216 (counter electrode) may be located on the opposite side of the substrate 212, and electrode 217 (reference electrode) may be located on one of electrodes 214 or 216 and separated therefrom by a dielectric material. Alternatively, in some embodiments, electrodes 214 (working electrode) and 216 (reference electrode) may be located on the opposite side of the substrate 212, and electrode 217 (counter electrode) may be located on one of electrodes 214 or 216 and separated therefrom by a dielectric material. In yet another embodiment, the reference electrode and counter electrode may be located on one side of the substrate 212, and the working electrode may be located on the opposite side. Optionally, a reference material layer 230 (e.g., Ag / AgCl) may be present on the electrode 217, and the position of the reference material layer 230 is not limited to those shown in Figures 2B and 2C.

[0029] Similar to sensor 200 shown in Figure 2A, the active regions 218 of 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 regions 218 of sensors 201 and 202. Furthermore, analyte sensors 201 and 202 may be operable to assay at least alcohol by any of the following detection techniques: coulometry, amperometry, voltammetry, electrochemistry by potentiometry, or iontophoresis.

[0030] Continuing with reference to Figures 2A and 2B, similar to the analyte sensor 200, the film 220 can also cover the active region 218 and other sensor components in sensors 201 and 202. In some embodiments, an additional electrode 217 may be covered by the film 220. In Figures 2B and 2C, electrodes 214, 216 and 217 are depicted as all covered by the film 220, but it should be understood that in some embodiments, only the working electrode 214 may be covered, or in some embodiments, only the working electrode 214 and one other electrode may be covered. Furthermore, the thickness of the film 220 on each of electrodes 214, 216 and / or 217 may be the same or different, and the amount of surface area of ​​each of electrodes 214, 216 and / or 217 covered by the film 220 may also be the same or different. As in the case of the two-electrode analyte sensor configuration (Figure 2A), one or both sides of the analyte sensors 201 and 202 may be covered with the film 220 in the sensor configurations of Figures 2B and 2C, or the entire analyte sensors 201 and 202 may be covered. Therefore, the three-electrode sensor configurations shown in Figures 2B and 2C should be understood as non-limiting examples of embodiments disclosed herein, with alternative electrode and / or layer configurations within the scope of this disclosure.

[0031] Figure 3 shows an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode, suitable for use in the alcohol sensor described herein. As shown in Figure 3, the sensor 300 includes working electrodes 304 and 306, respectively, located on opposing surfaces of a substrate 302. An active region 310 is located on the surface of the working electrode 304, and an active region 312 is located on the surface of the working electrode 306. Collectively, one or more enzymes may be present in the active regions 310 and 312 configured for at least the detection of 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, and the other active region may be 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, and the reference electrode 321 is electrically insulated from the working electrode 306 by a dielectric layer 323. The outer dielectric layers 330 and 332 are positioned on the reference electrode 321 and the counter electrode 320, respectively. The film 340 can cover at least the active regions 310 and 312 according to various embodiments. Other components of the analyte sensor 300 may also be optionally covered with the film 340, and as described above, one or both sides, or a portion thereof, of the analyte sensor 300 may be covered with the film 340. Similar to the analyte sensors 200, 201, and 202, the sensor 300 may be operable to assay at least alcohol by any of the detection techniques of coulometry, amperometry, voltammetry, potentiometry, or iontophoresis.

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

[0033] The above description relating to Figures 2 and 3 is primarily directed toward an analyte sensor configuration having two working electrodes, but it should be understood through the extension of the disclosure herein that more than two working electrodes can be incorporated without issue. Additional working electrodes allow for additional active regions, and the corresponding detection capability is given to such a sensor having such features.

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

[0035] According to various embodiments of this disclosure, electron transfer agents may be present in one or more active regions 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 electron transfer agents within each active region can determine the redox potential observed in the alcohol analyte.

[0036] Suitable electron transfer agents may include electroreducible and electrooxidizing ions, complexes, or molecules (e.g., quinones) having 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. Patents 6,134,461 and 6,605,200, which are incorporated herein by reference in their entirety. Additional examples include those described in U.S. Patents 6,736,957, 7,501,053 and 7,754,093, the respective disclosures 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., polyvinylferrocene or hexacyanoferrate), cobalt, and its metallocene compounds. Suitable ligands for metal complexes may 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 o-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 electron transfer agent selected for use in the alcohol-responsive active region described herein is an osmium complex.

[0037] According to various embodiments of this 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, the enzymes may each be chemically bonded or otherwise immobilized on a single polymer. In other embodiments, such as when multiple enzymes are used, one or more enzymes may be polymerized on a first polymer and one or more other enzymes may be polymerized on a second polymer so that both the polymers and enzymes form the active regions 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 combination thereof. Exemplary copolymers suitable for inclusion in the active region include, for example, those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile. If multiple active regions exist, one or more polymers within each active region may be the same or different. Any combination of the aforementioned polymers may also be used without departing from the scope of this disclosure.

[0039] According to various embodiments of this disclosure, electron transfer agents can be covalently bonded to one or more polymers in one or more active regions. The method of covalent bonding is not considered to be particularly limited. Covalent bonding of electron transfer agents to one or more polymers can occur by polymerizing monomer units having covalently bonded electron transfer agents, or, if one or more polymers have already been synthesized, by reacting the electron transfer agents with them separately. According to some embodiments, a bifunctional spacer can covalently bond electron transfer agents to one or more polymers in an active region, where a first functional group is reactive with one or more polymers (e.g., a functional group that can quaternize 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 exemplary embodiments, one or more polymers within the active region of the alcohol sensor disclosed herein may be poly(4-vinylpyridine), of which some monomer units are functionalized with alkylcarboxylate side chains, some monomer units are attached to the osmium electron transfer agent with amide spacer groups, and some monomer units are not functionalized. That is, the polymer may be a redox polymer of an osmium-decorated poly(vinylpyridine)-based polymer, referred to herein as "X7".

[0041] Similarly, according to some or other various embodiments of the present disclosure, one or more enzymes within one or more active regions may be covalently bonded to one or more polymers. If multiple enzymes are present in a single active region, all of the multiple enzymes may be covalently bonded to a single polymer or to two or more distinct polymers, which may be the same or different. For example, if two or more enzymes are covalently bonded to separate and different polymers, one polymer can be coated onto the other to form an active region. In other embodiments, only some of the multiple enzymes may be covalently bonded to one or more polymers. For example, a first enzyme may be covalently bonded to a first polymer, and a second enzyme may be acovalently bonded to either the first or second polymer.

[0042] Covalent bonding of enzymes to polymers can occur via crosslinkers introduced with appropriate crosslinking agents. Suitable crosslinkers for reaction with free amino groups in enzymes may include, for example, polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanogen chloride, N-hydroxysuccinimide, imide esters, epichlorohydrin, or their derivatized variants. Suitable crosslinkers for reaction with free carboxylic acid groups in enzymes may include, for example, carbodiimides. Crosslinking is generally intermolecular, but can be intramolecular in some embodiments.

[0043] As described above, electron transfer agents and / or one or more enzymes can bind to one or more polymers within the active region by means other than covalent bonding. In some embodiments, electron transfer agents and / or one or more enzymes can bind to one or more polymers ionically or coordinationally. For example, a charged polymer can ionically bond with an electron transfer agent or enzyme that is conversely charged. In yet another embodiment, electron transfer agents and / or one or more enzymes can be physically encombined within one or more polymers without binding to them.

[0044] In some embodiments, incorporating stabilizers into the active region of the alcohol sensor described herein can improve the function of the sensor and achieve desired sensitivity and stability. Such stabilizers may include, for example, antioxidants and / or companion proteins for stabilizing 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 suitable albumin), glutaraldehyde-bridged albumin, catalase, glutaraldehyde-bridged catalase, other enzyme antioxidants, and any combination thereof. The stabilizers may be conjugated or unconjugated and may or may not be chemically bound to the active region. The amount of stabilizer may vary depending on the type of stabilizer selected, but can be about 1% to about 50% by weight of the total active region (i.e., the combined weight of the components of the active region). This includes any values ​​and subsets between them, for example, 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 membrane or biocompatible membrane may be positioned in at least a portion of the active region. Suitable membranes may consist of crosslinked polymers containing heterocyclic nitrogen groups, such as polyvinylpyridine and / or polyvinylimidazole polymers. Embodiments also include membranes made of polyurethane, polyether urethane, or chemically related materials, and membranes made of silicone.

[0046] In some embodiments, the membrane may be formed in situ by crosslinking a polymer in a buffer (e.g., an alcohol buffer or other biological buffer such as HEPES) that is modified with an amphoteric moiety, a nonpyridine copolymer component, and optionally another moiety that is either hydrophilic or hydrophobic and / or has other desirable properties. The modified polymer can be prepared from a precursor polymer containing heterocyclic nitrogen groups. For example, the precursor polymer may 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 a specific type of alcohol 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 membrane can be applied to the active area by means of deposition of the membrane solution, powder coating of the membrane solution, etc., and any combination thereof, by placing one or more droplets of the membrane solution on at least one working electrode of the alcohol sensor, for example by immersing the sensor tail in the membrane solution, by spraying the membrane solution onto the sensor tail, by heat pressing or melting a membrane of any size (including discrete or all layers, etc.).

[0048] In general, the film thickness is controlled by the concentration of the film solution, the number of droplets of the film solution applied, the number of times the sensor tail is immersed in the film solution, the amount of film solution sprayed onto the sensor tail, and any combination of these factors. In some embodiments, the films described herein may have a thickness in the range of about 0.1 micrometers (μm) to about 1000 μm, encompassing any value and subsets between them, e.g., about 1 μm to about 500 μm, or about 10 μm to about 100 μm. As described above, the film can cover one or more active regions, and in some embodiments, the active regions may have a thickness of about 0.1 μm to about 10 μm, encompassing any value and subsets between them. Furthermore, the active regions may have a size of about 0.001 mm 2 ~about 1mm 2 This range may include any values ​​and subsets between them. However, it should be understood that thicker or thinner films, thicker and thinner active regions, and larger or smaller active regions are also intended in this application.

[0049] In some embodiments, the film 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 film may consist of a polyvinylpyridine-co-styrene polymer in which some of the pyridine nitrogen atoms are functionalized with uncrosslinked poly(ethylene glycol) tails and some of the pyridine nitrogen atoms are functionalized with alkylsulfonic acid groups. Other membrane compounds may, alone or in combination with the aforementioned membrane compounds, include suitable copolymers of 4-vinylpyridine and styrene and amine-free polyether arms. Any combination of the aforementioned membrane polymers may also be used without departing from the scope of this disclosure.

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

[0051] In some embodiments, the membrane compositions for use as mass transfer limiting layers of the present disclosure may include polydimethylsiloxane (PDMS), polydimethylsiloxane diglycidyl ether (PDMS-DGE), aminopropyl-terminated polydimethylsiloxane, and any combination thereof, for use as leveling agents (e.g., to reduce the contact angle of the membrane composition or sensing element composition). Branched versions having similar terminal chemical structures are also suitable for the present disclosure. Further examples of certain leveling agents may include those found in U.S. Patent 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 its grammatical variations refer to any kind of interaction between atoms or molecules that enables compounds to form bonds with one another, including, but not limited to, covalent bonds, ionic bonds, dipole-dipole interactions, hydrogen bonds, London dispersion forces, and any combination thereof. For example, in situ polymerization of a membrane can form crosslinks between one or more polymers of the membrane and one or more polymers within the active region. In some embodiments, crosslinking of the membrane to the active region facilitates a reduction in the occurrence of delamination of the membrane from the sensor.

[0053] As stated above, this disclosure provides various methods, systems, and apparatus for detecting at least alcohol (e.g., alcohol concentration or level) in a person's bodily fluids. The detection may be in vitro or in vivo, utilizing the active region detection chemistry described herein to overcome one or more hurdles in the manufacture of a desirable alcohol sensor.

[0054] Alcohols can be detected using one or more enzymes, such as alcohol oxidase. Typical examples include alcohol oxidase, which interacts with alcohols, mostly primary alcohols, to form acetaldehyde (C2H4O) and hydrogen peroxide (H2O2). Other primary and secondary alcohols react to form aldehydes with corresponding higher or lower carbon content. Alcohol oxidase catalyzes only the forward conversion of alcohol to acetaldehyde and contains a strongly bound flavin cofactor. Therefore, theoretically, alcohol oxidase can be used in analyte sensors to detect alcohol by analyzing either the acetaldehyde or hydrogen peroxide product produced by the enzymatic reaction. However, this approach has two problems. Firstly, 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 in terms of promoting alcohol oxidation, thereby rendering the sensor ineffective in analyzing alcohol. Furthermore, if acetaldehyde and hydrogen peroxide are sequestered or quenched with other agents (which further complicates the sensing composition of the active region), there will be no species, or a sufficient number, available for electrochemical detection. Secondly, alcohol oxidase does not freely exchange electrons with redox mediators other than molecular oxygen. Therefore, electron transfer agents bound to polymers within the sensor's active region, such as osmium or other transition metal complexes described herein, are ineffective in cycling alcohol oxidase from an inactive reducing state to an oxidizing state that reacts with alcohol.

[0055] Alcohol dehydrogenases are another group of enzymes that promote the conversion of alcohols (mostly primary alcohols) to aldehydes or ketones. A typical example is alcohol dehydrogenase, which converts nicotinamide adenine dinucleotide (NAD). +In the presence of ), it interacts with ethanol to form acetaldehyde (C2H4O) and reduced nicotinamide adenine dinucleotide (NADH). Other primary and secondary alcohols react to form corresponding aldehydes with higher or lower carbon content. Unlike alcohol oxidases, alcohol dehydrogenases not only catalyze the forward conversion of alcohols to aldehydes (collectively referred to herein as acetaldehyde, although other aldehydes with higher or lower carbon content may also be produced) but also carry out the reverse reaction. Further unlike alcohol oxidases, alcohol dehydrogenases do not contain bound cofactors and therefore require exogenous cofactors to activate the enzyme in relation to promoting alcohol oxidation. In addition, the activity of alcohol dehydrogenases is controlled, at least in part, based on product inhibition by NADH and acetaldehyde (see Figure 4). These properties typically make alcohol dehydrogenases unsuitable for use in the design of detection chemistry for alcohol sensors.

[0056] Embodiments of the present disclosure utilize an oxidoreductase scheme for detecting alcohol levels using one or more embodiments of the analyte sensors of the present disclosure. More specifically, the detection chemistry described herein utilizes two enzymes that interact cooperatively with each other in at least one active region on a working electrode, where one of the enzymes is KRT. As used herein, the terms “cooperative enzyme” or “cooperative enzyme system,” and their grammatical variations, refer to at least two enzymes that can interact cooperatively (jointly) with each other. KRT is a type of oxidoreductase known to have NADH-dependent or NAD(P)(H)-dependent catalytic activity and is primarily capable of reducing aldehydes and ketones. Typically, KRT is relatively inefficient as an alcohol dehydrogenase because it lacks the metal cofactor used by alcohol dehydrogenases to hold and position the alcohol group. Furthermore, KRT usually prefers NAD(P)H to NADH. Therefore, their effective use in detecting individual alcohol levels has not been considered beneficial.

[0057] Embodiments of the present disclosure provide coenzyme systems suitable for use in the detection of alcohol, and the coenzyme systems utilize KRT. The KRT described for use in the sensing chemistry of the present disclosure for detecting alcohol includes wild-type (naturally occurring) and non-natural engineered peptide chains that exhibit KRT functionality. For example, the Ald KRT family may be suitable for use in embodiments of the present disclosure. Without being bound by theory, it is further contemplated that changes in one or more residues of the active site of KRT may change its reaction mechanism and thus its affinity for the detection of alcohol. That is, certain KRTs may be more effective at alcohol detection compared to other KRTs depending on their chemical composition.

[0058] More specifically, the coenzyme 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) + ) into aldehyde (e.g., acetaldehyde) and reduced NADH or reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H), respectively. Aldehyde functions as a representative molecule indicating the presence of alcohol. NADH or NAD(P)H may be reduced under the mediation of diaphorase, and the electrons transferred during this process provide the basis for alcohol detection at one or more working electrodes.

[0059] Figure 4A shows the cooperative reaction between KRT and diaphorase mediated by the NAD + cofactor for the detection of alcohol. For the detection of alcohol, NAD(P) +Figure 4B shows the cooperative reaction between KRT and diaphorase mediated by a cofactor. In Figures 4A and 4B, diaphorase is chemically bonded (e.g., by covalent bonds) to a polymer placed on the working electrode (e.g., in the active region of an alcohol sensor), and KRT is chemically bonded (e.g., by covalent bonds) to the same polymer (e.g., via a crosslinking agent) or another polymer. For example, in one embodiment, diaphorase may be chemically bonded to a first polymer placed on the working electrode, and KRT may be chemically bonded to a second polymer placed on the first polymer. The membrane is positioned to cover the entire active region and contains a specific cofactor (e.g., NAD + or NAD(P) + Either of the above may be non-covalently bonded to the polymer in the active region, but the membrane may be otherwise restricted. 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 the polymer placed on the working electrode. For example, X7 comprises both a covalently bonded polymer and an electron transfer agent as described above. In some embodiments, the active region comprises an X7 polymer to which diaphorase and KRT are chemically bonded, and these are covered by a membrane.

[0060] KRT and diaphorase may be present in amounts ranging from approximately 1% to approximately 50% by weight of the total active region (i.e., the combined weight of the components of the active region), including any values ​​and subsets between them, for example, approximately 1% to approximately 40% by weight of the total active region, or approximately 10% to approximately 40% by weight.

[0061] As can be seen from Figures 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, i.e., the amount of alcohol present (i.e., alcohol concentration or level). The correlation between the working electrode current and the alcohol concentration can be determined by referring to a lookup table of currents at known alcohol concentrations or by using a calibration curve.

[0062] Accordingly, in some embodiments, the Disclosure provides an alcohol-responsive active region based on a cooperative enzyme system of ketoreductase and diaphorase. More specifically, the Disclosure provides an analytic sensor and system comprising at least a working electrode and a sensor tail comprising at least one alcohol-responsive active region disposed on the surface of the working electrode, wherein the alcohol-responsive active region comprises an enzyme system comprising at least two enzymes that can act cooperatively to facilitate the detection of alcohol, one of which is ketoreductase. The at least one alcohol-responsive active region may be further covered by a membrane. Furthermore, at least the sensor tail of the analytic sensor may be configured for at least partial insertion into tissue, such as transdermally, subcutaneously, or intravenously, so that the analysis can be performed in vivo. In other embodiments, the analytic sensor (including the sensor tail) may be fully embedded in tissue. Accordingly, the Disclosure provides a method for detecting alcohol using the above-described analytic sensor. 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 is wearing the analyte sensor (e.g., for more than one day, e.g., up to about one month or longer). The signal can be detected (e.g., by electrochemical detection) from the alcohol-responsive active region of the analyte sensor, which is proportional to the alcohol concentration.

[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 the 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 that can act in coordination to facilitate the detection of alcohol, the first enzyme being ketoreductase.

[0064] Embodiment B: A method comprising detecting a signal proportional to the concentration of alcohol using an analyte sensor, wherein the analyte sensor comprises a sensor tail comprising at least a working electrode and configured to be implanted in tissue; and at least one alcohol-responsive active region disposed on the surface of the working electrode, wherein the at least one alcohol-responsive active region comprises an enzyme system comprising at least a first enzyme and a second enzyme that can act in coordination to facilitate the detection of alcohol, the first enzyme being ketoreductase.

[0065] Embodiment C: An electrode assembly comprising at least one working electrode and at least one alcohol-responsive active region disposed on the 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 that can act in coordination to facilitate the detection of alcohol, the first enzyme being ketoleductase.

[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 that can act in coordination to facilitate the detection of alcohol, wherein the first enzyme is ketoreductase.

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

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

[0069] Element 2: Ketreductase is aldoketreductase. Element 3: The ketoductase is KRED-P1-A04, KRED-P2-C11, KRED-P2-G03, or KRED-P2-H07, manufactured by Codexis.

[0070] Element 4: A film is placed on the at least one alcohol-responsive active region. Element 5: The membrane is located on the at least one alcohol-responsive active region, and the membrane is 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 first enzyme, ketoreductase, 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. As a non-limiting example, exemplary combinations applicable to A, B, C, D, and E include the following:

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

[0074] In addition to the advantages of the alcohol sensor described above, and to facilitate a better understanding of the embodiments described herein compared to other alcohol detection chemistry, the following examples of various representative embodiments are given. The following examples should not be construed as limiting or defining the scope of the invention.

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

[0076] Preparation of alcohol sensors. Various experimental KRT-containing alcohol-responsive active regions were prepared using a two-layer active region system. The active regions were coated onto a carbon working electrode (note that other types of electrode surfaces can be used according to embodiments of this disclosure). Here, a first layer composition (Table 1 below) was first coated directly onto the working electrode, and a second layer composition (Table 2 below) was coated on top of the first layer composition. The first and second layers contain active regions. Following the deposition of the first layer composition, the first layer composition was cured overnight at room temperature (RT) (approximately 25°C). After curing the first layer composition, the second layer composition was deposited and cured overnight at RT. Subsequently, the PVP film was applied to the working electrode using a coating solution containing 40:1 polyvinylpyridine-co-styrene (15) (i.e., containing 15% styrene) and PEGDGE400. The film was deposited on the active region (3 × 5 mm / second immersion), cured overnight in RT, and then cured 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 crosslinking agent (PEGDGE400) in Table 2, without departing from the scope of this disclosure. The layered active region used in this example is intended to facilitate the testing and comparison of numerous KRT sensor samples.

[0077] [Table 1]

[0078] [Table 2]

[0079] Using KRT obtained from Codexis, headquartered in Redwood City, California, various sensors were prepared as provided in Example 1 above. The Codexis® enzyme codes ("Codexis® Ref.") and their respective cofactors (NAD+ or NADP+) for the 24 KRTs tested are shown in Table 3, and will be referred to hereafter as Samples A1 to A24 ("ID").

[0080] [Table 3]

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

[0082] Example 2: Example 2 evaluates the functionality of alcohol sensors including KRT IDs A1, A11, A15, and A16 compared to a control alcohol dehydrogenase active region system.

[0083] Preparation of alcohol sensors. Experimental A1, A11, A15, and A16KRT alcohol-responsive active regions were prepared using a monolayer active region system and compared to a control ADH-containing sensor. The ADH was obtained from Sigma-Aldrich Corp., headquartered in St. Louis, Missouri, under product number A3263. ADC control was performed using NAD. + Cofactors were utilized. The active region was coated onto a carbon working electrode with a monolayer composition (Table 4 below). Following the deposition of the active region, it was cured overnight in RT. Subsequently, the PVP film was 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 PEGDGE400. The film was deposited onto the active region (immersion at 3 × 5 mm / second), cured overnight in RT, and then cured at 56°C for 48 hours in a dry vial.

[0084] [Table 4]

[0085] Beaker calibration of sensors A1-A24. Alcohol detection analysis of sensors including A1, A11, A15, and A16KRT prepared according to Example 2 was performed by immersing the electrodes in 100 mM PBS buffer of RT and various concentrations of ethanol (2, 4, 6, 10, 15, 20, 30, and 40 mM ethanol). Figure 6A shows the responses of A1, A11, A15, and A16, respectively. As shown, each KRT shows a measurable response to increasing ethanol concentration. Figure 6B shows the linear sensitivity response of the A1, A11, A25, and A16KRT sensors based on beaker calibration. Positive driving forces are shown particularly for A1, A11, and A15.

[0086] Beaker Stability. The beaker stability (long-term stability) of sensors A1, A11, A15, and A16 in 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 detecting alcohol), as shown in Table 5. However, the signal drop of sensor A15 was significantly smaller than that of the other KRT sensors tested.

[0087] [Table 5]

[0088] Aldehyde inhibition. The performance of sensors A1, A11, A15, and A16 in Example 2 was compared with that of the ADH control sensor in Example 2 due to aldehyde inhibition (e.g., acetaldehyde inhibition as described above). The sensors were incubated over time in 30 mM EtOH in 100 mM PBS at 33°C 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 negligible or nonexistent for sensors A15 and A16 up to approximately 20 hours. Sensors A1 and A11 showed slightly greater inhibition. The ADH control sensor showed substantial inhibition. The percentage decrease in signal due to aldehyde inhibition for the tested sensors is shown in Table 6.

[0089] [Table 6]

[0090] Example 3. A standard kinetic assay performed in RT was performed on KRT IDs A15 and A16 and compared with an ADH control (ADH as described in Example 2 above). Cofactor NAD + (or NADP) +The reaction was performed using ) 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 (orders of magnitude smaller than that of A15 and A16) (both concentrations in PBS buffer). The results of the kinetic assay are shown graphically in Figure 8. The numerical results are shown in Table 7. In particular, despite the substantially smaller amount of ADH control enzyme compared to A15 and A16 KRT enzymes, it yielded approximately 260% higher kinetic activity, although A15 and A16 showed considerably higher resistance to aldehyde inhibition (see Figure 7).

[0091] [Table 7]

[0092] Example 4. Standard kinetic assays performed in RT were carried out on KRT IDs A15 and A16 in various types of alcohol and compared with the ADH control (ADH described in Example 2 above). Cofactor NAD(P) + The reaction was performed using the reduced NAD(P)H, and the absorbance ("OD") at 340 nm was measured. The tests for A15 and A16 in the kinetic assays are shown in Figures 9A and 9B. The tests were performed in ethanol (EtOH), isopropyl alcohol (IPA), propanol (nPA), butanol (1-BuA), and methanol (MeOH). "Blank" excludes the KRT enzyme. The results of the KRT kinetic assays are shown graphically in Figures 9A and 9B. Figure 9A is for A15, and 9B is for A16. Numerical results are shown in Table 8. This also includes results measured with the ADH control (not shown graphically). In particular, KRT showed substantially greater enzymatic activity in IPA compared to the ADH control, further demonstrating that the KRT sensor described herein can be used with alcohols other than primary alcohols.

[0093] [Table 8]

[0094] Example 5. In this example, a KRT-containing sensor having additional polymers and / or crosslinking agents compared to the components of Example 2 was evaluated. IPA response. Following the results of Example 4, the effect of IPA on A15 and A16 was further illustrated. Sensors A15 and A16 were prepared according to Example 2, and in some examples, 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 for about 3.5 hours in 30 mM EtOH in 100 mM PBS buffer at 33°C, and then spiked (buffer changed and IPA added) while increasing the concentration of IPA (1, 2, and 3 mM IPA). The results are shown in Figures 10A and 10B. Figure 10A is for A15, and 10B is for A16. The additional polymers and / or crosslinkers for each sensor tested are shown in Table 9, based on the legend in Figures 10A and 10B. Here, the concentration is expressed as mg / mL of the total active range, and the "-" symbol indicates that no additional components were added.

[0095] [Table 9]

[0096] As shown in Figures 10A and 10B, the response to 1 mM IPA was approximately 12 times greater than the response to 30 mM EtOH. Furthermore, as shown in Figure 10A, the A15 sensor containing glutaraldehyde crosslinking agent generally showed higher sensitivity to IPA, with the A15 sensor containing only glutaraldehyde (and without PVI) showing the highest sensitivity. Similar results are shown in Figure 10B for the A16 sensor. However, lower concentrations of glutaraldehyde (0.5 mg / mL) showed higher sensitivity to IPA compared to higher concentrations of glutaraldehyde (1 mg / mL).

[0097] 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 Figures 11A and 11B. Figure 11A is for A15, and 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 crosslinking agent 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, the active region appeared cloudy when both PVI and glutaraldehyde were present. Table 10 shows the sensor dropout values ​​experienced by the A15 sensors over 5 days.

[0098] [Table 10]

[0099] Example 6. In this example, a KRT-containing sensor with an additional polymer was compared to an ADH control sensor. Beaker calibration. Alcohol detection analysis of A15 sensors prepared according to Example 2 and further containing 8 mg / mL of PVI in the active region 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 sensors were compared to ADH control sensors prepared according to Example 2. Figure 12A shows the normalized signal responses of each A15 sensor and ADH control sensor containing an additional PVI polymer (labeled A15+8PVI). As shown, the A15 sensors exhibit higher ethanol sensitivity (i.e., signal loss is considerably greater in the ADH control after each higher concentration of EtOH). This is further shown in Figure 12B, which shows the linear sensitivity responses of the two sensors tested.

[0100] Beaker stability. The beaker stability (long-term stability) of the A15 and ADH control sensors in Example 6 was evaluated in 30 mM EtOH in 100 mM PBS at 33°C. The results shown in Figure 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, showing that the sensor's stability in EtOH solvent alone was higher than that in diluted EtOH solvent.

[0102] Example 8. The beaker stability (long-term stability) of analyte sensors having different membrane compositions (different from those in Example 2), containing an active region prepared according to Example 2 by adding 1 mg / mL of glutaraldehyde, was evaluated in 30 mM EtOH in 100 mM PBS buffer at 33°C. Three distinct membrane compositions were prepared and coated onto the A15 active region (further containing glutaraldehyde) containing either 1%, 2%, or 4% PEGDGE400 in PVP. The signal stability results are shown in Figure 15, and they followed very similar courses. The short-term stability of the tested membranes was greater than the long-term stability based on this composition. The numerical signal degradation over 5 and 10 days is shown in Table 11.

[0103] [Table 11]

[0104] Example 9. The beaker stability (long-term stability) of analyte sensors having different membrane compositions (different from those in Example 2), and containing an active region prepared according to Example 2 with the addition of 1 mg / mL of glutaraldehyde, was evaluated in 30 mM EtOH in 100 mM PBS buffer at 33°C. Three distinct membrane compositions were prepared and coated onto the A15 active region (further containing glutaraldehyde) containing either PVP without a crosslinking agent, polyvinylpyridine-co-styrene (30) without a crosslinking agent (i.e., having 30% styrene) ("PVPSty30"), or PVPSty30 containing 4% PEGDGE400 as a crosslinking agent. The signal stability results are shown in Figure 16. The addition of crosslinking agents does not appear to have a significant effect on the stability of the KRT sensor.

[0105] Overall, embodiments of this disclosure demonstrate that a collaborative enzyme system comprising KRT enzymes can be implemented as an alcohol sensor for detecting individual alcohol levels, particularly in vivo (but may also be used for in vivo measurements). Of the KRTs tested in this disclosure, the KRT sensor containing A15 exhibited the greatest stability (beaker stability), while A16 exhibited the least product inhibition. Both had much higher specific activity to IPA than to EtOH (e.g., 200 times for A15 and 700 times for A16).

[0106] Unless otherwise specified, all numbers representing quantities, etc., in this specification and related claims should be understood in all cases as being modified by the term "approximately." Therefore, unless otherwise indicated, the numerical parameters described in the following specification and appended claims are approximations that may vary depending on the desired characteristics to be obtained by embodiments of the present invention. Each numerical parameter should be interpreted by applying ordinary rounding techniques, at least in light of the reported number of significant figures, not as an attempt to limit the application of the doctrine of equivalents to the claims.

[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 an embodiment of the present invention, a number of implementation-specific decisions must be made to achieve the developer's objectives, such as compliance with system-related, business-related, government-related, and other constraints. These will vary depending on the implementation and may vary from case to case. While the developer's efforts may be time-consuming, such efforts are routine for those skilled in the art and will be of interest to the present disclosure.

[0108] While various systems, tools, and methods are described herein in terms of "including" various components or steps, systems, tools, and methods can also be "essentially made up of" or "consisting of" various components and steps.

[0109] As used herein, the phrase “at least one” that precedes a set of items and separates any of the items, accompanied by the terms “and” or “or,” applies to the entire list, rather than to each member of the list (i.e., each item). The phrase “at least one of ~” may mean 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. For example, the phrase “at least one of A, B, and C” or “at least one of A, B, or C” refers 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, respectively.

[0110] Accordingly, the disclosed systems, tools, and methods are well-suited to achieving the stated objectives and benefits, as well as those inherent therein. The teachings of this disclosure can be modified and implemented in different equivalent ways, which will be obvious to those skilled in the art who are interested in the teachings herein; therefore, the specific embodiments disclosed above are merely illustrative. Furthermore, except as described in the appended claims, it is not intended to limit the scope to the structural or design details shown herein. Accordingly, it is obvious that the specific exemplary embodiments disclosed above can be modified, combined, or altered, and all such variations are considered to be within the scope of this disclosure. The systems, tools, and methods disclosed exemplary herein can be adequately implemented without elements not specifically disclosed herein and / or any optional elements disclosed herein. While systems, tools, and methods are described in terms of "including" various components or steps, systems, tools, and methods can also "essentially consist of" or "consist of" various components and steps. All numbers and scopes disclosed above are subject to some variation. Whenever a numerical range with lower and upper limits is disclosed, any number within that range and any range included therein are specifically disclosed. In particular, all range values ​​disclosed herein (in the form of "about a to about b," or equivalently "about a to b," or equivalently "about a~b") should be understood to describe all numbers and ranges included in the broader range of values. Furthermore, unless explicitly and clearly defined by the patentee, the terms in the claims have their original, ordinary meanings. In the event of any inconsistency in the use of a word or term between this specification and one or more patent documents or other documents that may be incorporated herein by reference, the definitions in accordance with this specification shall prevail.

Claims

1. (a) A sensor, working electrode, At least one alcohol-responsive active region disposed on the surface of the working electrode, comprising at least (i) ketoreductase, (ii) diaphorase, and (iii) oxidized nicotinamide adenine dinucleotide (NAD+) or oxidized nicotinamide adenine dinucleotide phosphate (NAD(P)+) as a cofactor, and Mass transfer limiting membrane disposed in at least a portion of the at least one alcohol-responsive active region The reaction between ketoreductase and diaphorase is mediated by the cofactor, generating a signal proportional to the alcohol concentration. The sensor is adapted to provide continuous alcohol monitoring in vivo, and (b) A processor that is communicatively connected to the sensor. A sensor control device including a sensor control device.

2. The sensor control device according to claim 1, wherein the ketoreductase in the sensor is aldo ketoreductase.

3. The sensor control device according to claim 1, wherein the ketoreductase in the sensor is KRED-P1-A04, KRED-P2-C11, KRED-P2-G03, or KRED-P2-H07.

4. The sensor control device according to claim 1, wherein the ketoreductase and diaphorase in the sensor are covalently bonded to a polymer.

5. The sensor control device according to claim 1, wherein oxidized nicotinamide adenine dinucleotide (NAD+) or oxidized nicotinamide adenine dinucleotide phosphate (NAD(P)+) is covalently bonded to the polymer.

6. The sensor control device according to claim 4 or 5, wherein the polymer is polyvinylpyridine, polyvinylimidazole, copolymers thereof, or combinations thereof.

7. The sensor control device according to claim 1, wherein the sensor further comprises an electron transfer agent.

8. The sensor control device according to claim 7, wherein the electron transfer agent of the sensor comprises a transition metal complex.

9. The sensor control device according to claim 8, wherein the transition metal complex of the sensor is a ruthenium-containing complex or an osmium-containing complex.

10. The sensor control device according to claim 8, wherein the electron transfer agent of the sensor comprises an osmium complex covalently bonded to a polymer, and the polymer is a poly(vinylpyridine)-based polymer.

11. The sensor control device according to claim 1, further comprising a stabilizer.

12. The sensor control device according to claim 1, wherein the mass transfer limiting membrane comprises polyvinylpyridine, polyvinylimidazole, copolymers thereof, or combinations thereof.

13. The sensor control device according to claim 1, wherein the mass transfer limiting membrane comprises polyvinylpyridine.

14. The sensor control device according to claim 1, wherein the mass transfer limiting membrane comprises polyvinylpyridine-co-styrene.

15. The sensor control device according to claim 1, wherein the mass transfer limiting membrane comprises polyvinylimidazole.

16. The sensor control device according to claim 1, wherein the mass transfer limiting film has a thickness of about 0.1 μm to about 100 μm.

17. The sensor control device according to claim 1, wherein the mass transfer limiting membrane is covalently bonded to at least one alcohol-responsive active region.

18. An analyte detection system comprising the sensor control device according to claim 1 for monitoring the alcohol level in a target.

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