Analyte sensors employing multiple enzymes and methods associated therewith

Abstract

To provide an analyte sensor configured for analysis of multiple analytes.SOLUTION: An analyte sensor comprises: a sensor tail configured for insertion into a tissue, the sensor tail comprising at least a working electrode; and first and second active areas disposed on the sensor tail, the first and second active areas comprising at least two different enzymes for measuring the concentration of at least one analyte; where the first active area is coated with a first membrane polymer and a second membrane polymer that differ from one another.SELECTED DRAWING: Figure 2A

Description

Background Art

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

[0002] Any analyte can be suitable for physiological analysis if the appropriate chemistry for detecting the analyte can be identified. For this purpose, amperometric sensors configured to assay glucose in vivo have been developed and improved in recent years. Other analytes generally affected by physiological dysregulation and similarly desirable for in vitro or in vivo monitoring include, but are not limited to, lactate, oxygen, pH, A1c, ketones, drug levels, and the like.

[0003] Monitoring of an individual's analytes may be performed periodically or continuously over a period of time. Periodic analyte monitoring can be done by taking samples of body fluids such as blood at set time intervals and analyzing them in vitro. Continuous analyte monitoring can be done using one or more sensors that remain at least partially embedded within the individual's tissue, such as intradermally, subcutaneously, or intravenously, so that the analysis can be performed in vivo. The embedded sensors can collect analyte data at any instructed rate, depending on the individual's specific health needs and / or previously measured analyte levels.

[0004] Periodic in vitro analyte monitoring can be sufficient to determine the physiological state of many individuals. However, in vitro analyte monitoring can be inconvenient or painful for some people. Additionally, there is no way to recover lost data if analyte measurements are not obtained at the appropriate times.

[0005] Continuous analyte monitoring using sensors implanted in the body may be a more desirable approach for individuals with severe analyte dysregulation and / or rapidly fluctuating analyte levels, but may also be beneficial for other individuals. Continuous analyte monitoring by implanted sensors can be advantageous, but there are challenges associated with these types of measurements. Intravenous analyte sensors have the advantage of providing analyte concentrations directly from the blood, but are invasive and can be painful for the individual to wear, especially over long periods. Subcutaneous, interstitial, or dermal analyte sensors are often less painful for the individual to wear and can often provide sufficient measurement accuracy.

[0006] In vivo analyte sensors are typically configured to analyze a single analyte to provide a specific analysis and often use enzymes to provide analytical specificity. However, due to the physiological interactions between various combinations of analytes, in certain cases, the analysis of multiple analytes of interest may be desirable. Currently, in vivo analysis of multiple analytes may require the use of a corresponding number of analyte sensors configured for the analysis of each analyte. This approach can be inconvenient as the individual may need to wear multiple analyte sensors. Additionally, multiple analyte sensors can be a prohibitively costly burden for the individual or insurance company. Also, during such detection protocols, there is a higher likelihood that one of the independent analyte sensors will fail.

[0007] The in vivo analyte sensor may also include a membrane disposed over at least the embedded portion of the analyte sensor. In one aspect, the membrane may improve the biocompatibility of the analyte sensor. In another aspect, the membrane may be permeable or semi-permeable to the analyte of interest and restrict the overall analyte flux to the active region of the analyte sensor. That is, the membrane may function as a mass transfer limiting membrane. Limiting the access of the analyte to the active region of the sensor using a mass transfer limiting membrane can avoid sensor overload (saturation) and thereby improve detection performance and accuracy. Such membranes are highly specialized to limit the mass transfer of a particular analyte and may allow other substances to permeate the membrane at significantly different rates. The different membrane permeabilities for various potential analytes are a major barrier to developing analyte sensors configured for the analysis of multiple analytes. That is, if the membrane permeability values are different, the sensitivities to multiple analytes can vary significantly, thereby complicating the analysis. The different sensitivities to multiple analytes may be partially offset by using different sized active regions (e.g., a smaller active region for analytes with high sensitivity / permeability and a larger active region for analytes with low sensitivity / permeability). However, this approach has significant manufacturing challenges and may not be applicable in all cases.

Brief Description of the Drawings

[0008] The accompanying drawings are included to illustrate certain aspects of the present disclosure and should not be considered 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.

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DETAILED DESCRIPTION OF THE INVENTION

[0009] Detailed Description The present disclosure generally describes analyte sensors and methods that use multiple enzymes for detection, and more specifically, analyte sensors and methods in which multiple enzymes can function independently or cooperatively to detect one or more analytes.

[0010] As described above, an analyte sensor is typically used to detect a single analyte. If detection of multiple analytes is desired, a corresponding number of analyte sensors can be used. This approach may be undesirable, especially due to cost concerns, the need for an individual to wear multiple analyte sensors, and the increased likelihood of failure of individual sensors.

[0011] Some analyte sensors utilize an enzymatic reaction as a basis for detecting the analyte of interest. Since enzymes often exhibit reaction specificity for a particular substrate or related classes of substrates, they can provide an analyte sensor with a detection chemistry configured to analyze a single analyte of interest. Thus, an analyte sensor for analyzing a single analyte typically incorporates only the corresponding single enzyme to facilitate an appropriate enzymatic reaction for detection. Currently, it can be quite difficult to incorporate multiple enzymes into one analyte sensor to provide a detection function for multiple analytes. The reasons include differences in analyte sensitivity and the potential incompatibility of one or more enzymes with a given set of analysis conditions.

[0012] In contrast to an analyte sensor featuring a single enzyme, the present disclosure describes an analyte sensor in which multiple enzymes are present in the (one or more) active regions of the sensor. By incorporating multiple enzymes into the analyte sensor in various ways described herein, numerous advantages can be realized. In some sensor configurations of the present disclosure, the multiple enzymes can facilitate the independent detection of multiple analytes such as glucose and lactate. Membranes configured to provide permeability tailored to multiple analytes are also described herein, which can facilitate analyte detection by a single analyte sensor by leveling the sensitivity of the sensor to each analyte. In other sensor configurations of the present disclosure, multiple enzymes are selected to function cooperatively to facilitate the detection of a single analyte for which it might be problematic or impossible to assay using a single enzyme. In any case, the number of electrodes required to detect a given analyte or set of analytes can be reduced. Furthermore, the present disclosure can provide sensors that are small in size and reduce the complexity of the measurement electronics. Thus, analyte sensors using multiple enzymes in various configurations can facilitate the efficient detection of one or more analytes according to the disclosure herein.

[0013] Analyte sensors containing multiple enzymes can function with higher stability in the presence of a suitable stabilizer, whether the enzymes operate independently or cooperatively. 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. On the other hand, albumin is not thought to exhibit a function of removing reactive species, and as a result, their ability to stabilize the response of the analyte sensors of the present disclosure is surprising.

[0014] Before describing the analyte sensor of the present disclosure in more detail, a brief overview of a suitable in vivo analyte sensor configuration and a sensor system using such an analyte sensor will first be provided to better understand the embodiments of the present disclosure. It should be understood that according to various embodiments of the present disclosure, any of the sensor systems and analyte sensor configurations described below may feature multiple enzymes.

[0015] Figure 1 shows a diagram of an exemplary detection system into which an analyte sensor of the present disclosure can be incorporated. As shown, detection system 100 includes a sensor control device 102 and a reader device 120 configured to communicate with each other via a local communication path or link that is wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. According to some embodiments, the reader device 120 can constitute an output medium for displaying an analyte concentration and a warning or notification determined by the sensor 104 or an associated processor, and can also enable one or more user inputs. The reader device 120 can be a multi-purpose smartphone or a dedicated electronic reader device. Although only one reader device 120 is shown, in certain cases, multiple reader devices 120 can exist. The reader device 120 can also communicate with a remote terminal 170 and / or a trusted computer system 180 via communication paths / links 141 and / or 142, which are also wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. In addition or alternatively, the reader device 120 can communicate with a network 150 (e.g., a cellular phone network, the Internet, or a cloud server) via a communication path / link 151. The network 150 can be further communicatively connected to the remote terminal 170 via a communication path / link 152 and / or to the trusted computer system 180 via a communication path / link 153. Alternatively, the sensor 104 can communicate directly with the remote terminal 170 and / or the trusted computer system 180 without the presence of an intervening reader device 120. For example, according to some embodiments, the sensor 104 can communicate with the remote terminal 170 and / or the trusted computer system 180 via a direct communication link to the network 150, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is hereby incorporated by reference in its entirety.Any suitable electronic communication protocol such as Near Field Communication (NFC), Radio Frequency Identification (RFID), BLUETOOTH® or BLUETOOTH® Low Energy protocol, WiFi® can be used for each of the communication paths or links. The remote terminal 170 and / or the trusted computer system 180 can be accessed, according to some embodiments, by individuals other than the primary user who are interested in the user's analyte levels. The reader device 120 can include a display 122 and an optional input component 121. The display 122 can include a touch screen interface, according to some embodiments.

[0016] The sensor control device 102 includes a sensor housing 103 that can accommodate a circuit and a power source for operating the sensor 104. Optionally, the power source and / or the active circuit may be omitted. A processor (not shown) may be communicatively connected to the sensor 104, and the processor is physically disposed within the sensor housing 103 or the reader device 120. The sensor 104 projects from the lower side of the sensor housing 103 and extends through the adhesive layer 105. The adhesive layer 105 is adapted to adhere the sensor housing 103 to a tissue surface such as the skin, according to some embodiments.

[0017] Sensor 104 is adapted to be at least partially inserted into the tissue of interest, such as within the dermis or subcutaneous layer of the skin. Sensor 104 may include a sensor tail that is long enough to insert to a desired depth within a given tissue. The sensor tail may include at least one working electrode and one or more active regions (detection regions / spots or detection layers) disposed on the at least one working electrode and that are active for detecting one or more analytes of interest. Generally, according to one or more embodiments of the present disclosure, the one or more active regions may include a plurality of enzymes. According to some embodiments, the active region may include a polymeric material to which at least some of the enzymes are covalently bound. In various embodiments of the present disclosure, the analyte may be monitored in any biological fluid of the subject, such as dermal fluid, interstitial fluid, plasma, blood, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid. In certain embodiments, the analyte sensor of the present disclosure can be adapted to assay dermal fluid or interstitial fluid.

[0018] In some embodiments, sensor 104 can automatically transfer data to reader device 120. For example, analyte concentration data can be stored in memory until it is transmitted (e.g., every minute, every 5 minutes, or at other predetermined intervals) when the data is acquired, and can be communicated automatically and periodically, such as at a specific frequency or after a specific period of time has elapsed. In other embodiments, sensor 104 can communicate with reader device 120 in a non-automatic manner, rather than according to a set schedule. For example, data can be communicated from sensor 104 using RFID technology when the sensor electronics enter the communication range of reader device 120. The data may remain stored in the memory of sensor 104 until it is communicated to reader device 120. Thus, the patient does not need to be constantly in proximity to reader device 120 and instead can upload data at a convenient time. In yet other embodiments, a combination of automatic and non-automatic data transfer can be implemented. For example, data transfer can be automatically continued until reader device 120 moves out of the communication range of sensor 104.

[0019] To facilitate the introduction of sensor 104 into the tissue, an introducer may be temporarily present. In an exemplary embodiment, the introducer may include a needle or similar sharp object. In alternative embodiments, it should be recognized that other types of introducers, such as sheaths or blades, may exist. More specifically, the needle or other introducer may be temporarily present in the vicinity of sensor 104 prior to tissue insertion and then withdrawn. While present, the needle or other introducer may facilitate the insertion of sensor 104 into the tissue by opening an access path for sensor 104 to follow. For example, according to one or more embodiments, the needle may facilitate penetration of the epidermis as an access path to the dermis, enabling the implantation of sensor 104. After opening the access path, the needle or other introducer can be withdrawn to avoid imparting a sharp hazard. In an exemplary embodiment, a suitable needle may be solid or hollow, beveled or non-beveled, and / or have a circular or non-circular cross-section. In a more particular embodiment, a suitable needle may be comparable to an acupuncture needle that may have a cross-sectional diameter of about 250 microns in cross-sectional diameter and / or tip design. However, it should be recognized that a suitable needle may have a larger or smaller cross-sectional diameter if required for a particular application.

[0020] In some embodiments, the tip of the needle (while present) may be angled on the end of sensor 104 such that the needle first penetrates the tissue and opens an access path for sensor 104. In other exemplary embodiments, sensor 104 may be present within the lumen or groove of the needle, and the needle, likewise, opens an access path for sensor 104. In either case, the needle is withdrawn after facilitating the insertion of the sensor.

[0021] The analyte sensors described herein may feature multiple enzymes on the active region 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 of an analyte sensor can use a two-electrode or three-electrode detection motif. A sensor configuration featuring a single working electrode will be described below with reference to FIGS. 2A-2C. Thereafter, a sensor configuration featuring multiple working electrodes will be described separately with reference to FIG. 3. The multiple enzymes can be incorporated into any of the sensor configurations described below, and specific configurations suitable for incorporating multiple enzymes will be described in further detail below.

[0022] When a single working electrode is present in the analyte sensor, the three-electrode detection motif can include a working electrode, a counter electrode, and a reference electrode. The related two-electrode detection motif can include a working electrode and a second electrode, where the second electrode functions as both 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 regions of the analyte sensor may be in contact with the working electrode. The one or more active regions may include multiple enzymes according to embodiments of the present disclosure, and the multiple enzymes may be present in a single active region and / or multiple active regions. In some embodiments, as will be described in further detail below, the various electrodes can be at least partially stacked (layered) on top of each other. In some or other embodiments, the various electrodes may be laterally spaced apart from each other on the sensor tail. Similarly, the related active regions on each electrode can be stacked vertically on top of each other or laterally spaced apart. In either case, the various electrodes can be electrically insulated from each other by a dielectric material or a similar insulator.

[0023] Figure 2A shows a diagram of an exemplary two - electrode analyte sensor configuration having a single working electrode, suitable for use in some embodiments of the disclosure herein. As shown, the analyte sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter / reference electrode 216. Alternatively, the working electrode 214 and the counter / reference electrode 216 can be disposed on the same side of the substrate 212 with a dielectric material inserted therebetween (the configuration is not shown). 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 can include a plurality of spots or a single spot configured for the detection of one or more target analytes. Generally, a plurality of enzymes can be present in the active region 218 (i.e., in a single spot or a plurality of spots).

[0024] Continuing to refer to FIG. 2A, the membrane 220, according to some embodiments, can cover at least the active region 218 and optionally can cover a portion or all of the working electrode 214 and / or the counter / reference electrode 216, or the entire analyte sensor 200. One or both sides of the analyte sensor 200 can be covered by the membrane 220. The membrane 220 can include one or more polymeric membrane materials having the ability to restrict the analyte flux to the active region 218. As further described herein, the composition of the membrane 220 can be changed by something. The analyte sensor 200 can be operable to assay one or more analytes by any of the electrochemical detection techniques of coulometry, amperometry, voltammetry, or potentiometry.

[0025] Figures 2B and 2C show diagrams of exemplary three - electrode analyte sensor configurations having a single working electrode that are suitable for use in some embodiments of the disclosure herein. The three - electrode analyte sensor configuration using a single working electrode can be made similar to that shown for the analyte sensor 200 of Figure 2A, except that additional electrode 217 is included in analyte sensors 201 and 202 (Figures 2B and 2C). With the additional electrode 217, the counter / reference electrode 216 can function either as a counter electrode or a reference electrode, and the additional electrode 217 serves the other electrode function. The working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed either on the working electrode 210 or on the electrode 216, with a dielectric separation layer sandwiched in between. For example, as shown in Figure 2B, dielectric layers 219a, 219b, and 219c separate the electrodes 214, 216, and 217 from each other and provide electrical insulation. Alternatively, at least one of the electrodes 214, 216, and 217 can be disposed on the opposite face of the substrate 212, as shown in Figure 2C. Thus, in some embodiments, the electrode 214 (working electrode) and the electrode 216 (counter electrode) can be disposed on opposite faces of the substrate 212, and the electrode 217 (reference electrode) can be disposed on one of the electrodes 214 or 216 and be separated therefrom by a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) may be present on the electrode 217, and the position of the reference material layer 230 is not limited to that shown in Figures 2B and 2C. Similar to the sensor 200 shown in Figure 2A, the active regions 218 of the analyte sensors 201 and 202 can include multiple spots or a single spot configured for the detection of one or more target analytes. Generally, multiple enzymes can be present in the active regions 218 of the analyte sensors 201 and 202. Further, the analyte sensors 201 and 202 can be operable to assay one or more analytes by any of the electrochemical detection techniques of coulometry, amperometry, voltammetry, or potentiometry.

[0026] Similar to the analyte sensor 200, the membrane 220 can also cover the active regions 218, as well as other sensor components, in the analyte sensors 201 and 202. In some embodiments, the additional electrode 217 can be covered by the membrane 220. In FIGS. 2B and 2C, all of the electrodes 214, 216, and 217 are depicted as being covered by the membrane 220, although it should be recognized that in some embodiments, only the working electrode 214 may be covered. Further, the thickness of the membrane 220 at each of the electrodes 214, 216, and 217 may be the same or different. As in the case of the two-electrode analyte sensor configuration (FIG. 2A), in the sensor configurations of FIGS. 2B and 2C, one or both sides of the analyte sensors 201 and 202 can be covered by the membrane 220, or the entire analyte sensors 201 and 202 can be covered. Thus, the three-electrode sensor configurations shown in FIGS. 2B and 2C should be understood as non-limiting examples of the embodiments disclosed herein with alternative electrode and / or layer configurations within the scope of the present disclosure.

[0027] Next, the analyte sensor configuration having a plurality of working electrodes will be described in more detail. The following description is mainly directed to the analyte sensor configuration having two working electrodes, although it should be understood that by extending the disclosure herein, three or more working electrodes can also be incorporated without problem. The additional working electrodes can enable an analyte sensor having such features to have additional active regions and corresponding sensing capabilities.

[0028] FIG. 3 shows a diagram of an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode that is suitable for use in some embodiments of the disclosure herein. As shown in FIG. 3, the analyte sensor 300 includes working electrodes 304 and 306 disposed on opposing surfaces of a substrate 302. An active region 310 is disposed on the surface of the working electrode 304, and an active region 312 is disposed on the surface of the working electrode 306. Generally, a plurality of enzymes may be present in the active regions 310 and 312, and each of the active regions 310, 312 includes one or more enzymes. For example, in certain embodiments, 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 insulated from the working electrode 304 by a dielectric layer 322, and the reference electrode 321 is electrically insulated from the working electrode 306 by a dielectric layer 323. Outer dielectric layers 330 and 332 are disposed on the reference electrode 321 and the counter electrode 320, respectively. The membrane 340 can cover at least the active regions 310 and 312 according to various embodiments. Other components of the analyte sensor 300 may similarly be covered by the membrane 340, and as described above, one or both sides, or a portion thereof, of the analyte sensor 300 may be covered by the membrane 340. Similar to the analyte sensors 200, 201, and 202, the analyte sensor 300 may be operable to assay one or more analytes by any of the electrochemical detection techniques of coulometry, amperometry, voltammetry, or potentiometry.

[0029] Alternative analyte sensor configurations having a plurality of working electrodes and different from those shown in FIG. 3 may feature a counter / reference electrode and / or an arrangement of layers and / or membranes different from those explicitly shown, instead of separate counter and reference electrodes 320, 321. For example, the arrangement of the counter electrode 320 and the reference electrode 321 can be reversed from that shown in FIG. 3. Further, the working electrodes 304 and 306 do not necessarily have to be present on opposing surfaces of the substrate 302 in the manner shown in FIG. 3.

[0030] An analyte sensor configuration characterized by a working electrode having separated active regions is shown in FIGS. 6A and 6B and will be further described below. According to various embodiments of the present disclosure, an electron transfer agent may be present in one or more active regions of any of the analyte sensors or analyte sensor configurations disclosed herein. A suitable electron transfer agent may facilitate the transfer of electrons to the working electrode when the analyte (enzyme substrate) undergoes a redox reaction. The choice of electron transfer agent within each active region may determine the redox potential observed for each. If multiple active regions are present, the electron transfer agents within each active region may be the same or different.

[0031] Suitable electron transfer agents can include electroreductive and electrooxidative ions, complexes, or molecules (e.g., quinones) having a redox potential several hundred millivolts above or below the redox potential of a standard calomel electrode (SCE). According to some embodiments, suitable electron transfer agents can include low potential osmium complexes such as those described in U.S. Pat. Nos. 6,134,461 and 6,605,200, which are hereby incorporated by reference in their entirety. Additional examples include those described in U.S. Pat. Nos. 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are hereby incorporated by reference in their entirety. Other suitable electron transfer agents can include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt (e.g., including its metallocene compounds). Suitable examples of electron transfer mediators and polymer-bound electron transfer mediators can include those described in U.S. Pat. Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are hereby incorporated by reference in their entirety. Ligands suitable for metal complexes can include, for example, bidentate or higher dentate ligands such as bipyridine, biimidazole, phenanthroline, or pyridyl(imidazole). Other suitable bidentate ligands can include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. To achieve a complete coordination sphere, any combination of monodentate, bidentate, tridentate, tetradentate, or higher dentate ligands can be present in the metal complex.

[0032] According to various embodiments of the present disclosure, the polymer may be present in any active region of the analyte sensor or analyte sensor configuration disclosed herein. Suitable polymers for inclusion in the active region include, but are not limited to, polyvinylpyridine (e.g., poly(4-vinylpyridine)), polyvinylimidazole (e.g., poly(1-vinylimidazole)), or any copolymer thereof. Exemplary copolymers that may be suitable for inclusion in the active region include those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile. If multiple active regions are present, the polymers within each active region may be the same or different.

[0033] According to various embodiments of the present disclosure, the electron transfer agent may 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 having the covalently bonded electron transfer agent, or, if the polymer has already been synthesized, the electron transfer agent can be reacted separately with the polymer. According to some embodiments, a bifunctional spacer can covalently bond the electron transfer agent to the polymer within the active region, where the first functional group is reactive 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 is reactive with the electron transfer agent (e.g., a functional group that reacts with a ligand coordinating a metal ion).

[0034] Similarly, according to some or various other embodiments of the present disclosure, enzymes within one or more active regions can be covalently bound to a polymer. When multiple enzymes are present in a single active region, in some embodiments, all of the multiple enzymes can be covalently bound to the polymer, and in other embodiments, only some of the multiple enzymes can be covalently bound to the polymer. For example, a first enzyme can be covalently bound to the polymer, and a second enzyme can be non-covalently bound to the polymer. According to more specific embodiments, the covalent binding of the enzyme to the polymer can occur via a cross-linking agent introduced with a suitable cross-linking agent. Suitable cross-linking agents for reaction with free amino groups in the enzyme (e.g., with free amines in lysine) can include, for example, polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanogen chloride, N-hydroxysuccinimide, imido esters, epichlorohydrin, or derivatized variants thereof. Cross-linking agents suitable for reaction with free carboxylic acid groups in the enzyme can include, for example, carbodiimides. Cross-linking is generally intermolecular, but in some embodiments can be intramolecular.

[0035] The electron transfer agent and / or enzyme can also be bound to the polymer within the active region by means other than covalent bonding. In some embodiments, the electron transfer agent and / or enzyme can be ionically or coordinatively bound to the polymer. For example, a charged polymer can be ionically bound to an oppositely charged electron transfer agent or enzyme. In still other embodiments, the electron transfer agent and / or enzyme can be physically entrained within the polymer without binding to the polymer.

[0036] Next, various configurations suitable for disposing multiple enzymes in the analyte sensor of the present disclosure will be described in further detail. The multiple enzymes can be deposited within one or more active regions of the sensor. The active region can range in size from about 0.01 mm 2 to about 1 mm 2 although larger or smaller active regions are also contemplated herein.

[0037] In some embodiments, multiple enzymes can be disposed within separate active regions on a single working electrode. When multiple enzymes are so disposed, each active region can facilitate the detection of a separate analyte, as described below. At least one of the active regions can generate a signal independently of the other active regions.

[0038] According to some embodiments, an analyte sensor of the present disclosure having multiple active regions on a single working electrode can include a sensor tail including at least the working electrode, and at least two active regions disposed on the surface of the working electrode. Each active region includes an analyte-responsive enzyme and a polymer, and the analyte-responsive enzymes in each active region are different. 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 enable the generation of a signal from the first active region independently of the signal from the second active region. In more specific embodiments, such an analyte sensor can include a single working electrode having at least two active regions. To facilitate electron transfer, an electron transfer agent can be incorporated within each active region.

[0039] An alternative sensor configuration can include a single active region that includes both a first analyte-responsive enzyme and a second analyte-responsive enzyme, along with an electron transfer agent. Each enzyme can covalently bind to a separate portion of the polymer within the single active region. The single active region can facilitate the detection of an analyte in a manner similar to that described below for separate active regions, provided that the detection chemicals for facilitating electron transfer of each analyte are not overly diluted within the single active region. Such a sensor configuration can be particularly feasible when the analytes assayed with the first and second analyte-responsive enzymes have comparable membrane permeability values.

[0040] In more specific embodiments, the sensor tail can be configured for insertion into tissue. Suitable tissue is not considered to be particularly limited and is described in more detail above. Similarly, considerations for disposing the sensor tail at a specific location within the tissue are described above.

[0041] 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 100 mV, or at least about 150 mV, or at least about 200 mV. The upper limit of the separation between the redox potentials is determined in vivo by the working electrochemical window. By sufficiently separating the magnitudes of the redox potentials of the active regions from each other, the electrochemical reaction can occur within the first active region without substantially inducing an electrochemical reaction within the second active region. Thus, a signal from the first active region can be generated independently at or above its corresponding redox potential. In contrast, above the redox potential of the second active region, electrochemical reactions can occur within both active regions. Thus, a signal obtained above the redox potential of the second active region can include signal contributions from both the first active region and the second active region, and the signal is a composite signal. Next, the signal contribution from the second active region above its redox potential can be determined by subtracting the signal obtained only from the first active region above its corresponding redox potential from the composite signal. Similar considerations also apply to the analysis of signal contributions from a single active region containing two different enzymes that generate signals at different redox potentials.

[0042] In more specific embodiments, the first and second active regions may include different electron transfer agents in order to sufficiently separate the magnitudes of the redox potentials when the active regions are located on the same working electrode. 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 electron transfer agent and the second electron transfer agent are different. According to various embodiments of the present disclosure, the redox potentials of the first and second active regions can be sufficiently separated by changing the metal center and / or ligand present in a given electron transfer agent. According to even more specific embodiments, the first electron transfer agent may be covalently bonded to the polymer in the first active region, and the second electron transfer agent may be covalently bonded to the polymer in the second active region. The modes of covalent bonding of the first electron transfer agent and the second electron transfer agent may be the same or different. In selecting an electron transfer agent suitable for use in combination with the first analyte-responsive enzyme and the second analyte-responsive enzyme included within a single active region, similar considerations apply in accordance with the above disclosure.

[0043] In more specific embodiments of the present disclosure, the analyte-responsive enzyme of each active region may be covalently bonded (or immobilized in another way) to the polymer within each active region. In even more specific embodiments, the analyte-responsive enzyme and the electron transfer agent within each active region may be covalently bonded to the polymer within each active region. When included in a single active region, the first analyte-responsive enzyme and the first electron transfer agent may be covalently bonded to a first portion of the polymer, and the second analyte-responsive enzyme and the second electron transfer agent may be covalently bonded to a second portion of the polymer. The polymers of the first portion and the second portion may be the same or different.

[0044] Ideally, the first and second active regions disposed on a single working electrode can be configured to achieve a steady-state current rapidly when operating the analyte sensor at a given potential. The rapid achievement of the steady-state current can be facilitated by selecting, for each active region, an electron transfer agent that rapidly changes its oxidation state when exposed to a potential above its redox potential. Making the active regions as thin as possible can also facilitate the rapid achievement of the steady-state current. For example, suitable thicknesses for the first and second active regions can range from about 0.1 micron to about 10 microns. In some or other embodiments, combining one or more conductive materials, such as carbon nanotubes, graphene, or metal nanoparticles, within one or more of the active regions can facilitate the rapid achievement of the steady-state current. Suitable amounts of the conductive particles can range from about 0.1 wt% to about 50 wt%, or about 1 wt% to about 50 wt%, or about 0.1 wt% to about 10 wt%, or about 1 wt% to about 10 wt% of the active region. Stabilizers can also be used to promote the stability of the response.

[0045] It should also be understood that the sensitivity (output current) of the analyte sensor to each analyte can be altered by varying the coverage (area or size) of the active regions, the area ratio of the active regions to each other, the nature and thickness of the mass transfer limiting membrane covering the active regions, and any combination thereof. Altering these parameters can be readily accomplished by one of ordinary skill in the art given the benefit of the disclosure herein.

[0046] The foregoing description has been directed primarily to analyte sensors configured to detect two different analytes, but it should be understood that the concepts described above can be extended to detect three or more analytes using a corresponding number of active regions disposed on a single working electrode. Specifically, in further embodiments of the present disclosure, an analyte sensor using three or more active regions and a corresponding number of different enzymes (and electron mediators) therein can be used to detect the same number of different analytes. If the redox potential of each active region is sufficiently separated from the redox potential of other active regions, the signal contributions from each active region can be analyzed in a manner related to the methods described above to provide the concentration of each analyte.

[0047] In a more specific embodiment, the first active region may include a glucose-responsive enzyme such as glucose oxidase in addition to the appropriate electron mediator and polymer described in more detail above, and the second active region may include a lactate-responsive enzyme such as lactate oxidase. According to a particular embodiment, an analyte sensor suitable for detecting glucose and lactate may include a working electrode on which the first and second active regions are disposed, and a mass transfer limiting membrane covering the first and second active regions on the working electrode. The second active region includes a polymer, albumin, and a lactate-responsive enzyme (e.g., lactate oxidase) covalently bonded to the polymer, and the first active region includes a glucose-responsive enzyme (e.g., glucose oxidase) covalently bonded to the polymer. Different first and second electron mediators may be present in each active region. In a more specific embodiment, the mass transfer limiting membrane may include at least a crosslinked polyvinylpyridine homopolymer or copolymer. The composition of the mass transfer limiting membrane may be the same or different when the mass transfer limiting membrane covers each active region. In a particular embodiment, the mass transfer limiting membrane covering the first active region may be a single component (including a single membrane polymer), and the mass transfer limiting membrane covering the second active region may be a multi-component (including two or more different membrane polymers, one of which is a polyvinylpyridine homopolymer or copolymer), and may be either a bilayer or a homogeneous mixture.

[0048] Similarly, it should be understood that some analyte sensors of the present disclosure having two or more active regions disposed on a given working electrode may include two or more analyte-responsive enzymes in at least one active region. According to more specific embodiments, two or more analyte-responsive enzymes within a given active region may interact cooperatively to produce a signal proportional to the concentration of a single analyte. Thus, analyte-responsive enzymes need not necessarily be present in a 1:1 ratio for the selection of a particular analyte. Analyte sensors comprising enzymes that interact cooperatively are described in more detail below.

[0049] Accordingly, a multi-analyte detection method using an analyte sensor characterized by a plurality of enzymes disposed on a single working electrode is also described herein. In various embodiments, such a method may include exposing the analyte sensor to a fluid containing at least one analyte. The analyte sensor includes at least a working electrode, particularly a single working electrode, and a sensor tail including at least two active regions disposed on the surface of the working electrode. Each active region includes an analyte-responsive enzyme and a polymer, and the analyte-responsive enzymes in each active region are different. 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 enable the generation of a signal from the first active region independent of the generation of a signal from the second active region. The method further includes obtaining a first signal at a redox potential above that of the first active region such that the first signal is proportional to the concentration of the first analyte; obtaining a second signal at a redox potential above that of the second active region such that the second signal is a composite signal including a signal contribution from the first active region and a signal contribution from the second active region; and obtaining a differential signal proportional to the concentration of the second analyte by subtracting the first signal from the second signal.

[0050] In more specific embodiments, 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 100 mV, or at least about 150 mV, or at least about 200 mV in order to be sufficiently separated for the independent generation of signals from the first active region.

[0051] In some or other more specific embodiments, the fluid is a biological fluid and the analyte sensor is exposed to the biological fluid within an individual's body. Biological fluids suitable for analysis using an analyte sensor having at least two different active regions disposed on a given working electrode can include any of the biological fluids described in more detail above.

[0052] In some embodiments, the signal associated with each active region can be correlated to the corresponding analyte concentration by referring to a lookup table or calibration curve for each analyte. The lookup table for each analyte can be created by analyzing a plurality of samples having known analyte concentrations and recording the sensor response at each concentration of each analyte. Similarly, the calibration curve for each analyte can be determined by plotting the analyte sensor response for 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.

[0053] The processor can determine which sensor response value in the lookup table is closest to that measured for a sample having an unknown analyte concentration and report the analyte concentration accordingly. In some or other embodiments, if the sensor response value of a sample having an unknown analyte concentration is between the values recorded in the lookup table, the processor can interpolate between the two lookup table values to estimate the analyte concentration. The interpolation can assume a linear concentration variation between the two values reported in the lookup table. Interpolation can be used when the response of the sensor is sufficiently different (such as a variation of about 10% or more) from a given value in the lookup table.

[0054] Similarly, according to some or other various embodiments, the processor can input the sensor response value of a sample having an unknown analyte concentration into the corresponding calibration curve. Next, the sensor can then report the analyte concentration accordingly.

[0055] Embodiments of an analyte sensor having two different active regions disposed on a given working electrode can use the sensor configurations related to those shown in FIGS. 2A - 2C and above. However, it should be understood that a suitable analyte sensor can also be characterized by multiple working electrodes, such as the sensor configuration shown in FIG. 3, where at least one of the working electrodes has at least two different active regions that are different from each other. It should also be understood that other analyte sensor configurations having more than two different active regions disposed on the surface of a given working electrode are also within the scope of the present disclosure. For example, the position, orientation, or function of the working electrode and the counter electrode and / or reference electrode may be different from those shown in the drawings of the present application.

[0056] FIG. 4 shows an exemplary analyte sensor configuration suitable for use in some embodiments of the present disclosure where two different active regions are disposed on the surface of a single working electrode. The analyte sensor configuration of FIG. 4 is most similar to that of FIG. 2C and can be better understood by referring to FIG. 2C. Where appropriate, reference numerals common to FIG. 2C are used in FIG. 4 for clarity purposes, and for brevity, features having common structure and / or function are not described in further detail. It should also be understood in this case that other analyte sensor configurations can similarly incorporate the features described below for FIG. 4.

[0057] Referring to FIG. 4, the analyte sensor 400 includes active regions 218a and 218b on the surface of the working electrode 214. The active region 218a includes a first electron transfer agent and a first analyte-responsive enzyme that can be covalently bonded to a polymer containing the active region 218a. The active region 218b similarly includes a second electron transfer agent and a second analyte-responsive enzyme that can be covalently bonded to a polymer containing the active region 218b. The first electron transfer agent and the second electron transfer agent may have different compositions so as to provide a separation of the redox potentials of the first active region 218a and the second active region 218b. In certain embodiments, the active region 218b may include a lactate-responsive enzyme such as lactate oxidase, and the active region 218a may include a glucose-responsive enzyme such as glucose oxidase.

[0058] The redox potentials of the first active region 218a and the second active region 218b can be sufficiently separated from each other to enable the generation of a signal from the first active region 218a independently of the signal generation from the second active region 218b. Thus, the analyte sensor 400 can operate at a first potential at which a redox reaction occurs within the first active region 218a but not within the second active region 218b. Accordingly, the first analyte (e.g., glucose) can be selectively detected at a potential above the redox potential of the first active region 218a, provided that 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 referring to a look-up table or a calibration curve.

[0059] Above the redox potential of the second active region 218b, a separate redox reaction can occur simultaneously or almost simultaneously within both the first active region 218a and the second active region 218b. As a result, the signal generated above the redox potential of the second active region 218b can include a composite signal having signal contributions from both the first active region 218a and the second active region 218b. To determine the concentration of the second analyte (e.g., lactate) from the composite signal, the signal from the first active region 218a above the corresponding redox potential can be subtracted from the composite signal to provide a differential signal related only to the second active region 218b. Once the differential signal is determined, the concentration of the second analyte can be determined by referring to a lookup table or a calibration curve.

[0060] As described above, similar considerations also apply to separating a first signal and a second signal from a single active region containing two different analyte-responsive enzymes to determine the concentrations of two different analytes that differ from each other.

[0061] In some or other embodiments of the present disclosure, multiple enzymes may be present in a single active region. Unlike sensor configurations where multiple enzymes function independently to detect different analytes, particularly sensor configurations that are spaced apart in separate active regions on the surface of a working electrode, according to some embodiments of the present disclosure, multiple enzymes disposed in a single active region function cooperatively, particularly in the presence of a stabilizer, to facilitate the detection of a single analyte. As used herein, the term "cooperatively" and its grammatical variations refer to a combined enzymatic reaction in which the product of a first enzymatic reaction serves as the substrate for a second enzymatic reaction, and the second enzymatic reaction functions as the basis for measuring the concentration of the substrate (analyte) that reacted during the first enzymatic reaction. In cases where a single enzyme cannot facilitate detection, it may be desirable to utilize two enzymes that act cooperatively with each other to detect a given analyte of interest. Situations where a single enzyme may be ineffective in promoting the detection of an analyte include, for example, situations where the enzyme is inhibited by one or more products of the enzymatic reaction or situations where, when disposed within an analyte sensor, it cannot cycle between an oxidized state and a reduced state.

[0062] As also disclosed herein, multiple enzymes disposed in separate active regions may similarly cooperate and interact to facilitate the detection of a single analyte. When multiple enzymes are disposed in separate active regions, one of the active regions can be separated from the working electrode such that electron transfer to and from the working electrode occurs only from that one active region.

[0063] In a more specific embodiment, an analyte sensor characterized by at least two enzymes that cooperate and interact with each other may include a sensor tail including at least a working electrode; and at least one active region disposed on the surface of the working electrode. The at least one active region includes a first enzyme, a second enzyme, and a polymer. The first and second enzymes can cooperate and interact with each other. The first enzyme can convert an 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 bound to the polymer in the at least one active region. As will be described in more detail below, the analysis of the signal (e.g., the current measured at a fixed input voltage) resulting from the reaction of the first product to the second product can provide a basis for detecting the analyte and measuring its concentration.

[0064] More specifically, the active region may include an electron transfer agent as described above, and only the second enzyme can exchange electrons with the electron transfer agent. In that case, the first enzyme can indirectly transfer electrons to the second enzyme as described below. Thus, a more specific embodiment of the present disclosure may be characterized by a first enzyme that is not covalently bound to the polymer (and thus has a low potential to exchange electrons with the electron transfer agent) and a second enzyme that is covalently bound to the polymer (to facilitate electron exchange with the electron transfer agent). The electron transfer agent can be covalently bound to the polymer in the active region in any case. Coordination bonds are also included within the scope of covalent bonds according to the disclosure herein.

[0065] According to some embodiments, a stabilizer may be present in the active region. Stabilizers particularly suitable for analyte sensors including enzymes that cooperate and interact with each other include, for example, catalase and albumin.

[0066] According to some embodiments, the sensor satellite can be configured to be inserted into the target tissue. Thus, according to some embodiments, an analyte sensor that includes enzymes that can cooperate and interact with each other in a given active region can be adapted to analyze the concentration of an analyte in a biological fluid in vivo. Here again, what the biological fluid is is not particularly limited.

[0067] As described above, the sensor configuration incorporating two enzymes that can cooperate and interact with each other can include those in which at least one active region includes an electron transfer agent covalently bonded to a polymer. Also in this case, according to the disclosure of the present specification, coordination bonds are also included within the scope of covalent bonds. In such embodiments, at least the second enzyme can also be covalently bonded to the polymer. In some embodiments, the first enzyme is not covalently bonded to the polymer. In other embodiments, both the first enzyme and the second enzyme can 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 the first enzyme leaching from at least one active region.

[0068] Analyte sensor configurations suitable for incorporating two enzymes that cooperate and interact with each other in one or more active regions on the working electrode can be the same as those shown in FIGS. 2A-2C and described in more detail above. Enzymes that can cooperate and interact with each other (i.e., cooperative enzymes or cooperative enzyme pairs) can also be incorporated into analyte sensor configurations having multiple working electrodes (FIG. 3) or multiple active regions (FIG. 4) disposed on a given working electrode. Any of the analyte sensors disclosed herein having a pair of cooperative enzymes disposed directly on the surface of the working electrode can employ any of the aforementioned analyte sensor configurations. An analyte sensor configuration having two or more enzymes that cooperate and interact with each other in multiple active regions, where one of the active regions is remote from the working electrode, will be further described below with reference to FIGS. 6A and 6B.

[0069] In a more specific configuration of the analyte sensor including coenzymes both disposed directly on the working electrode, the first enzyme can be alcohol oxidase (AOX), and the second enzyme can be xanthine oxidase (XOX). With this pair of enzymes, the analyte sensor can function to detect alcohol, particularly ethanol, according to one or more embodiments. The cooperation between alcohol oxidase and xanthine oxidase for detecting ethanol and other alcohols when both enzymes are disposed on the working electrode will be described in more detail below (see FIG. 5A). In a more specific embodiment of the present disclosure, xanthine oxidase can be covalently bonded to the polymer in the active region, but alcohol oxidase is not covalently bonded to the polymer. In an even more specific embodiment, both xanthine oxidase and the electron transfer agent can be covalently bonded to the polymer, but alcohol oxidase is not covalently bonded to the polymer. Along with this pair of enzymes, catalase may be present as a stabilizer.

[0070] Another pair of coenzymes that may be suitable for use in the disclosure herein, where both enzymes are disposed directly on the surface of the working electrode, are β-hydroxybutyrate dehydrogenase and diaphorase. This pair of coenzymes can be used for the detection of ketone bodies, and β-hydroxybutyric acid is a representative molecule indicating the presence of ketones. In the sensor configuration including this pair of coenzymes, β-hydroxybutyrate dehydrogenase can convert β-hydroxybutyric acid and nicotinamide adenine dinucleotide (NAD + ) to acetoacetic acid and reduced nicotinamide adenine dinucleotide (NADH). Next, NADH undergoes reduction under the mediation of diaphorase, and the electrons transferred during this process provide the basis for ketone detection at the working electrode. The concerted reaction between β-hydroxybutyric acid and diaphorase (mediated by the NAD + cofactor) for detecting ketones is shown in FIG. 5B. Along with the pair of coenzymes, albumin may be present as a stabilizer.

[0071] Still other alternative enzymatic detection chemistries for ketones are shown in FIGS. 5C and 5D. As shown in FIG. 5C, β-hydroxybutyrate dehydrogenase can convert β-hydroxybutyric acid and NAD + to acetoacetic acid and NADH. Instead of the electron transfer to the working electrode being completed by diaphorase (see FIG. 5B), reduced NADH oxidase (NADHOx(red)) undergoes a reaction to form the corresponding oxidized form (NADHOx(Ox)). Next, NADHOx(red) is reformed by reaction with oxygen molecules to produce superoxide, which can then be converted to hydrogen peroxide via superoxide dismutase (SOD). According to various embodiments, SOD can be covalently bound to the polymer of the active region. Next, hydrogen peroxide undergoes a reaction at the working electrode to provide a signal that can be correlated with the amount of ketone present. FIG. 5D shows another alternative enzymatic detection chemistry in which β-hydroxybutyrate dehydrogenase can again convert β-hydroxybutyric acid and NAD + to acetoacetic acid and NADH. The detection cycle in this case is completed by the oxidation of poly-1,10-phenanthroline-5,6-dione at the working electrode. As with other sensing chemistries disclosed herein, including albumin in the active region can provide a surprising improvement in response stability.

[0072] Creatinamide hydrolase and sarcosine oxidase are another pair of coenzymes that may be suitable for use in the disclosure herein when both enzymes are placed directly on the working electrode. Creatinamide hydrolase produces sarcosine and urea from creatine. Next, sarcosine oxidase can catalyze the reaction of sarcosine to form glycine, formaldehyde, and hydrogen peroxide. Thus, the detection of hydrogen peroxide at the working electrode can serve as a basis for quantifying creatine and / or sarcosine.

[0073] The detection of ethanol and other alcohols using alcohol oxidase and xanthine oxidase via a coupled enzyme reaction will be described in more detail. Alcohol oxidase interacts with ethanol to form acetaldehyde and hydrogen peroxide. Other alcohols react to form the corresponding aldehydes with a higher or lower number of carbon atoms. Advantageously, alcohol oxidase catalyzes only the forward conversion from ethanol to acetaldehyde (in contrast to reversibly conducting the reaction as in the case of alcohol dehydrogenase), which can be advantageous for using this enzyme in an analyte sensor. Further, since alcohol oxidase contains a tightly bound flavin cofactor, it is not always necessary to combine an exogenous cofactor with alcohol oxidase to activate the enzyme to promote alcohol oxidation.

[0074] In principle, only alcohol oxidase can be used for ethanol detection in an analyte sensor by assaying either the acetaldehyde or hydrogen peroxide product generated in the enzymatic reaction. However, this approach has two problems. First, both acetaldehyde and hydrogen peroxide inhibit alcohol oxidase. Therefore, if these compounds are not removed from the sensor environment, alcohol oxidase becomes inactive in promoting ethanol oxidation, whereby the analyte sensor ceases to function for assaying ethanol. Further, when acetaldehyde and hydrogen peroxide are sequestered or quenched with other agents, there are no species available for electrochemical detection. Second, alcohol oxidase does not freely exchange electrons with redox mediators other than oxygen molecules. Thus, electron transfer agents associated with polymers within the active region of the analyte sensor, such as osmium and other transition metal complexes described herein, are ineffective in cycling alcohol oxidase from an inactive reduced state to an oxidized state that is reactive with ethanol. Therefore, alcohol oxidase can optionally be covalently attached to the polymer, but there is no particular advantage to doing so for the electron transfer process. That is, covalent attachment of alcohol oxidase to the polymer does not assist in facilitating electron transfer by the electron transfer agent.

[0075] A coordinated combination of alcohol oxidase and xanthine oxidase directly on the working electrode, particularly together in a given active region, can overcome at least some of the aforementioned problems associated with ethanol detection using an analyte sensor that uses alcohol oxidase. Acetaldehyde and other aldehydes can function as substrates for xanthine oxidase, and acetaldehyde is enzymatically converted to acetic acid. Thus, xanthine oxidase can remove acetaldehyde from the sensor environment, thereby preventing acetaldehyde-based inactivation of alcohol oxidase. To remove hydrogen peroxide, catalase may be present in the active region (e.g., as a catalase-hydrogen peroxide complex), thereby preventing inactivation of alcohol oxidase by this species. Further, unlike alcohol oxidase, xanthine oxidase can exchange electrons with osmium and other transition metal complexes associated with the polymer within the active region of the analyte sensor. Thus, xanthine oxidase can cycle between its oxidized and reduced forms, thereby enabling the analyte sensor to maintain an active sensing state. Accordingly, the detection of ethanol in the aforementioned analyte sensor is based on the enzymatic reaction of xanthine oxidase with acetaldehyde, which is an enzymatic reaction product of ethanol. Further, by configuring the enzymes within the analyte sensor in the aforementioned manner, alcohol oxidase may be reoxidized by oxygen molecules to maintain its activity.

[0076] Figure 5A shows a coenzyme reaction cycle related to ethanol detection using alcohol oxidase and xanthine oxidase directly disposed on a working electrode according to various embodiments of the present disclosure. Xanthine oxidase is covalently bound to a polymer in the active region of the analyte sensor, and alcohol oxidase is non-covalently bound to the polymer in the active region. In addition to xanthine oxidase, an osmium complex or other transition metal complex capable of exchanging electrons with this enzyme is also covalently bound to the polymer. As shown in Figure 5A, ethanol reacts with oxidized (active) alcohol oxidase in the presence of a flavin cofactor (FAD - already bound to alcohol oxidase), thereby forming reduced alcohol oxidase, acetaldehyde, and hydrogen peroxide. The reduced alcohol oxidase can be re-oxidized with an oxygen molecule as shown to return the alcohol oxidase to its catalytically active oxidized form.

[0077] Continuing to refer to Figure 5A, the acetaldehyde enzymatically formed from ethanol then undergoes a subsequent reaction with oxidized xanthine oxidase in the presence of the flavin cofactor that is inherently present with the enzyme. Acetic acid is produced in this process and the xanthine oxidase changes to a reduced state. Next, the reduced xanthine oxidase can react with a transition metal electron transfer agent bound to the polymer to transfer electrons to the working electrode, thereby generating a current and regenerating the oxidized xanthine oxidase. Although not shown in Figure 5A, hydrogen peroxide is separately removed from the sensor environment by catalase present in the active region.

[0078] As can be understood from FIG. 5A, the amount of enzymatically formed acetaldehyde is proportional to the amount of ethanol originally present. Therefore, the current generated at the working electrode during the xanthine oxidase oxidation of acetaldehyde can be proportional to the amount of acetaldehyde present and thus to the amount of ethanol. The correlation between the working electrode current and the ethanol concentration can be made by referring to a look-up table of currents at known ethanol concentrations or by using a calibration curve. These concepts are explained in more detail above.

[0079] Similarly, when analyzing ketones, the current generated at the working electrode can be proportional to the amount of β-hydroxybutyric acid that is oxidized to form acetoacetone (FIGS. 5B-5D). Thus, the correlation of the current at the working electrode can be made in a manner related to that provided above for ethanol (e.g., using a calibration curve or a look-up table).

[0080] Accordingly, in a more specific embodiment, the present disclosure provides an alcohol sensor based on the co-enzymatic reaction of alcohol oxidase and xanthine oxidase. More specifically, the alcohol sensor can include at least a working electrode and a sensor tail including at least one active region disposed on the surface of the working electrode, where the at least one active region includes alcohol oxidase, xanthine oxidase, catalase, a polymer, and an electron transfer agent. According to a particular embodiment, the electron transfer agent and xanthine oxidase can be covalently bound to the polymer, while the alcohol oxidase is not covalently bound to the polymer. The alcohol oxidase and xanthine oxidase cooperate and interact to be able to generate a signal proportional to the alcohol concentration at the working electrode. More specifically, both the alcohol oxidase and xanthine oxidase are disposed directly on the working electrode to achieve the foregoing.

[0081] According to more specific embodiments, the catalase within at least one active region of the alcohol sensor is not covalently bound to the polymer. The catalase can be present in an amount ranging from about 1 wt% to about 50 wt% of the polymer, more specifically from about 1 wt% to about 10 wt% of the polymer, or from about 1 wt% to about 5 wt% of the polymer.

[0082] Accordingly, the present disclosure also provides a detection method based on a coenzyme reaction in which a coenzyme pair is disposed directly on the surface of a working electrode. According to various embodiments, the detection method can include exposing an analyte sensor to a fluid containing an analyte, the analyte sensor including a sensor tail including at least a working electrode and at least one active region disposed on the surface of the working electrode, the at least one active region including a first enzyme, a second enzyme, and a polymer. The first enzyme and the second enzyme can interact cooperatively, the second enzyme is covalently bound to the polymer, and is non-reactive with the analyte. The method further 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 and generate a signal at the working electrode; and correlating the signal to the concentration of the analyte in the fluid.

[0083] According to more specific embodiments, when performing the method described above, an electron transfer agent can also be covalently bound to the polymer. Suitable electron transfer agents are described in more detail above. In some or other embodiments, particularly when a covalently bound electron transfer agent is present, the first enzyme is not covalently bound to the polymer in at least one active region.

[0084] In more specific embodiments, the ethanol detection method of the present disclosure may include exposing an analyte sensor to a fluid containing ethanol, particularly a biological fluid, wherein the analyte sensor includes at least a working electrode and a sensor tail including at least one active region disposed on the surface of the working electrode and containing alcohol oxidase, xanthine oxidase, catalase, a polymer, and an electron transfer agent. The electron transfer agent and xanthine oxidase are covalently bonded to the polymer, and the alcohol oxidase is not covalently bonded to the polymer. The alcohol oxidase and xanthine oxidase can interact cooperatively. The method further includes reacting ethanol with alcohol oxidase to form acetaldehyde; reacting acetaldehyde with xanthine oxidase to form acetic acid and generating a signal at the working electrode; and correlating the signal with the ethanol concentration in the fluid. According to some embodiments, the fluid can be a biological fluid, and the analyte sensor can be exposed to the biological fluid in vivo.

[0085] Two different enzymes within a single active region of an analyte sensor can cooperate and interact with each other to determine analyte concentration, but it should be understood that the enzymes can also function independently of each other to detect alternative analytes in other embodiments. For example, if the second enzyme of the analyte sensor described above is xanthine oxidase, instead of using the analyte sensor to detect ethanol, the analyte sensor can be used instead to detect any of a variety of substrates that have an affinity for xanthine oxidase. Alternative substrates for xanthine oxidase can include, for example, hypoxanthine, xanthine, uric acid, purines, pterins, and similar compounds. When the analyte sensor is used in this way, if alcohol is absent, alcohol oxidase remains unused and / or can be inactivated by acetaldehyde / hydrogen peroxide if these species are not removed by xanthine oxidase or other species. Thus, a sensor containing cooperative enzymes can also be considered capable of detecting multiple analytes, one analyte from a cooperative enzyme pair, and at least a second analyte from one of the members of the independently acting cooperative enzyme pair. Whether such a sensor assays a single analyte or multiple analytes can be determined based on the environment to which the sensor is exposed.

[0086] As noted above, multiple enzymes disposed in separate active regions can also cooperate and interact to facilitate the detection of a single analyte. In some cases, both of the multiple enzymes can be disposed directly on the surface of the working electrode, as described in more detail above. In an alternative analyte sensor configuration that includes multiple enzymes in separate active regions, one of the active regions can be isolated from the working electrode such that electron transfer to the working electrode occurs from only one of the active regions. That is, as described in further detail below, the active region isolated from the working electrode can facilitate an enzymatic reaction of the analyte of interest to produce a reaction product (substrate) that reacts with the enzyme of the active region that is in direct contact with the working electrode. Next, a signal associated with the enzymatic reaction occurring in the active region in direct contact with the working electrode provides the basis for detecting the analyte. The correlation between the signal and the analyte concentration can be achieved in a manner similar to that described in more detail above.

[0087] More specifically, FIG. 5E shows a cooperative enzyme reaction cycle associated with the detection of ethanol using glucose oxidase and xanthine oxidase in accordance with various embodiments of the present disclosure, which is further mediated by catalase when only xanthine oxidase or both xanthine oxidase and catalase are disposed on the surface of the working electrode. The cooperative enzyme reaction cycle shown in FIG. 5E is dependent on glucose and ethanol co-existing in the fluid with each other during the analysis, as described below. Since glucose is a ubiquitous biological nutrient, it often co-exists with other analytes when assaying biological fluids. However, if a particular fluid being analyzed lacks glucose, a particular embodiment of the present disclosure can be characterized by adding glucose to the fluid and using the cooperative enzyme reaction of glucose oxidase and xanthine oxidase to facilitate the detection of ethanol or another alcohol.

[0088] Before further describing the coenzyme reaction shown in FIG. 5E, an exemplary analyte sensor configuration characterized by at least one active region isolated from the working electrode will first be described in further detail. As described above, all of the analyte sensor configurations shown in FIGS. 2A-4 are characterized by one or more working electrodes having one or more active regions disposed directly on the surface of each working electrode. In contrast, FIGS. 6A, 6B, and 6C show diagrams of working electrodes 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 (spaced apart or remote). The working electrode configurations shown in FIGS. 6A, 6B, and 6C can substitute for any of the specific working electrode configurations shown in FIGS. 2A-4. That is, the working electrode configurations shown in FIGS. 6A, 6B, and 6C can be combined in any suitable manner with counter electrodes and / or reference electrodes, membranes, substrates, and similar structures within the analyte sensor.

[0089] As shown in FIG. 6A, the working electrode 400 has an active region 402 disposed directly on its surface. The active region 402 includes a first enzyme covalently bonded to a first polymer. Typically, an electron transfer agent is also present in the active region 402 and is also covalently bonded to the polymer. The active region 402 is covered by a membrane 404. The membrane 404 can also cover the surface of the working electrode 400, as well as other portions of the analyte sensor in which the working electrode 400 is present. The membrane 404 isolates the active region 406 from the working electrode 400, such that electron exchange between the two is impeded. The active region 406 includes a second enzyme covalently bonded to a second polymer, but no separate electron transfer agent is present. FIG. 6A shows the active region 406 disposed directly above the active region 402, and it should be understood that in alternative configurations compatible with the present disclosure, they can be laterally spaced apart from each other. A membrane 408 provides mass transfer limiting properties by covering the active region 406, and optionally other sensor components. Similarly, as shown in FIG. 6B, the membrane 404 does not necessarily extend the same lateral distance on the working electrode 400 as the membrane 408 does. In fact, the membrane 404 in FIG. 6B covers the active region 402, but only covers a portion of the surface of the working electrode 400, and the membrane 408 covers the active region 406, the surface of the membrane 404, and the remaining portion of the surface of the working electrode 400 not covered by the membrane 404. The active regions 402 and 406 can also be laterally offset from each other in some embodiments, as shown in FIG. 6C. Here, the active region 406 is again isolated from the working electrode 400 by the membrane 404.

[0090] The membrane 408 is permeable to the analyte and any additional components required to facilitate the enzymatic reaction in the active region 406. In contrast, the membrane 404 is permeable to the product formed in the active region 406. That is, the analyte reacts in the active region 406 to form a first product, which then diffuses through the membrane 404 and subsequently reacts further in the active region 402 to form a second product. Subsequently, the second product is detectable based on electron exchange with the working electrode 400.

[0091] Optionally, if glucose detection is desired, a lead 410 may be extended between the active regions 402 and 406. In an alcohol sensor characterized by detection based on the coenzyme reaction of glucose oxidase and xanthine oxidase, glucose oxidase is present in the active region 406 and xanthine oxidase is present in the active region 402. Continuing to refer to FIGS. 6A, 6B, and 6C and referring back to FIG. 5E, glucose oxidase is present in the active region 406 and converts exogenous glucose to D-glucono-1,5-lactone-1,5-dione and hydrogen peroxide. Different from the alcohol sensor (FIG. 5A) characterized by detection based on the coenzyme reaction between alcohol oxidase and xanthine oxide, catalase plays a more active role in the coenzyme reaction shown in FIG. 5E. That is, catalase reacts with hydrogen peroxide to form a catalase-hydrogen peroxide complex (the same peroxide removal function shown by catalase in the coenzyme reaction of alcohol oxidase and xanthine oxidase), and subsequently the complex reacts with ethanol to form acetaldehyde. The acetaldehyde formed in the active region 406 when ethanol is reacted with the catalase-hydrogen peroxide complex diffuses through the membrane 404, which separates the active region 406 from the active region 402. Alternatively, catalase may be present in the active region 402, in which case the hydrogen peroxide formed in the active region 406 diffuses through the membrane 404 into the active region 402, forms a catalase-hydrogen peroxide complex in the active region 402, and may oxidize ethanol to acetaldehyde in the active region 402. When acetaldehyde is formed in the active region 402, the coenzyme reaction may continue as shown in FIG. 5E. The membrane 404 may include crosslinked polyvinyl pyridine that is permeable to acetaldehyde. Next, acetaldehyde reacts with xanthine oxidase in the active region 402 to form acetic acid in the same manner as described above for FIG. 5A.

[0092] Accordingly, the alcohol sensor of the present disclosure includes at least a sensor tail including a working electrode; a first active region disposed on the surface of the working electrode, including 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 covering the first active region, including a first membrane polymer and being permeable to acetaldehyde; a second active region disposed on the first membrane, including glucose oxidase, catalase, and a second polymer, wherein the glucose oxidase is covalently bonded to the second polymer; and a second membrane covering the second active region, including a second membrane polymer and being permeable to glucose and alcohol. Here, the glucose oxidase and the xanthine oxidase can cooperate and interact to generate a signal proportional to the alcohol concentration at the working electrode. In a more specific embodiment, the alcohol can be ethanol.

[0093] According to some embodiments, the first membrane polymer and the second membrane polymer may be different from each other. According to some embodiments, the first membrane polymer can be crosslinked polyvinylpyridine. In an embodiment of the present disclosure, the crosslinked polyvinylpyridine easily permits the permeation of acetaldehyde. The second membrane polymer can be a crosslinked polyvinylpyridine-co-styrene polymer, wherein a part of the pyridine nitrogen atoms are functionalized with non-crosslinked poly(ethylene glycol) tails and a part of the pyridine nitrogen atoms are functionalized with alkylsulfonic acid groups. Such a second membrane polymer easily permits the permeation of both glucose and ethanol.

[0094] According to some embodiments, catalase is not covalently bonded to the first polymer or the second polymer in the first active region or the second active region. Catalase can be physically constrained within the first active region and the second active region by any one of the first polymer, the second polymer, the first membrane polymer, or the second membrane polymer.

[0095] Similarly, a method for analyzing ethanol or another alcohol using the aforementioned analyte sensor that includes cooperatively interacting glucose oxidase and xanthine oxidase can include oxidizing glucose with 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 xanthine oxidase to form acetic acid and generate a signal at a working electrode; and correlating the signal with the alcohol concentration in the fluid. According to various embodiments of the present disclosure, the fluid can include a biological fluid. The correlation between the signal and the alcohol concentration in the fluid can be performed using any suitable correlation technique outlined in more detail above.

[0096] In still other embodiments of the present disclosure, a plurality of enzymes can be disposed within the active regions of separate working electrodes. Thus, the signals associated with the enzyme reactions occurring within each active region can be measured separately by responding to each working electrode simultaneously or at different times. Next, the signals associated with each active region can be correlated with the concentration of a separate analyte.

[0097] As described above, a membrane (i.e., a mass transport limiting membrane) can cover one or more active regions of an analyte sensor to enhance biocompatibility and modify the analyte flux to the active regions. Such a membrane can be present in any of the analyte sensors disclosed herein. Since different analytes can exhibit different permeability values within a given membrane, an analyte sensor configured to analyze multiple analytes can exhibit different sensitivities to each analyte. One approach for addressing different sensitivity values can include utilizing different membrane thicknesses across each active region. While this approach is feasible, it can be difficult to implement from a manufacturing perspective. That is, it can be difficult to vary the membrane thickness at different locations using typical dip coating techniques used for membrane deposition. Another possible approach is to use active regions of different sizes for each analyte.

[0098] An analyte sensor having an active region configured to assay different analytes on separate working electrodes can address the aforementioned problems associated with different analyte sensitivities. That is, the following disclosure provides various ways in which the membrane permeability can be varied on each working electrode to equalize the analyte membrane permeability at each location. That is, the disclosure herein enables the independent variation of the permeability and sensitivity of the analyte at each working electrode. According to the disclosure herein, a mass transfer limiting membrane comprising two or more different membrane polymers can provide a more equalized analyte permeability at each working electrode. Specific membrane configurations that may be suitable for equalizing the analyte permeability on one or more working electrodes include bilayer membranes and mixed membranes, each of which comprises two or more different membrane polymers. Surprisingly, bilayer membranes and mixed membranes comprising membrane polymers that are individually unsuitable for promoting the permeability of a given analyte can provide satisfactory performance when arranged in a bilayer membrane or a mixed membrane, as described below. This approach may be advantageous compared to varying the size of the active region on each working electrode to provide an equivalent sensitivity value for each analyte.

[0099] Thus, in some embodiments, an analyte sensor characterized by two or more enzymes disposed on separate working electrodes can include a sensor tail comprising at least a first working electrode and a second working electrode, a first active region located on the surface of the first working electrode, a second active region located on the surface of the second working electrode, a multi-component membrane covering the first active region, and a homogeneous membrane covering the second active region. The first active region includes a first polymer and a first analyte-responsive enzyme that reacts with a first analyte, and the second active region 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 are reactive with different analytes. The multi-component membrane includes 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.

[0100] The specific configuration of the multi-component film described above may include a bilayer film in some embodiments, or a mixture of membrane polymers in other embodiments. Surprisingly, as will be described in more detail below, the bilayer film and the mixed film can function to equalize the permeability of the analyte.

[0101] The analyte sensor of the present disclosure having two different active regions disposed on separate working electrodes can use a sensor configuration similar to that described above in FIG. 3 or variations thereof. For example, in some embodiments, the counter / reference electrode can be replaced with a separate counter electrode and reference electrode within an analyte sensor having two or more working electrodes. Similarly, the layer configuration and arrangement within an analyte sensor having two different active regions disposed on separate working electrodes may differ from that shown in FIG. 3. Further details regarding the film arrangement on each active region are provided below with reference to FIG. 7.

[0102] According to a more specific embodiment of the present disclosure, an analyte sensor having a plurality of working electrodes may include an active region in which 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 in a first active region and a second analyte responsiveness covalently bonded to a polymer in a second active region. Again, in certain embodiments, the first analyte-responsive enzyme can be a glucose-responsive enzyme such as glucose oxidase, and the second analyte-responsive enzyme can be a lactate-responsive enzyme such as lactate oxidase.

[0103] In yet another more specific embodiment, an analyte sensor having a plurality of working electrodes may include a sensor tail configured for insertion into tissue. In some embodiments, the bilayer film can cover the first active region on one working electrode. The bilayer film includes a first film polymer and a second film polymer that are layered on top of each other on the active region. In more specific embodiments, the first film polymer can be disposed directly on the active region of the first working electrode, and the second film polymer can be disposed on top of the first film polymer to define the bilayer film. In such embodiments, the second film polymer is present in a homogeneous film located on the second working electrode. Such a bilayer configuration can be prepared, in some embodiments, by coating only the first film polymer on the first working electrode (e.g., by spray coating, painting, inkjet printing, roller coating, etc.), and then coating the second film polymer simultaneously on both working electrodes (e.g., by dip coating or a similar technique). In other embodiments, the bilayer film can be configured as described above with the first film polymer disposed on the second working electrode.

[0104] FIG. 7 shows an exemplary schematic view of a portion of an analyte sensor having two working electrodes and a bilayer film covering one of the two working electrodes, which is suitable for use in some embodiments of the disclosure herein. As shown in FIG. 7, the analyte sensor features a sensor tail 600 having working electrodes 614a and 614b disposed on opposite faces of a substrate 612. An active region 618a is disposed on working electrode 614a, and an active region 618b is disposed on working electrode 614b. Active regions 618a and 618b contain different analyte-responsive enzymes and are configured to assay for different analytes in accordance with the disclosure herein. FIG. 7 shows that active regions 618a and 618b are disposed substantially opposite each other with respect to substrate 612, but it should be understood that active regions 618a and 618b can be laterally spaced (shifted) from each other on opposite faces of substrate 612. The laterally spaced configuration of active regions 618a and 618b can be particularly advantageous for covering each of active regions 618a and 618b with a mass transport limiting membrane, as described below.

[0105] As further shown in FIG. 7, the active region 618a is covered by a membrane layer 620. The membrane layer 620 is a homogeneous membrane containing a single membrane polymer. The active region 618b is covered by a bilayer membrane 621 including a membrane layer 621a in direct contact with the active region 618b and a membrane layer 621b covering the membrane layer 621a. The membrane layers 621a and 621b contain different membrane polymers. As described above, in certain embodiments, the membrane layer 620 and the membrane layer 621b may contain the same membrane polymer.

[0106] According to one or more embodiments, an analyte sensor having a plurality of active regions on separate working electrodes, with one of the active regions covered by a bilayer membrane, may exhibit a normalized or independently variable analyte permeability. That is, the analyte sensor may have sensitivities closer to each other for two different analytes than in the absence of the bilayer membrane. In such an analyte sensor configuration, an active region covered by a homogeneous membrane (e.g., the membrane layer 620 in FIG. 7) may exhibit an analyte permeability characteristic of that particular membrane polymer for a first analyte. Surprisingly, the bilayer membrane (e.g., the bilayer membrane 621 in FIG. 7) may include a membrane polymer that does not adversely affect the permeability of a second analyte (i.e., a membrane polymer having a neutral effect on permeability), thereby allowing the other membrane polymer including the bilayer membrane to exhibit its characteristic permeability for the second analyte as if the first membrane polymer were not present. Thus, according to various embodiments, a membrane polymer having a neutral effect on permeability and a membrane polymer including a homogeneous membrane may constitute the same polymer.

[0107] In some or other particular embodiments, the membrane polymer having a neutral effect on permeability may include the inner layer of the bilayer membrane. Thus, according to such embodiments, the inner layer of the bilayer membrane and the homogeneous membrane may constitute the same membrane polymer. In other particular embodiments, the outer layer of the bilayer membrane and the homogeneous membrane may constitute the same membrane polymer.

[0108] In certain embodiments, 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. Thus, according to such embodiments, the first active region containing the glucose-responsive enzyme may be covered with a bilayer membrane, and the second active region containing the lactate-responsive enzyme may be overacted by a homogeneous (single-component membrane polymer) membrane. In an even more specific embodiment, the second active region may include a polymer, albumin, and a lactate-responsive enzyme covalently bound to the polymer. In an even more specific embodiment, the homogeneous membrane covering the second active region may include at least a crosslinked polyvinylpyridine homopolymer or copolymer, and the bilayer membrane covering the first active region may also include a polyvinylpyridine homopolymer or copolymer.

[0109] In other embodiments of the present disclosure, the multi-component membrane may include a mixture (homogeneous blend) of a first membrane polymer and a second membrane polymer. Such an analyte sensor configuration may be similar in appearance to that shown in FIG. 7, except that the bilayer membrane 621 is replaced with a mixed membrane including a homogeneous blend of two different membrane polymers. Similar to the analyte sensor including a bilayer membrane disposed on one of the active regions, a homogeneous membrane including one of the first membrane polymer or the second membrane polymer of the mixed membrane may cover the other active region on the second working electrode.

[0110] Similar to the bilayer membrane, a mixed membrane containing a membrane polymer that has a neutral effect on the permeability of the second analyte can allow the mixed membrane to exhibit a permeability characteristic of the other membrane polymer in the mixture with respect to the second analyte. Thus, according to various embodiments of the present disclosure, one of the membrane polymers of the homogeneous membrane and the membrane polymer of the mixed membrane can be selected such that the permeability of the second analyte through the mixed membrane is not substantially changed by that membrane polymer. In certain embodiments, the first active region can include a glucose-responsive enzyme such as glucose oxidase, and the second active region can include a lactate-responsive enzyme such as lactate oxidase. Thus, according to such embodiments, the first active region containing the glucose-responsive enzyme can be covered with a mixed membrane, and the second active region containing the lactate-responsive enzyme can be overacted by a homogeneous (single-component membrane polymer) membrane. In even more specific embodiments, the second active region can include a polymer, albumin, and a lactate-responsive enzyme covalently bonded to the polymer. In a more specific embodiment, the homogeneous membrane covering the second active region can include at least a crosslinked polyvinylpyridine homopolymer or copolymer, and the mixed membrane covering the first active region can also include a polyvinylpyridine homopolymer or copolymer.

[0111] As noted above, the bilayer membrane and the mixed membrane can equalize analyte permeability in the analyte sensor of the present disclosure, where two or more active regions can be spatially separated from each other and covered with different mass transfer limiting membranes. Specifically, the bilayer membrane and the mixed membrane of the present disclosure can equalize analyte permeability in an analyte sensor having separate working electrodes and including two or more active regions having different enzymes, with at least one active region disposed at each working electrode. Thus, 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.

[0112] Accordingly, a method for using an analyte sensor that includes two working electrodes can include exposing the analyte sensor to a fluid that includes at least one analyte. The analyte sensor includes a sensor tail that includes at least a first working electrode and a second working electrode. A first active region is disposed on the surface of the first working electrode, and a second active region is disposed on the surface of the second working electrode. The first active region includes a first polymer and a first analyte-responsive enzyme that reacts with a first analyte, and the second active region 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. A multi-component film covers the first active region, and a homogeneous film covers the second active region. The multi-component film includes at least a first film polymer and a second film polymer that are different from each other, and the homogeneous film includes one of the first film polymer or the second film polymer. The method further includes obtaining a first signal at a redox potential that is greater than or equal to that of the first active region, obtaining a second signal at a redox potential that is greater than or equal to that of the second active region, and correlating the first signal to the concentration of the first analyte in the fluid and the second signal to 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.

[0113] According to more specific embodiments, the first signal and the second signal can be measured at different times. Thus, in such embodiments, 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 a first channel and a second channel, in which case the potential can be applied to both electrodes simultaneously.

[0114] Embodiments disclosed herein include the following. A. An analyte sensor comprising two active regions having different analyte-responsive enzymes. The analyte sensor includes a sensor tail including at least a working electrode; and at least two active regions disposed on the surface of the working electrode, each active region including an analyte-responsive enzyme and a polymer, and the analyte-responsive enzymes in each active region being different. Here, 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, and a signal can be generated from the first active region independently of the generation of a signal from the second active region.

[0115] B. A method for assaying two or more analytes using a first active region and a second active region containing different analyte-responsive enzymes. The method includes exposing an analyte sensor to a fluid containing at least one analyte; the analyte sensor including a sensor tail including at least a working electrode and at least two active regions disposed on the surface of the working electrode, each active region including an analyte-responsive enzyme and a polymer; the analyte-responsive enzymes in each active region being different; each active region having a redox potential, the redox potential of the first active region being sufficiently separated from the redox potential of the second active region, and a signal being able to be generated from the first active region independently of the generation of a signal from the second active region; obtaining a first signal proportional to the concentration of a first analyte at a potential above the redox potential of the first active region; obtaining a second signal at a potential above the redox potential of the second active region, the second signal being a composite signal including 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 differential signal proportional to the concentration of a second analyte.

[0116] C. An analyte sensor comprising two or more enzymes that can cooperate and interact with each other. The analyte sensor includes a sensor tail including at least a working electrode; and at least one active region disposed on the surface of the working electrode, the at least one active region including a first enzyme, a second enzyme, and a polymer, the first enzyme and the second enzyme being able to cooperate and interact with each other; the first enzyme being able to convert an analyte into a first product, the second enzyme being able to convert the first product into a second product and generate a signal at the working electrode; the second enzyme being covalently bonded to the polymer and being non-reactive with the analyte.

[0117] D. A method for assaying an analyte using two or more enzymes that can cooperate and interact with each other. The method includes exposing an analyte sensor to a fluid containing the analyte; the analyte sensor including a sensor tail including at least a working electrode and at least one active region disposed on the surface of the working electrode, the at least one active region including a first enzyme, a second enzyme, and a polymer; the first enzyme and the second enzyme being able to cooperate and interact with each other, the second enzyme being covalently bonded to the polymer and being non-reactive 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 and generate a signal at the working electrode; and correlating the signal with the concentration of the analyte in the fluid.

[0118] E. An alcohol sensor. The alcohol sensor includes a sensor tail including at least a working electrode; and at least one active region disposed on the surface of the working electrode, the at least one active region including alcohol oxidase, xanthine oxidase, catalase, a polymer, and an electron transfer agent; the electron transfer agent and xanthine oxidase being covalently bonded to the polymer, the alcohol oxidase not being covalently bonded to the polymer; the alcohol oxidase and xanthine oxidase being able to cooperate and interact to generate a signal proportional to the alcohol concentration at the working electrode.

[0119] F. A method for detecting alcohol. This method includes exposing an analyte sensor to a fluid containing ethanol; the analyte sensor includes a sensor tail including at least a working electrode and at least one active region disposed on the surface of the working electrode, and the at least one active region includes alcohol oxidase, xanthine oxidase, catalase, a polymer, and an electron transfer agent; the electron transfer agent and xanthine oxidase are covalently bonded to the polymer, and alcohol oxidase is not covalently bonded to the polymer; and alcohol oxidase and xanthine oxidase can cooperate and interact with each other; reacting ethanol with alcohol oxidase to form acetaldehyde; reacting acetaldehyde with xanthine oxidase to form acetic acid and generating a signal at the working electrode; and correlating the signal with the ethanol concentration in the fluid.

[0120] G. An analyte sensor including two or more working electrodes covered with different mass transfer limiting membranes. The analyte sensor includes a sensor tail including 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 including 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 including 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 covering the first active region, the multi-component membrane including at least a first membrane polymer and a second membrane polymer that are different from each other; and a homogeneous membrane covering the second active region and having a composition different from that of the multi-component membrane, the homogeneous membrane including one of the first membrane polymer and the second membrane polymer.

[0121] H. A method for assaying two or more analytes using two working electrodes covered with different mass transfer limiting membranes. The method includes exposing an analyte sensor to a fluid containing at least one analyte; the analyte sensor includes a sensor tail including at least a first working electrode and a second working electrode; a first active region is disposed on the surface of the first working electrode, the first active region includes a first polymer and a first analyte-responsive enzyme that reacts with a first analyte, a second active region is disposed on the surface of the second working electrode, the second active region 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 a multi-component film covers the first active region, a homogeneous film covers the second active region, the multi-component film includes at least a first film polymer and a second film polymer that are different from each other, the homogeneous film includes one of the first film polymer or the second film polymer and has a different composition from the multi-component film; obtaining a first signal proportional to the concentration of the first analyte in the fluid at an oxidation-reduction potential equal to or higher than that of the first active region; obtaining a second signal proportional to the concentration of the second analyte in the fluid at an oxidation-reduction potential equal to or higher than that of the second active region; 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.

[0122] I. An alcohol sensor comprising glucose oxidase and xanthine oxidase that cooperate and interact. The alcohol sensor includes a sensor tail including at least a working electrode; a first active region disposed on the surface of the working electrode, the first active region including 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 covering the first active region, the first membrane including a first membrane polymer and being permeable to acetaldehyde; a second active region disposed on the first membrane, the second active region including glucose oxidase, catalase, and a second polymer; the glucose oxidase being covalently bonded to the second polymer; a second membrane covering the second active region, the second membrane including a second membrane polymer and being permeable to glucose and alcohol; and the glucose oxidase and the xanthine oxidase being able to cooperate and interact to generate a signal proportional to the alcohol concentration at the working electrode.

[0123] A method for detecting alcohol using a cooperative interaction between J. glucose oxidase and xanthine oxidase. The method includes exposing an analyte sensor to a fluid containing ethanol and glucose; the analyte sensor including a sensor tail comprising: at least a working electrode; a first active region disposed on the 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 covering 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 covering the second active region, the second membrane comprising a second membrane polymer and being permeable to glucose and alcohol; the ability of glucose oxidase and xanthine oxidase to cooperate and interact with each other; 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 and generating a signal at the working electrode; and correlating the signal with the alcohol concentration in the liquid.

[0124] Each of embodiments A and B may have one or more of the following additional elements in any combination: Element 1: The sensor tail is configured for insertion into tissue.

[0125] Element 2: The redox potential of the first active region is at least about 100 mV away from the redox potential of the second active region. Element 3: The first active region includes a first electron transfer agent, the second active region includes a second electron transfer agent, and the first and second electron transfer agents are different.

[0126] Element 4: 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. Element 5: The analyte-responsive enzyme in each active region is covalently bonded to the polymer.

[0127] Element 6: The analyte sensor further includes a mass transfer limiting membrane that covers at least at least two active regions. Element 7: At least one of the at least two active regions includes two or more analyte-responsive enzymes, and the two or more analyte-responsive enzymes cooperate and interact to generate a signal proportional to the concentration of a single analyte.

[0128] Element 8: The fluid is a biological fluid, and the analyte sensor is exposed to the biological fluid in vivo. Element 9: The mass transfer limiting membrane covers at least the at least two active regions.

[0129] Element 10: The analyte sensor includes glucose oxidase as the first enzyme and lactate oxidase as the second enzyme. Each of Embodiments C and D may have one or more of the following additional elements in any combination.

[0130] Element 11: The first enzyme is alcohol oxidase and the second enzyme is xanthine oxidase. Element 12: At least one active region further includes catalase.

[0131] Element 13: Catalase is not covalently bonded to the polymer. Element 14: Alcohol oxidase is not covalently bonded to the polymer. Element 15: The first enzyme is not covalently bonded to the polymer.

[0132] Element 16: At least one active region includes an electron transfer agent covalently bonded to the polymer. Element 17: The sensor tail is configured for insertion into tissue.

[0133] Element 18: The analyte sensor further includes a mass transfer limiting membrane that covers at least the at least one active region. Element 19: The fluid is a biological fluid, and the analyte sensor is exposed to the biological fluid in vivo.

[0134] Element 20: The mass transfer limiting membrane covers at least one active region. Each of Embodiments E and F may have one or more of the following additional elements in any combination.

[0135] Element 21: Catalase is not covalently bonded to the polymer. Element 22: The sensor tail is configured for insertion into tissue. Element 23: The alcohol sensor further includes a mass transfer limiting membrane that covers at least the at least one active region.

[0136] Element 24: The fluid is a biological fluid, and the analyte sensor is exposed to the biological fluid in vivo. Element 25: The mass transfer limiting membrane covers at least one active region.

[0137] Each of Embodiments G and H may have one or more of the following additional elements in any combination. Element 26: The multi-component membrane includes a bilayer membrane.

[0138] Element 27: The first membrane polymer is disposed directly on the first active region, and the second membrane polymer is disposed on the first membrane polymer to define a bilayer membrane, and the second membrane polymer is also present in the homogeneous membrane.

[0139] Element 28: The multi-component membrane includes a mixture of the first membrane polymer and the second membrane polymer. Element 29: The sensor tail is configured for insertion into tissue. Element 30: Each active region further includes an electron transfer agent covalently bonded to the polymer.

[0140] Element 31: The first analyte-responsive enzyme is covalently bound to the polymer in the first active region, and the second analyte-responsive enzyme is covalently bound to the polymer in the second active region. Element 32: The electron transfer agent is covalently bound to the polymer in each active region.

[0141] Element 33: The analyte-responsive enzyme in each active region is covalently bound to the polymer. Element 34: The fluid is a biological fluid, and the analyte sensor is exposed to the biological fluid in vivo.

[0142] Element 35: The first signal and the second signal are measured at different times. Element 36: The first signal and the second signal are measured simultaneously via the first channel and the second channel.

[0143] Each of Embodiments I and J may have one or more of the following additional elements in any combination. Element 37: Catalase is not covalently bound to the first polymer or the second polymer.

[0144] Element 38: The sensor tail is configured for insertion into tissue. Element 39: The first membrane polymer and the second membrane polymer are different from each other. Element 40: Here, the first membrane polymer includes crosslinked polyvinylpyridine.

[0145] Element 41: The fluid is a biological fluid, and the analyte sensor is exposed to the biological fluid in vivo. As non-limiting examples, exemplary combinations applicable to A - J include the following.

[0146] Analyte sensors of A combined 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 to 4; 2 and 5; 2 and 6; 2 and 7; 3 to 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 to 5; as well as 2, 5, and 6. Analyte sensors of B combined with elements 2 and 3; 2 to 4; 2 and 5; 2 and 6; 2 and 7; 2 and 8; 3 to 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 to 5; 2 to 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 to 8 and 9; as well as any one of 2 to 8 and 10.

[0147] 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 to 14; 11 to 14 and 16; 11 to 14 and 17; 11 to 14 and 18; 15 to 17; 15 to 18; and an analyte sensor of C in combination with 15, 17, and 18. 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 19; 11, 12, 14, and 20; 11 to 14; 11 to 14 and 19; 11 to 14 and 20; 11 to 14 and 16; 11 to 14, 16, and 19; 11 to 14, 16, and 20; 11 to 14 and 17; 11 to 14, 17, and 19; 11 to 14, 17, and 20; 11 to 14 and 18; 11 to 14, 18, and 19; 11 to 14, 18, and 20; 15 to 17; 15 to 17 and 19; 15 to 17 and 20; 15 to 18; 15 to 18 and 19; 15 to 18 and 20; 15, 17, and 18; 15, 17, 18, and 19; 15, 17, 18, and 20; any one of 11 to 16 and 19; and a method of D in combination with any one of 11 to 16 and 20.

[0148] Elements 21 and 22; 21 and 23; 22 and 23; and an analyte sensor of E in combination with 21 to 23. Elements 21 and 22; 21 and 23; 22 and 23; 21 to 23; 21 and 24; and a method of F in combination with 21 and 25.

[0149] An analyte sensor of G in combination with 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.

[0150] A method of H in combination with 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, 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.

[0151] An analyte sensor of I in combination with elements 37 and 38; 37 and 39; 37 and 40; 38 and 39; 38 and 40; and 39 and 40. A method of J in combination with elements 37 and 39; 37 and 40; 37 and 41; 39 and 40; 39 and 41; and 40 and 41.

[0152] Further embodiments disclosed herein include the following. A1: An analyte sensor having a multi - component film. The analyte sensor is a sensor tail configured to be inserted into tissue, the sensor tail including at least a working electrode; and first and second active regions disposed on the sensor tail and including at least two different enzymes for measuring the concentration of at least one analyte; the first active region being covered with a first membrane polymer and a second membrane polymer different from each other.

[0153] B1: An analyte sensor having two active regions on a working electrode and configured to detect different analytes. The analyte sensor is configured to be inserted into tissue and includes a sensor tail configured to include at least the working electrode; and at least two active regions disposed on the sensor tail, each active region including an enzyme, an electron transfer agent, and a polymer, the enzymes of each active region being different and responsive to different analytes; each active region having a redox potential, the redox potential of the first active region being sufficiently separated from the redox potential of the second active region such that a signal can be generated from the first active region independently of signal generation from the second active region.

[0154] Embodiment A1 can have one or more of the following additional elements in any combination. Element 1’: The first active region is covered with a mixture of a first membrane polymer and a second membrane polymer, and one of the first membrane polymer and the second membrane polymer covers the second active region as a homogeneous membrane.

[0155] Element 2’: The first active region is covered with a bilayer membrane including a first membrane polymer disposed on a second membrane polymer, and the second membrane polymer covers the second active region as a homogeneous membrane. Element 3’: The first active region includes a first enzyme of at least two different enzymes, and the second active region includes a second enzyme of at least two different enzymes.

[0156] Element 4’: The first enzyme is non-reactive with at least one analyte, and the first and second enzymes can cooperate and interact to generate a signal proportional to the concentration of the at least one analyte.

[0157] Element 5’: The second enzyme can convert at least one analyte into a product that reacts with the first enzyme, such that the first enzyme can react with the product to generate a signal at the working electrode.

[0158] Element 6': The first enzyme is xanthine oxidase, the second enzyme is glucose oxidase, and at least one of the first active region and the second active region further contains catalase.

[0159] Element 7': Catalase is present in the first active region. Element 8': The first active region is disposed directly on the working electrode and further contains an electron transfer agent. Element 9': 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.

[0160] Element 10': The sensor tail includes a first working electrode and a second working electrode. The first active region is disposed on the surface of the first working electrode, the second active region is disposed 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.

[0161] Element 11': Each of the first and second active regions has a redox potential. The redox potential of the first active region is sufficiently separated from the redox potential of the second active region, enabling the generation of a signal from the first active region independently of the generation of a signal from the second active region.

[0162] Element 12': The redox potential of the first active region is at least about 100 mV separated from the redox potential of the second active region. Element 13': The signal from the first active region corresponds to the first analyte concentration, and the signal from the second active region corresponds to the second analyte concentration.

[0163] Element 14': The first active region contains a first electron transfer agent, and the second active region contains a second electron transfer agent different from the first electron transfer agent. Embodiment B1 can have one or more of the following additional elements in any combination.

[0164] Element 15’: The redox potential of the first active region is at least about 100 mV away from the redox potential of the second active region. Element 16’: The first active region contains a first electron transfer agent, and the second active region contains a second electron transfer agent different from the first electron transfer agent.

[0165] Element 17’: The first and second active regions are covered with a mass transfer limiting membrane, the first active region is covered with a single membrane polymer, and the second active region is covered with two or more different membrane polymers.

[0166] To facilitate a better understanding of the embodiments described herein, the following examples of various representative embodiments are provided. The following examples should not be construed as limiting or defining the scope of the present invention.

[0167] Example Example 1: Detection of glucose and lactate using an analyte sensor having two different active regions on a single working electrode. Two solutions containing different poly(vinylpyridine) - bound transition metal complexes were prepared. The structure of the polymer of the first solution is shown in Formula 1, and the structure of the polymer of the second solution is shown in Formula 2. Further details regarding these polymers are provided in commonly owned U.S. Patent No. 6,605,200, which is incorporated by reference above. The subscripts of each monomer represent exemplary atomic ratios.

[0168] [Chemical formula]

[0169] The redox potential of the polymer of Formula 1 relative to the Ag / AgCl reference was -50 mV, and the redox potential of the polymer of Formula 2 relative to the same reference was +220 mV (a separation of 270 mV, see Figure 8). In addition to the transition metal complexes that function as electron transfer agents respectively, the polymer of Formula 1 contained glucose oxidase (GOX) covalently bonded thereto, and the polymer of Formula 2 contained lactose oxidase (LOX) covalently bonded thereto after deposition and curing on the working electrode. Crosslinking was achieved using polyethylene glycol diglycidyl ether (PEGDE400). Solutions containing the polymer of Formula 1 and the polymer of Formula 2 were prepared as specified in Tables 1 and 2 below.

[0170]

Table 1

[0171]

Table 2

[0172] To deposit each active region, approximately 20 nL of each solution was deposited onto the carbon working electrode to form two separate and distinct spots, each having an area of approximately 0.1 mm 2 One spot contained the glucose oxidase formulation and the other spot contained the lactose oxidase formulation. After deposition, the working electrode was cured at 25 °C overnight.

[0173] After curing, a film was deposited on the working electrode. The film polymer was filed on June 13, 2018, titled "Temperature-Insensitive Membrane Materials and Analyte As described in U.S. Provisional Patent Application No. 62 / 684,438 entitled "(Sensors Containing the Same)", it was a polyvinylpyridine copolymer having polyether side chain functional groups free of amines. The deposition of the film was achieved by dip-coating the electrode three times in a solution containing 4 mL of the film polymer (120 mg / mL) and 0.35 mL of PEG1000 (200 mg / mL). The film can be deposited by alternately using spray coating, screen printing, or a similar process. After deposition, the electrode was cured overnight at 25 °C and then further cured at 56 °C for 2 days in a dried vial.

[0174] After manufacture, the electrode was analyzed by cyclic voltammetry in a buffer solution free of both glucose and lactate. The resulting cyclic voltammogram is shown in Figure 8. Since there was no current contribution from either glucose or lactate, Figure 8 shows the anodic and cathodic peaks characteristic of the two osmium complexes. The redox potentials reported above were calculated from the average of the cathodic and anodic peaks of each osmium complex.

[0175] To analyze glucose and lactate, the electrode was placed at a potential above the average redox potential of the first polymer, specifically +40 mV (E1 in Figure 8). At this potential, oxidation of the osmium complexes in the first polymer and glucose may occur, but oxidation of the osmium complexes in the second polymer or lactate does not occur. To oxidize both osmium complexes, as well as both glucose and lactate, the electrode was placed at a potential above the average redox potential of the second polymer, specifically +250 mV (E2 in Figure 8).

[0176] The analysis of glucose and lactate was carried out by immersing the electrodes in a buffer containing 5 mM glucose and 5 mM lactate, and E1 and E2 potentials were applied continuously. Figure 9 shows four replicates of the electrode response in a 5 mM glucose / 5 mM lactate buffer when cycled between E1 and E2. As shown, the current at E1 is about 5 nA, which is due to the oxidation of glucose, and the current at E2 is about 10.5 nA, which is due to the oxidation of both glucose and lactate. Taking the difference in the currents measured at E1 and E2 gives a contribution of about 5.5 nA at E2 due to the oxidation of lactate. The unknown glucose and lactate concentrations can be analyzed similarly by comparison with a lookup table or calibration curve.

[0177] Example 2A: Detection of ethanol using an analyte sensor having two different enzymes (XOX / AOX) operating cooperatively on a single working electrode. A spotting solution of the formulation shown in Table 3 was prepared. All components were dissolved in 10 mM HEPES buffer at pH 8. Crosslinking was achieved using polyethylene glycol diglycidyl ether.

[0178]

Table 3

[0179] Approximately 15 nL of the solution was deposited as a single spot having an area of about 0.05 mm 2 on the carbon working electrode. After deposition, the working electrode was cured at 25 °C overnight. After curing, a poly(4-vinylpyridine) (PVP) membrane was deposited on the working electrode from a coating solution containing 100 mg / mL PVP and 100 mg / mL PEGDE400. Deposition of the membrane was achieved by dip-coating the electrode three times in the coating solution. After deposition, the electrode was cured at 25 °C overnight and then further cured at 56 °C for 2 days in a dry vial. Spray coating, screen printing, or a similar process can be used alternately to deposit the membrane.

[0180] Ethanol analysis was performed by immersing the electrodes in ethanol-containing PBS solutions each containing various concentrations of ethanol. FIG. 10 shows three replicates of the response of an electrode containing both alcohol oxidase and xanthine oxidase at the detection spot upon exposure to various ethanol concentrations. As shown, the current response increased within a few minutes after exposure to the new ethanol concentration and then stabilized. FIG. 11A shows an exemplary plot of the average current response versus ethanol concentration. FIG. 11B shows data corresponding to a single sensor. As shown, the sensor response was approximately linear over the ethanol concentration range of 0 to 10 mM.

[0181] Example 2B: Detection of ethanol using an analyte sensor having two different enzymes (XOX / GOX) operating in concert on a single working electrode. A first spotting solution having the formulation shown in Table 4 was prepared. All components were dissolved in 10 mM HEPES buffer at pH 8. Crosslinking was achieved using polyethylene glycol diglycidyl ether.

[0182]

Table 4

[0183] Approximately 15 nL of the first spotting solution was deposited onto the carbon working electrode as a single spot (XOX spot) having an area of approximately 0.05 mm 2 . After deposition, the working electrode was cured at 25° C. overnight.

[0184] After curing, a poly(4-vinylpyridine) (PVP) membrane was deposited onto the working electrode and the XOX spot from a coating solution containing 100 mg / mL PVP and 100 mg / mL PEGDE400. Deposition of the membrane was achieved by dip-coating the electrode in the coating solution three times. Spray coating, screen printing, or similar processes can be used alternately to deposit the membrane. After deposition, the electrode was cured at 25° C. overnight and then further cured at 56° C. for 2 days in a dry vial.

[0185] A second spotting solution having the formulation shown in Table 5 was prepared. All components were dissolved in 10 mM HEPES buffer at pH 8. Crosslinking was achieved using polyethylene glycol diglycidyl ether.

[0186] [Table 5]

[0187] Approximately 15 nL of the second spotting solution was deposited onto the PVP membrane from above as a single spot (GOX spot) having an area of approximately 0.05 mm 2 . After deposition, curing was carried out overnight at 25 °C.

[0188] After curing, a second membrane was deposited onto the GOX spot and the PVP membrane. The membrane polymer in this case was a crosslinked polyvinylpyridine-co-styrene polymer, with some of the pyridine nitrogen atoms functionalized with non-crosslinked poly(ethylene glycol) tails and some of the pyridine nitrogen atoms functionalized with alkylsulfonic acid groups. The membrane at this position was deposited from a coating solution containing 35 mg / mL of crosslinked polyvinylpyridine-co-styrene polymer and 100 mg / mL PEGDE400. Deposition of the membrane was achieved by dip-coating the electrode into the coating solution three times. Spray coating, screen printing, or similar processes can be used alternately to deposit the membrane. After deposition, the electrode was cured overnight at 25 °C and then further cured at 56 °C for 2 days in a drying vial.

[0189] Ethanol analysis was performed by immersing the electrodes in ethanol-containing PBS solutions each containing ethanol at various concentrations. FIG. 12A shows two replicates of the response when an electrode containing glucose oxidase and xanthine oxidase, layered in separate active regions and separated by a membrane, was exposed to various ethanol concentrations. Catalase is within the active region together with glucose oxidase. As shown, the current response increased within a few minutes after exposure to the new ethanol concentration and then stabilized. Good reproducibility was obtained for the two replicates observed. FIG. 13 shows an exemplary plot of the average current response versus ethanol concentration. The curve shape was similar to that obtained using AOX / XOX (FIG. 11A, Example 2A).

[0190] FIG. 12B shows comparative response data when exposed to various ethanol concentrations between electrodes containing glucose oxidase and xanthine oxidase, layered in separate active regions and separated by a membrane. Catalase is present in the active region separately. As shown, the sensor response was greater when catalase was included in the active region containing xanthine oxidase.

[0191] Example 3: Comparison of the response of the analyte sensor to lactate in the presence of various mass transfer limiting membranes. In this example, the following membrane formulations were coated onto a carbon working electrode containing lactate oxidase in its active region. The active region was deposited using a lactate oxidase formulation as described in Example 1. However, the polymer of formula 1 was used instead of the polymer of formula 2 in the formulation, and the concentration was adjusted to the concentrations specified in Table 6 below.

[0192]

Table 6

[0193] Deposition and curing of the active region were carried out as described in Example 1. However, instead of a single spot having an area of 0.1 mm in Example 1 2 each having an area of 0.01 mm 2Six spots having the area of were deposited. Unless otherwise stated below, the film deposition was carried out by dip coating (immersion of the electrode 1 - 5 times and waiting time of about 10 minutes between immersions). After completion of dip coating, the film was cured at 25°C for 24 hours and then cured at 56°C for 48 hours in a dry vial.

[0194] The electrode response was 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 was raised to +40 mV vs. Ag / AgCl and then the current was continuously monitored. To measure the response at various lactate concentrations, sodium lactate was added to the buffer in 1 mM increments up to a maximum of 5 mM. To determine the response stability, the current was measured over a long period such as 2 weeks in 5 mM sodium lactate.

[0195] Membrane Polymers 1A and 1B: The first membrane polymers tested were cross-linked polyvinylpyridine-co-styrene polymers, where some of the pyridine nitrogen atoms were functionalized with non-cross-linked poly(ethylene glycol) tails and some of the pyridine nitrogen atoms were functionalized with alkylsulfonic acid groups. To affect the cross-linking of this membrane polymer, two different cross-linking agents were used, namely glycerol triglycidyl ether (Gly3 - formulation 1) and polyethylene glycol diglycidyl ether 400 (PEGDGE400 - formulation 2). Formulation 1 contained 4 mL of the membrane polymer (140 mg / mL) in 80:20 ethanol:HEPES buffer, 1 mL of Gly3 (35 mg / mL) in 80:20 ethanol:HEPES buffer, and 0.0132 mL of aminopropyl-terminated polydimethylsiloxane (PDMS) (100 mg / mL) in ethanol. Formulation 2 contained 4 ml of the membrane polymer (140 mg / ml) in 80:20 ethanol:HEPES buffer, 0.2 mL of PEGDGE400 (100 mg / ml) in 80:20 ethanol:HEPES buffer, and 0.0132 ml of aminopropyl-terminated polydimethylsiloxane (PDMS) (100 mg / mL) in ethanol. The corresponding cross-linked polymers are shown herein as Polymer 1A and 1B, respectively.

[0196] Figure 14 shows an exemplary plot of the response of electrodes covered with Polymers 1A and 1B to a 5 mM lactate solution. As shown, neither formulation provided a stable sensor response over time. The sensor current provided by Polymer 1A (formulation 1) decreased slowly over a two-week measurement period, whereas the sensor current provided by Polymer 1B (formulation 2) first increased during the first week of lactate exposure and then decreased. In contrast, both of these membranes provided a stable response in the presence of the glucose analyte (data not shown).

[0197] Membrane Polymer 2: The second membrane polymer tested was polyvinylpyridine (PVP) crosslinked with polyethylene glycol diglycidyl ether 1000 (PEGDGE1000). This membrane polymer is designated as Polymer 2 herein. The membrane formulation (Formulation 3) contained 4.3 mL of PVP (100 mg / mL) in 80:20 ethanol:HEPES buffer, 0.25 mL of PEGDGE1000 (200 mg / mL) in 80:20 ethanol:HEPES buffer, and 0.0132 mL of PDMS (100 mg / mL) in ethanol.

[0198] Figure 15 shows an exemplary plot of 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 did not provide a stable current response over time. The response decreased significantly in the first 48 hours, followed by relatively stable operation. Furthermore, the sensitivity was well below the target value of approximately 1 nA / mM. Similar to Polymers 1A and 1B, Polymer 2 provided a stable current response in the presence of glucose analyte (data not shown).

[0199] Membrane Polymer 3: The third membrane polymer tested was polyvinylpyridine (PVP) crosslinked with PEGDGE400. This membrane polymer is designated as Polymer 3 herein. The membrane formulation (Formulation 4) contained 4.3 mL of PVP (100 mg / mL) in 80:20 ethanol:HEPES buffer, 0.23 mL of PEGDGE400 (100 mg / mL) in 80:20 ethanol:HEPES buffer, and 0.0132 mL of PDMS (100 mg / mL) in ethanol.

[0200] Figure 16 shows an exemplary plot of the response of an electrode coated with Polymer 3 (Formulation 4) to a 5 mM lactate solution. Different from Polymer 2 crosslinked with the high molecular weight variant of the same crosslinking agent, Polymer 3 surprisingly resulted in a stable current response over time. Furthermore, the current responded rapidly and achieved a stable current when the amount of added lactate was increased in 1 mM increments (Figure 17).

[0201] Membrane Polymer 4: The fourth membrane polymer tested was PVP containing 3-4 wt% non-crosslinked PEG side chains, which was then crosslinked with PEGDGE1000. Thus, the tested membrane polymer contained both non-crosslinked PEG chains and crosslinked PEG1000 chains. This membrane polymer is designated as Polymer 4 herein. The membrane formulation (Formulation 5) contained 4.3 mL of polymer (100 mg / mL) in 80:20 ethanol:HEPES buffer, 0.025 mL of PEGDGE1000 (200 mg / mL) in 80:20 ethanol:HEPES buffer, and 0.0132 mL of PDMS (100 mg / mL) in ethanol.

[0202] Figure 18 shows an exemplary plot of the response of an electrode coated with Polymer 4 (Formulation 5) to a 5 mM lactate solution. Unlike Polymers 2 and 3, Polymer 4 surprisingly provided a stable current response over time. Furthermore, the current responded rapidly and achieved a stable value when the amount of lactate added was increased in 1 mM increments (Figure 19).

[0203] Two-layer membranes containing Polymer 2 and 1B or Polymer 2 and 1A: Formulation 3 (Polymer 2) was coated onto the electrode surface by repeating the dip-coating operation. Spray coating, screen printing, or a similar process can be used alternately to deposit the membrane. Next, Formulation 2 (Polymer 1B) was coated onto the deposited crosslinked PVP layer by repeating the dip-coating operation. There was a 10-minute waiting time between successive immersions. After all dipping operations were completed, the sensor was cured at 25 °C for 24 hours and then at 56 °C for 48 hours in a drying vial. As shown above, none of these membrane polymers provided satisfactory performance when used alone.

[0204] Figure 20 shows an exemplary plot of the response of an electrode covered with a bilayer membrane containing a lower layer of cross-linked PVP (Polymer 2) and an upper layer of cross-linked Polymer 1B to a 5 mM lactate solution. Unlike Polymer 1B or PVP cross-linked with the same cross-linking agent (Polymer 2), neither of these polymers alone provides acceptable performance. Nevertheless, the bilayer membrane containing these membrane polymers surprisingly provided a current response that was stable over time with an acceptable level of sensitivity. The response data in Figure 20 were for electrodes immersed twice in Formulation 3 (Polymer 2) and four times in Formulation 2 (Polymer 1B).

[0205] The amount (thickness) of each membrane polymer in the bilayer membrane can potentially change the sensor performance as shown below for Polymer 2 and Polymer 1A. Thus, the Gly3 cross-linked variant of Polymer 1B (i.e., Polymer 1A) may provide acceptable performance when combined in a bilayer membrane with Polymer 2, even though neither of these polymers alone provided acceptable performance.

[0206] Figure 21 shows an exemplary plot of the response of an electrode covered with a bilayer membrane containing a lower layer of cross-linked PVP (Polymer 2) and an upper layer of cross-linked Polymer 1A to a 5 mM lactate solution. The electrodes were dip-coated various numbers of times with Formulation 1 (Polymer 1A) and Formulation 3 (Polymer 2). The cross-linking agent for PVP (Polymer 2) in this case remained PEGDGE1000, while the cross-linking agent for Polymer 1A was Gly3. This indicates that this cross-linking agent is also suitable for use in bilayer membrane configurations. As shown in Figure 21, by dip-coating the electrodes twice with Formulation 3 and four times with Formulation 1, a good balance of sensitivity and stable current response was obtained. Changing the number of dip-coating operations changed the thickness of each component of the bilayer membrane and the mutual mass ratio of the membrane polymers. As shown in Figure 21, if the PVP layer is too thin (0 or 1 immersion of Polymer 2), the sensitivity is high but the response stability is low. On the other hand, if it is too thick (3 or more immersions), in some cases the electrode has low sensitivity and low response stability.

[0207] Mixed membrane containing membrane polymers 1B and 3: The mixed membrane formulation (formulation 6) was prepared by mixing 1.5 mL of PVP (100 mg / mL) in 80:20 ethanol:HEPES buffer, 2.5 mL of the copolymer (140 mg / mL) used to prepare formulations 1A and 1B in 80:20 ethanol:HEPES buffer, 0.175 mL of PEGDGE400 (100 mg / mL) in 80:20 ethanol:HEPES buffer, and 0.0132 mL of PDMS (100 mg / mL) in ethanol. Thus, after crosslinking, formulation 6 contains polymer 1B and polymer 3, which are crosslinked with PEG400 respectively.

[0208] Figure 22 shows an exemplary plot of the response of an electrode covered with a mixed membrane containing crosslinked PVP (polymer 3) and crosslinked polymer 1B to a 5 mM lactate solution. Similar to the bilayer membrane containing one of the same components (polymer 1B), the mixed membrane provided a stable current response over time and an acceptable level of sensitivity. Furthermore, the current responded rapidly and reached a stable value when the amount of additional lactate was increased in 1 mM increments (Figure 23).

[0209] Figure 24 shows an exemplary plot of the response of sensors covered with mixed membranes containing crosslinked PVP (polymer 3) and crosslinked polymer 1B in various ratios. As shown in Figure 24, the sensitivity increases with a higher amount of polymer 1B, but the response stability decreases.

[0210] Example 4: Performance of a sensor containing two working electrodes covered with a bilayer mass transfer limiting membrane. In this example, the first working electrode containing glucose oxidase and the second working electrode containing lactate oxidase were covered with a bilayer membrane. The active region containing glucose oxidase was deposited using the glucose oxidase formulation as described in Example 1 (Table 1). The active region containing lactate oxidase was deposited using the lactate oxidase formulation as described in Example 3 (Table 6). The deposition and curing of the active region were performed as described in Example 1. However, instead of a single spot with an area of 0.1 mm in Example 1 2 each had an area of 0.01 mm2 Five spots having the area of 2 were deposited. Membrane polymer formulations corresponding to Formulation 2 (Polymer 1B) and Formulation 4 (Polymer 3) from Example 3 were used to deposit the bilayer membranes in this example. That is, Polymer 3 was deposited on the second working electrode characterized by lactate oxidase. Selective deposition onto the second working electrode was achieved by a modified slot coating procedure. Next, curing was carried out at 25 °C for 24 hours. Thereafter, the entire assembly (i.e., both working electrodes, the PVP coating on the second working electrode, as well as the counter electrode and reference electrode) was dip-coated with Formulation 2. Curing was carried out again at 25 °C for 24 hours, followed by baking at 56 °C for 48 hours in a dry environment. Thus, a homogeneous membrane was deposited on the first working electrode (glucose-responsive), and a bilayer membrane was deposited on the second working electrode (lactate-responsive). Crosslinked PVP (Polymer 3) was in contact with the lactate-responsive active region on the second working electrode.

[0211] This sensor was used to simultaneously assay 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 at 37 °C for 2 weeks. In this test, the sensor was held at +40 mV versus Ag / AgCl. Figure 25 shows an exemplary plot of the sensor response for each working electrode upon exposure to 30 mM glucose and 5 mM lactate. As shown, the response of the sensor remained very stable over the observation period.

[0212] Next, glucose and lactate were added stepwise to 100 mM PBS at 37 °C to measure the responsiveness of the sensor to each analyte. In this test, the sensor was held at +40 mV versus Ag / AgCl again. Glucose was added in the concentration range of 0 - 30 mM, and lactate was added in the concentration range of 0 - 5 mM. Figure 26 shows an exemplary plot of the sensor response to various concentrations of glucose and lactate. As shown in Figure 26, the sensor response was rapid for both analytes and remained stable at a given analyte concentration.

[0213] ​ Example 5: Detection of ketones using an analyte sensor having coordinately interacting diaphorase and β-hydroxybutyrate dehydrogenase. In this example, the membrane formulations shown in Table 7 below were coated on a carbon working electrode. On the working electrode, depositions were made to place six spots each having an area of approximately 0.01 mm 2 . After deposition, the working electrode was cured overnight at 25 °C. Thereafter, a PVP membrane was applied to the working electrode via dip coating using a coating solution formulated with 4 mL of 100 mg / mL PVP, 0.2 mL of 100 mg / mL PEGDGE400, and 0.0132 mL of 100 mg / mL PDMS. Membrane curing was carried out at 25 °C for 24 hours, followed by 48 hours at 56 °C in a drying vial.

[0214] [Table 7]

[0215] The electrode was immersed in 100 mM PBS buffer (pH = 7.4) at 33 °C and ketone analysis was performed by introducing various amounts of β-hydroxybutyric acid (addition of β-hydroxybutyrate at a total of 0, 1, 2, 3, 4, 6, and 8 mM). Figure 27 shows four replicates of the response of an electrode containing diaphorase, NAD + , and β-hydroxybutyrate dehydrogenase when exposed to various β-hydroxybutyric acid concentrations. As shown, the current response increased within a few minutes after exposure to the new β-hydroxybutyric acid concentration and then stabilized. Figure 28 shows an exemplary plot of the average current response versus β-hydroxybutyric acid concentration for the electrode of Figure 27. The ketone sensor also showed a stable response over an extended measurement time, as shown in Figure 29. Figure 29 shows an exemplary plot of the current response of the electrode of Figure 27 when exposed to 8 mM β-hydroxybutyric acid in 100 mM PBS at 33 °C for 2 weeks. The average signal loss during the measurement period was only 3.1%.

[0216] Example 6: Comparison of lactate sensor responses with various sensor configurations. To assay the performance of lactate-responsive sensors characterized by various permutations of formulations, two different lactate oxidase / polymer formulations for active region deposition and two different membrane polymer formulations for mass transport limiting membrane deposition were prepared. The details of the formulations and the processes used to prepare the analyte sensors are shown below. Generally, the analyte sensors were prepared in a manner similar to that described above.

[0217]

Table 8

[0218]

Table 9

[0219] To deposit each active region, approximately 20 nL of each solution was deposited onto the carbon working electrode to form six individual spots, each having an area of approximately 0.01 mm 2 . Formulation A was applied four times and Formulation B was applied six times to form the spots. After deposition, the working electrode was cured overnight at 25 °C. Formulation A corresponds to that used to deposit the active region of the glucose-responsive analyte sensor, except that lactate oxidase was used instead of glucose oxidase.

[0220] Formulations for mass transport limiting membrane deposition: Membrane polymer formulations were prepared in aqueous solution formulations as specified in Tables 10 and 11 below.

[0221]

Table 10

[0222]

Table 11

[0223] Using dip coating, a mass transfer limiting membrane was deposited on each active region prepared as described above. Formulation C was deposited using 4 dips and Formulation D was deposited using 44 dips. An approximately 10-minute waiting time was used between dips. After completion of the dip coating, the membrane was cured at 25 °C for 24 hours and subsequently cured at 56 °C for 48 hours in a drying vial. Spray coating, screen printing, or a similar process can be used alternately to deposit the mass transfer limiting membrane. Formulation C corresponds to that used to deposit a mass transfer limiting membrane within a glucose-responsive analyte sensor.

[0224] A lactate-responsive analyte sensor was prepared using the deposition conditions specified above. All possible combinations of active regions and mass transfer limiting membranes were prepared and 8 sensors were fabricated 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 working potential held at +40 mV versus Ag / AgCl. The tested combinations of active regions and mass transfer limiting membranes are shown in Table 12. The test results are shown in Figure 30.

[0225]

Table 12

[0226] As shown in Figure 30, lactate-responsive analyte sensors having active regions and mass transfer limiting membranes formulated similarly to those that worked well in glucose-responsive analyte sensors (Group 1) yielded inadequate performance when exposed to lactate. As shown, the signal intensity was less than 0.5 nA for all samples tested. This is an undesirably low value for a functional lactate-responsive sensor. In Formulation C (Group 2), when polyvinylpyridine and a different crosslinking agent were used instead of polyvinylpyridine-co-styrene and the Gly3 crosslinking agent, the signal intensity was even lower.

[0227] Furthermore, as shown in FIG. 30, the incorporation of human serum albumin significantly improved the sensor performance. For example, sample group 3 showed significantly higher signal intensity than that achieved with either sample of group 1 or group 2. However, there was significant variation in the initial signal intensity among the samples in this group (>4 nA variation). Furthermore, the signal intensity steadily decreased from the initially observed maximum signal intensity. Due to the variability of the response and the low signal stability over time, it is similarly considered unlikely that this combination of sample groups is suitable for a viable lactate-responsive analyte sensor.

[0228] Surprisingly, the combination of the active region containing human serum albumin and the mass transfer limiting membrane containing crosslinked polyvinylpyridine homopolymer (group 4) resulted in an acceptable combination of high signal intensity and extended signal stability over time. As shown in FIG. 30, all replicate sensors in group 4 had initial signal intensities that clustered within 1 nA of each other between 4 nA and 5 nA. This level of signal intensity and variability is within the range where a commercially viable lactate-responsive analyte sensor can be developed. Furthermore, the signal intensity changed by less than a few tenths of an nA over 190 hours of signal observation. This is also within a range that may be suitable for the development of a commercially viable sensor.

[0229] As shown in FIG. 31, the current observed for the sensors in group 4 responded rapidly and reached a stable value when increasing amounts of lactate were added in 1 mM increments to a PBS solution initially free of lactate.

[0230] Unless otherwise specified, all numbers expressing quantities etc. in this specification and the related claims should be understood as being modified in all cases by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximations that may vary depending on the desired characteristics sought to be obtained by the embodiments of the present invention. At a minimum, and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be construed in light of the reported significant digits and by applying ordinary rounding techniques.

[0231] Analyte sensor ignition lock Vehicle failsafes such as ignition locks may be used to prevent an operator from operating a vehicle when there is a malfunction or when the vehicle is not in a state where it can be safely operated. Operating a vehicle in a malfunctioning state can pose a significant risk to the operator and the general 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 by the use of alcohol. Such a locking device connects a breath alcohol analyzer or an optical sensor to the vehicle's ignition system, and the driver must pass a blood alcohol concentration test before starting the vehicle.

[0232] 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, since other impairments and conditions can also afflict the operator, it is necessary to closely monitor to ensure that the operator does not operate the vehicle when impaired. For example, an operator driving while hypoglycemic due to diabetes may experience dizziness, confusion, headache, loss of consciousness, seizures, and delayed reflexes, any of which can endanger the operator's life and the lives of those inside or near the vehicle.

[0233] Analyte monitoring systems have been developed to facilitate long-term monitoring of analytes in body fluids (e.g., blood). Some analyte monitoring systems are designed to detect and monitor blood glucose levels, which is useful for the treatment of diabetic conditions. However, other analyte monitoring systems are designed to detect and monitor other analytes present in the operator's body fluid, and abnormal analyte levels detected by the operator may indicate that the operator is not fit to safely operate a vehicle at present.

[0234] The following description relates to an analyte monitoring and vehicle control system used to prevent vehicle operation when the operator's analyte level exceeds a predetermined threshold. By appropriately deploying the sensor control device 102 (FIG. 1), the user can rationally track and monitor the levels and trends of body fluid analytes. When some analyte levels exceed a specific threshold, there may be a physical or cognitive impairment that prevents the user from safely operating the vehicle. In such cases, the user needs to take appropriate measures to bring the analyte level back to a safe range before attempting to operate the vehicle. However, in some cases, the user may feel that there is no problem operating the vehicle at all, but even so, they may have an unsafe analyte level that can suddenly trigger the onset of a dangerous physical disorder. In such cases, it may be advantageous to install a fail-safe system that prevents or warns of the possibility of the user operating the vehicle and putting themselves and others at risk.

[0235] FIG. 32 is a schematic diagram of an exemplary analyte monitoring and vehicle control system 3200 according to one or more embodiments of the present disclosure. As shown, the analyte monitoring and vehicle control system 3200 (hereinafter, “system 3200”) includes a sensor control device 102, which is deployed on a user or “operator” 3202, or can be delivered to a target monitoring location on the body of the operator 3202, such as the back of the arm. As described above, the sensor control device 102 includes a sensor 104 (FIG. 1), which, when appropriately deployed, is transdermally disposed within the skin to detect and monitor an analyte present in the body fluid of the operator 3202. An adhesive patch 105 (FIG. 1) applied to the bottom of the sensor control device 102 adheres to the skin and secures the sensor control device 102 in a predetermined position during operation.

[0236] The system 3200 is described herein as including an on-body sensor control device 102 for detecting and reporting analyte levels, but the system 3200 can alternatively incorporate an in vitro analyte sensor (e.g., a self-monitoring blood glucose “SMBG” meter) without departing from the scope of the present disclosure. Accordingly, the term “sensor control device” should be interpreted herein to include not only an on-body sensor system as primarily described above, but also a conventional handheld sensor system.

[0237] As shown, system 3200 can further include a reader device 120, and sensor control device 102 can communicate with reader device 120 via a local communication path or link to automatically, periodically, or as needed by operator 3202 provide analyte concentration data. Reader device 120 can communicate with a control module 3204, which communicates with the electrical system of vehicle 3206 and is powered by the vehicle battery or, alternatively, by a separate battery. In such embodiments, data transmitted from sensor control device 102 to reader device 120 can then be transmitted by reader device 120 to control module 3204 for processing. However, in other embodiments, sensor control device 102 can communicate directly with control module 3204 via any wireless communication protocol such as BLUETOOTH®. In such embodiments, reader device 120 may or may not be necessary in system 3200.

[0238] In the illustrated embodiment, vehicle 3206 is depicted as an automobile. However, as used herein, the term "vehicle" is used in a broad sense and is meant to include any type of conveyance that can be operated by a human user or "operator," including, but not limited to, any type of automobile, truck, sport utility vehicle, aircraft, ship, spacecraft, and / or any other means of transportation, or combinations thereof. Examples of vehicle 3206 include, but are not limited to, any type of automobile, truck, sport utility vehicle, aircraft, ship, spacecraft, and / or any other means of transportation, or combinations thereof.

[0239] The control module 3204 may include a communication interface for communicating information with the sensor control device 102 and / or the reader device 120. In the case of an exemplary BLUETOOTH (registered trademark) compatible sensor control device 102 and / or reader device 120, when the sensor control device 102 approaches the vehicle 3206, it can enter a pairing mode. During pairing, the control module 3204 can 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 can automatically detect the presence of the sensor control device 102 and enable communication therebetween or with the reader device 120.

[0240] In some embodiments, the control module 3204 can communicate with a vehicle user interface 3208 included in the vehicle 3206, such as an infotainment system, a touch screen display, or an information display. In such embodiments, the control module 3204 can communicate visually with the operator 3202 via the vehicle user interface 3208 and can also communicate aurally with the operator 3202 via an audio speaker included in the vehicle 3206. However, in other embodiments, the control module 3204 can be configured to communicate with the reader device 120 so as to be able to communicate with the operator 3202.

[0241] As shown, control module 3204 can be, or can include, a computer system 3210 configured or programmed to control various operations and / or systems of vehicle 3206 based on real-time measured analyte levels of operator 3202 obtained by sensor control device 102. The operation of vehicle 3206 is controlled, disabled, or altered by disabling one or more critical systems of vehicle 3206 or by activating a warning system within vehicle 3206. When the real-time measured analyte levels of operator 3202 are within a predetermined safe range, it may be considered safe for operator 3202 to operate vehicle 3206. However, if the real-time measured analyte levels of operator 3202 are outside a predetermined safe range or exceed a predetermined threshold, computer system 3210 can be programmed to control, disable, or alter the operation of vehicle 3206.

[0242] In some embodiments, for example, computer system 3210 can be configured to disable various critical vehicle systems when the detected analyte levels of operator 3202 are outside a predetermined range or exceed a predetermined threshold, and thus, when identifying operator 3202 as impaired from safely operating vehicle 3206, gradually and safely disable the operation of the vehicle. Critical vehicle systems of vehicle 3206 that can be disabled include the ignition system (e.g., energy switching / control system), the transmission system (or gearbox), the fuel system, the energy supply system (e.g., battery, capacitor, conversion / reaction battery, etc.). When an elevated or decreased (unsafe) analyte level is detected, computer system 3210 can prevent critical vehicle systems from functioning or operating. As a result, operator 3202 cannot start or operate vehicle 3206, thereby preventing operator 3202 from endangering themselves and / or others.

[0243] In other embodiments, or in addition thereto, computer system 3210 may be configured to activate various non-critical vehicle systems when the detected analyte level of operator 3202 exceeds or is above a predetermined threshold. Non-critical vehicle systems that may be activated include, for example, vehicle horns, vehicle lights, or audible warning systems installed in vehicle 3206. In such embodiments, activation of non-critical vehicle systems can alert law enforcement and others (e.g., operators of adjacent vehicles, bystanders, pedestrians, etc.) of operator 3202, who may be driving in an impaired state, and thus enable legal action and notify others of potentially dangerous situations to quickly address related issues.

[0244] In yet other embodiments, or in addition thereto, computer system 3210 may be configured to automatically call one or more emergency contacts when the analyte level of operator 3202 is outside a predetermined safe operating range or exceeds a predetermined threshold. In such embodiments, computer system 3210 can operate via reader device 120 (e.g., a cellular phone) or a cellular or satellite communication system (e.g., OnStar®) incorporated into vehicle 3206. In other embodiments, or in addition thereto, computer system 3210 may 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 operator 3202 is outside a predetermined safe operating range or exceeds a predetermined threshold. Examples of emergency contacts include, but are not limited to, a spouse, a parent, a healthcare provider (such as a physician), a hospital, 911, or any combination thereof.

[0245] In some embodiments, system 3200 may further include one or more proximity sensors 3212 configured to detect the presence of operator 3202, and more specifically, sensor control device 102. In such embodiments, proximity sensors 3212 may be configured to monitor the overall area of driver's seat 3214 within vehicle 3206. If sensor control device 102 is detected within the area of driver's seat 3214 by proximity sensors 3212, it may provide a positive indication that operator 3202 is in driver's seat 3214 and may be attempting to operate vehicle 3206. In such a case, a signal may be sent to control module 3204 to alert computer system 3210 that operator 3202 is in vehicle 3206 and may be attempting to operate vehicle 3206. If the real-time measured analyte level of operator 3202 is within a predetermined safe range or below a predetermined level, computer system 3210 may enable operator 3202 to operate vehicle 3206. However, if the real-time measured analyte level of operator 3202 is outside a predetermined safe range or exceeds a predetermined threshold, computer system 3210 may control, disable, or modify the operation of vehicle 3206 as outlined above. As will be appreciated, proximity sensors 3212 may be advantageous in preventing operation of vehicle 3206 only when an impaired operator 3202 is in driver's seat 3214 and ready to operate vehicle 3206. As a result, a user wearing sensor control device 102 may ride in vehicle 3206 as a passenger in any condition without affecting the operation of control module 3204 or vehicle 3206.

[0246] In some embodiments, the control module 3204 may further include a vehicle state detection module 3216 configured to detect the current state of the vehicle 3206, including whether the vehicle 3206 is currently moving or stationary. Further, the vehicle state detection module 3216 may be configured to determine whether a motor within the vehicle 3206 is currently operating or stopped. In one or more embodiments, the vehicle state detection module 3216 can provide a state signal to the control module 3204, and the control module 3204 can use the state signal to determine which vehicle operations to activate or deactivate when the operator 3202's real-time measured analyte level is outside a predetermined safe range or exceeds a predetermined threshold. For example, if the state signal indicates that the vehicle 3206 is stationary, the control module 3204 can deactivate the vehicle fuel system, transmission system, ignition system, or any combination thereof. In contrast, if the state signal indicates that the vehicle 3206 is moving, the control module 3204 can activate the vehicle horn, flash the vehicle lights, or emit a warning sound indicating that the operator 3202 is not normal to the operator 3202 and / or people around the operator 3202.

[0247] In some embodiments, when the operator 3202 enters the vehicle 3206 or when the control module 3204 pairs with the sensor control device 102 and / or the reader device 120, an app may be launched on the reader device 120 or the vehicle user interface 3208. And a digital dashboard showing the current analyte level, trends, historical data, and predicted analyte levels may be displayed on the reader device 120 and / or the vehicle user interface 3208. However, if the current analyte level is outside a predetermined safe operating range, the computer system 3210 may be programmed to disable one or more critical vehicle systems to prevent the operator 3202 from operating the vehicle 3206. In such embodiments, a visual or audible warning may be issued by the control module 3204 to notify the operator 3202 of the reason why the vehicle 3206 will not start. More specifically, a visual warning (e.g., a written message) may be generated and displayed on the reader device 120 or the vehicle user interface 3208, or an audible warning (e.g., an audio message) may be communicated through a speaker in the reader device or the vehicle 3206.

[0248] 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 operation of the vehicle 3206 may be blocked until the current analyte level is obtained. If the current analyte level is within the safety limit, the computer system 3210 may enable the operation of the vehicle 3206. In some aspects, and 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 (e.g., after 1 hour, 2 hours, 5 hours, etc.).

[0249] In some embodiments, the control module 3204 can be configured to issue visual or auditory recommendations or instructions to the operator 3202 that may help return the measured analyte level to a safe range. In such embodiments, such visual or auditory recommendations can prompt the user to take some action that may result in returning the analyte level to a safe range. Further, in some embodiments, the operator 3202 may be able to communicate verbally with the control module 3204 by issuing a verbal response or command. This can prove advantageous in preventing distraction of the operator of the vehicle 3206.

[0250] In some embodiments, the settings of the control module 3204 can be customized by the operator 3202 such that the user can make an informed decision after a non-safe analyte level is detected and a visual or auditory warning is issued by the control module 3204. More specifically, in at least one embodiment, the control module 3204 can include a bypass function that allows the operator 3202 to operate the vehicle 3206 even if a non-safe analyte level is measured. In such embodiments, the operator 3202 can operate the vehicle 3206 by acknowledging that the operator 3202 may be operating the vehicle 3206 in an impaired or dangerous health condition.

[0251] In some embodiments, computer system 3210 may be configured or programmed to calculate a prediction timeline if the analyte level of operator 3202 deviates from a predetermined safe range or may exceed a predetermined threshold. In such embodiments, control module 3204 may be configured to issue a visual or auditory warning to operator 3202 indicating approximately how much time operator 3202 has until reaching an unsafe analyte level and a potentially dangerous medical condition can occur. Multiple warnings may be provided to indicate that the operator has left a specific time increment before reaching an unsafe analyte level. For example, visual or auditory warnings may be issued when an unsafe analyte level is likely to be reached within one hour, within 30 minutes, within 10 minutes, within 5 minutes, within 1 minute, and any time increment therebetween. Further, a visual or auditory warning may be issued if the operator's analyte level reaches an unsafe level or exceeds a predetermined threshold.

[0252] In some embodiments, when an unsafe analyte level is measured while the operator 3202 is operating the vehicle 3206, the control module 3204 may be configured to issue one or more warnings (visual or audible) to warn the operator 3202 about the unsafe analyte level. In some cases, the volume of the stereo within the vehicle 3206 can be automatically decreased to enable the operator 3202 to hear the audible warning. In such embodiments, the control module 3204 may be configured to propose one or more corrective actions to the operator 3202. Examples of corrective actions include, but are not limited to, decelerating and stopping the vehicle 3206, identifying the location of a nearby convenience store or pharmacy and driving there, and identifying the location of a nearby hospital or medical facility. If the vehicle 3206 is an autonomous vehicle and the current analyte level potentially places the operator 3202 in a dangerous condition, the control module 3204 can automatically direct the vehicle 3206 to a medical facility for treatment. Alternatively, or in addition, the control module 3204 can gradually decelerate or limit the speed of the vehicle 3206 when an unsafe analyte level is detected, thus stopping the operator 3202 and allowing the operator 3202 to address the problem before continuing to operate the vehicle 3206.

[0253] System 3200 may be useful in several different scenarios to protect the operator 3202 during operation and / or people around the operator 3202. In some applications, System 3200 may be voluntarily incorporated by the operator to detect impairments in real time. In other applications, System 3200 may be required by the owner of the vehicle 3206 to detect impairments of the operator 3202. In such applications, the owner of the vehicle 3206 can be a transportation company or a trucking company. In yet other applications, System 3200 may be legally imposed on the operator 3202 to detect impairments.

[0254] The embodiments disclosed herein include the following. An analyte monitoring and vehicle control system including a sensor control device having a sensor for detecting and monitoring one or more analytes present in an operator's body, and a control module in communication with the sensor control device and an electrical system of a vehicle, wherein the control module includes a computer system programmed to receive and process data provided by the sensor control device, and wherein vehicle operation is controlled or disabled by the computer system when a real-time measured analyte level of the operator exceeds a predetermined safety threshold.

[0255] L. Detecting and monitoring one or more analytes present in an operator's body using a sensor control device having a sensor, receiving and processing data provided by the sensor control device in a control module in communication with the sensor control device and an electrical system of a vehicle; and controlling or disabling vehicle operation using a computer system of the control module when a real-time measured analyte level of the operator exceeds a predetermined safety threshold.

[0256] Each of Embodiments K and L may have one or more of the following additional elements in any combination: Element 1: The sensor control device is coupled to an operator and is placed transcutaneously under the operator's skin to detect and monitor an analyte present in the operator's body fluid. Element 2: The sensor control device includes an in vitro analyte sensor. Element 3: The sensor control device further comprises a reader device that receives data from the sensor control device and transmits the data to a control module. Element 4: The vehicle includes a transporter selected from the group consisting of an automobile, a self-driving vehicle, a truck, a sports utility vehicle, an aircraft, a ship, a spacecraft, or any combination thereof. Element 5: The sensor control device pairs with the control module for communication when the operator approaches the vehicle. Element 6: The vehicle further comprises a vehicle user interface included in the vehicle and communicating with the control module. Element 7: The operation of the vehicle is disabled by disabling one or more critical systems of the vehicle, and the critical systems are selected from the group consisting of an ignition system, a transmission system, a fuel system, and an energy supply system. Element 8: 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: The vehicle further comprises one or more proximity sensors installed in the vehicle to monitor the area of the driver's seat and detect the presence of the operator. Element 10: The control module further includes a vehicle state detection module that detects the current state of the vehicle. Element 11: The control module generates a visual or audible warning that can be perceived by the operator when the operator's real-time measured analyte level is outside a predetermined safety threshold. Element 12: Visual or audible warnings are generated at specific time increments before reaching an unsafe analyte level. Element 13: The visual or audible warning includes one or more proposed corrective actions communicated to the operator. Element 14: The control module includes a bypass function that enables the operator to operate the vehicle when the operator's real-time measured analyte level exceeds a predetermined threshold.

[0257] Element 15: Further includes receiving data from a sensor control device and transmitting the data to a control module using a reader device that communicates with the sensor control device and the control module. Element 16: Disabling the operation of the vehicle includes disabling one or more critical systems of the vehicle, and the critical systems are selected from the group consisting of an ignition system, a transmission system, a fuel system, and an energy supply system. Element 17: 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: Further includes monitoring the area of the driver's seat of the vehicle using one or more proximity sensors attached to the vehicle and detecting the presence of the operator. Element 19: Further includes detecting the current state of the vehicle using a vehicle state detection module included in the control module. Element 20: Further includes generating a visual or audible warning perceptible to the operator using the control module when the real-time measured analyte level of the operator exceeds a predetermined threshold.

[0258] One or more exemplary embodiments incorporating various features are presented herein. For clarity, not all features of a physical implementation are described or shown in this application. In developing a physical implementation incorporating embodiments of the present invention, it will be understood that numerous implementation-specific decisions must be made in order to achieve the developer's goals, such as compliance with system-related, business-related, government-related, and other constraints. These vary by implementation and, in some cases, by circumstance. Although the developer's efforts may be time-consuming, such efforts will be routine for those of ordinary skill in the art and will be to the benefit of this disclosure.

[0259] Although various systems, tools, and methods are described herein in terms of "including" various components or steps, the systems, tools, and methods can also be "consisting essentially of" or "consisting of" various components and steps.

[0260] As used herein, the phrase "at least one" preceding a series of items and accompanied by the term "and" or "or" which separates any of the items, modifies the entire list rather than each member of the list (i.e., each item). The phrase "at least one of" allows the meaning of including at least one of any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. By way of example, the 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.

[0261] Accordingly, the disclosed systems, tools, and methods are well suited to achieve the recited objectives and advantages, as well as those inherent therein. Since the teachings of the present disclosure can be modified and implemented in different equivalent manners that will be apparent to those skilled in the art having the benefit of the teachings herein, the specific embodiments disclosed above are merely illustrative. Further, it is not intended to be limited to the details of the structure or design shown herein, except as described in the appended claims. Thus, it is apparent that the specific exemplary embodiments disclosed above can be changed, combined, or modified, and all such variations are considered to be within the scope of the present disclosure. The systems, tools, and methods exemplarily disclosed herein can be appropriately implemented without elements specifically disclosed herein and / or in the absence of any optional elements disclosed herein. The systems, tools, and methods are described from the perspective of "including" various components or steps, but the systems, tools, and methods can also "consist essentially of" or "consist of" various components and steps. All numbers and ranges disclosed above may vary somewhat. Whenever a numerical range with a lower limit and an upper limit is disclosed, any number and any included range within that range are specifically disclosed. In particular, all range values disclosed herein (in the form of "about a to about b", or equivalently "about a to b", or equivalently "about a~b") should be understood as describing all numbers and ranges included in a broader range of values. Also, unless expressly and clearly defined by the patentee, the terms of the claims have their ordinary and original meaning. In case of any conflict in the usage of words or terms between this specification and one or more patent documents or other documents that can be incorporated herein by reference, the definitions according to this specification shall be adopted.

Claims

**Claim 1** An analyte sensor, comprising a sensor tail configured to be inserted into tissue, the sensor tail including at least a working electrode; and first and second active regions disposed on the sensor tail, the first and second active regions including at least two different enzymes for measuring the concentration of at least one analyte; The first active region is covered with a first membrane polymer and a second membrane polymer different from each other, the analyte sensor. **Claim 2** The first active region is covered with a mixture of a first membrane polymer and a second membrane polymer, and one of the first membrane polymer and the second membrane polymer covers the second active region as a homogeneous membrane, the analyte sensor according to claim 1. **Claim 3** The first active region is covered with a bilayer membrane including a first membrane polymer disposed on the second membrane polymer, and the second membrane polymer covers the second active region as a homogeneous membrane, the analyte sensor according to claim 1. **Claim 4** The first active region includes a first enzyme among at least two different enzymes, and the second active region includes a second enzyme among at least two different enzymes, the analyte sensor according to claim 1. **Claim 5** The first enzyme is non-reactive with the at least one analyte, and the first and second enzymes can cooperate and interact to generate a signal proportional to the concentration of the at least one analyte, the analyte sensor according to claim 4. **Claim 6** The second enzyme can convert the at least one analyte into a product that reacts with the first enzyme, so that the first enzyme can react with the product to generate a signal at the working electrode, the analyte sensor according to claim 5. **Claim 7** The first enzyme is xanthine oxidase, the second enzyme is glucose oxidase, and at least one of the first active region and the second active region further includes catalase, the analyte sensor according to claim 5. **Claim 8** The analyte sensor according to claim 7, wherein catalase is present in the first active region. **Claim 9** The first active region is disposed directly on the working electrode and further includes an electron transfer agent, the analyte sensor according to claim 5. **Claim 10** 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, the analyte sensor according to claim 5.

11. The sensor tail includes a first working electrode and a second working electrode. The first active region is disposed on the surface of the first working electrode, and the second active region is disposed 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. The analyte sensor according to claim 4.

12. 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 enable the generation of a signal from the first active region independently of the generation of a signal from the second active region. The analyte sensor according to claim 4.

13. The redox potential of the first active region is at least about 100 mV away from the redox potential of the second active region. The analyte sensor according to claim 12.

14. The signal from the first active region corresponds to the first analyte concentration, and the signal from the second active region corresponds to the second analyte concentration. The analyte sensor according to claim 12.

15. The first active region includes a first electron transfer agent, and the second active region includes a second electron transfer agent different from the first electron transfer agent. The analyte sensor according to claim 12.

16. An analyte sensor, A sensor tail configured to be inserted into tissue and including at least a working electrode; and At least two active regions disposed on the sensor tail, each active region including at least two active regions including an enzyme, an electron transfer agent, and a polymer, The enzymes of each active region are different and are responsive to different analytes; 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 enable the generation of a signal from the first active region independently of the generation of a signal from the second active region. An analyte sensor.

17. The redox potential of the first active region is at least about 100 mV away from the redox potential of the second active region. The analyte sensor according to claim 16.

18. The first active region includes a first electron transfer agent, and the second active region includes a second electron transfer agent different from the first electron transfer agent. The analyte sensor according to claim 16.

19. The first and second active regions are covered with a mass transfer limiting membrane, the first active region is covered with a single membrane polymer, and the second active region is covered with two or more different membrane polymers. The analyte sensor according to claim 16.

20. A control module that communicates with an analyte sensor and an electrical system of a vehicle, further comprising a computer system programmed to receive and process data provided by the analyte sensor. The analyte sensor according to claim 16, wherein when the operator's real-time measured analyte level exceeds a predetermined safety threshold, the operation of the vehicle is controlled or disabled by the computer system.

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