Analyte sensors using multiple enzymes and methods related thereto

Abstract

The present disclosure relates to analyte sensors using multiple enzymes and methods related thereto. A plurality of enzymes may be present in one or more active regions of the electrochemical sensor to facilitate analysis of one or more analytes. Multiple enzymes may act independently to detect several analytes or collectively to detect a single analyte. A sensor configuration includes a first active region and a second active region wherein a redox potential of the first active region is substantially separated from a redox potential of the second active region to allow for independent signal generation. Some configurations may have active regions coated with a multi-component film containing two or more different film polymers. Sensor configurations having multiple enzymes capable of synergistically interacting include those where a first enzyme converts an analyte to a first product and a second enzyme converts the first product to a second product, thereby generating a signal on a working electrode proportional to the analyte concentration.

Description

[0001] This application is a divisional application of an application with a filing date of January 28, 2020, application number 202080011303.7, and invention name “Analyte sensor using multiple enzymes and related methods thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 797,566, filed on January 28, 2019, entitled “Analysis Sensor Employing Multiple Enzymes and Methods Associated Therewith.” Background Art

[0004] Detection of various analytes in an individual is sometimes critical to monitoring their health and well-being. Deviations from normal analyte levels can often indicate an underlying physiological condition, such as a metabolic condition or disease, or exposure to a specific environmental condition.

[0005] Any analyte may be suitable for physiological analysis, provided that a suitable chemical can be identified to sense the analyte. To this end, amperometric sensors configured for in vivo glucose determination have been developed and improved in recent years. Other analytes that are commonly subject to physiological disorders include, but are not limited to, lactate, oxygen, pH, A1e, ketones, drug levels, etc., which may also be desirable for in vitro or in vivo monitoring.

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

[0007] Periodic ex vivo analyte monitoring is sufficient to determine the physiological status of many individuals. However, ex vivo analyte monitoring may be inconvenient or painful for some people. In addition, if the analyte measurement is not obtained at the appropriate time, there is no way to recover the lost data.

[0008] For individuals with severe analyte imbalances and / or rapidly fluctuating analyte levels, continuous analyte monitoring with an implanted sensor may be a more ideal approach, although it may also benefit other individuals. Although continuous analyte monitoring with an implanted sensor may be advantageous, there are challenges associated with these types of measurements. Intravenous analyte sensors have the advantage of providing analyte concentrations directly from the blood, but they are invasive and sometimes painful for individuals to wear, especially for long periods of time. Subcutaneous, interstitial or skin analyte sensors can generally be less painful for individuals to wear and can provide adequate measurement accuracy in many cases.

[0009] In vivo analyte sensors are typically configured to analyze a single analyte to provide a specific analysis, typically using enzymes to provide analytical specificity. However, physiological interactions between various combinations of analytes may also make multi-analyte analysis desirable in some cases. Currently, in vivo analysis of multiple analytes may require the use of a corresponding number of analyte sensors configured to analyze each analyte. This approach may be inconvenient because multiple analyte sensors are required to be worn by an individual. In addition, multiple analyte sensors may impose an unacceptable cost burden on an individual or insurance provider. During this sensing protocol, there is also an increased chance that one of the independent analyte sensors will fail.

[0010] In vivo analyte sensor can also include a membrane disposed on at least the implanted portion of the analyte sensor. In one aspect, the membrane can improve the biocompatibility of the analyte sensor. On the other hand, the membrane can be permeable or semi-permeable for the analyte of interest, and the total analyte flow rate is limited to the active area of ​​the analyte sensor. That is, the membrane can be used as a mass transfer limiting membrane. Limiting the analyte to enter the active area of ​​the sensor with a mass transfer limiting membrane can help avoid sensor overload (saturation), thereby improving detection performance and accuracy. This membrane is highly specific for limiting the mass transfer of a specific analyte, and other substances penetrate through the membrane at significantly different rates. The different membrane permeabilities of various potential analytes represent a significant obstacle to the development of analyte sensors configured for measuring multiple analytes. That is, different membrane permeability values ​​may lead to significantly different sensitivities of multiple analytes, thereby complicating the analysis. The different sensitivities to multiple analytes can sometimes be partially compensated by using active areas of different sizes (e.g., smaller active areas for analytes with high sensitivity / permeability and larger active areas for analytes with lower sensitivity / permeability), but this method may have significant manufacturing challenges and may not be applicable to all situations. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following drawings are included to illustrate certain aspects of the present disclosure and should not be considered to be exclusive embodiments. The disclosed subject matter is capable of considerable modification, alteration, combination, and equivalents in form and function without departing from the scope of the present disclosure.

[0012] Figure 1 A diagram of an illustrative sensing system that may incorporate the analyte sensors of the present disclosure is shown.

[0013] Figure 2A A diagram showing an illustrative two-electrode analyte sensor configuration with a single working electrode that is compatible for use in some embodiments disclosed herein. Figure 2B and 2C A diagram showing an illustrative three-electrode analyte sensor configuration with a single working electrode that is compatible for use in some embodiments disclosed herein.

[0014] Figure 3 A diagram of an illustrative analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode is shown, which may be compatible for use in some embodiments of the present disclosure.

[0015] Figure 4 An exemplary analyte sensor configuration suitable for use with some embodiments of the disclosure herein is shown in which two distinct active regions are disposed on the surface of a single working electrode.

[0016] Figure 5A Shown are the coordinated enzymatic reaction cycles associated with ethanol detection using alcohol oxidase and xanthine oxidase directly on the working electrode, according to various embodiments of the present disclosure.

[0017] Figure 5B Shown are coordinated enzymatic reaction cycles associated with ketone detection using β-hydroxybutyrate dehydrogenase, nicotinamide adenine dinucleotide, and diaphorase directly on a working electrode, according to various embodiments of the present disclosure.

[0018] Figure 5C Shown are coordinated enzymatic reaction cycles associated with ketone detection using β-hydroxybutyrate dehydrogenase, nicotinamide adenine dinucleotide, NADH oxidase, and superoxide dismutase directly on a working electrode, according to various embodiments of the present disclosure.

[0019] Figure 5D Shown are coordinated enzymatic reaction cycles associated with ketone detection using β-hydroxybutyrate dehydrogenase, nicotinamide adenine dinucleotide, and poly-1,10-phenanthroline-5,6-dione directly on a working electrode, according to various embodiments of the present disclosure.

[0020] Figure 5EThe coordinated enzymatic reaction cycle associated with ethanol detection using glucose oxidase, catalase, and xanthine oxidase is shown, according to various embodiments of the present disclosure, wherein the glucose oxidase is remote from the working electrode and the xanthine oxidase is directly on the working electrode.

[0021] Fig. 6A and 6B A diagram of an exemplary working electrode is shown in which a first active region is disposed directly on the surface of the working electrode and a second active region is separated from the working electrode by a membrane and is suitable for use in some embodiments disclosed herein. Figure 6C A diagram of an illustrative working electrode is shown in which a first active region and a second active region are laterally spaced apart from each other on the working electrode, and one of the active regions is separated from the working electrode by a membrane.

[0022] Figure 7 A schematic diagram of a portion of an analyte sensor having two working electrodes and featuring a bilayer membrane coating one of the two working electrodes that is compatible for use with some embodiments disclosed herein is shown.

[0023] Figure 8 Shown are exemplary cyclic voltammograms obtained for analyte-free buffer solutions using working electrodes containing two different osmium complexes as electron transfer mediators. Fig. 9 Display when Figure 8 Four replicates of the electrode response in 5 mM glucose / 5 mM lactate buffer as the electrode was cycled between E1 and E2 potentials.

[0024] Fig.10 Shown are triplicate responses of an electrode containing both alcohol oxidase and xanthine oxidase in the active area when exposed to different ethanol concentrations.

[0025] Fig.11A Shows Fig.10 Illustrative graph of the average current response of the electrode to ethanol concentration. Fig. 11B An exemplary graph of the current response versus ethanol concentration for a single electrode is shown.

[0026] Fig. 12A Shown are two replicates of the response of an electrode containing glucose oxidase and xanthine oxidase layered in separate active zones and separated by a membrane, with catalase present in the active zone containing glucose oxidase, when exposed to different concentrations of ethanol. Fig. 12BShown are comparative response data between electrodes containing glucose oxidase and xanthine oxidase layered in separate active zones and separated by a membrane, with catalase present separately in the active zone, upon exposure to varying concentrations of ethanol.

[0027] Fig.13 Shows Fig. 12A and Fig. 12B Illustrative graph of the average current response of the electrode to ethanol concentration.

[0028] Fig.14 Exemplary curves are shown for the response of electrodes coated with polymers 1A and 1B to 5 mM lactate solution.

[0029] Fig.15 An exemplary curve is shown for the response of an electrode coated with polymer 2 to a 5 mM lactate solution.

[0030] Fig.16 An exemplary curve is shown for the response of an electrode coated with polymer 3 to a 5 mM lactate solution.

[0031] Fig.17 Exemplary curves showing the response of an electrode coated with polymer 3 to lactate solutions with different lactate concentrations.

[0032] Fig.18 An exemplary curve is shown for the response of an electrode coated with polymer 4 to a 5 mM lactate solution.

[0033] Fig.19 Exemplary curves showing the response of an electrode coated with polymer 4 to lactate solutions with different lactate concentrations.

[0034] Fig. 20 An exemplary curve is shown for the response of an electrode coated with a bilayer membrane comprising a lower layer of cross-linked PVP (polymer 2) and an upper layer of cross-linked polymer 1B to a 5 mM lactate solution.

[0035] Fig.21 Illustrative curves are shown for the response of an electrode coated with a bilayer membrane comprising a lower layer of cross-linked PVP (Polymer 2) and an upper layer of cross-linked Polymer 1A, wherein the electrode was dip-coated with Formulations 1 and 3 for variable numbers of times, to a 5 mM lactate solution.

[0036] Fig. 22 An exemplary curve is shown for the response of an electrode coated with a hybrid film comprising cross-linked PVP (Polymer 2) and cross-linked Polymer IB to a 5 mM lactate solution.

[0037] Fig.23Exemplary curves showing the response of an electrode coated with a hybrid film comprising cross-linked PVP (Polymer 2) and cross-linked Polymer IB to different lactate concentrations.

[0038] Fig.24 Illustrative curves are shown for the response of electrodes coated with mixed films containing various ratios of cross-linked PVP (Polymer 2) and cross-linked Polymer IB.

[0039] Fig.25 An exemplary curve is shown for the response of a sensor containing two working electrodes to a 30 mM glucose / 5 mM lactate solution, wherein the lactate-responsive working electrode is coated with a bilayer film and the glucose-responsive working electrode is coated with a homogeneous film.

[0040] Fig.26 Exemplary curves are shown for the response of a sensor comprising two working electrodes to solutions containing different concentrations of glucose and lactate, wherein the lactate-responsive working electrode is coated with a bilayer membrane and the glucose-responsive working electrode is coated with a homogeneous membrane.

[0041] Fig. 27 Shown are the responses of four replicates of an electrode containing diaphorase, NAD+, and β-hydroxybutyrate dehydrogenase when exposed to different β-hydroxybutyrate concentrations.

[0042] Fig.28 Shows Fig. 27 Illustrative graph of the average current response of an electrode to β-hydroxybutyrate concentration.

[0043] Fig.29 Shows when Fig. 27 Example curve of the current response of an electrode exposed to 8 mM β-hydroxybutyrate in 100 mM PBS at 33°C for 2 weeks.

[0044] Fig.30 An illustrative graph showing sensor performance for Groups 1-4 in Example 6 is shown.

[0045] Fig.31 Exemplary curves showing the responses of the analyte sensors of Group 1 of Example 6 to lactate solutions having different lactate concentrations are shown.

[0046] Fig.32 is a schematic diagram of an example analyte monitoring and vehicle control system according to one or more embodiments of the present disclosure. DETAILED DESCRIPTION

[0047] The present disclosure generally describes analyte sensors and methods that employ multiple enzymes for detection, and more particularly describes analyte sensors and methods in which multiple enzymes can function independently or synergistically to detect one or more analytes.

[0048] As described above, analyte sensors are typically used to detect a single analyte. If multiple analytes need to be detected, a corresponding number of analyte sensors may be used. This approach may be undesirable, particularly due to cost issues, the necessity for an individual to wear multiple analyte sensors, and the increased likelihood of individual sensor failure.

[0049] Some analyte sensors utilize enzyme reactions as the basis for detecting the analyte of interest. Since enzymes generally display reaction specificity to a particular substrate or substrates of related species, they can provide analyte sensors with detection chemistry that is configured to analyze a single analyte of interest. Therefore, analyte sensors for analyzing a single analyte generally incorporate only the corresponding single enzyme to promote a suitable enzyme reaction for ease of detection. Currently, in order to provide detection capabilities for multiple analytes, it may be quite difficult to incorporate multiple enzymes into analyte sensors. Reasons include differences in analyte sensitivity and potential incompatibility of one or more enzymes with a given set of analytical conditions.

[0050] In contrast to analyte sensors characterized by a single enzyme, the present disclosure describes analyte sensors in which multiple enzymes are present in one or more active regions of the sensor. By incorporating multiple enzymes in the analyte sensor in various ways as described herein, many advantages can be achieved. In some sensor configurations of the present disclosure, multiple enzymes can promote independent detection of multiple analytes, such as glucose and lactate. Also described herein is a membrane configured to provide customized permeability for multiple analytes, which can promote analyte detection with a single analyte sensor by leveling the sensitivity level of the sensor to each analyte. In other sensor configurations of the present disclosure, multiple enzymes can be selected to act in synergy to promote detection of a single target analyte, otherwise it may be problematic or impossible to measure using a single enzyme. In any case, fewer electrodes may be required to detect a given analyte or analyte group compared to other feasible ones. In addition, the present disclosure can provide a sensor size smaller than other possible sensor sizes and a smaller sensor measurement electronics complexity. Therefore, according to the disclosure herein, an analyte sensor using multiple enzymes of multiple different configurations can promote effective detection of one or more analytes.

[0051] Analyte sensors containing multiple enzymes, whether operating independently or in concert, can function with enhanced stability in the presence of an appropriate stabilizer. Stabilizers that can be used include, for example, catalase or albumin (e.g., bovine serum albumin or human serum albumin). Catalase is known for its ability to remove reactive species such as peroxides from biological environments. In contrast, albumins are thought not to exhibit the function of removing reactive species, so their ability to stabilize the response of the analyte sensors of the present disclosure is surprising.

[0052] Before describing the analyte sensor of the present disclosure in more detail, a brief overview of suitable in vivo analyte sensor configurations and sensor systems employing analyte sensors is first provided so that embodiments of the present disclosure can be better understood. It should be understood that any sensor system and analyte sensor configuration described below can feature a variety of enzymes according to various embodiments of the present disclosure.

[0053] Figure 1 Shown is a diagram of an illustrative sensing system that can be combined with the analyte sensor of the present disclosure.As shown, sensing system 100 includes sensor control device 102 and reading device 120, which are configured to communicate with each other through local communication path or link, and the local communication path or link can be wired or wireless, unidirectional or bidirectional and encrypted or non-encrypted.According to some embodiments, reader device 120 can constitute an output medium for viewing analyte concentration and an alarm or notification determined by sensor 104 or a processor associated therewith, and allows one or more users to input.Reader device 120 can be a multi-purpose smart phone or a dedicated electronic reader instrument.Although only one reader device 120 is shown, multiple reader devices 120 can be present in some cases.Reader device 120 can also communicate with remote terminal 170 and / or trusted computer system 180 via one or more communication paths / links 141 and / or 142 respectively, and the communication path / link can also be wired or wireless, unidirectional or bidirectional and encrypted or non-encrypted. Reader device 120 may also or alternatively communicate with network 150 (e.g., a mobile phone network, the Internet, or a cloud server) via communication path / link 151. Network 150 may also be communicatively coupled to remote terminal 170 via communication path / link 152 and / or to trusted computer system 180 via communication path / link 153. Alternatively, 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, according to some embodiments, sensor 104 may communicate with remote terminal 170 and / or trusted computer system 180 via a direct communication link to network 150, as described in U.S. Patent Application Publication 2011 / 0213225, which is incorporated herein by reference in its entirety. Any suitable electronic communication protocol may be used for each of the communication paths or links, such as near field communication (NFC), radio frequency identification (RFID), or Low energy protocols, WiFi, etc. According to some embodiments, the remote terminal 170 and / or the trusted computer system 180 may be accessed by individuals other than the primary user who are interested in the user's analyte level. The reader device 120 may include a display 122 and an optional input component 121. According to some embodiments, the display 122 may include a touch screen interface.

[0054] 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 can be omitted. A processor (not shown) can be communicatively coupled to the sensor 104, wherein the processor is physically located within the sensor housing 103 or the reader device 120. According to some embodiments, the sensor 104 protrudes from the underside of the sensor housing 103 and extends through an adhesive layer 105 that is suitable for adhering the sensor housing 103 to a tissue surface, such as skin.

[0055] Sensor 104 is suitable for at least partially inserting into a tissue of interest, such as the dermis or subcutaneous layer of the skin. Sensor 104 may include a sensor tail of sufficient length for inserting into a desired depth in a given tissue. The sensor tail may include at least one working electrode and one or more active regions (sensing regions / points or sensing layers) located on at least one working electrode, and the active region is active for sensing one or more analytes of interest. According to one or more embodiments of the present disclosure, one or more active regions may contain a variety of enzymes together. According to some embodiments, the active region may include a polymer material, and at least some enzymes are covalently bound to the polymer material. In various embodiments of the present disclosure, analytes can be monitored in any biological fluid of interest, such as epithelial fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, etc. In a specific embodiment, the analyte sensor of the present disclosure may be suitable for measuring skin fluid or interstitial fluid.

[0056] In some embodiments, the sensor 104 can automatically forward data to the reader device 120. For example, the analyte concentration data can be automatically and periodically transmitted, such as with the acquisition of data at a specific frequency, or after a specific time period, wherein the data is stored in the memory until the transmission (e.g., every minute, five minutes or other predetermined time period). In other embodiments, the sensor 104 can communicate with the reader device 120 in a non-automatic manner and not according to a set schedule. For example, when the sensor electronics are brought into the communication range of the reader device 120, RFID technology can be used to transmit data from the sensor 104. The data can remain stored in the memory of the sensor 104 until it is transmitted to the reader device 120. Therefore, the patient does not have to keep in close proximity with the reader device 120 all the time, but can upload data at a convenient time. In other embodiments, a combination of automatic and non-automatic data transmission can be implemented. For example, data transmission can continue automatically until the reader device 120 is no longer in the communication range of the sensor 104.

[0057] The 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 tip. It should be appreciated that other types of introducers, such as a sheath or a blade, may be present in alternative embodiments. More specifically, a needle or other introducer may temporarily reside near the sensor 104 before the tissue is inserted and then be withdrawn thereafter. Although present, a needle or other introducer may facilitate the insertion of the sensor 104 into the tissue by opening the entry path followed by the sensor 104. For example, according to one or more embodiments, the needle may facilitate the penetration of the epidermis as an entry path to the dermis to allow the implantation of the sensor 104 to occur. After opening the entry path, the needle or other introducer may be withdrawn so that it does not represent a sharp hazard. In an exemplary embodiment, the cross-section of a suitable needle may be solid or hollow, beveled or non-beveled, and / or circular or non-circular. In a more specific embodiment, a suitable needle may be comparable to an acupuncture needle in cross-sectional diameter and / or tip design, and an acupuncture needle may have a cross-sectional diameter of about 250 microns. However, it will be appreciated that a suitable needle may have a larger or smaller cross-sectional diameter if desired for a particular application.

[0058] In some embodiments, the tip of the needle (when present) may be angled above the end of the sensor 104 so that the needle first penetrates the tissue and opens an access path for the sensor 104. In other illustrative embodiments, the sensor 104 may be positioned within a lumen or recess of the needle, which similarly opens an access path for the sensor 104. In either case, after facilitating sensor insertion, the needle is subsequently withdrawn.

[0059] The analyte sensors described herein may feature multiple enzymes on one or more active areas of a single working electrode or on two or more separate working electrodes. According to various embodiments of the present disclosure, a single working electrode configuration for an analyte sensor may employ a two-electrode or three-electrode detection motif. Figures 2A-2C A sensor structure featuring a single working electrode is described. Figure 3 Sensor configurations featuring multiple working electrodes are described separately. Multiple enzymes can be introduced into any of the sensor configurations described below, and specific configurations suitable for introducing multiple enzymes are described in further detail below.

[0060] When there is a single working electrode in the analyte sensor, 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, wherein the second electrode is used as a counter electrode and a reference electrode (i.e., a counter / reference electrode). In both the two-electrode and three-electrode detection motifs, one or more active areas of the analyte sensor may contact the working electrode. According to an embodiment of the present disclosure, one or more active areas may include a variety of enzymes, wherein a variety of enzymes are present in a single active area and / or a plurality of active areas. In certain embodiments, various electrodes may be at least partially stacked (layered) on top of each other, as described in further detail below. In some or other embodiments, each electrode may be laterally spaced apart from each other on the sensor tail. Similarly, the related active areas on each electrode may be vertically stacked on top of each other or laterally spaced apart. In either case, various electrodes may be electrically isolated from each other by dielectric materials or similar insulators.

[0061] Figure 2A A diagram of an illustrative two-electrode analyte sensor configuration with a single working electrode is shown, which is compatible for use in some embodiments disclosed herein. As shown, the analyte sensor 200 includes a substrate 212 disposed between a working electrode 214 and a pair / reference electrode 216. Alternatively, the working electrode 214 and the pair / reference electrode 216 can be located on the same side of the substrate 212, with a dielectric material (configuration not shown) inserted therebetween. The active region 218 is disposed 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 points or a single point, which is configured to detect one or more analytes of interest. Collectively, a variety of enzymes may be present in the active region 218 (i.e., in a single point or in multiple points).

[0062] Still refer to Figure 2AAccording to some embodiments, the membrane 220 coats at least the active area 218, and may optionally coat some or all of the working electrode 214 and / or the counter / reference electrode 216, or the entirety of the analyte sensor 200. One or both sides of the analyte sensor 200 may be coated with the membrane 220. The membrane 220 may include one or more polymeric membrane materials having the ability to restrict the flow of the analyte to the active area 218. As further described herein, the composition of the membrane 220 may vary depending on the characteristics of the one or more analytes. The analyte sensor 200 may be operable to measure one or more analytes by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

[0063] Figure 2B and 2C A diagram showing an illustrative three-electrode analyte sensor configuration with a single working electrode that is compatible for use in some embodiments disclosed herein. A three-electrode analyte sensor configuration employing a single working electrode may be similar to Figure 2A The configuration of the analyte sensor 200 shown in FIG. 1 is different from that of the analyte sensor 201 and 202. Figure 2B and 2C ) in addition to including the additional electrode 217. With the additional electrode 217, the counter / reference electrode 216 can then be used as a counter electrode or a reference electrode, and the additional electrode 217 performs another electrode function not otherwise described. The working electrode 214 continues to perform its original function. The additional electrode 217 can be arranged on the working electrode 214 or the electrode 216 with a separation layer of dielectric material therebetween. For example, as Figure 2B As shown, dielectric layers 219a, 219b, and 219c separate electrodes 214, 216, and 217 from each other and provide electrical insulation. Alternatively, at least one of electrodes 214, 216, and 217 may be located on opposite sides of substrate 212, such as Figure 2C Thus, in some embodiments, electrode 214 (working electrode) and electrode 216 (counter electrode) may be located on opposite sides of substrate 212, wherein electrode 217 (reference electrode) is located on one of electrodes 214 or 216 and is separated therefrom by a dielectric material. Reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, and the location of reference material layer 230 is not limited to Figure 2B and 2C As described in Figure 2AAs with the illustrated sensor 200, the active area 218 in the analyte sensors 201 and 202 may include multiple spots or a single spot configured to detect one or more analytes of interest. In general, a plurality of enzymes may be present in the active area 218 of the analyte sensors 201 and 202. In addition, the analyte sensors 201 and 202 may be operable to measure one or more analytes by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

[0064] Similar to analyte sensor 200, membrane 220 may also coat active areas 218 and other sensor components in analyte sensors 201 and 202. In some embodiments, additional electrodes 217 may be coated with membrane 220. Figure 2B and 2C All electrodes 214, 216, and 217 have been depicted as being coated with film 220, but it should be appreciated that in some embodiments, only working electrode 214 may be coated. Furthermore, the thickness of film 220 at each of electrodes 214, 216, and 217 may be the same or different. As in a two-electrode analyte sensor configuration ( Figure 2A ), in Figure 2B and 2C In the sensor configuration of FIG. 2 , one or both sides of the analyte sensors 201 and 202 may be coated with the membrane 220 , or the entirety of the analyte sensors 201 and 202 may be coated. Figure 2B and 2C The three-electrode sensor configuration shown in should be understood as not limiting the embodiments disclosed herein, with alternative electrode and / or layer configurations remaining within the scope of the present invention.

[0065] Analyte sensor configurations with multiple working electrodes will now be described in more detail. Although the following description is primarily directed to analyte sensor configurations with two working electrodes, it should be understood that more than two working electrodes may be successfully incorporated by extension of the present disclosure. Additional working electrodes may allow additional active area(s) and corresponding sensing capabilities to be imparted to analyte sensors having such features.

[0066] Figure 3 A diagram of an illustrative analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode is shown, which may be compatible for use in some embodiments of the present disclosure. Figure 3As shown, the analyte sensor 300 includes working electrodes 304 and 306 disposed on opposite sides of a substrate 302. An active region 310 is disposed on the surface of the working electrode 304, and an active region 312 is disposed on the surface of the working electrode 306. Collectively, a plurality of enzymes may be present in the active regions 310 and 312, wherein each active region 310, 312 comprises one or more enzymes. For example, in a particular embodiment, a glucose-responsive enzyme may be present in the active region 310, and a lactate-responsive enzyme may be present in the active region 312. The counter electrode 320 is electrically isolated from the working electrode 304 by a dielectric layer 322, and the reference electrode 321 is electrically isolated from the working electrode 306 by a dielectric layer 323. External dielectric layers 330 and 332 are located on the reference electrode 321 and the counter electrode 320, respectively. According to various embodiments, a membrane 340 may coat at least the active regions 310 and 312. Other components of analyte sensor 300 may also be coated with membrane 340, and as described above, one or both sides of analyte sensor 300, or a portion thereof, may be coated with membrane 340. Similar to analyte sensors 200, 201, and 202, analyte sensor 300 may be operable to measure one or more analytes by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

[0067] Having multiple working electrodes and being different from Figure 3 The alternative analyte sensor configurations shown may feature counter / reference electrodes rather than separate counter and reference electrodes 320, 321, and / or feature layer and / or membrane arrangements different from those explicitly shown. For example, the positions of the counter electrode 320 and the reference electrode 321 may be different from those shown. Figure 3 In addition, working electrodes 304 and 306 do not need to be Figure 3 The manner shown in FIG. 1 is located on the opposite side of the substrate 302.

[0068] An analyte sensor characterized by a working electrode having an active region remote from the working electrode is disposed in Fig. 6A and 6B and discussed further below.

[0069] According to various embodiments of the present disclosure, an electron transfer agent may be present in one or more active regions of any analyte sensor or analyte sensor configuration disclosed herein. When the analyte (enzyme substrate) undergoes an oxidation-reduction reaction, a suitable electron transfer agent may facilitate the transfer of electrons to the working electrode. The selection of the electron transfer agent in each active region may determine the redox potential observed for each active region. When multiple active regions are present, the electron transfer agent in each active region may be the same or different.

[0070] Suitable electron transfer agents may include electroreducible and electrooxidizable ions, complexes or molecules (e.g., quinones) whose redox potential is several hundred millivolts higher or lower than the redox potential of the 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. Other examples include those described in U.S. Patents 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are incorporated herein by reference in their entirety. Other suitable electron transfer agents may include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt, including, for example, metallocene compounds thereof. Suitable examples of electron transfer mediators and polymer-bound electron transfer mediators may include those described in U.S. Patents 8,444,834,8,268,143 and 6,605,201, the disclosure of which is incorporated herein by reference in its entirety. Suitable ligands for metal complexes may also include, for example, bidentate or higher coordination ligands, such as bipyridine, biimidazole, phenanthroline or pyridyl (imidazole). Other suitable bidentate ligands may include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes or adjacent diaminoaromatics. Any combination of monodentate, bidentate, tridentate, tetradentate or higher dentate ligands may be present in metal complexes to achieve a full coordination layer.

[0071] According to various embodiments of the present disclosure, polymers may be present in each active region of any analyte sensor or analyte sensor configuration disclosed herein. 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)), or any copolymer thereof. Exemplary copolymers suitable for inclusion in the active region include those containing monomeric units, such as styrene, acrylamide, methacrylamide, or acrylonitrile. When there are multiple active regions, the polymers in each active region may be the same or different.

[0072] According to various embodiments of the present disclosure, the electron transfer agent can be covalently bonded to the polymer in each active region. The mode of covalent bonding is not considered to be particularly limited. The covalent bonding of the electron transfer agent to the polymer can occur by polymerizing monomer units of the covalently bonded electron transfer agent, or the electron transfer agent can react with the polymer separately after the polymer has been synthesized. According to some embodiments, the bifunctional spacer can covalently bond the electron transfer agent to the polymer in the active region, wherein the first functional group reacts with the polymer (e.g., a functional group capable of quaternizing a pyridine nitrogen atom or an imidazole nitrogen atom) and the second functional group reacts with the electron transfer agent (e.g., a functional group reacting with a ligand of a coordinated metal ion).

[0073] Similarly, according to some or other various embodiments of the present disclosure, the enzyme in one or more active regions can be covalently bonded to a polymer. When there are multiple enzymes in a single active region, in some embodiments, all multiple enzymes can be covalently bonded to a polymer, and in other embodiments, only a portion of multiple enzymes can be covalently bonded to a polymer. For example, a first enzyme can be covalently bound to a polymer, and a second enzyme can be non-covalently associated with a polymer. According to a more specific embodiment, the covalent bonding of an enzyme to a polymer can be carried out by introducing a cross-linking agent having a suitable cross-linking agent. Suitable cross-linking agents for reacting with free amino groups in an enzyme (for example, reacting with free amines in lysine) can include cross-linking agents, such as polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin or its derivative variants. Suitable cross-linking agents for reacting with free carboxylic acid groups in an enzyme can include, for example, carbodiimide. Cross-linking is usually intermolecular, but can be intramolecular in some embodiments.

[0074] The electron transfer agent and / or enzyme may also be associated with the polymer in the active region by means other than covalent bonding. In some embodiments, the electron transfer agent and / or enzyme may be associated with the polymer ionically or by coordination. For example, a charged polymer may be associated with an electron transfer agent or enzyme ion with an opposite charge. In other embodiments, the electron transfer agent and / or enzyme may be physically entrained within the polymer without being bonded thereto.

[0075] Various configurations suitable for arranging multiple enzymes in the analyte sensor of the present disclosure will now be described in more detail. The multiple enzymes can be deposited in one or more active regions of the sensor. The size of the active region can range from about 0.01 mm to about 0.1 mm. 2 To about 1mm 2 , although larger or smaller active areas are also considered here.

[0076] In some embodiments, multiple enzymes can be arranged in separate active areas on a single working electrode. When multiple enzymes are arranged in this manner, each active area can facilitate detection of a single analyte, as described below. At least one active area can generate a signal independently of other active areas.

[0077] According to some embodiments, an analyte sensor of the present disclosure having multiple active areas on a single working electrode may include: a sensor tail, including at least one working electrode and at least two active areas disposed on the surface of the working electrode. Each active area contains an analyte-responsive enzyme and a polymer, and the analyte-responsive enzyme in each active area is different. Each active area has a redox potential, and the redox potential of the first active area is sufficiently separated from the redox potential of the second active area to allow the generation of a signal from the first active area independently of the generation of a signal from the second active area. In a more specific embodiment, such an analyte sensor may include a single working electrode having at least two active areas. An electron transfer agent may be incorporated into each active area to promote electron transfer.

[0078] An alternative sensor configuration may include a single active region that contains both a first analyte responsive enzyme and a second analyte responsive enzyme and an electron transfer agent. Each enzyme may be covalently bound to a different portion of a polymer in a single active region. Assuming that the sensing chemical used to promote electron transfer of each analyte is not overly diluted in a single active region, a single active region may promote analyte detection in a manner similar to that described below for a separate active region. Such a sensor configuration may be particularly feasible when the analytes to be determined with the first and second analyte responsive enzymes have comparable membrane permeability values.

[0079] In more specific embodiments, the sensor tail may be configured for insertion into tissue. Suitable tissues are not considered to be particularly limiting, and are described in more detail above. Similarly, considerations for deploying the sensor tail at a specific location within the tissue are presented above.

[0080] In a more specific embodiment, the redox potential associated with the first active region can be separated from the redox potential of the second active region by at least about 100mV, or at least about 150mV, or at least about 200mV. The upper limit of the interval between the redox potentials is determined by the working electrochemical window in the body. By making the redox potentials of the active regions sufficiently separated from each other in size, electrochemical reactions can occur in the first active region without substantially causing electrochemical reactions in the second active region. Therefore, the signal from the first active region can be independently generated at or above its corresponding redox potential. In contrast, at or above the redox potential of the second active region, electrochemical reactions may occur in both active regions. In this way, the signal generated at or above the redox potential of the second active region can include signal contributions from the first active region and the second active region, and the signal is a composite signal. Then, the signal contribution from the second active region at or above its corresponding redox potential can be determined by subtracting the signal obtained only from the first active region at or above its corresponding redox potential from the composite signal. Similar considerations apply to analyzing the signal contribution from a single active region containing two different enzymes that generate signals at different redox potentials.

[0081] In a more specific embodiment, when the active regions are located on the same working electrode, the first and second active regions may contain different electron transfer agents to provide redox potentials that are sufficiently separated in size. More specifically, the first active region may include a first electron transfer agent, the second active region may include a second electron transfer agent, and the first and second electron transfer agents are different. According to various embodiments of the present invention, the metal center and / or ligand present in a given electron transfer agent may be changed to provide sufficient separation of the redox potentials of the first and second active regions. According to a more specific embodiment, the first electron transfer agent may be covalently bonded to a polymer in the first active region, and the second electron transfer agent may be covalently bonded to a polymer in the second active region. The covalent bonding modes of the first electron transfer agent and the second electron transfer agent may be the same or different. According to the above disclosure, similar considerations apply to selecting an electron transfer agent suitable for use in combination with a first analyte-responsive enzyme and a second analyte-responsive enzyme contained in a single active region.

[0082] In a more specific embodiment of the present invention, the analyte responsive enzyme in each active region can be covalently bonded (or otherwise fixed) to the polymer in each active region. In a more specific embodiment, the analyte responsive enzyme and the electron transfer agent in each active region can be covalently bonded to the polymer in each active region. When included in a single active region, the first analyte responsive enzyme and the first electron transfer agent can be covalently bonded to the first part of the polymer, and the second analyte responsive enzyme and the second electron transfer agent can be covalently bonded to the second part of the polymer. The polymers in the first part and the second part can be the same or different.

[0083] Ideally, the first and second active regions located on a single working electrode can be configured to quickly obtain a steady-state current when operating the analyte sensor at a given potential. By selecting an electron transfer agent for each active region, it is possible to facilitate rapid steady-state current, which rapidly changes its oxidation state when exposed to a potential equal to or higher than its redox potential. Making the active region as thin as possible can also facilitate rapid steady-state current. For example, the suitable thickness of the first and second active regions can be in the range of about 0.1 micron to about 10 microns. In some or other embodiments, incorporating conductive materials such as carbon nanotubes, graphene or metal nanoparticles in one or more active regions can facilitate rapid steady-state current. The suitable amount of conductive particles can be in the range of about 0.1% to about 50% by weight of the active region, or about 1% to about 50% by weight, or about 0.1% to about 10% by weight, or about 1% to about 10% by weight. Stabilizers can also be used to improve response stability.

[0084] It should also be understood that the sensitivity (output current) of the analyte sensor to each analyte can be changed by changing the coverage (area or size) of the active areas, the area ratio of the active areas relative to each other, the nature and thickness of the mass transfer limiting membrane coating the active areas, and any combination thereof. These parameters can be easily changed by one of ordinary skill in the art once having the benefit of this disclosure.

[0085] Although the foregoing description is primarily directed to an analyte sensor configured to detect two different analytes, it will be appreciated that the above concepts can be extended to detect more than two analytes using a corresponding number of active areas located on a single working electrode. Specifically, in additional embodiments of the present disclosure, an analyte sensor employing more than two active areas and a corresponding number of different enzymes (and electron transfer agents) therein can be used to detect the same number of different analytes. Assuming that the redox potential of each active area is sufficiently separated from the redox potentials of the other active areas, the signal contribution from each active area can be analyzed in a manner related to that described above to provide the concentration of each analyte.

[0086] In a more specific embodiment, in addition to the suitable electron transfer agent and polymer discussed in more detail above, the first active area may include a glucose responsive enzyme, such as glucose oxidase, and the second active area may include a lactate responsive enzyme, such as lactate oxidase. According to a specific embodiment, an analyte sensor suitable for detecting glucose and lactic acid may include a working electrode having a first active area and a second active area disposed thereon, and a mass transfer limiting membrane coated on the first and second active areas on the working electrode, wherein the second active area includes a polymer, albumin, and a lactate responsive enzyme (such as lactate oxidase) covalently bonded to the polymer, and the first active area includes a glucose responsive enzyme (such as glucose oxidase) covalently bonded to the polymer. The first and second electron transfer agents different from each other may be present in each active area. In a more specific embodiment, the mass transfer limiting membrane may at least include a cross-linked polyvinyl pyridine homopolymer or copolymer. In the case where the mass transfer limiting membrane coats each active area, the components of the mass transfer limiting membrane may be the same or different. In a particular embodiment, the mass transfer limiting membrane coating the first active area can be a single component (comprising a single membrane polymer), and the mass transfer limiting membrane coating the second active area can be a multicomponent (comprising two or more different membrane polymers, one of which is a polyvinylpyridine homopolymer or copolymer), or a double layer or a homogeneous mixture.

[0087] Similarly, it should also be understood that some analyte sensors of the present disclosure having two or more active areas located on a given working electrode may include two or more analyte-responsive enzymes in at least one active area. According to a more specific embodiment, two or more analyte-responsive enzymes in a given active area can interact synergistically to produce a signal proportional to the concentration of a single analyte. Therefore, the analyte-responsive enzyme does not necessarily have to be present in a 1:1 ratio with a given selected analyte. Analyte sensors containing synergistically interacting enzymes are described in further detail below.

[0088] Therefore, the present invention also describes a multi-analyte detection method using an analyte sensor characterized by multiple enzymes arranged on a single working electrode. In various embodiments, such methods may include: exposing the analyte sensor to a fluid including at least one analyte. The analyte sensor includes a sensor tail, which includes at least one working electrode, in particular a single working electrode, and at least two active areas arranged on the surface of the working electrode. Each active area contains an analyte-responsive enzyme and a polymer, and the analyte-responsive enzyme in each active area is different. Each active area has a redox potential, and the redox potential of the first active area is sufficiently separated from the redox potential of the second active area to allow the generation of a signal from the first active area independently of the generation of a signal from the second active area. The method further includes: obtaining a first signal at or above the redox potential of the first active area, so that the first signal is proportional to the concentration of the first analyte; obtaining a second signal at or above the redox potential of the second active area, so that the second signal is a composite signal including a signal contribution from the first active area and a signal contribution from the second active area; and subtracting the first signal from the second signal to obtain a difference signal, which is proportional to the concentration of the second analyte.

[0089] In more specific embodiments, the redox potential associated with the first active region may be separated from the redox potential of the second active region by at least about 100 mV, or at least about 150 mV, or at least about 200 mV to provide sufficient separation for independent generation of a signal from the first active region.

[0090] In some or other more specific embodiments, the fluid is a biological fluid and the analyte sensor is exposed to the in vivo biological fluid within an individual. Suitable biological fluids for analysis using an analyte sensor having at least two different active areas located on a given working electrode can include any of the biological fluids discussed in more detail above.

[0091] In some embodiments, the signal associated with each active area can be related to the corresponding analyte concentration by reference to a lookup table or calibration curve for each analyte. The lookup table for each analyte can be populated by measuring multiple samples with known analyte concentrations and recording the sensor response of each analyte at each concentration. Similarly, the calibration curve for each analyte can be determined by plotting the analyte sensor response of each analyte as a function of concentration. According to some embodiments, the calibration curve of the analyte sensor of the present disclosure can be linear.

[0092] The processor may determine which sensor response value in the lookup table is closest to the sensor response value measured for the sample with the unknown analyte concentration and then report the analyte concentration accordingly. In some or other embodiments, if the sensor response value for the sample with the unknown analyte concentration is between the recorded values ​​in the lookup table, the processor may interpolate between the two lookup table values ​​to estimate the analyte concentration. The interpolation may assume a linear concentration change between the two values ​​reported in the lookup table. Interpolation may be employed when the sensor response differs from a given value in the lookup table by a sufficient amount, such as a change of about 10% or greater.

[0093] Likewise, according to some or other various embodiments, the processor may input the sensor response values ​​for a sample having an unknown analyte concentration into a corresponding calibration curve. The sensor may then report the analyte concentration accordingly.

[0094] Embodiments of analyte sensors having two distinct active areas disposed on a given working electrode may employ the same Figures 2A-2C However, it should be understood that suitable analyte sensors may also feature multiple working electrodes, such as Figure 3 The sensor configuration shown in the figure, wherein at least one working electrode has at least two active areas that are different from each other. It should also be understood that other analyte sensor configurations with two or more different active areas arranged on the surface of a given working electrode also fall within the scope of the present disclosure. For example, the position, orientation or functionality of the working electrode and the counter electrode and / or the reference electrode may be different from that shown in the drawings herein.

[0095] Figure 4 An exemplary analyte sensor configuration suitable for use with some embodiments of the disclosure herein is shown in which two distinct active regions are disposed on the surface of a single working electrode. Figure 4 Analyte sensor configuration and Figure 2C The analyte sensor configuration of has the greatest similarity and may be better understood by reference thereto. Where appropriate, for clarity, the analyte sensor configuration of Figure 4 Used from Figure 2C , and for the sake of brevity, features having common structures and / or functions are not described in further detail. Again, it should be understood that other analyte sensor configurations can be similarly combined with the following specific Figure 4 Describe the characteristics.

[0096] refer to Figure 4, the analyte sensor 400 includes active areas 218a and 218b on the surface of the working electrode 214. The active area 218a includes a first electron transfer agent and a first analyte responsive enzyme, which can be covalently bonded to the polymer constituting the active area 218a. The active area 218b similarly includes a second electron transfer agent and a second analyte responsive enzyme, which can be covalently bonded to the polymer comprising the active area 218b. The first electron transfer agent and the second electron transfer agent can be different in composition so as to provide separation of the redox potentials of the first active area 218a and the second active area 218b. In a specific embodiment, the active area 218b may include a lactate responsive enzyme, such as lactate oxidase, and the active area 218a may include a glucose responsive enzyme, such as glucose oxidase.

[0097] The redox potentials of the first active region 218a and the second active region 218b can be sufficiently separated from each other to allow the generation of a signal from the first active region 218a independently of the generation of a signal from the second active region 218b. Therefore, the analyte sensor 400 can be operated at a first potential at which a redox reaction occurs within the first active region 218a but not within the second active region 218b. Therefore, a first analyte (e.g., glucose) can be selectively detected at or above the redox potential of the first active region 218a, as long as the applied potential is not high enough to promote a redox reaction with the second active region 218b. The concentration of the first analyte can be determined from the sensor response of the first active region 218a by reference to a lookup table or a calibration curve.

[0098] At or above the redox potential of the second active region 218b, separate redox reactions may occur simultaneously or nearly simultaneously in the first active region 218a and the second active region 218b. As a result, the signal generated at or above the redox potential of the second active region 218b may include a composite signal having contributions from the signals of the first active region 218a and the second active region 218b. In order to determine the concentration of the second analyte (e.g., lactate) from the composite signal, the signal from the first active region 218a at or above its corresponding redox potential may be subtracted from the composite signal to provide a difference signal associated only with the second active region 218b. Once the difference signal is determined, the concentration of the second analyte may be determined by reference to a lookup table or calibration curve.

[0099] As previously discussed, similar considerations apply to separating the first signal and the second signal from a single active zone containing two different analyte-reactive enzymes in order to determine the concentrations of the first and second analytes that are different from each other.

[0100] In some or other embodiments of the present disclosure, multiple enzymes may be present in a single active area. According to some embodiments of the present disclosure, different from where multiple enzymes act independently to detect different analytes, particularly different from being spaced apart in separate active areas on the working electrode surface, multiple enzymes arranged in a single active area can work synergistically to promote the detection of a single analyte, particularly in the presence of a stabilizer. As used herein, the term "synergistic" and its grammatical variants refer to coupled enzyme reactions, wherein the product of the first enzymatic reaction becomes the substrate of the second enzymatic reaction, and the second enzymatic reaction serves as the basis for measuring the concentration of the substrate (analyte) reacted during the first enzymatic reaction. When a single enzyme cannot promote detection, it may be necessary to utilize two mutually synergistic enzymes to detect a given target analyte. Wherein a single enzyme may be invalid for promoting analyte detection, including, for example, those situations in which the enzyme is inhibited by one or more products of the enzyme reaction or cannot circulate between an oxidized state and a reduced state when arranged in an analyte sensor.

[0101] As also disclosed herein, multiple enzymes disposed in separate active regions can also interact synergistically to facilitate detection of a single analyte. When multiple enzymes are located in separate active regions, one of the active regions can be isolated from the working electrode so that transfer of electrons from and to the working electrode occurs only from one of the active regions.

[0102] In a more specific embodiment, an analyte sensor characterized by at least two enzymes that interact cooperatively with each other may include: a sensor tail, which includes at least one working electrode; and at least one active area disposed on the surface of the working electrode. The at least one active area includes a first enzyme, a second enzyme, and a polymer. The first and second enzymes can interact cooperatively so that the first enzyme can convert the analyte into a first product, and the second enzyme can convert the first product into a second product to generate a signal at the working electrode. The second enzyme does not react with the analyte. At least the second enzyme is covalently bonded to the polymer in at least one active area. Analysis of the signal (e.g., the current measured at a fixed input voltage) generated by the reaction of the first product into the second product can provide a basis for detecting the analyte and measuring its concentration, as explained in further detail below.

[0103] More specifically, the active region may include an electron transfer agent, such as those described above, and only the second enzyme is able to exchange electrons with the electron transfer agent, in which case the first enzyme can indirectly transfer electrons to the second enzyme, as described below. Therefore, a more specific embodiment of the present disclosure may be characterized by a first enzyme and a second enzyme, the first enzyme not being covalently bonded to a polymer (making it less likely to exchange electrons with an electron transfer agent), the second enzyme being covalently bonded to a polymer (to facilitate exchanging electrons with an electron transfer agent). In either case, the electron transfer agent may be covalently bonded to the polymer of the active region. Coordinate bonding is also included within the scope of covalent bonding according to the disclosure herein.

[0104] According to some embodiments, a stabilizer may be present in the active region. Stabilizers particularly suitable for use in analyte sensors containing cooperatively interacting enzymes include, for example, catalase and albumin.

[0105] According to some embodiments, the sensor tail can be configured to be inserted into a tissue of interest. Thus, according to some embodiments, an analyte sensor comprising enzymes that can cooperatively interact with each other in a given active region can be suitable for analyzing analyte concentrations in biological fluids in vivo. The characteristics of the biological fluid are also not particularly limited.

[0106] As described above, the sensor configuration incorporating two enzymes capable of synergistic interaction may include those in which at least one active region comprises an electron transfer agent covalently bonded to a polymer. Similarly, according to the disclosure herein, coordinate bonding is also included in the scope of covalent bonding. In such an embodiment, at least the second enzyme may also be covalently bonded to the polymer. In some embodiments, the first enzyme is not covalently bonded to the polymer. In other embodiments, the first enzyme and the second enzyme may be covalently bonded to the polymer in at least one active region. Covalent bonding of the first enzyme to the polymer may be desirable, for example, to reduce the possibility of leaching of the first enzyme from at least one active region.

[0107] An analyte sensor configuration suitable for combining two synergistic enzymes in one or more active regions on a working electrode can be used with Figures 2A-2C The enzymes that can synergistically interact with each other (i.e., synergistic enzymes or synergistic enzyme pairs) can also be incorporated into a system having multiple working electrodes ( Figure 3 ) or having multiple active areas arranged on a given working electrode ( Figure 4 ) in an analyte sensor configuration. Any analyte sensor disclosed herein having a cooperative enzyme pair directly located on the working electrode surface can employ any of the aforementioned analyte sensor configurations. Fig. 6A and 6BFurther discussed are analyte sensor configurations having two or more enzymes cooperatively interacting with each other in multiple active regions, wherein one of the active regions is distal to a working electrode.

[0108] In a more specific configuration of an analyte sensor containing cooperative enzymes both disposed directly on a working electrode, the first enzyme may be an alcohol oxidase (AOX) and the second enzyme may be a xanthine oxidase (XOX). According to one or more embodiments, using this pair of enzymes, the analyte sensor may have the function of detecting alcohol, particularly ethanol. The cooperative nature of alcohol oxidase and xanthine oxidase for detecting ethanol and other alcohols will be explained in further detail below (see Figure 5A ), the two enzymes are disposed on the working electrode. In a more specific embodiment of the invention, the xanthine oxidase may be covalently bonded to the polymer in the active region, and the alcohol oxidase is not covalently bonded to the polymer. In a more specific embodiment, the xanthine oxidase and the electron transfer agent may both be covalently bonded to the polymer, and the alcohol oxidase is not covalently bonded to the polymer. Catalase may be present as a stabilizer with the pair of enzymes.

[0109] Another pair of synergistic enzymes that can be applied to the disclosure herein is β-hydroxybutyrate dehydrogenase and diaphorase, wherein both enzymes are directly disposed on the surface of the working electrode. This synergistic enzyme pair can be used for ketone body detection, wherein β-hydroxybutyrate is a representative molecule, indicating the presence of ketones. In a sensor configuration containing this pair of synergistic enzymes, β-hydroxybutyrate dehydrogenase can convert β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD+) into acetoacetate and reduced nicotinamide adenine dinucleotide (NADH). NADH can then be reduced under the mediation of diaphorase, and the transfer of electrons in this process provides the basis for detecting ketones on the working electrode. Figure 5B The synergistic reaction between β-hydroxybutyrate and diaphorase (mediated by the NAD+ cofactor) for the detection of ketones is shown in Figure 1. Albumin can be present as a stabilizer with this synergistic enzyme pair.

[0110] Figure 5C and 5D Other alternative enzyme detection chemistries for ketones are shown. Figure 5C As shown, β-hydroxybutyrate dehydrogenase can convert β-hydroxybutyrate and NAD+ into acetoacetate and NADH. Electron transfer to the working electrode is not completed by diaphorase (see Figure 5B), the reduced NADH oxidase (NADHox(reduced)) reacts to form the corresponding oxidized form (NADHox(oxidized)). NADHOX(reduced) can then reform superoxide by reacting with molecular oxygen, which can then be converted to hydrogen peroxide under the mediation of superoxide dismutase (SOD). According to various embodiments, SOD can be covalently bonded to the polymer in the active region. The hydrogen peroxide can then undergo a reaction at the working electrode to provide a signal that can be related to the amount of ketone present. Figure 5D Another alternative enzyme detection chemistry is shown, where β-hydroxybutyrate dehydrogenase can again convert β-hydroxybutyrate and NAD+ to acetoacetate and NADH. In this case, the detection cycle is completed by oxidation of poly-1,10-phenanthroline-5,6-dione at the working electrode. As with other sensing chemistries disclosed herein, the inclusion of albumin in the active region can provide surprising improvements in response stability.

[0111] Creatinamide hydrolase and sarcosine oxidase are another pair of synergistic enzymes that can be applied to the disclosure herein when the two enzymes are placed directly on the working electrode. Creatinamide hydrolase produces sarcosine and urea from creatine. Sarcosine oxidase can in turn catalyze the reaction of sarcosine to form glycine, formaldehyde, and hydrogen peroxide. Therefore, detection of hydrogen peroxide at the working electrode can serve as the basis for quantifying creatine and / or sarcosine.

[0112] The detection of ethanol and other alcohols by a coordinated enzymatic reaction using alcohol oxidase and xanthine oxidase will now be described in further detail. Alcohol oxidase interacts with ethanol to form acetaldehyde and hydrogen peroxide. Other alcohols react to form aldehydes having correspondingly higher or lower carbon numbers. Advantageously, alcohol oxidase catalyzes only the forward conversion of ethanol to acetaldehyde (as opposed to performing the reaction reversibly, such as is the case with alcohol dehydrogenase), which may facilitate the use of the enzyme in an analyte sensor. In addition, alcohol oxidase contains a strongly bound flavin cofactor, such that an exogenous cofactor does not have to be bound to the alcohol oxidase in order for the enzyme to have activity promoting alcohol oxidation.

[0113] In principle, alcohol oxidase can be used alone for ethanol detection in analyte sensors by analyzing the acetaldehyde or hydrogen peroxide products produced in the enzyme reaction. However, there are two problems with this approach. First, both acetaldehyde and hydrogen peroxide have an inhibitory effect on alcohol oxidase. Therefore, if these compounds are not removed from the sensor environment, alcohol oxidase becomes inactive to promote ethanol oxidation, thereby rendering the analyte sensor ineffective for measuring ethanol. In addition, if acetaldehyde and hydrogen peroxide are chelated or quenched by other reagents, there is no longer any material available for electrochemical detection. Second, alcohol oxidase cannot freely exchange electrons with redox mediators other than molecular oxygen. Therefore, electron transfer agents bound to polymers in the active region of the analyte sensor, such as osmium and other transition metal complexes discussed herein, are ineffective for alcohol oxidase to cycle from an inactive reduced state to an oxidized state that reacts with ethanol. Therefore, although alcohol oxidase can optionally be covalently bonded to a polymer, doing so has no particular benefit to the electron transfer process. That is, covalent bonding of alcohol oxidase to a polymer cannot help promote electron transfer with an electron transfer agent.

[0114] The synergistic combination of alcohol oxidase and xanthine oxidase directly on the working electrode, especially together in a given active area, can overcome at least some of the aforementioned challenges associated with the detection of ethanol by an analyte sensor using alcohol oxidase. Acetaldehyde and other aldehydes can serve as substrates for xanthine oxidase, wherein acetaldehyde is enzymatically converted into acetic acid. Therefore, xanthine oxidase can remove acetaldehyde from the sensor environment, thereby excluding the inactivation of alcohol oxidase based on acetaldehyde. Catalase can be present in the active area to remove hydrogen peroxide (e.g., as a catalase-hydrogen peroxide complex), thereby excluding the inactivation of alcohol oxidase by the substance. In addition, unlike alcohol oxidase, xanthine oxidase can exchange electrons with osmium and other transition metal complexes associated with the polymer in the active area of ​​the analyte sensor. Therefore, xanthine oxidase can circulate between its oxidized form and reduced form, thereby allowing the analyte sensor to maintain an active sensing state. Therefore, the detection of ethanol in the aforementioned analyte sensor is based on the enzymatic reaction of xanthine oxidase with acetaldehyde, which is the enzymatic reaction product of ethanol. Furthermore, by configuring the enzyme in the analyte sensor in the aforementioned manner, the alcohol oxidase can undergo reoxidation with molecular oxygen to maintain its activity.

[0115] Figure 5A A synergistic enzymatic reaction cycle associated with ethanol detection using alcohol oxidase and xanthine oxidase placed directly on a working electrode is shown according to various embodiments of the present disclosure. The xanthine oxidase is covalently bonded to a polymer in the active region of the analyte sensor, and the alcohol oxidase is non-covalently associated with the polymer in the active region. In addition to the xanthine oxidase, an osmium complex or other transition metal complex capable of exchanging electrons with the enzyme is also covalently bonded to the polymer. Figure 5AAs shown, ethanol reacts with oxidized (active) alcohol oxidase in the presence of a flavin cofactor (FAD-already bound to the alcohol oxidase) to form reduced alcohol oxidase, acetaldehyde, and hydrogen peroxide. The reduced alcohol oxidase can be reoxidized with molecular oxygen, as shown, to restore the alcohol oxidase to its catalytically active oxidized form.

[0116] Still reference Figure 5A , the acetaldehyde enzymatically formed from the ethanol then subsequently reacts with the oxidized form of xanthine oxidase in the presence of the flavin cofactor naturally present with the enzyme. Acetic acid is formed in the process and the xanthine oxidase is converted to a reduced state. The reduced xanthine oxidase can then react with a transition metal electron transfer agent associated with the polymer to transfer electrons to the working electrode, thereby generating an electric current and regenerating the oxidized form of xanthine oxidase. Although in Figure 5A Not shown, hydrogen peroxide is removed from the sensor environment solely by catalase present in the active region.

[0117] from Figure 5A It can be seen that the amount of acetaldehyde formed enzymatically is proportional to the amount of ethanol initially present. Therefore, the current generated at the working electrode during xanthine oxidase oxidation of acetaldehyde can be proportional to the amount of acetaldehyde present, and by extension, the amount of ethanol. Correlation of the working electrode current with ethanol concentration can be performed by reference to a lookup table of currents at known ethanol concentrations or by utilizing a calibration curve, the concepts of which are described in more detail above.

[0118] Similarly, when analyzing ketones, the current generated at the working electrode can be proportional to the amount of β-hydroxybutyrate that is oxidized to form acetylacetone ( Figures 5B-5D ). Thus, correlation of the current at the working electrode can occur in the same manner as the correlation provided above for ethanol (eg, using a calibration curve or lookup table).

[0119] Therefore, in a more specific embodiment, the present disclosure provides an alcohol sensor based on the synergistic enzymatic reaction of alcohol oxidase and xanthine oxidase. More specifically, the alcohol sensor can include a sensor tail, the tail including at least one working electrode and at least one active area disposed on the surface of the working electrode, wherein the at least one active area includes alcohol oxidase, xanthine oxidase, catalase, a polymer and an electron transfer agent. According to a specific embodiment, the electron transfer agent and the xanthine oxidase can be covalently bonded to the polymer, and the alcohol oxidase is not covalently bonded to the polymer. The alcohol oxidase and the xanthine oxidase can interact synergistically to produce a signal at the working electrode that is proportional to the alcohol concentration. More specifically, both the alcohol oxidase and the xanthine oxidase are directly placed on the working electrode to achieve the above purpose.

[0120] According to more specific embodiments, the catalase in at least one active region of the alcohol sensor is not covalently bonded to the polymer. The catalase may be present in an amount ranging from about 1% to about 50% by weight of the polymer, more particularly about 1% to about 10% by weight of the polymer, or about 1% to about 5% by weight of the polymer.

[0121] Therefore, the present disclosure also provides a detection method based on a cooperative enzyme reaction, wherein the cooperative enzyme pair is directly placed on the surface of the working electrode. According to various embodiments, the detection method may include: exposing an analyte sensor to a fluid containing an analyte, the analyte sensor including a sensor tail, the sensor tail including at least one working electrode and at least one active area arranged on the surface of the working electrode, the at least one active area comprising a first enzyme, a second enzyme and a polymer. The first enzyme and the second enzyme are capable of cooperative interaction, wherein the second enzyme is covalently bonded to the polymer and does not react with the analyte. The method also includes: reacting the analyte with the first enzyme to form a first product; reacting the first product with the second enzyme to form a second product, thereby generating a signal at the working electrode; and correlating the signal with the concentration of the analyte in the fluid.

[0122] According to more specific embodiments, when carrying out the aforementioned method, the electron transfer agent can also be covalently bonded to the polymer. Suitable electron transfer agents are described in more detail above. In some or other embodiments, the first enzyme is not covalently bonded to the polymer in at least one active region, particularly when there is a covalently bonded electron transfer agent.

[0123] In a more specific embodiment, the ethanol detection method of the present disclosure may include: exposing an analyte sensor to a fluid, particularly a biological fluid, containing ethanol, the analyte sensor comprising a sensor tail, the sensor tail comprising at least one working electrode and at least one active area, the at least one active area being disposed on the surface of the working electrode and comprising alcohol oxidase, xanthine oxidase, catalase, a polymer and an electron transfer agent. The electron transfer agent and the xanthine oxidase are covalently bonded to the polymer, and the alcohol oxidase is not covalently bonded to the polymer. The alcohol oxidase and the xanthine oxidase are capable of synergistic interaction. The method also includes: reacting ethanol with the alcohol oxidase to form acetaldehyde; reacting acetaldehyde with the xanthine oxidase to form acetic acid, thereby generating a signal at the working electrode; and correlating the signal with the ethanol concentration in the fluid. According to some embodiments, the fluid may be a biological fluid, and the analyte sensor may be exposed to the biological fluid in vivo.

[0124] Although two different enzymes in a single active area of ​​an analyte sensor can interact cooperatively with each other to determine the analyte concentration, it should be understood that in other embodiments, enzymes can also work independently of each other to detect alternative analytes. For example, in the case of xanthine oxidase as the second enzyme in the above-mentioned analyte sensor, instead of using the analyte sensor to detect ethanol, the analyte sensor can be used to detect any of a wide range of substrates compatible with xanthine oxidase. The alternative substrates of xanthine oxidase can include, for example, hypoxanthine, xanthine, uric acid, purine, pterin and similar compounds. When the analyte sensor is used in this way, if there is no alcohol, the alcohol oxidase can remain unused, and / or if acetaldehyde / hydrogen peroxide is not removed by xanthine oxidase or other substances, the alcohol oxidase is inactivated by acetaldehyde / hydrogen peroxide. Therefore, sensors containing cooperative enzymes can also be considered to be able to detect multiple analytes, and at least the second analyte from one of the members of the cooperative enzyme pair works independently. It can be determined based on the environment to which the sensor is exposed whether such a sensor measures a single analyte or measures multiple analytes.

[0125] As mentioned above, multiple enzymes arranged in the active area of ​​separation can also interact synergistically to promote the detection of a single analyte. In some cases, multiple enzymes can all be directly located on the surface of the working electrode, as discussed in more detail above. In the alternative analyte sensor configuration comprising multiple enzymes in the active area of ​​separation, one of the active areas can be isolated from the working electrode so that the electron transfer to the working electrode occurs only from one of the active areas. That is, as discussed in more detail below, the active area isolated from the working electrode can promote the enzyme reaction of the analyte of interest to produce a reaction product (substrate), which itself reacts with the enzyme in the active area of ​​direct contact with the working electrode. The signal associated with the enzyme reaction occurring in the active area directly contacting the working electrode then provides the basis for detecting the analyte. The association of the signal with the analyte concentration can be realized in a manner similar to that discussed in more detail above.

[0126] More specifically, Figure 5E Shown are the coordinated enzymatic reaction cycles associated with ethanol detection using glucose oxidase and xanthine oxidase, as further mediated by catalase, when only xanthine oxidase or xanthine oxidase and catalase are placed on the surface of a working electrode, according to various embodiments of the present disclosure. Figure 5EThe illustrated cooperative enzymatic reaction cycle relies on the coexistence of glucose and ethanol in the fluid during analysis, as described below. Since glucose is a ubiquitous biological nutrient, it is often found to coexist with other analytes when measuring biological fluids. However, if the particular fluid undergoing analysis lacks glucose, certain embodiments of the present disclosure may feature the addition of glucose to the fluid to facilitate the detection of ethanol or another alcohol using the cooperative enzymatic reaction of glucose oxidase and xanthine oxidase.

[0127] In further discussion Figure 5E Before describing the cooperative enzymatic reaction shown in , an exemplary analyte sensor configuration featuring at least one active region isolated from a working electrode will first be described in more detail. As previously described, Figure 2A-4 The analyte sensor configurations shown all feature one or more working electrodes having one or more active regions disposed directly on the surface of each working electrode. Fig. 6A , 6B 6C show diagrams of a working electrode in which a first active region is disposed directly on a surface of the working electrode and a second active region is separated (spaced apart or remote) from the working electrode by a membrane. Fig. 6A , 6B and the working electrode configuration shown in 6C can be replaced by Figure 2A-4 any particular working electrode configuration shown in . That is, Fig. 6A , 6B The working electrode configurations shown in FIG. 6C may be combined in any suitable manner with counter and / or reference electrodes, membranes, substrates, and the like in an analyte sensor.

[0128] like Fig. 6A As shown, the working electrode 400 has an active area 402 disposed directly on its surface. The active area 402 includes a first enzyme covalently bound to a first polymer. Typically, an electron transfer agent is also present in the active area 402, wherein the electron transfer agent is also covalently bonded to the polymer. The active area 402 is coated with a thin film 404. The membrane 404 can also coat the surface of the working electrode 400, as depicted, as well as other portions of the analyte sensor in which the working electrode 400 is present. The membrane 404 isolates the active area 406 from the working electrode 400, so that the exchange of electrons between the two is prevented. The active area 406 includes a second enzyme covalently bound to a second polymer, but there is no separate electron transfer agent. Although Fig. 6A Active region 406 is shown disposed directly above active region 402, but it should be understood that they may be laterally spaced apart from one another in alternative configurations that are also compatible with the present disclosure. Membrane 408 is coated over active region 406 and optionally other sensor components to provide mass transport limiting properties. Similarly, as Figure 6BAs shown, membrane 404 need not extend the same lateral distance as membrane 408 on working electrode 400. In fact, Figure 6B In the embodiment, film 404 coats active region 402, but only coats a portion of the surface of working electrode 400, and film 408 coats active region 406, the surface of film 404, and the remaining portion of the surface of working electrode 400 not coated by film 404. In some embodiments, active regions 402 and 406 may also be laterally offset from each other, such as Figure 6C As shown, the active area 406 is again isolated from the working electrode 400 by the membrane 404.

[0129] Membrane 408 is permeable to the analyte and any additional components required to facilitate the enzyme reaction in active region 406. In contrast, membrane 404 is permeable to the products formed in active region 406. That is, the analyte reacts in active region 406 to form a first product, which then diffuses through membrane 404 and then further reacts in active region 402 to form a second product. Subsequently, the second product can be detected based on the exchange of electrons with working electrode 400.

[0130] Optionally, if glucose sensing is desired, a wire 410 may extend between active areas 402 and 406 .

[0131] In the alcohol sensor featuring detection based on the cooperative enzymatic reaction of glucose oxidase and xanthine oxidase, glucose oxidase is present in active region 406, while xanthine oxidase is present in active region 402. Figure 5E , continue to refer to Fig. 6A , 6B and 6C, glucose oxidase is present in active region 406 and converts exogenous glucose into D-gluconolactone-1,5-dione and hydrogen peroxide. An alcohol sensor ( Figure 5A ) is different, catalase in Figure 5E402. In some embodiments, catalase plays a more active role in the cooperative enzymatic reaction described in . That is, catalase reacts with hydrogen peroxide to form a catalase-hydrogen peroxide complex (the same peroxide scavenging function exhibited by catalase in the cooperative enzymatic reaction of alcohol oxidase and xanthine oxidase), which then reacts with ethanol to form acetaldehyde. Acetaldehyde formed in active region 406 when ethanol reacts with the catalase-hydrogen peroxide complex diffuses through membrane 404, which separates active region 406 from active region 402. Alternatively, catalase may be present in active region 402, in which case hydrogen peroxide formed in active region 406 may diffuse into active region 402 via membrane 404, form a catalase-hydrogen peroxide complex in active region 402, and oxidize ethanol to acetaldehyde in active region 402. Once acetaldehyde is formed in active region 402, the cooperative enzymatic reaction may be as follows Figure 5E Membrane 404 may comprise cross-linked polyvinylpyridine, which is permeable to acetaldehyde. Acetaldehyde is then reacted with xanthine oxidase in active zone 402 in a manner similar to that described above. Figure 5A reacts in a manner to form acetic acid.

[0132] Therefore, the alcohol sensor of the present disclosure may include: a sensor tail, which includes at least one working electrode; a first active area disposed on the surface of the working electrode, the first active area comprising xanthine oxidase, catalase, a first polymer and an electron transfer agent; wherein the xanthine oxidase and the electron transfer agent are covalently bonded to the first polymer; a first membrane, which coats the first active area, the first membrane comprising a first membrane polymer and acetaldehyde permeable; a second active area disposed on the first membrane, the second active area comprising glucose oxidase, catalase and a second polymer; wherein the glucose oxidase is covalently bonded to the second polymer; and a second membrane coating the second active area, the second membrane comprising a second membrane polymer and permeable to glucose and alcohol; wherein the glucose oxidase and the xanthine oxidase are capable of synergistically interacting to generate a signal proportional to the alcohol concentration at the working electrode. In a more specific embodiment, the alcohol may be ethanol.

[0133] According to some embodiments, the first membrane polymer and the second membrane polymer can be different from each other. According to some embodiments, the first membrane polymer can be cross-linked polyvinyl pyridine. In embodiments of the present disclosure, cross-linked polyvinyl pyridine easily permeates acetaldehyde. The second membrane polymer can be a cross-linked polyvinyl pyridine-styrene copolymer, in which a portion of the pyridine nitrogen atoms are functionalized with non-cross-linked poly (ethylene glycol) tails, and a portion of the pyridine nitrogen atoms are functionalized with alkyl sulfonic acid groups. This second membrane polymer is easily permeable to both glucose and ethanol.

[0134] According to some embodiments, the catalase is not covalently bonded to the first polymer or the second polymer in the first active region or the second active region. The catalase can be physically constrained within the first active region and the second active region by any of the first polymer, the second polymer, the first membrane polymer, or the second membrane polymer.

[0135] Similarly, a method for analyzing ethanol or another alcohol using the aforementioned analyte sensor, including synergistically interacting glucose oxidase and xanthine oxidase, may include: exposing the analyte sensor to a fluid containing ethanol and glucose; oxidizing glucose with glucose oxidase to produce hydrogen peroxide; forming a catalase-hydrogen peroxide complex; oxidizing alcohol with the catalase-hydrogen peroxide complex to form acetaldehyde; reacting acetaldehyde with xanthine oxidase to form acetic acid, thereby generating a signal at the working electrode; and correlating the signal with the alcohol concentration in the fluid. According to various embodiments of the present disclosure, the fluid may include a biological fluid. The correlation of the signal with the alcohol concentration in the fluid may be performed using any suitable correlation technique outlined in more detail above.

[0136] In other embodiments of the present disclosure, multiple enzymes can be arranged in the active regions of separate working electrodes. Therefore, the signal associated with the enzyme reaction occurring in each active region can be measured separately by interrogating each working electrode simultaneously or at different times. The signal associated with each active region can then be correlated with the concentration of a separate analyte.

[0137] As described above, a membrane (i.e., a mass transfer limiting membrane) can coat one or more active areas in an analyte sensor to increase biocompatibility and change the flux of analytes to the active area. Such a membrane may be present in any analyte sensor disclosed herein. Because different analytes can show varying permeability values ​​in a given membrane, an analyte sensor configured to analyze multiple analytes can show different sensitivities to each analyte. A method for solving different sensitivity values ​​may involve utilizing different film thicknesses on each active area. Although feasible, from a manufacturing point of view, this method may be difficult to implement. That is, using a typical dip coating technique for film deposition may be difficult to change the film thickness at different locations. Another possible method is to use active areas of different sizes for each analyte.

[0138] An analyte sensor having an active area configured to measure different analytes on a separate working electrode can solve the aforementioned problems associated with dissimilar analyte sensitivity. That is, the disclosure below provides various ways in which the membrane permeability can be changed on each working electrode to level the analyte membrane permeability at each position. That is, the disclosure herein allows the analyte permeability and sensitivity at each working electrode to be changed independently. According to the disclosure herein, a mass transfer limiting membrane comprising two or more different membrane polymers can provide a more balanced analyte permeability at each working electrode. Specific membrane configurations that can be suitable for leveling the analyte permeability on one or more working electrodes include bilayer membranes and mixed membranes, each of which includes two or more different membrane polymers. Surprisingly, when located in a bilayer membrane or mixed membrane, a bilayer membrane and a mixed membrane comprising a membrane polymer that is not suitable for promoting the permeability of a given analyte alone can provide satisfactory performance, as described below. Compared with changing the size of the active area on each working electrode to provide a comparable sensitivity value for each analyte, this method may be advantageous.

[0139] Therefore, in some embodiments, an analyte sensor characterized by two or more enzymes arranged on separate working electrodes may include: a sensor tail, which includes at least a first working electrode and a second working electrode, a first active area located on the surface of the first working electrode, a second active area located on the surface of the second working electrode, a multi-component membrane coating the first active area, and a homogeneous membrane coating the second active area. The first active area includes a first polymer and a first analyte-responsive enzyme that reacts with a first analyte, and the second active area includes a second polymer and a second analyte-responsive enzyme that reacts with a second analyte. The first analyte-responsive enzyme and the second analyte-responsive enzyme are different and react with different analytes. The multi-component membrane contains at least a first membrane polymer and a second membrane polymer that are different from each other. The homogeneous membrane includes one of the first membrane polymer and the second membrane polymer.

[0140] Specific configurations of the above-described multi-component membranes may include bilayer membranes in some embodiments, or mixtures of membrane polymers in other embodiments. Surprisingly, bilayer membranes and mixed membranes may function to level analyte permeability, as explained in further detail below.

[0141] The analyte sensor of the present disclosure having two different active areas located on separate working electrodes can be used in the same manner as described above. Figure 3, or variations thereof. For example, in some embodiments, a counter / reference electrode can replace a separate counter electrode and reference electrode in an analyte sensor that carries two or more working electrodes. Similarly, the layer configuration and arrangement within an analyte sensor having two different active areas located on a separate working electrode can be similar to the sensor configuration described in . Figure 3 Further details on the membrane arrangement on each active area are given below in Figure 7 supply.

[0142] According to more specific embodiments of the present disclosure, an analyte sensor having multiple working electrodes may include an active region, wherein an electron transfer agent is covalently bonded to a polymer in each active region. In some or other embodiments, such an analyte sensor may be characterized by a first analyte-responsive enzyme covalently bonded to a polymer of a first active region and a second analyte-responsive enzyme covalently bonded to a polymer of a second active region. In addition, in a particular embodiment, the first analyte-responsive enzyme may be a glucose-responsive enzyme, such as glucose oxidase, and the second analyte-responsive enzyme may be a lactate-responsive enzyme, such as lactate oxidase.

[0143] In other more specific embodiments, an analyte sensor having multiple working electrodes can include a sensor tail configured for insertion into tissue.

[0144] In some embodiments, the double-layer film can be coated on the first active area on one of the working electrodes. The double-layer film includes a first film polymer and a second film polymer stacked on each other on the active area. In a more specific embodiment, the first film polymer can be directly disposed on the active area of ​​the first working electrode, and the second film polymer can be disposed on the first film polymer to define the double-layer film. In such an embodiment, the second film polymer is present in a homogeneous film located on the second working electrode. In some embodiments, this double-layer configuration can be prepared by coating only the first film polymer on the first working electrode (e.g., by spraying, smearing, inkjet printing, roller coating, etc.), and then coating the second film polymer on the two working electrodes at the same time (e.g., by dip coating or similar techniques). In other embodiments, the double-layer film can be configured as described above, wherein the first film polymer is located on the second working electrode.

[0145] Figure 7 A schematic diagram of a portion of an analyte sensor having two working electrodes and featuring a bilayer membrane coating one of the two working electrodes, the bilayer membrane being compatible for use with some embodiments disclosed herein. Figure 7As shown, the analyte sensor is characterized by a sensor tail 600 having working electrodes 614a and 614b disposed on opposite sides of a substrate 612. An active region 618a is disposed on the working electrode 614a, and an active region 618b is disposed on the working electrode 614b. According to the disclosure herein, the active regions 618a and 618b contain different analyte-responsive enzymes and are configured to measure different analytes. Although Figure 7 Active regions 618a and 618b are shown disposed generally opposite one another relative to substrate 612, but it is understood that active regions 618a and 618b may be laterally spaced (offset) from one another on opposite sides of substrate 612. Laterally spaced configurations for active regions 618a and 618b may be particularly advantageous for coating each active region 618a and 618b with a mass transfer limiting membrane, as discussed below.

[0146] like Figure 7 As further shown, active region 618a is coated by membrane layer 620. Membrane layer 620 is a homogeneous membrane comprising a single membrane polymer. Active region 618b is coated by a double layer membrane 621, which comprises membrane layer 621a in direct contact with active region 618b and membrane layer 621b covering membrane layer 621a. Membrane layers 621a and 621b comprise different membrane polymers. As described above, in certain embodiments, diaphragm layer 620 and diaphragm layer 621b may comprise the same diaphragm polymer.

[0147] According to one or more embodiments, an analyte sensor having multiple active areas on separate working electrodes, where one of the active areas is coated with a bilayer membrane, can exhibit balanced or independently variable analyte permeability. That is, the analyte sensor can have sensitivity to two different analytes that are closer to each other than in the absence of a bilayer membrane. In this analyte sensor configuration, the analyte permeability of the analyte sensor can be adjusted by using a homogeneous membrane (e.g., Figure 7 The active area coated with the membrane layer 620 in the double layer membrane (e.g., Figure 7 The bilayer membrane 621 in the embodiment may include a membrane polymer that does not negatively affect the permeability of the second analyte (i.e., a membrane polymer with a neutral permeability effect), thereby allowing the other membrane polymers comprising the bilayer membrane to exhibit their characteristic permeability to the second analyte as if the first membrane polymer was not present. Thus, according to various embodiments, the membrane polymer with a neutral permeability effect and the membrane polymer comprising the homogeneous membrane may constitute the same polymer.

[0148] In some or other specific embodiments, the membrane polymer having a neutral permeability influence can constitute the inner layer of the bilayer membrane. Thus, according to such embodiments, the inner layer of the bilayer membrane and the homogeneous membrane can constitute the same membrane polymer. In other specific embodiments, the outer layer of the bilayer membrane and the homogeneous membrane can constitute the same membrane polymer.

[0149] In a specific embodiment, the first active area may include a glucose responsive enzyme, such as glucose oxidase, and the second active area may include a lactate responsive enzyme, such as lactate oxidase. Therefore, according to such an embodiment, the first active area containing the glucose responsive enzyme can be coated with a double-layer membrane, and the second active area containing the lactate responsive enzyme can be coated with a homogeneous (single-component membrane polymer) membrane. In a more specific embodiment, the second active area may include a polymer, albumin, and a lactate responsive enzyme covalently bonded to the polymer. In a more specific embodiment, the homogeneous film coating the second active area may include at least a cross-linked polyvinyl pyridine homopolymer or copolymer, and the double-layer membrane coating the first active area may also include a polyvinyl pyridine homopolymer or copolymer.

[0150] In other embodiments of the present invention, the multi-component membrane may include a mixture (homogeneous blend) of a first membrane polymer and a second membrane polymer. In addition to replacing the bilayer membrane 621 with a mixed membrane of two different membrane polymers contained in a homogenous mixture, this analyte sensor configuration may be similar in appearance to Figure 7 As with the analyte sensor comprising a bilayer membrane disposed on one of the active regions, a homogeneous film comprising one of the first membrane polymer or the second membrane polymer of the mixed membrane may coat another active region on the second working electrode.

[0151] Similar to a double-layer membrane, a mixed membrane containing a membrane polymer that neutrally affects the permeability of a second analyte can allow the mixed membrane to show permeability to a second analyte, which is mainly the feature of other membrane polymers in the mixture. Therefore, according to various embodiments of the present disclosure, a membrane polymer of a homogeneous membrane and a mixed membrane polymer can be selected so that the permeability of the second analyte through the mixed membrane is substantially not changed by the membrane polymer. In a particular embodiment, the first active region may include a glucose responsive enzyme, such as glucose oxidase, and the second active region may include a lactate responsive enzyme, such as lactate oxidase. Therefore, according to such an embodiment, the first active region containing a glucose responsive enzyme can be coated with a mixed membrane, and the second active region containing a lactate responsive enzyme can be coated with a homogeneous (single component membrane polymer) membrane. In a more specific embodiment, the second active region may include a polymer, albumin, and a lactate responsive enzyme covalently bonded to a polymer. In a more specific embodiment, the homogeneous membrane coating the second active region may include at least a cross-linked polyvinylpyridine homopolymer or copolymer, and the mixed membrane coating the first active region may also include a polyvinylpyridine homopolymer or copolymer.

[0152] As described above, bilayer membranes and hybrid membranes can level the analyte permeability levels in the analyte sensors of the present invention, wherein two or more active areas are spatially separated from each other and can be coated with different mass transfer limiting membranes. Specifically, the bilayer membranes and hybrid membranes of the present disclosure can level the analyte permeability in analyte sensors having separate working electrodes and comprising two or more active areas with different enzymes, wherein at least one active area is located at each working electrode. Therefore, such membranes can advantageously allow the sensor sensitivity to be varied independently for each analyte. The membrane thickness and / or the relative ratio of the first membrane polymer to the second membrane polymer represent other parameters that can be varied to adjust the characteristic permeability of the analyte at each working electrode.

[0153] Therefore, a method of using an analyte sensor comprising two working electrodes may include exposing the analyte sensor to a fluid comprising at least one analyte. The analyte sensor includes a sensor tail, which includes at least a first working electrode and a second working electrode. The first active area is disposed on the surface of the first working electrode, and the second active area is disposed on the surface of the second working electrode. The first active area includes a first polymer and a first analyte-responsive enzyme that reacts with the first analyte, and the second active area includes a second polymer and a second analyte-responsive enzyme that reacts with the second analyte. The first analyte-responsive enzyme and the second analyte-responsive enzyme are different. A multi-component membrane coats the first active area, and a homogeneous membrane coats the second active area. The multi-component membrane includes at least a first membrane polymer and a second membrane polymer that are different from each other, and the homogeneous membrane includes one of the first membrane polymer or the second membrane polymer. The method also includes obtaining a first signal at or above the redox potential of the first active area, obtaining a second signal at or above the redox potential of the second active area, and correlating the first signal with the concentration of the first analyte in the fluid, and correlating the second signal with the concentration of the second analyte in the fluid. The first signal is proportional to the concentration of the first analyte in the fluid and the second signal is proportional to the concentration of the second analyte in the fluid.

[0154] According to more specific embodiments, the first signal and the second signal can be measured at different times. Therefore, in such an embodiment, the potential can be applied alternately to the first working electrode and the second working electrode. In other embodiments, the first signal and the second signal can be measured simultaneously via the first channel and the second channel, in which case the potential can be applied to both electrodes simultaneously.

[0155] Embodiments disclosed herein include:

[0156] A. The analyte sensor comprises two active regions with different analyte-responsive enzymes. The analyte sensor comprises: a sensor tail comprising at least one working electrode; and at least two active regions disposed on a surface of the working electrode, each active region comprising an analyte-responsive enzyme and a polymer, wherein the analyte-responsive enzyme in each active region is different; and wherein each active region has a redox potential, and the redox potential of the first active region is sufficiently separated from the redox potential of the second active region to allow generation of a signal from the first active region independently of generation of a signal from the second active region.

[0157] B. A method for determining two or more analytes using a first active region and a second active region containing different analyte-responsive enzymes. The method comprises: exposing an analyte sensor to a fluid containing at least one analyte; wherein the analyte sensor comprises a sensor tail, the sensor tail comprising at least one working electrode and at least two active regions disposed on a surface of the working electrode, each active region comprising an analyte-responsive enzyme and a polymer; wherein the analyte-responsive enzyme in each active region is different; and wherein each active region has a redox potential, and the redox potential of the first active region is sufficiently separated from the redox potential of the second active region to allow generation of a signal from the first active region independently of generation of a signal from the second active region; obtaining a first signal at or above the redox potential of the first active region, the first signal being proportional to the concentration of the first analyte; obtaining a second signal at or above the redox potential of the second active region, the second signal being a composite signal comprising a signal contribution from the first active region and a signal contribution from the second active region; and subtracting the first signal from the second signal to obtain a difference signal, the difference signal being proportional to the concentration of the second analyte.

[0158] C. An analyte sensor comprising two or more enzymes capable of synergistically interacting with each other. The analyte sensor comprises: a sensor tail comprising at least one working electrode; and at least one active region disposed on a surface of the working electrode, the at least one active region comprising a first enzyme, a second enzyme, and a polymer, wherein the first enzyme and the second enzyme are capable of synergistically interacting; wherein the first enzyme is capable of converting an analyte into a first product, and the second enzyme is capable of converting the first product into a second product to generate a signal at the working electrode; and wherein the second enzyme is covalently bonded to the polymer and does not react with the analyte.

[0159] D. A method for determining an analyte using two or more enzymes that can synergistically interact with each other. The method comprises: exposing an analyte sensor to a fluid containing an analyte; wherein the analyte sensor comprises a sensor tail, the sensor tail comprising at least one working electrode and at least one active area disposed on a surface of the working electrode, the at least one active area comprising a first enzyme, a second enzyme, and a polymer; wherein the first enzyme and the second enzyme can synergistically interact, and the second enzyme is covalently bonded to the polymer and does not react with the analyte; reacting the analyte with the first enzyme to form a first product; reacting the first product with the second enzyme to form a second product, thereby generating a signal at the working electrode; and correlating the signal with the concentration of the analyte in the fluid.

[0160] E. Alcohol sensor. The alcohol sensor comprises: a sensor tail comprising at least one working electrode; and at least one active region disposed on a surface of the working electrode, the at least one active region comprising an alcohol oxidase, a xanthine oxidase, a catalase, a polymer, and an electron transfer agent; wherein the electron transfer agent and the xanthine oxidase are covalently bonded to the polymer, and the alcohol oxidase is not covalently bonded to the polymer; and wherein the alcohol oxidase and the xanthine oxidase are capable of synergistically interacting to generate a signal at the working electrode that is proportional to the alcohol concentration.

[0161] F. A method for detecting alcohol. The method comprises: exposing an analyte sensor to a fluid containing ethanol; wherein the analyte sensor comprises a sensor tail, the sensor tail comprising at least one working electrode and at least one active area disposed on a surface of the working electrode, the at least one active area comprising an alcohol oxidase, a xanthine oxidase, a catalase, a polymer, and an electron transfer agent; wherein the electron transfer agent and the xanthine oxidase are covalently bonded to the polymer, and the alcohol oxidase is not covalently bonded to the polymer; and wherein the alcohol oxidase and the xanthine oxidase are capable of synergistically interacting; reacting the ethanol with the alcohol oxidase to form acetaldehyde; reacting the acetaldehyde with the xanthine oxidase to form acetic acid, thereby generating a signal at the working electrode; and correlating the signal to the concentration of ethanol in the fluid.

[0162] G. An analyte sensor comprising two or more working electrodes coated with different mass transfer limiting membranes. The analyte sensor comprises: a sensor tail comprising at least a first working electrode and a second working electrode; a first active region disposed on the surface of the first working electrode, the first active region comprising a first polymer and a first analyte-responsive enzyme that reacts with a first analyte; a second active region disposed on the surface of the second working electrode, the second active region comprising a second polymer and a second analyte-responsive enzyme that reacts with a second analyte; wherein the first analyte-responsive enzyme and the second analyte-responsive enzyme are different; a multi-component membrane coating the first active region, the multi-component membrane comprising at least a first membrane polymer and a second membrane polymer that are different from each other; and a homogeneous membrane coating the second active region and being different in composition from the multi-component membrane, the homogeneous membrane comprising one of the first membrane polymer and the second membrane polymer.

[0163] H. A method for determining two or more analytes using two working electrodes coated with different mass transfer limiting membranes. The method comprises: exposing an analyte sensor to a fluid containing at least one analyte; wherein the analyte sensor comprises a sensor tail, the sensor tail comprising at least a first working electrode and a second working electrode; wherein a first active area is disposed on a surface of the first working electrode, the first active area comprises a first polymer and a first analyte-responsive enzyme that reacts with the first analyte, and a second active area is disposed on a surface of the second working electrode, the second active area comprises a second polymer and a second analyte-responsive enzyme that reacts with the second analyte; wherein the first analyte-responsive enzyme and the second analyte-responsive enzyme are different; and wherein the multi-component membrane coats the first active area. region, and a homogeneous membrane coats the second active region, the multi-component membrane comprises at least a first membrane polymer and a second membrane polymer that are different from each other, and the homogeneous membrane comprises one of the first membrane polymer or the second membrane polymer and is different in composition from the multi-component membrane; obtaining a first signal at or above the redox potential of the first active region, the first signal being proportional to the concentration of the first analyte in the fluid; obtaining a second signal at or above the redox potential of the second active region, the second signal being proportional to the concentration of the second analyte in the fluid; and correlating the first signal with the concentration of the first analyte in the fluid, and correlating the second signal with the concentration of the second analyte in the fluid.

[0164] I. An alcohol sensor comprising synergistically interacting glucose oxidase and xanthine oxidase. The alcohol sensor comprises: a sensor tail comprising at least one working electrode; a first active region disposed on a surface of the working electrode, the first active region comprising xanthine oxidase, catalase, a first polymer, and an electron transfer agent; wherein the xanthine oxidase and the electron transfer agent are covalently bonded to the first polymer; a first membrane coating the first active region, the first membrane comprising a first membrane polymer and being permeable to acetaldehyde; a second active region disposed on the first membrane, the second active region comprising glucose oxidase, catalase, and a second polymer; wherein the glucose oxidase is covalently bonded to the second polymer; and a second membrane coating the second active region, the second membrane comprising a second membrane polymer and being permeable to glucose and alcohol; wherein the glucose oxidase and the xanthine oxidase are capable of synergistically interacting to produce a signal at the working electrode that is proportional to the alcohol concentration.

[0165] J. A method for detecting alcohol using a synergistic interaction between glucose oxidase and xanthine oxidase. The method comprises: exposing an analyte sensor to a fluid containing ethanol and glucose; wherein the analyte sensor comprises a sensor tail, the sensor tail comprising: at least one working electrode; a first active region disposed on a surface of the working electrode, the first active region comprising xanthine oxidase, catalase, a first polymer, and an electron transfer agent, the xanthine oxidase and the electron transfer agent being covalently bonded to the first polymer; a first membrane coating the first active region, the first membrane comprising a first membrane polymer and being permeable to acetaldehyde; a second active region disposed on the first membrane, the second active region comprising glucose glucose oxidase, catalase, and a second polymer, wherein the glucose oxidase is covalently bonded to the second polymer; and a second membrane coating a second active area, the second membrane comprising a second membrane polymer and being permeable to glucose and alcohol; wherein the glucose oxidase and xanthine oxidase are capable of synergistically interacting; oxidizing glucose with the glucose oxidase to produce hydrogen peroxide; forming a catalase-hydrogen peroxide complex; oxidizing the alcohol with the catalase-hydrogen peroxide complex to form acetaldehyde; reacting the acetaldehyde with the xanthine oxidase to form acetic acid, thereby generating a signal at a working electrode; and correlating the signal to the alcohol concentration in the fluid.

[0166] Each of embodiments A and B may have one or more of the following additional elements in any combination:

[0167] Element 1: wherein the sensor tail is configured for insertion into tissue.

[0168] Element 2: wherein the redox potential of the first active region is at least about 100 mV away from the redox potential of the second active region.

[0169] Element 3: wherein the first active region comprises a first electron transfer agent, the second active region comprises a second electron transfer agent, and the first and second electron transfer agents are different.

[0170] Element 4: wherein the first electron transfer agent is covalently bonded to the polymer in the first active region, and the second electron transfer agent is covalently bonded to the polymer in the second active region.

[0171] Element 5: wherein the analyte-responsive enzyme in each active region is covalently bonded to the polymer.

[0172] Element 6: wherein the analyte sensor further comprises: a mass transfer limiting membrane coating at least two active regions.

[0173] Element 7: wherein at least one of the at least two active zones comprises two or more analyte-responsive enzymes, the two or more analyte-responsive enzymes interact synergistically to generate a signal proportional to the concentration of a single analyte.

[0174] Element 8: wherein the fluid is a biological fluid and the analyte sensor is exposed to the in vivo biological fluid.

[0175] Element 9: wherein the mass transfer limiting membrane coats at least the at least two active regions.

[0176] Element 10: wherein the analyte sensor comprises glucose oxidase as a first enzyme and lactate oxidase as a second enzyme.

[0177] Each of embodiments C and D may have one or more of the following additional elements in any combination:

[0178] Element 11: wherein the first enzyme is alcohol oxidase and the second enzyme is xanthine oxidase.

[0179] Element 12: wherein the at least one active region further comprises catalase.

[0180] Element 13: wherein the catalase is not covalently bonded to the polymer.

[0181] Element 14: wherein the alcohol oxidase is not covalently bound to the polymer.

[0182] Element 15: wherein the first enzyme is not covalently bonded to the polymer.

[0183] Element 16: wherein the at least one active region comprises an electron transfer agent covalently bonded to the polymer.

[0184] Element 17: Wherein, the sensor tail is configured for insertion into tissue.

[0185] Element 18: wherein the analyte sensor further comprises: a mass transfer limiting membrane coating at least the at least one active region.

[0186] Element 19: wherein the fluid is a biological fluid and the analyte sensor is exposed to the biological fluid in vivo.

[0187] Element 20: wherein a mass transfer limiting membrane coats at least one active region.

[0188] Each of embodiments E and F may have one or more of the following additional elements in any combination:

[0189] Element 21: wherein the catalase is not covalently bonded to the polymer.

[0190] Element 22: wherein the sensor tail is configured for insertion into tissue.

[0191] Element 23: wherein the alcohol sensor further comprises: a mass transfer limiting membrane coating at least at least one active region.

[0192] Element 24: wherein the fluid is a biological fluid and the analyte sensor is exposed to the in vivo biological fluid.

[0193] Element 25: wherein a mass transfer limiting membrane coats at least one active region.

[0194] Each of Embodiments G and H may have one or more of the following additional elements in any combination:

[0195] Element 26: wherein the multi-component membrane comprises a double-layer membrane.

[0196] Element 27: wherein the first membrane polymer is disposed directly on the first active area and the second membrane polymer is disposed on the first membrane polymer to define the bilayer membrane, the second membrane polymer also being present in the homogeneous membrane.

[0197] Element 28: wherein the multicomponent membrane comprises a mixture of the first membrane polymer and the second membrane polymer.

[0198] Element 29: Wherein, the sensor tail is configured for insertion into tissue.

[0199] Element 30: wherein each active region further comprises an electron transfer agent covalently bonded to the polymer.

[0200] Element 31: wherein the first analyte responsive enzyme is covalently bonded to the polymer in the first active region, and the second analyte responsive enzyme is covalently bonded to the polymer in the second active region.

[0201] Element 32: A polymer in which an electron transfer agent is covalently bonded to each active region.

[0202] Element 33: wherein the analyte-responsive enzyme in each active region is covalently bonded to the polymer.

[0203] Element 34: wherein the fluid is a biological fluid and the analyte sensor is exposed to the in vivo biological fluid in vivo.

[0204] Element 35: wherein the first signal and the second signal are measured at different times.

[0205] Element 36: wherein the first signal and the second signal are measured simultaneously via a first channel and a second channel.

[0206] Each of embodiments I and J may have one or more of the following additional elements in any combination:

[0207] Element 37: wherein the catalase is not covalently bonded to the first polymer or the second polymer.

[0208] Element 38: Wherein, the sensor tail is configured for insertion into tissue.

[0209] Element 39: wherein the first membrane polymer and the second membrane polymer are different from each other.

[0210] Element 40: wherein the first membrane polymer comprises cross-linked polyvinyl pyridine.

[0211] Element 41: wherein the fluid is a biological fluid and the analyte sensor is exposed to the in vivo biological fluid.

[0212] As non-limiting examples, exemplary combinations applicable to AJ include:

[0213] Analyte sensors of A with elements 1 and 2; 1 and 3; 1, 3 and 4; 1 and 5; 1 and 6; 1 and 7; 2 and 3; 2-4; 2 and 5; 2 and 6; 2 and 7; 3-5; 3 and 4; 3 and 5; 3 and 6; 3 and 7; 5 and 6; 6 and 7; 2, 3 and 5; 2, 3 and 6; 2-5; and combinations of 2, 5 and 6. Method B is a combination of elements 2 and 3; 2-4; 2 and 5; 2 and 6; 2 and 7; 2 and 8; 3-5; 3 and 4; 3 and 5; 3 and 6; 3 and 7; 3 and 8; 5 and 6; 5 and 8; 6 and 7; 6 and 8; 7 and 8; 2, 3 and 5; 2, 3, 5 and 8; 2, 3 and 6; 2, 3, 6 and 8; 2-5; 2-5 and 8; 2, 5 and 6; 2, 5, 6 and 8; 2, 3 and 8; 3, 5 and 8; 2, 6 and 8; 2, 5 and 8; any one of 2-8 and 9; and any one of 2-8 and 10.

[0214] Analyte sensors of C with elements 11 and 12; 11, 12 and 13; 12 and 13; 11 and 14; 11 and 15; 11 and 17; 11 and 18; 12 and 13; 12 and 14; 12 and 15; 12 and 16; 12 and 17; 12 and 18; 15 and 16; 15 and 17; 15 and 18; 16 and 17; 16 and 18; 17 and 18; 11, 12 and 13; 11, 12 and 14; 11-14; 11-14 and 16; 11-14 and 17; 11-14 and 18; 15-17; 15-18; and combinations of 15, 17 and 18. Method D with elements 11 and 12; 11, 12 and 13; 12 and 13; 11 and 14; 11 and 15; 11 and 17; 11 and 18; 11 and 19; 11 and 20; 12 and 13; 12 and 14; 12 and 15; 12 and 16; 12 and 17; 12 and 18; 12 and 19; 12 and 20; 15 and 16; 15 and 17; 15 and 18; 15 and 19; 15 and 20; 16 and 17; 16 and 18; 16 and 19; 16 and 20; 17 and 18; 17 and 19; 17 and 20; 18 and 19; 18 and 20; 19 and 20; 11, 12 and 13; 11, 12, 13 and 19; 11, 12, 13 and 20; 11, 12 and 14; 11, 12, 14 and 1 9; 11, 12, 14 and 20; 11-14; 11-14 and 19; 11-14 and 20; 11-14 and 16; 11-14, 16 and 19; 11-14, 16 and 20; 11-14 and 17; 11-14, 17 and 19; 11-14, 17 and 20; 11-14 and 18; 11-14, 18 and 19; 11-14, 18 and 20; 15-17; 15-17 and 19; 15-17 and 20; 15-18; 15-18 and 19; 15-18 and 20; 15, 17 and 18; 15, 17, 18 and 19; 15, 17, 18 and 20; any one of 11-16 and 19; and any combination of 11-16 and 20.

[0215] The analyte sensor of E combines elements 21 and 22; 21 and 23; 22 and 23; and 21-23, and the method of F combines elements 21 and 22; 21 and 23; 22 and 23; 21-23; 21 and 24; 21 and 25.

[0216] Analyte sensors of G with combinations of elements 26 and 27; 26 and 29; 26, 27 and 29; 26 and 30; 26, 27 and 30; 26 and 31; 26, 27 and 31; 28 and 29; 28 and 30; 28-30; 28 and 31; 29 and 30; 29 and 31; and 30 and 31. The method of H with combinations of elements 26 and 27; 26 and 29; 26, 27 and 29; 26 and 30; 26, 27 and 30; 26 and 31; 26, 27 and 31; 28 and 29; 28 and 30; 28-30; 28 and 31; 29 and 30; 29 and 31; 30 and 31; 26 and 33; 26, 27 and 33; 26 and 34; 26, 27 and 34; 26 and 35; 26, 27 and 35; 26 and 36; 26, 27 and 36; 28 and 33; 28 and 34; 28 and 35; 28 and 36; 30 and 33; 30 and 34; 30 and 35; 30 and 36; 33 and 34; 33 and 35; 33 and 36; 34 and 35; and 34 and 36.

[0217] Analyte Sensor I with combinations of elements 37 and 38; 37 and 39; 37 and 40; 38 and 39; 38 and 40; and 39 and 40. Method J with combinations of elements 37 and 39; 37 and 40; 37 and 41; 39 and 40; 39 and 41; and 40 and 41.

[0218] Additional embodiments disclosed herein include:

[0219] A1: An analyte sensor with a multi-component membrane. The analyte sensor comprises: a sensor tail configured to be inserted into a tissue, the sensor tail comprising at least a working electrode; and first and second active regions disposed on the sensor tail, the first and second active regions comprising at least two different enzymes for measuring the concentration of at least one analyte; wherein the first active region is coated with a first membrane polymer and a second membrane polymer that are different from each other.

[0220] B1: An analyte sensor having two active regions on a working electrode and configured to sense different analytes. The analyte sensor comprises: a sensor tail configured to be inserted into a tissue and comprising at least one working electrode; and at least two active regions disposed on the sensor tail, each active region comprising an enzyme, an electron transfer agent, and a polymer;

[0221] wherein the enzyme in each active zone is different and responsive to a different analyte; and wherein each active zone has a redox potential, and the redox potential of a first active zone is sufficiently separated from the redox potential of a second active zone to allow generation of a signal from the first active zone independent of generation of a signal from the second active zone.

[0222] Embodiment A1 may have one or more of the following additional elements in any combination:

[0223] Element 1 ': wherein the first active area is coated by a mixture of the first membrane polymer and the second membrane polymer, and one of the first membrane polymer and the second membrane polymer coats the second active area as a homogeneous film.

[0224] Element 2': wherein the first active area is coated by a double layer membrane comprising the first membrane polymer disposed on the second membrane polymer, and the second membrane polymer coats the second active area as a homogenous film.

[0225] Element 3': wherein the first active zone comprises a first enzyme of the at least two different enzymes, and the second active zone comprises a second enzyme of the at least two different enzymes.

[0226] Element 4': wherein the first enzyme is non-reactive with the at least one analyte, and the first and second enzymes are capable of synergistically interacting to generate a signal proportional to the concentration of the at least one analyte.

[0227] Element 5': wherein the second enzyme is capable of converting the at least one analyte into a product that reacts with the first enzyme, such that the first enzyme is capable of reacting the product to produce a signal at the working electrode.

[0228] Element 6': wherein the first enzyme is xanthine oxidase, and the second enzyme is glucose oxidase, and at least one of the first active region and the second active region further comprises catalase.

[0229] Element 7': wherein the catalase is present in the first active region.

[0230] Element 8': wherein the first active region is directly disposed on the working electrode and further comprises an electron transfer agent.

[0231] Element 9': wherein the first membrane polymer is disposed directly on the first active region, the second active region is disposed directly on the first membrane polymer, and the second membrane polymer is disposed directly on the second active region.

[0232] Element 10': wherein the sensor tail includes a first working electrode and a second working electrode, the first active area is arranged on the surface of the first working electrode, the second active area is arranged on the surface of the second working electrode, the first enzyme reacts with the first analyte to generate a signal proportional to the concentration of the first analyte, and the second enzyme reacts with the second analyte to generate a signal proportional to the concentration of the second analyte.

[0233] Element 11': wherein each of the first and second active regions has a redox potential, and the redox potential of the first active region is sufficiently separated from the redox potential of the second active region to allow generation of a signal from the first active region independent of generation of a signal from the second active region.

[0234] Element 12': wherein the redox potential of the first active region is at least about 100 mV away from the redox potential of the second active region.

[0235] Element 13': wherein the signal from the first active area corresponds to the first analyte concentration and the signal from the second active area corresponds to the second analyte concentration.

[0236] Element 14': wherein the first active region comprises a first electron transfer agent, and the second active region comprises a second electron transfer agent different from the first electron transfer agent.

[0237] Embodiment B1 may have one or more of the following additional elements in any combination:

[0238] Element 15': wherein the redox potential of the first active region is at least about 100 mV away from the redox potential of the second active region.

[0239] Element 16': wherein the first active region comprises a first electron transfer agent, and the second active region comprises a second electron transfer agent different from the first electron transfer agent.

[0240] Element 17': wherein the first and second active regions are coated by a mass transfer limiting membrane, the first active region is coated by a single membrane polymer and the second active region is coated by two or more different membrane polymers.

[0241] To facilitate a better understanding of the embodiments described herein, the following examples of various representative embodiments are given. The following embodiments should in no way be construed as limiting or defining the scope of the present invention.

[0242] Example

[0243] Example 1: Detection of glucose and lactate using an analyte sensor with two different active areas on a single working electrode. Two solutions containing different poly(vinylpyridine)-bound transition metal complexes were prepared. The polymer structure of the first solution is shown in Formula 1 and the polymer structure of the second solution is shown in Formula 2. Further details about these polymers are provided in co-owned U.S. Patent 6,605,200, which is incorporated by reference above. The subscripts for each monomer represent exemplary atomic ratios.

[0244]

[0245] The oxidation-reduction potential of the polymer of formula 1 is -50 mV relative to the Ag / AgCl reference, and the oxidation-reduction potential of the polymer of formula 2 is +220 mV relative to the same reference (270 mV separation, see Figure 8 ). In addition to the transition metal complex used as the respective electron transfer agent, after being deposited on the working electrode and cured, the polymer of formula 1 includes glucose oxidase (GOX) covalently bonded thereto and the polymer of formula 2 includes lactate oxidase (LOX) covalently bonded thereto. Crosslinking is accomplished using polyethylene glycol diglycidyl ether (PEGDE400). Solutions containing the polymer of formula 1 and the polymer of formula 2 are formulated as specified in Tables 1 and 2 below.

[0246] Table 1

[0247]

[0248]

[0249] Table 2

[0250]

[0251] To deposit each active area, approximately 20 nL of each solution was deposited on the carbon working electrode to form two discrete, separated spots, each with a spacing of approximately 0.1 mm. 2 One spot contained a glucose oxidase preparation and the other contained a lactate oxidase preparation. After deposition, the working electrode was cured overnight at 25 °C.

[0252] After curing, a film is deposited on the working electrode. The membrane polymer is a polyvinylpyridine copolymer with amine-free polyether side chain functionalization, as described in U.S. Provisional Patent Application 62 / 684,438, entitled "Temperature-Insensitive Membrane Materials and Analyte Sensors Containing the Same" filed on June 13, 2018. The film deposition is completed by dipping the electrode three times in a solution containing 4 mL of membrane polymer (120 mg / ml_) and 0.35 mL of PEG1000 (200 mg / ml_). Spraying, screen printing or similar processes can be used to deposit the film alternatively. After deposition, the electrode is cured overnight at 25 ° C and then further cured at 56 ° C in a dried vial for two days.

[0253] After fabrication, the electrode was analyzed by cyclic voltammetry in a buffer solution containing neither glucose nor lactate. The resulting cyclic voltammogram is shown in Figure 8, which shows the anodic and cathodic peaks characteristic of the two osmium complexes, since there is no current contribution from glucose or lactate. The redox potentials reported above were calculated from the average of the cathodic and anodic peaks for each osmium complex.

[0254] For analysis of glucose and lactate, the electrode was placed at a potential above the average oxidation-reduction potential of the first polymer, specifically +40 mV ( Figure 8 At this potential, oxidation of the osmium complex in the first polymer and glucose may occur, but oxidation of the osmium complex in the second polymer or lactate does not occur. To oxidize the osmium complex and glucose and lactate, the electrode is placed at a potential above the average oxidation-reduction potential of the second polymer, specifically +250 mV ( Figure 8 in E2).

[0255] Glucose and lactate analysis was performed by immersing the electrode in a buffer solution containing 5 mM glucose and 5 mM lactate, and applying E1 and E2 potentials sequentially. Fig. 9 Four replicates of the electrode response in 5mM glucose / 5mM lactate buffer are shown when cycling between E1 and E2. As shown, the current at E1 is about 5nA, which is due to glucose oxidation, and the current at E2 is about 10.5nA, which is due to glucose and lactate oxidation. Taking the difference between the currents measured at E1 and E2, a contribution of about 5.5nA is obtained from lactate oxidation at E2. Unknown glucose and lactate concentrations can be similarly analyzed by comparison with a lookup table or calibration curve.

[0256] Example 2A: Detection of ethanol using an analyte sensor with two different enzymes operating in concert on a single working electrode (XOX / AOX). A spotting solution was prepared with the formulation shown in Table 3. All components were dissolved in 10 mM HEPES buffer at pH 8 and cross-linking was accomplished using polyethylene glycol diglycidyl ether.

[0257] Table 3

[0258]

[0259] 15 nL of the solution was deposited on the carbon working electrode as a surface area of ​​approximately 0.05 mm 2 After deposition, the working electrode was cured at 25 °C overnight.

[0260] After curing, a poly(4-vinylpyridine) (PVP) film was deposited on the working electrode from a coating solution containing 100 mg / ml PVP and 100 mg / ml PEGDE400. Film deposition was accomplished by dip-coating the electrode three times in the coating solution. After deposition, the electrode was cured overnight at 25°C and then further cured for two days at 56°C in a dry vial. Spray coating, screen printing or similar processes may alternatively be used to deposit the film.

[0261] Ethanol analysis was performed by immersing the electrode in ethanol-containing PBS solutions, where each PBS solution contained different concentrations of ethanol. Fig.10 Shown are three replicate responses of an electrode containing alcohol oxidase and xanthine oxidase in the sensing spot when exposed to different ethanol concentrations. As shown, after exposure to a new ethanol concentration, the current response increases over the course of several minutes after stabilization. Fig.11A An exemplary graph of average current response versus ethanol concentration is shown. Fig. 11B The corresponding data for a single sensor are shown. As shown, the sensor response is approximately linear over the ethanol concentration range of 0-10 mM.

[0262] Example 2B: Detection of ethanol using an analyte sensor with two different enzymes operating in concert on a single working electrode (XOX / GOX). A first spot solution was prepared having the formulation shown in Table 4. All components were dissolved in 10 mM HEPES buffer at pH 8. Cross-linking was accomplished using polyethylene glycol diglycidyl ether.

[0263] Table 4

[0264]

[0265] 15 nL of the first dot solution was deposited on the carbon working electrode as a dot with an area of ​​about 0.05 mm 2 After deposition, the working electrode was cured at 25°C overnight.

[0266] After curing, a poly(4-vinylpyridine) (PVP) film was deposited on the working electrode and XOX dots from a coating solution containing 100 mg / ml PVP and 100 mg / ml PEGDE400. Film deposition was accomplished by dipping the electrode three times in the coating solution. Spray coating, screen printing or similar processes may alternatively be used to deposit the film. After deposition, the electrode was cured overnight at 25°C and then further cured for two days at 56°C in a dried vial.

[0267] A second spotting solution was prepared with the formulation shown in Table 5. All components were dissolved in 10 mM HEPES buffer, pH 8. Cross-linking was accomplished with polyethylene glycol diglycidyl ether.

[0268] Table 5

[0269]

[0270] 15 nL of the second spotting solution was deposited from above on the PVP membrane as a single spot (GOX spot) with an area of ​​about 0.05 mm2. After deposition, it was cured at 25°C overnight.

[0271] After curing, a second film is deposited on the GOX points and the PVP film. The film polymer in this case is a cross-linked polyvinylpyridine-styrene copolymer, in which a portion of the pyridine nitrogen atoms are functionalized with non-cross-linked poly(ethylene glycol) tails and a portion of the pyridine nitrogen atoms are functionalized with alkyl sulfonic acid groups. The film at this location is deposited from a coating solution containing 35mg / ml_cross-linked polyvinylpyridine-to-styrene polymer and 100mg / ml_PEGDE400. Film deposition is completed by dipping the electrode three times in the coating solution. Spraying, screen printing or similar processes can be used to deposit the film alternatively. After deposition, the electrode is cured overnight at 25°C and then further cured for two days at 56°C in a dried vial.

[0272] Ethanol analysis was performed by immersing the electrode in ethanol-containing PBS solutions, where each PBS solution contained different concentrations of ethanol. Fig. 12A Two replicates of the response of an electrode containing glucose oxidase and xanthine oxidase, layered in separate active regions and separated by a membrane, with catalase in the active region with glucose oxidase, are shown when exposed to different concentrations of ethanol. As shown, after exposure to a new ethanol concentration, the current response increases over the course of several minutes before stabilizing thereafter. Good reproducibility of the two replicates was observed. Fig.13 An illustrative plot of the average current response versus ethanol concentration is shown. The curve shape is similar to that obtained using AOX / XOX ( Fig.11A , Example 2A).

[0273] Fig. 12B Shown is comparative response data between electrodes containing glucose oxidase and xanthine oxidase layered in separate active regions and separated by a membrane, with catalase present separately in the active region, upon exposure to varying concentrations of ethanol. As shown, the sensor response was greater when catalase was included in the active region containing xanthine oxidase.

[0274] Example 3: Comparative response of analyte sensors to lactate in the presence of various mass transfer limiting membranes. For this example, the following membrane formulation was coated onto a carbon working electrode containing lactate oxidase in its active region. The active region was deposited using the lactate oxidase formulation described in Example 1, except that the polymer of Formula 1 was substituted for the polymer of Formula 2 in the formulation, and the concentration was adjusted to the concentration specified in Table 6 below.

[0275] Table 6

[0276]

[0277] Active area deposition and curing were performed as described in Example 1, except that six 0.01 mm 2 area of ​​the point, instead of 0.1 mm in Example 1 2 A single point of area. Unless otherwise stated below, film deposition was performed by dip coating (1-5 electrode dips with a waiting time of about 10 minutes between dips). After dip coating was completed, the film was cured at 25°C for 24 hours and then at 56°C for 48 hours in a dry vial.

[0278] The electrode response is measured by placing the active area of ​​the electrode in a beaker containing 100 mM pH = 7.5 phosphate buffered saline at 37°C. The potential is raised to +40 mV relative to Ag / AgCl, and the current is continuously monitored thereafter. To determine the response at different lactate concentrations, sodium lactate is added to the buffer solution in increments of 1 mM to 5 mM. To determine the stability of the response, the current is measured in 5 mM sodium lactate over an extended period of time, such as two weeks.

[0279] Membrane polymers 1A and 1B: The first test membrane polymer was a cross-linked polyvinylpyridine-co-styrene polymer in which a portion of the pyridine nitrogen atoms were functionalized with non-cross-linked poly(ethylene glycol) tails and a portion of the pyridine nitrogen atoms were functionalized with alkyl sulfonic acid groups. Two different cross-linking agents were used to effect cross-linking of the membrane polymer: glycerol triglycidyl ether (Gly3 - Formulation 1) and polyethylene glycol diglycidyl ether 400 (PEGDGE400 - Formulation 2). Formulation 1 contained 4 ml of membrane polymer in 80:20 ethanol HEPES buffer (140 mg / ml), 1 ml of Gly3 in 80:20 ethanol:HEPES buffer (35 mg / ml) and 0.0132 ml of aminopropyl terminated polydimethylsiloxane (PDMS) in ethanol solution (100 mg / ml). Formulation 2 contains 4 ml of _ membrane polymer in 80:20 ethanol: HEPES buffer (140 mg / mL), 0.2 mL of PEGDGE400 in 80:20 ethanol: HEPES buffer (100 mg / mL) and 0.0132 mL of aminopropyl-terminated polydimethylsiloxane (PDMS) in ethanol solution (100 mg / mL). The corresponding cross-linked polymers are referred to as polymers 1A and 1B, respectively, herein.

[0280] Fig.14 Shown are exemplary curves of the response of electrodes coated with polymers 1A and 1B to 5 mM lactate solutions. As shown, neither formulation provides a stable sensor response over time. The sensor current provided by polymer 1A (formulation 1) slowly decreases over a two-week measurement period, while the sensor current provided by polymer 1B (formulation 2) initially increases and then decreases within the first week of lactate exposure. In contrast, the two membranes provide a stable response in the presence of glucose analytes (data not shown).

[0281] Membrane polymer 2: The second membrane polymer tested was polyvinylpyridine (PVP) cross-linked with polyethylene glycol diglycidyl ether 1000 (PEGDGE1000). This membrane polymer is referred to herein as polymer 2. The membrane preparation (formulation 3) contained 4.3 ml of PVP in 80:20 ethanol HEPES buffer (100 mg / ml), 0.25 ml of PEGDGE1000 in 80:20 ethanol: HEPES buffer (200 mg / ml), and 0.0132 ml of PDMS in ethanol solution (100 mg / ml).

[0282] Fig.15An exemplary curve is shown for the response of an electrode coated with polymer 2 (formulation 3) to a 5 mM lactate solution. Similar to polymers 1A and 1B, polymer 2 also fails to provide a stable current response over time. There is a large drop in response over the first 48 hours, followed by relatively stable performance. In addition, the sensitivity is well below the target value of about 1 nA / mM. Similar to polymers 1A and 1B, polymer 2 provides a stable current response in the presence of glucose analyte (data not shown).

[0283] Membrane polymer 3: The third membrane polymer tested was polyvinylpyridine (PVP) cross-linked with PEGDGE 400. This membrane polymer is referred to herein as polymer 3. The membrane preparation (Formulation 4) contained 4.3 mL of PVP in 80:20 ethanol:HEPES buffer (100 mg / mL), 0.23 mL of PEGDGE 400 in 80:20 ethanol:HEPES buffer (100 mg / mL), and 0.0132 mL of PDMS in ethanol solution (100 mg / mL).

[0284] Fig.16 An exemplary curve is shown for the response of an electrode coated with polymer 3 (formulation 4) to a 5 mM lactate solution. Unlike polymer 2 cross-linked with a higher molecular weight variant of the same cross-linker, polymer 3 surprisingly provides a stable current response over time. Furthermore, when increasing amounts of lactate are added in increments of 1 mM, the current responds rapidly and reaches a stable current ( Fig.17 ).

[0285] Membrane polymer 4 : The fourth membrane polymer tested was PVP containing 3-4wt% non-crosslinked PEG side chains, which were then crosslinked with PEGDGE1000. Thus, the membrane polymer tested contained non-crosslinked PEG chains and crosslinked PEG1000 chains. This membrane polymer is referred to herein as polymer 4. The membrane preparation (preparation 5) contained 4.3 ml of polymer in 80:20 ethanol HEPES buffer (100 mg / ml), 0.025 ml of PEGDGE1000 in 80:20 ethanol: HEPES buffer (200 mg / ml), and 0.0132 ml of PDMS in ethanol solution (100 mg / ml).

[0286] Fig.18 An exemplary curve of the response of an electrode coated with polymer 4 (formulation 5) to a 5 mM lactate solution is shown. Unlike polymers 2 and 3, polymer 4 surprisingly provides a stable current response over time. In addition, when increasing amounts of lactate are added in increments of 1 mM, the current responds rapidly and reaches a stable value ( Fig.19 ).

[0287] Bilayer film comprising polymer 2 and 1B or polymer 2 and 1A : Formulation 3 (polymer 2) is applied to the electrode surface by repeated dip-coating operations. Spray coating, screen printing or similar processes can alternatively be used to deposit the film. Formulation 2 (polymer 1B) is then applied to the deposited cross-linked PVP layer by repeated dip-coating operations. There is a 10 minute waiting time between consecutive dips. After all dip-coating operations are completed, the sensor is cured at 25°C for 24 hours and then cured at 56°C for 48 hours in a dried vial. As shown above, none of these membrane polymers provide satisfactory performance when used alone.

[0288] Fig. 20 An exemplary curve is shown for the response of an electrode coated with a bilayer membrane comprising a lower layer of cross-linked PVP (polymer 2) and an upper layer of cross-linked polymer 1 B to a 5 mM lactate solution. Unlike polymer 1 B or PVP (polymer 2) cross-linked with the same cross-linker, bilayer membranes comprising these membrane polymers surprisingly provide a stable current response over time at an acceptable level of sensitivity, even though neither polymer alone provides acceptable performance. Fig. 20 The response data in are for electrodes dipped twice in Formulation 3 (Polymer 2) and four times in Formulation 2 (Polymer 1B).

[0289] The amount (thickness) of each membrane polymer in a bilayer can alter sensor performance, as shown below for polymer 2 and polymer 1 A. Thus, a Gly3 cross-linked variant of polymer 1B (i.e., polymer 1A) can similarly provide acceptable performance when combined with polymer 2 into a bilayer, even though neither polymer alone provides acceptable performance.

[0290] Fig.21 An illustrative curve is shown of the response of an electrode coated with a bilayer membrane comprising a lower layer of cross-linked PVP (polymer 2) and an upper layer of cross-linked polymer 1A to a 5 mM lactate solution, where the electrode was dip-coated with Formulation 1 (polymer 1A) and Formulation 3 (polymer 2) for different times. In this case, the cross-linker for PVP (polymer 2) remained PEGDGE1000, but the cross-linker for polymer 1A was Gly3, suggesting that this cross-linker may also be suitable for bilayer membrane configurations. Fig.21 As shown, electrodes dipped twice in Formulation 3 and four times in Formulation 1 provided a good balance of sensitivity and stable current response. Changing the number of dip-coating operations changed the thickness of each component of the bilayer membrane, as well as the mass ratio of the membrane polymers to each other. Fig.21 As shown, if the PVP layer is too thin (0 or 1 polymer 2 dip coating), the sensitivity is high but the response stability is poor, while if it is too thick (3 or more dip coating), the electrode exhibits low sensitivity and poor response stability in some cases.

[0291] Mixed membranes containing membrane polymers 1B and 3: A combined membrane formulation (Formulation 6) was prepared by mixing 1.5 ml of PVP in 80:20 ethanol HEPES buffer (100 mg / ml), 2.5 ml of the copolymer used to prepare Formulations 1A and 1B in 80:20 ethanol: HEPES buffer (140 mg / ml), 0.175 ml of PEGDGE400 in 80:20 ethanol: HEPES buffer (100 mg / ml), and 0.0132 ml of PDMS in ethanol solution (100 mg / ml). Thus, after cross-linking, Formulation 6 contained polymer 1B and polymer 3, each of which was cross-linked with PEG400.

[0292] Fig. 22 An exemplary curve showing the response of an electrode coated with a hybrid film comprising cross-linked PVP (polymer 3) and cross-linked polymer 1B to a 5 mM lactate solution is shown. As with the bilayer film containing one of the same components (polymer 1B), the hybrid film provided a stable current response over time and an acceptable level of sensitivity. In addition, when increasing amounts of lactate were added in 1 mM increments, the current responded rapidly and reached a stable value ( Fig.23 ).

[0293] Fig.24 Exemplary curves of the response of sensors coated with mixed films containing various ratios of cross-linked PVP (Polymer 3) and cross-linked Polymer 1B are shown. Fig.24 As shown, a higher amount of polymer 1B increases the sensitivity but provides poorer response stability.

[0294] Example 4: Performance of a sensor comprising two working electrodes coated with a double layer of mass transfer limiting membrane. For this example, a first working electrode containing glucose oxidase and a second working electrode containing lactate oxidase were coated with a double layer of membrane. The active region containing glucose oxidase was deposited using the glucose oxidase preparation as described in Example 1 (Table 1). The active region containing lactate oxidase was deposited using the lactate oxidase preparation described in Example 3 (Table 6). The active region was deposited and cured as described in Example 1, except that each having a 0.01 mm 2 The five points of the area of ​​​​the example 1 have 0.1mm 2A single point of an area. In this example, a membrane polymer formulation corresponding to formulation 2 (polymer 1B) and formulation 4 (polymer 3) of Example 3 was used to deposit a double-layer membrane. That is, polymer 3 was deposited on a second working electrode having lactate oxidase characteristics. Selective deposition was achieved on the second working electrode by an improved coating process. Then a 24-hour cure was performed at 25°C. Then, the entire assembly (i.e., the two working electrodes, the PVP coating on the second working electrode, and the counter electrode and the reference electrode) was dip-coated with formulation 2. Cured again at 25°C for 24 hours, and then baked at 56°C in a dry environment for 48 hours. Therefore, a homogeneous film was deposited on the first working electrode (glucose response) and a double-layer membrane was deposited on the second working electrode (lactate response). The cross-linked PVP (polymer 3) was in contact with the lactate response active area on the second working electrode.

[0295] The sensor was used to simultaneously measure glucose and lactate in 100 mM PBS at 37° C. In the first experiment, the sensor was exposed to a 100 mM PBS solution containing 30 mM glucose and 5 mM lactate for 2 weeks at 37° C. For this test, the sensor was held at +40 mV vs. Ag / AgCl. Fig.25 An exemplary graph of the sensor response of each working electrode after exposure to 30 mM glucose and 5 mM lactate is shown. As shown, the sensor response remained very stable during the observation period.

[0296] Next, glucose and lactate were added incrementally in 100 mM PBS at 37°C to determine the sensor's responsiveness to each analyte. For this test, the sensor was again held at 40 mV vs. Ag / AgCl+. Glucose was added to a concentration range of 0-30 mM and lactate was added to a concentration range of 0-5 mM. Fig.26 Figure 2 shows exemplary curves of the sensor's response to different concentrations of glucose and lactate. Fig.26 As shown, the sensor responded quickly to both analytes and remained stable at a given analyte concentration.

[0297] Example 5: Detection of Ketones Using an Analyte Sensor with Synergistic Interaction of Diaphorase and β-Hydroxybutyrate Dehydrogenase. For this example, the film formulation shown in Table 7 below was coated onto a carbon working electrode. Deposition was performed to place six spots on the working electrode, each with approximately 0.01 mm 2After deposition, the working electrode was cured overnight at 25°C. Thereafter, the PVP film was applied to the working electrode by dip coating using a coating solution prepared from 4 ml_100 mg / ml_PVP, 0.2 ml_100 mg / ml_PEGDGE400 and 0.0132 ml_100 mg / ml_PDMS. Film curing was performed at 25°C for 24 hours and then at 56°C for 48 hours in a dried vial.

[0298] Table 7

[0299]

[0300] Ketone analysis was performed by immersing the electrode in 100 mM PBS buffer (pH = 7.4) at 33°C and introducing various amounts of β-hydroxybutyrate (0, 1, 2, 3, 4, 6 and 8 mM total β-hydroxybutyrate addition). Fig. 27 Shown are the responses of four replicates of an electrode containing diaphorase, NAD+, and β-hydroxybutyrate dehydrogenase when exposed to varying concentrations of β-hydroxybutyrate. As shown, after exposure to a new β-hydroxybutyrate concentration, the current response increases over the course of several minutes before stabilizing thereafter. Fig.28 Shows Fig. 27 The ketone sensor also exhibits a stable response over an extended measurement time, as shown in Figure 2. Fig.29 shown. Fig.29 Shows when Fig. 27 Example curve of the current response of an electrode exposed to 8 mM β-hydroxybutyrate in 100 mM PBS for 2 weeks at 33° C. The average signal loss during the measurement period was only 3.1%.

[0301] Example 6: Comparison of lactate sensor response for various sensor configurations. Two different lactate oxidase / polymer formulations for active region deposition and two different membrane polymer formulations for mass transfer limiting membrane deposition were prepared to determine the performance of lactate responsive sensors characterized by various variations of these formulations. Formulation details and methods used to prepare analyte sensors are provided below. In general, analyte sensors were prepared in a manner similar to that described above.

[0302] Table 8

[0303]

[0304] Table 9

[0305]

[0306]

[0307] To deposit each active area, approximately 20 nL of each solution was deposited on the carbon working electrode to form 6 discrete spots, each with a diameter of approximately 0.01 mm. 2 Formulation A was dispensed 4 times and formulation B was dispensed 6 times to form the dots. After deposition, the working electrode was cured overnight at 25°C. Formulation A corresponds to the formulation used to deposit the active area of ​​the glucose-responsive analyte sensor, except that lactate oxidase was used instead of glucose oxidase.

[0308] Formulations for mass transfer limited membrane deposition : Film polymer formulations were prepared in the aqueous solution formulations specified in Tables 10 and 11 below.

[0309] Table 10

[0310]

[0311] Table 11

[0312]

[0313] The mass transfer limiting membrane was deposited on each active area prepared as above using dip coating. Formulation C was deposited using 4 dip coatings and Formulation D was deposited using 4 dip coatings. A waiting time of about 10 minutes was used between two dip coatings. After the dip coating was completed, the film was cured at 25°C for 24 hours in a dry vial and then at 56°C for 48 hours. Spray coating, screen printing or similar processes can be used to deposit the mass transfer limiting membrane instead. Formulation C corresponds to the formulation used to deposit the mass transfer limiting membrane in the glucose responsive analyte sensor.

[0314] Lactic acid responsive analyte sensors were prepared using the deposition conditions described above. All possible combinations of active area and mass transfer limiting membrane were prepared, with 8 sensors made for each possible combination. After fabrication, each sensor was exposed to a 5 mM lactate solution in 100 mM phosphate buffered saline (PBS) at 37°C for 190 hours, with the operating potential maintained at +40 mV vs. Ag / AgCl. The tested combinations of active area and mass transfer limiting membrane are detailed in Table 12. The test results are given in Table 13. Fig.30 Shown in.

[0315] Table 12

[0316]

[0317] like Fig.30As shown, lactic acid response analyte sensors with active areas and mass transfer limiting membranes are similarly formulated to those successfully used for glucose response analyte sensors (group 1) and provide poor performance when exposed to lactic acid. As shown, the signal intensity of all test samples is less than 0.5nA, which is an undesirable low value for feasible lactic acid response sensors. When polyvinylpyridine and different cross-linking agents replace polyvinylpyridine-to-styrene and Gly3 cross-linking agents in preparation C (group 2), the signal intensity is even worse.

[0318] like Fig.30 As further shown, the incorporation of human serum albumin significantly improved sensor performance. For example, sample set 3 showed much higher signal intensities than achieved in either of the samples in set 1 or set 2. However, there was considerable variation in the initial signal intensity (>4 nA variation) in this set of samples. In addition, the signal intensity steadily decreased from the maximum signal intensity initially observed. The response variability and poor signal stability over time also make this sample set combination unlikely to be suitable for a viable lactate-responsive analyte sensor.

[0319] Surprisingly, the combination of an active region containing human serum albumin and a mass transfer limiting membrane comprising a cross-linked polyvinylpyridine homopolymer (Group 4) yielded an acceptable combination of high signal intensity and signal stability over time. Fig.30 As shown, all replicate sensors of Group 4 had initial signal intensities between 4 nA and 5 nA clustered within 1 nA of each other. This level of signal intensity and variability is within the range that a commercially viable lactate-responsive analyte sensor can be developed. Furthermore, over 190 hours of signal observation, the signal intensity varied by only a few tenths of a nA or less, which is also within a range suitable for developing a commercially viable sensor.

[0320] like Fig.31 As shown, when increasing amounts of lactate were added in 1 mM increments to the initially lactate-free PBS solution, the current response observed for the sensors of Group 4 was rapid and reached a stable value.

[0321] Unless otherwise indicated, all numbers indicating quantities and the like in this specification and the associated claims should be understood as being modified in all cases by the term "about". Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that may vary depending on the desired properties sought to be obtained by embodiments of the present invention. At the very least, and not attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0322] Analyte Sensor Ignition Lock

[0323] Vehicle failsafe devices, such as ignition locks, are sometimes used to prevent an operator from operating a vehicle while impaired or not in a safe position to operate the vehicle. Operating a vehicle while impaired could potentially pose a significant risk to the operator and the public. One common type of ignition lock is designed to prevent drunk driving, and more specifically, to prevent an individual from operating a vehicle while intoxicated with alcohol. Such a lock device connects a breathalyzer or optical sensor to the vehicle's ignition system, and the driver must successfully pass a blood alcohol level test before the vehicle can be started.

[0324] Intoxication is a type of impairment or condition that an operator may experience that renders the operator unfit or unable to operate a vehicle. However, other injuries and conditions may also afflict the operator and should also be closely monitored to ensure that the operator does not operate the vehicle while impaired. For example, an operator with diabetes and driving when hypoglycemia (i.e., low blood sugar) may potentially experience dizziness, confusion, headache, loss of consciousness, seizures, and delayed reflexes, any of which may endanger his / her own life and those in or near the vehicle.

[0325] Analyte monitoring systems have been developed to facilitate long-term monitoring of analytes in body fluids (e.g., blood). Some analyte monitoring systems are intended to detect and monitor blood glucose levels, which can aid in the treatment of diabetes. However, other analyte monitoring systems are intended to detect and monitor other analytes present in an operator's body fluids, and abnormal analyte levels detected in an operator's body may indicate that the operator is currently unfit to safely operate a vehicle.

[0326] The following discussion describes an analyte monitoring and vehicle control system for preventing operation of a vehicle when an operator analyte level exceeds a predetermined threshold. Proper deployment of the sensor control device 102 ( Figure 1 ) allows the user to intelligently track and monitor bodily fluid analyte levels and trends. When some analyte levels exceed certain thresholds, physical or cognitive impairments may occur, making the user unfit to safely operate the vehicle. In this case, the user should take appropriate measures to return the analyte level to a safe range before attempting to operate the vehicle. However, in some cases, the user may feel well operating the vehicle, but there may still be unsafe analyte levels that may suddenly trigger the onset of dangerous physical injury. In this case, it may be advantageous to install a fail-safe system that prevents the user from operating the vehicle and potentially putting themselves and / or others in danger or warns the user not to operate the vehicle and potentially putting themselves and / or others in danger.

[0327] Fig.323200) includes a sensor control device 102 that can be deployed on a user or "operator" 3202 and otherwise delivered to a target monitoring location on the operator's 3202 body (such as the back of the arm). As discussed above, the sensor control device 102 includes a sensor 104 ( Figure 1 ), and when properly deployed, the sensor 104 is transcutaneously positioned within the skin to detect and monitor analytes present in the bodily fluids of the operator 3202. An adhesive patch 105 ( Figure 1 ) adheres to the skin to secure the sensor control device 102 in place during operation.

[0328] Although the system 3200 is described herein as including an on-body sensor control device 102 to detect and report analyte levels, the system 3200 may alternatively incorporate an off-body analyte sensor (e.g., a self-monitoring blood glucose "SMBG" meter) without departing from the scope of the present disclosure. Thus, the term "sensor control device" should be interpreted herein to include not only on-body sensor systems as described above, but also conventional handheld sensor systems.

[0329] As shown, the system 3200 may also include a reader device 120, and the sensor control device 102 may communicate with the reader device 120 via a local communication path or link to automatically, periodically, or according to the expectations of the operator 3202 to provide analyte concentration data. The reader device 120 may communicate with a control module 3204, which communicates with the electrical system of the vehicle 3206 and is powered by the vehicle battery or otherwise by a separate battery. In such embodiments, data transmitted from the sensor control device 102 to the reader device 120 may then be transmitted by the reader device 120 to the control module 3204 for processing. However, in other embodiments, the sensor control device 102 may communicate with the reader device 120 via any wireless communication protocol (such as ) communicates directly with the control module 3204. In such embodiments, the reader device 120 may or may not be required in the system 3200.

[0330] In the illustrated embodiment, vehicle 3206 is depicted as an automobile. However, as used herein, the term "vehicle" is used broadly and is intended to include any kind of transportation vehicle that can be operated by a human user or "operator," but may also include autonomous vehicles used to transport humans. Examples of vehicle 3206 include, but are not limited to, any type of automobile, truck, sport utility vehicle, airplane, boat, spacecraft, and / or any other vehicle, or combinations thereof.

[0331] The control module 3204 may include a communication interface to transmit information to / from the sensor control device 102 and / or the reader device 120. In the case of the sensor control device 102 and / or the reader device 120 of the operator 3202, the pairing mode may be entered when the sensor control device 102 approaches the vehicle 3206. After pairing, the control module 3204 may be programmed and configured to automatically detect the presence of the sensor control device 102 and / or the reader device 120 and establish communication therewith. For example, when the operator 3202 approaches or enters the vehicle 3206, the control module 3204 may automatically detect the presence of the sensor control device 102 and enable communication therebetween or with the reader device 120.

[0332] In some embodiments, the control module 3204 may communicate with a vehicle user interface 3208 (such as an infotainment system, a touch screen display, or an information display) included in the vehicle 3206. In such embodiments, the control module 3204 may visually communicate with the operator 3202 via the vehicle user interface 3208, and may also be able to audibly communicate with the operator 3202 via audio speakers included in the vehicle 3206. However, in other embodiments, the control module 3204 may be configured to communicate with the reader device 120 to be able to communicate with the operator 3202.

[0333] As shown, the control module 3204 may be or otherwise include a computer system 3210 configured and programmed to control various operations and / or systems of the vehicle 3206 based on the analyte level of the operator 3202 measured in real time obtained by the sensor control device 102. Or control, disable or modify the operation of the vehicle 3206 by disabling one or more critical systems of the vehicle 3206 or by activating a warning system in the vehicle 3206. When the analyte level measured in real time by the operator 3202 is within a predetermined safety range, it can be considered safe for the operator 3202 to operate the vehicle 3206. However, when the analyte level of the operator 3202 measured in real time falls outside the predetermined safety range or exceeds a predetermined threshold, the computer system 3210 can then be programmed to control, disable or modify the operation of the vehicle 3206.

[0334] In some embodiments, for example, the computer system 3210 can be configured to disable various key vehicle systems when the detected analyte level of the operator 3202 falls outside a predetermined range or otherwise exceeds a predetermined threshold, thereby gradually and safely disabling the operation of the vehicle when the operator 3202 is identified as being impaired for the safe operation of the vehicle 3206. The key vehicle systems of the vehicle 3206 that may be disabled include an ignition system (e.g., an energy switching / control system), a transmission system (or a gearbox), a fuel system, an energy supply system (e.g., a battery, a capacitor, a conversion / reaction cell, etc.). When an increased or decreased (unsafe) analyte level is detected, the computer system 3210 can prevent key vehicle systems from functioning or operating. Therefore, the operator 3202 will not be able to start or operate the vehicle 3206, thereby preventing the operator 3202 from putting himself and / or others in danger.

[0335] In other embodiments, or in addition thereto, the computer system 3210 may be configured to activate various non-critical vehicle systems when the detected analyte level of the operator 3202 exceeds or crosses a predetermined threshold. Non-critical vehicle systems that may be activated include, for example, a vehicle horn, vehicle lights, or an audible warning system installed in the vehicle 3206. In such embodiments, activation of non-critical vehicle systems may alert law enforcement and others (e.g., operators of adjacent vehicles, bystanders, pedestrians, etc.) that the operator 3202 may be driving under the influence, thereby allowing law enforcement to quickly resolve any problems associated therewith and alerting others to a potentially dangerous situation.

[0336] In still other embodiments, or in addition thereto, the computer system 3210 can be configured to automatically place a call to one or more emergency contacts when the operator's analyte level falls outside of a predetermined safe operating range or otherwise exceeds a predetermined threshold. In such embodiments, the computer system 3210 can communicate with the operator 3202 through a reader device 120 (e.g., a cellular phone) or a cellular or satellite communication system incorporated into the vehicle 3206 (e.g., ) to operate. In other embodiments, or in addition thereto, the computer system 3210 can be configured to automatically send a message (e.g., a text or SMS message, an email, etc.) to an emergency contact when the analyte level of the operator 3202 falls outside a predetermined safe operating range or otherwise exceeds a predetermined threshold. Example emergency contacts include, but are not limited to, a spouse, a parent, a medical professional (e.g., a doctor), a hospital, 911, or any combination thereof.

[0337] In some embodiments, the system 3200 may also include one or more proximity sensors 3212 configured to detect the presence of the operator 3202 and, more specifically, the sensor control device 102. In such embodiments, the proximity sensor(s) 3212 may be configured to monitor the general area of ​​the driver's seat 3214 within the vehicle 3206. If the proximity sensor(s) 3212 detects the sensor control device 102 within the area of ​​the driver's seat 3214, this may provide a positive indication that the operator 3202 is in the driver's seat 3214 and may be attempting to operate the vehicle 3206. In such cases, a signal may be sent to the control module 3204, alerting the computer system 3210 that the operator 3202 is in the vehicle 3206 and may be attempting to operate the vehicle 3206. If the real-time measured analyte level of the operator 3202 is within a predetermined safety range or below a predetermined level, the computer system 3210 may allow the operator 3202 to operate the vehicle 3206. However, when the real-time measured analyte level of the operator 3202 falls outside of a predetermined safety range or exceeds a predetermined threshold, the computer system 3210 can control, disable, or modify the operation of the vehicle 3206, as generally described above. As will be appreciated, the proximity sensor(s) 3212 can be advantageous in preventing operation of the vehicle 3206 only when the impaired operator 3202 is in the driver's seat 3214 and is ready to operate the vehicle 3206. Thus, a user wearing the sensor control device 102 is able to ride in the vehicle 3206 as a passenger in any state without affecting the operation of the control module 3204 or the vehicle 3206.

[0338] In some embodiments, the control module 3204 may also include a vehicle state detection module 3216, which is configured to detect the current state of the vehicle 3206, including whether the vehicle 3206 is currently moving or stationary. In addition, the vehicle state detection module 3232 may be configured to determine whether the motor in the vehicle 3206 is currently operating or stopped. In one or more embodiments, the vehicle state detection module 3216 may provide a state signal to the control module 3204, and then the control module 3204 may use the state signal to determine which vehicle operations should be activated or disabled when the analyte level of the operator 3202 measured in real time falls outside a predetermined safety range or exceeds a predetermined threshold. For example, when the state signal indicates that the vehicle 3206 is stationary, the control module 3204 may disable the vehicle fuel system, transmission system, ignition system, or any combination thereof. On the contrary, when the state signal indicates that the vehicle 3206 is moving, the control module 3204 may activate the vehicle horn, flash the vehicle lights, or activate a sound warning to the operator 3202 and / or the people around the operator 3202 to inform the operator 3202 that it is damaged.

[0339] In some embodiments, once the operator 3202 enters the vehicle 3206 or when the control module 3204 is paired with the sensor control device 102 and / or the reader device 120, the application can be launched on the reader device 120 or the vehicle user interface 3208, and a digital dashboard can appear on the reader device 120 and / or the vehicle user interface 3208, which depicts the current analyte level, trend, historical data, and expected analyte level. However, if the current analyte level falls outside the predetermined safe operating range, the computer system 3210 can be programmed to disable one or more key vehicle systems to prevent the operator 3202 from operating the vehicle 3206. In such embodiments, a visual or audible alarm can be issued by the control module 3204 to notify the operator 3202 why the vehicle 3206 is not started. More specifically, a visual alarm (e.g., a written message) can be generated and displayed on the reader device 120 or the vehicle user interface 3208, or an audible alarm (e.g., a voice message) can be transmitted through a speaker in the reader device 120 or the vehicle 3206.

[0340] If not done automatically, the operator 3202 may be prompted to obtain the current analyte level when pairing the sensor control device 102 with the control module 3204. In some cases, the vehicle 3206 may be prevented from operating until the current analyte level is obtained. If the current analyte level is within safe limits, the computer system 3210 may allow operation of the vehicle 3206. In some aspects, unless done automatically, the control module 3204 may prompt the operator 3202 to obtain additional current analyte levels after operating the vehicle 3206 for a predetermined period of time (e.g., after 1 hour, 2 hours, 5 hours, etc.).

[0341] In some embodiments, the control module 3204 may be configured to issue visual or auditory suggestions or instructions to the operator 3202, which may help bring the measured analyte level back into a safe range. In such embodiments, such visual or auditory recommendations may prompt the user to take actions that may result in bringing the analyte level back into a safe range. Moreover, in some embodiments, the operator 3202 may be able to verbally communicate with the control module 3204 by issuing verbal responses or commands. This may prove advantageous in helping to prevent distracted operation of the vehicle 3206.

[0342] In some embodiments, the settings of the control module 3204 may be customized by the operator 3202 to allow the user to make an informed decision when an unsafe analyte level is detected and the control module 3204 has sounded a visual or audible alarm. More specifically, in at least one embodiment, the control module 3204 may include a bypass feature that the operator 3202 may enable to allow the operator 3202 to operate the vehicle 3206 even though an unsafe analyte level has been measured. In such embodiments, the operator 3202 may operate the vehicle 3206 by acknowledging that the operator 3202 may be operating the vehicle 3206 in an impaired or unsafe state of health.

[0343] In some embodiments, when the analyte level of the operator 3202 may deviate from a predetermined safe range or otherwise exceed a predetermined threshold, the computer system 3210 may be configured or otherwise programmed to calculate a predicted timeline. In such embodiments, the control module 3204 may be configured to issue a visual or audible alarm to the operator 3202, indicating how much time the operator 3202 has before reaching an unsafe analyte level and a potential unsafe medical condition may occur. Multiple alarms may be provided to indicate when the operator has a specific time increment remaining before reaching an unsafe analyte level. For example, when an unsafe analyte level is reached within an hour, within half an hour, within 10 minutes, within 5 minutes, within 1 minute, and within any time increment therebetween, a visual or audible alarm may be issued. In addition, once the operator's analyte level reaches an unsafe level or exceeds a predetermined threshold, a visual or audible alarm may be issued.

[0344] In some embodiments, if an unsafe analyte level is measured when the operator 3202 is operating the vehicle 3206, the control module 3204 can be configured to send one or more alarms (visual or auditory) to warn the operator 3202 of an unsafe analyte level. In some cases, the stereo volume in the vehicle 3206 can be automatically reduced so that the operator 3202 can hear the sound alarm. In such embodiments, the control module 3204 can be configured to suggest one or more corrective actions to the operator 3202. Example corrective actions include but are not limited to slowing down and stopping the vehicle 3206, locating and driving to a nearby convenience store or pharmacy, and locating a nearby hospital or medical institution. If the vehicle 3206 is an autonomous vehicle, and the current analyte level places the operator 3202 under potentially dangerous conditions, the control module 3204 can automatically guide the vehicle 3206 to a medical institution for treatment. Alternatively or in addition, when an unsafe analyte level is detected, the control module 3204 can gradually reduce or limit the speed of the vehicle 3206, thereby forcing the operator 3202 to stop and resolve the problem before continuing to operate the vehicle 3206.

[0345] System 3200 can be used in several different scenarios to protect operator 3202 and / or people around operator 3202 while driving. In some applications, system 3200 can be voluntarily incorporated by the operator to detect impairment in real time. In other applications, the owner of vehicle 3206 may require system 3200 to detect impairment of operator 3202. In such applications, the owner of vehicle 3206 may be a transportation or trucking company. In still other applications, system 3200 may be legally imposed on operator 3202 to detect impairment.

[0346] Embodiments disclosed herein include:

[0347] K. An analyte monitoring and vehicle control system comprising a sensor control device having a sensor for detecting and monitoring one or more analytes present in an operator, and a control module in communication with the sensor control device and an electrical system of a vehicle, the control module including a computer system programmed to receive and process data provided by the sensor control device, wherein the computer system controls or disables operation of the vehicle when the real-time measured analyte level of the operator exceeds a predetermined safety threshold.

[0348] L. A method comprising using a sensor control device having a sensor to detect and monitor one or more analytes present in an operator's body, using a control module that communicates with the sensor control device and an electrical system of a vehicle to receive and process data provided by the sensor control device; and controlling or disabling operation of the vehicle through a computer system of the control module when the real-time measured analyte level of the operator exceeds a predetermined safety threshold.

[0349] Each of embodiments K and L may have one or more of the following additional elements in any combination: Element 1: wherein the sensor control device is coupled to the operator and the sensor is transcutaneously positioned under the skin of the operator to detect and monitor analytes present in the operator's bodily fluids. Element 2: wherein the sensor control device includes an in vitro analyte sensor. Element 3: further comprising a reader device that receives data from the sensor control device and transmits the data to the control module. Element 4: wherein the vehicle includes a transportation vehicle selected from a car, an autonomous vehicle, a truck, a sport utility vehicle, an airplane, a boat, a spacecraft, or any combination thereof. Element 5: wherein the sensor control device is paired with the control module to communicate when the operator approaches the vehicle. Element 6: further comprising a vehicle user interface included in the vehicle and in communication with the control module. Element 7: wherein the operation of the vehicle is disabled by disabling one or more critical systems of the vehicle, the critical system being selected from the group consisting of an ignition system, a transmission system, a fuel system, and an energy supply system. Element 8: wherein the operation of the vehicle is controlled by at least one of activating one or more non-critical systems of the vehicle, calling or sending a message to one or more emergency contacts, and gradually reducing the speed of the vehicle. Element 9: Also included is one or more proximity sensors mounted on the vehicle to monitor the area of ​​the driver's seat of the vehicle and detect the presence of an operator. Element 10: Wherein the control module also includes a vehicle state detection module that detects the current state of the vehicle. Element 11: Wherein when the real-time measured analyte level of the operator falls outside a predetermined safety threshold, the control module generates a visual or audible alarm that can be perceived by the operator. Element 12: Wherein the visual or audible alarm is generated at specific time increments before an unsafe analyte level is reached. Element 13: Wherein the visual or audible alarm includes one or more recommended corrective actions communicated to the operator. Element 14: Wherein the control module includes a bypass feature that allows the operator to operate the vehicle when the real-time measured analyte level of the operator exceeds a predetermined threshold.

[0350] Element 15: Also includes receiving data from a sensor control device and transmitting the data to the control module using a reader device that communicates with the sensor control device and the control module. Element 16: Wherein disabling the operation of the vehicle includes disabling one or more critical systems of the vehicle, the critical system being selected from the group consisting of an ignition system, a transmission system, a fuel system, and an energy supply system. Element 17: Wherein controlling the operation of the vehicle includes at least one of activating one or more non-critical systems of the vehicle, calling or sending a message to one or more emergency contacts, and gradually reducing the speed of the vehicle. Element 18: Also includes monitoring the area of ​​the driver's seat of the vehicle and detecting the presence of an operator using one or more proximity sensors installed on the vehicle. Element 19: Also includes detecting the current state of the vehicle using a vehicle status detection module included in the control module. Element 20: Also includes generating a visual or audible alarm that can be perceived by the operator through the control module when the real-time measured analyte level of the operator exceeds a predetermined threshold.

[0351] One or more illustrative embodiments incorporating various features are presented herein. For clarity, not all features of physical implementations are described or shown in this application. It will be appreciated that in the development of physical implementations in conjunction with embodiments of the present invention, many implementation-specific decisions must be made to achieve the developer's goals, such as compliance with system-related, business-related, government-related, and other restrictions, which vary from implementation to implementation and from time to time. Although the developer's efforts may be time-consuming, such efforts will be routine work for those of ordinary skill in the art who benefit from this disclosure.

[0352] Although various systems, tools, and methods are described herein in terms of "comprising" various components or steps, the systems, tools, and methods may also "consist essentially of" or "consist of" the various components and steps.

[0353] As used herein, the phrase "at least one of" preceding a series of items, with the terms "and" or "or" separating any items, modifies the list as a whole rather than each member (i.e., each item) of the list. The phrase "at least one of" allows for a meaning that includes at least one of any one item, and / or at least one of any combination of items, and / or at least one of each item. For example, the phrases "at least one of A, B, and C" or "at least one of A, B, or C" each refer to only A, only B, or only C; any combination of A, B, and C; and / or at least one of each of A, B, and C.

[0354] Therefore, the disclosed systems, tools and methods are well suited for achieving the mentioned purposes and advantages and those inherent therein. The specific embodiments disclosed above are merely illustrative, as the teachings of the present disclosure can be modified and practiced in different but equivalent ways, which is clear to those skilled in the art who benefit from the teachings herein. In addition, except as described in the claims below, it is not intended to limit the details of the construction or design shown herein. It is therefore clear that the specific illustrative embodiments disclosed above can be changed, combined or modified, and all these changes are considered to be within the scope of the present disclosure. In the absence of any element not specifically disclosed herein and / or any optional element disclosed herein, the systems, tools and methods disclosed exemplarily herein can be appropriately implemented. Although systems, tools and methods are described in terms of "including", "comprising" various components or steps, systems, tools and methods can also be "essentially composed of" or "composed of" various components and steps. All numbers and ranges disclosed above can be different. Whenever a numerical range with a lower limit and an upper limit is disclosed, any numerical value and any included range falling within the range are specifically disclosed. In particular, each range of values ​​disclosed herein (in the form of "from about a to about b", or equivalently "from approximately a to b", or equivalently, "from approximately ab") should be understood to list each number and range encompassed within the broader numerical range. Moreover, unless otherwise clearly and explicitly defined by the patentee, the terms in the claims have their plain, ordinary meaning. Moreover, the indefinite articles "a" and "an" used in the claims are defined herein to mean the element or elements it introduces. If the usage of a word or term in this specification conflicts with one or more patents or other documents that may be incorporated herein by reference, the definition consistent with this specification should be adopted.

Claims

1. An analyte sensor, comprising: a sensor tail configured for insertion into tissue, the sensor tail comprising a first working electrode and a second working electrode; as well as A first active region and a second active region are disposed on the tail of the sensor, the first active region and the second active region are used to measure the concentration of the analyte, the first active region includes xanthine oxidase, and the second active region includes glucose oxidase and catalase; wherein the first active area is disposed on the surface of the first working electrode, The second active area is arranged on the surface of the first working electrode, or on the surface of the second working electrode, wherein the first active area is coated with a first membrane polymer and a second membrane polymer compositionally different from the first membrane polymer, The xanthine oxidase does not react with the analyte, and the glucose oxidase and xanthine oxidase interact to produce a signal proportional to the concentration of the analyte.

2. The analyte sensor according to claim 1, wherein The first active region is coated with a mixture of the first membrane polymer and the second membrane polymer, and one of the first membrane polymer and the second membrane polymer coats the second active region as a homogeneous film.

3. The analyte sensor of claim 1, wherein the first active area is coated with a bilayer membrane comprising the first membrane polymer disposed on the second membrane polymer, and the second membrane polymer coats the second active area as a homogenous film.

4. The analyte sensor of claim 1, wherein glucose oxidase is capable of converting the analyte into a product that reacts with xanthine oxidase, such that the xanthine oxidase is capable of reacting the product to generate a signal at the first working electrode. The analyte sensor of claim 1 , wherein the first active region further comprises catalase.

6. The analyte sensor according to claim 1, wherein: The first active region also includes an electron transfer agent.

7. The analyte sensor of claim 1, wherein the first membrane polymer is disposed directly on the first active area, the second active area is disposed directly on the first membrane polymer, and the second membrane polymer is disposed directly on the second active area.

8. The analyte sensor according to claim 1, wherein: The first active region and the second active region each have a redox potential, and the redox potential of the first active region is sufficiently separated from the redox potential of the second active region to allow generation of a signal from the first active region independent of generation of a signal from the second active region.

9. The analyte sensor of claim 8, wherein the redox potential of the first active region is separated from the redox potential of the second active region by at least 100 mV.

10. The analyte sensor according to claim 8, wherein: The signal from the first active area corresponds to a first analyte concentration and the signal from the second active area corresponds to a second analyte concentration.

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