Multienzyme analyte sensors.

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 technology]

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

[0002] Any analyte may be suitable for physiological analysis, provided that the appropriate chemistry for sensing the analyte can be identified. To this end, amperometric sensors configured to assay glucose in vivo have been developed and improved in recent years. Other analytes commonly 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, etc.

[0003] Analyte monitoring of an individual may occur periodically or continuously over a period of time. Periodic analyte monitoring may occur by taking samples of bodily fluids, such as blood, at set time intervals and analyzing them ex vivo. Continuous analyte monitoring may occur using one or more sensors that remain at least partially implanted within the individual's tissue, such as intradermally, subcutaneously, or intravenously, such that analysis may be performed in vivo. The implanted sensors may collect analyte data at any prescribed rate depending on the individual's particular health needs and / or previously measured analyte levels.

[0004] Periodic in vitro analyte monitoring may be sufficient to determine the physiological status of many individuals. However, in vitro analyte monitoring may be inconvenient or painful for some individuals. Furthermore, if analyte measurements are not taken in a timely manner, there is no way to recover lost data.

[0005] Continuous analyte monitoring with an in vivo implanted sensor 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. Although continuous analyte monitoring with an implanted sensor may be advantageous, there are challenges associated with these types of measurements. Intravenous analyte sensors have the advantage of providing analyte concentrations directly from the blood, but are invasive and may be painful for an individual to wear, especially over extended periods of time. Subcutaneous, interstitial, or dermal analyte sensors are often less painful for an 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, often using enzymes to provide analytical specificity. However, due to physiological interactions between various combinations of analytes, in certain cases, the analysis of multiple analytes 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 may be inconvenient as it requires an individual to wear multiple analyte sensors. Furthermore, multiple analyte sensors may represent an unacceptable cost burden for individuals or insurance companies. There is also an increased likelihood that one of the independent analyte sensors will fail during such a sensing protocol.

[0007] The in vivo analyte sensor may also include a membrane disposed over at least the implanted 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 area of ​​the analyte sensor. That is, the membrane may function as a mass-transport limiting membrane. The use of a mass-transport limiting membrane to restrict analyte access to the active area of ​​the sensor can avoid overloading (saturation) of the sensor, thereby improving detection performance and accuracy. Such membranes are highly specialized in restricting mass transport of a particular analyte, while allowing other substances to permeate the membrane at significantly different rates. Different membrane permeabilities of various potential analytes are a significant barrier to developing analyte sensors configured for analysis of multiple analytes. That is, different membrane permeability values ​​can result in significantly different sensitivities to multiple analytes, thereby complicating the analysis. Differential sensitivity to multiple analytes may be partially offset by using active areas of different sizes (e.g., smaller active areas for more sensitive / permeable analytes and larger active areas for less sensitive / permeable analytes), but this approach presents 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 as exclusive embodiments. The disclosed subject matter is capable of considerable modification, alteration, combinations, and equivalents in form and function without departing from the scope of the present disclosure. [Figure 1] 1 shows a diagram of an exemplary sensing system that can incorporate an analyte sensor of the present disclosure. [Figure 2A] FIG. 1 shows a diagram of an exemplary two-electrode analyte sensor configuration having a single working electrode suitable for use in some embodiments disclosed herein. [Figure 2B]FIG. 1 shows a diagram of an exemplary three-electrode analyte sensor configuration having a single working electrode suitable for use in some embodiments disclosed herein. [Figure 2C] Same as above. [Diagram 3] FIG. 1 shows a diagram of an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode suitable for use in some embodiments disclosed herein. [Figure 4] 1 illustrates an exemplary analyte sensor configuration suitable for use in some embodiments disclosed herein, in which two distinct active areas are disposed on the surface of a single working electrode. [Figure 5A] 1 illustrates a coordinated enzymatic reaction cycle associated with ethanol detection using alcohol oxidase and xanthine oxidase disposed directly on a working electrode according to various embodiments of the present disclosure. [Figure 5B] 1 shows a coordinated enzymatic reaction cycle associated with ketone detection using β-hydroxybutyrate dehydrogenase, nicotinamide adenine dinucleotide, and diaphorase disposed directly on a working electrode according to various embodiments of the present disclosure. [Figure 5C] 1 shows a coordinated enzymatic reaction cycle associated with ketone detection using β-hydroxybutyrate dehydrogenase, nicotinamide adenine dinucleotide, NADH oxidase, and superoxide dismutase disposed directly on a working electrode according to various embodiments of the present disclosure. [Figure 5D] 1 shows a coordinated enzymatic reaction cycle associated with ketone detection using β-hydroxybutyrate dehydrogenase, nicotinamide adenine dinucleotide, and poly-1,10-phenanthroline-5,6-dione disposed directly on a working electrode according to various embodiments of the present disclosure. [Figure 5E] 1 shows a coordinated enzymatic reaction cycle associated with ethanol detection using glucose oxidase, catalase, and xanthine oxidase, where glucose oxidase is remote from the working electrode and xanthine oxidase is located directly on the working electrode, according to various embodiments of the present disclosure. [Figure 6A]FIG. 1 shows a diagram of an exemplary working electrode in which a first active area is disposed directly on the surface of the working electrode and a second active area is separated from the working electrode by a membrane, suitable for use in some embodiments disclosed herein. [Figure 6B] Same as above. [Figure 6C] FIG. 1 shows a diagram of an exemplary working electrode in which a first active area and a second active area are laterally spaced apart from each other on the working electrode, with one of the active areas separated from the working electrode by a membrane. [Figure 7] FIG. 1 shows an exemplary schematic diagram of a portion of an analyte sensor suitable for use in some embodiments disclosed herein, the analyte sensor having two working electrodes and featuring a bilayer membrane covering one of the two working electrodes. [Figure 8] 1 shows exemplary cyclic voltammograms obtained in analyte-free buffer using a working electrode containing two different osmium complexes as electron transfer mediators. [Figure 9] Four replicates of the electrode response in 5 mM glucose / 5 mM lactate buffer are shown when the electrode of FIG. 8 is cycled between E1 and E2 potentials. [Figure 10] Three replicates of the response of an electrode containing alcohol oxidase and xanthine oxidase together in the active area upon exposure to various ethanol concentrations are shown. [Figure 11A] 11 shows an exemplary plot of average current response versus ethanol concentration for the electrodes of FIG. 10. [Figure 11B] 1 shows an exemplary plot of current response versus ethanol concentration for a single electrode. [Figure 12A] Two replicates of the response of an electrode containing glucose oxidase and xanthine oxidase layered into separate active regions and separated by a membrane upon exposure to various ethanol concentrations, with catalase present in the active region containing glucose oxidase. [Figure 12B]1 shows comparative response data upon exposure to various ethanol concentrations between electrodes containing glucose oxidase and xanthine oxidase layered in separate active regions and separated by a membrane, with catalase present separately in the active region. [Figure 13] 13 shows an exemplary plot of average current response versus ethanol concentration for the electrodes of FIG. 12. [Figure 14] 1 shows an exemplary plot of the response of electrodes coated with polymers 1A and 1B to a 5 mM lactate solution. [Figure 15] 1 shows an exemplary plot of the response of an electrode covered with polymer 2 to a 5 mM lactate solution. [Figure 16] 1 shows an exemplary plot of the response of an electrode covered with polymer 3 to a 5 mM lactate solution. [Figure 17] 1 shows an exemplary plot of the response of an electrode covered with polymer 3 to lactate solutions having various lactate concentrations. [Figure 18] 1 shows an exemplary plot of the response of an electrode covered with polymer 4 to a 5 mM lactate solution. [Figure 19] 1 shows an exemplary plot of the response of an electrode covered with polymer 4 to lactate solutions having various lactate concentrations. [Figure 20] 1 shows an exemplary plot of the response of an electrode covered with a bilayer membrane comprising a bottom layer of cross-linked PVP (Polymer 2) and a top layer of cross-linked Polymer 1B to a 5 mM lactate solution. [Figure 21] 1 shows an exemplary plot of the response of an electrode covered with a bilayer membrane comprising a bottom layer of cross-linked PVP (Polymer 2) and a top layer of cross-linked Polymer 1A to a 5 mM lactate solution, where the electrode was dip-coated various times with Formulation 1 and Formulation 3. [Figure 22] 1 shows an exemplary plot of the response of an electrode covered with a blend film containing cross-linked PVP (Polymer 2) and cross-linked Polymer 1B to a 5 mM lactate solution. [Diagram 23] 1 shows an exemplary plot of the response of an electrode covered with a blend film containing cross-linked PVP (Polymer 2) and cross-linked Polymer 1B to various lactate concentrations. [Figure 24] 1 shows exemplary plots of the response of an electrode coated with a blend film containing various ratios of cross-linked PVP (Polymer 2) and cross-linked Polymer 1B. [Diagram 25] 1 shows an exemplary plot of the response of a sensor containing two working electrodes to a 30 mM glucose / 5 mM lactate solution, where the lactate-responsive working electrode is covered with a bilayer membrane and the glucose-responsive working electrode is covered with a homogeneous membrane. [Figure 26] 1 shows exemplary plots of the response of a sensor containing two working electrodes to solutions containing various concentrations of glucose and lactate, where the lactate-responsive working electrode is covered with a bilayer membrane and the glucose-responsive working electrode is covered with a homogeneous membrane. [Figure 27] Shown are four replicates of the response of an electrode containing diaphorase, NAD+, and β-hydroxybutyrate dehydrogenase when exposed to various β-hydroxybutyrate concentrations. [Figure 28] 28 shows an exemplary plot of average current response vs. β-hydroxybutyric acid concentration for the electrodes of FIG. 27. [Figure 29] 28 shows an exemplary plot of the current response of the electrode of FIG. 27 upon exposure to 8 mM β-hydroxybutyrate in 100 mM PBS at 33° C. for 2 weeks. [Diagram 30] 13 shows exemplary plots of sensor performance for Groups 1-4 of Example 6. [Diagram 31] 13 shows an exemplary plot of the response of Group 1 analyte sensors from Example 6 to lactate solutions having various lactate concentrations. [Diagram 32] FIG. 1 is a schematic diagram of an exemplary analyte monitoring and vehicle control system in accordance with one or more embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 may function independently or in concert to detect one or more analytes.

[0010] As mentioned 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 may be used. This approach may be undesirable due to, among other things, cost concerns, the need for an individual to wear multiple analyte sensors, and the increased likelihood of individual sensor failure.

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

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

[0013] Analyte sensors containing multiple enzymes, whether the enzymes operate independently or in concert, may function with greater stability in the presence of an appropriate stabilizer. Stabilizers that can be used include, for example, catalase or albumin (e.g., bovine serum albumin or human serum albumin). Catalase is known for its ability to remove reactive species, such as peroxides, from biological environments. Albumins, on the other hand, are not believed to exhibit the ability to remove 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 sensors of the present disclosure in more detail, a brief overview of suitable in vivo analyte sensor configurations and sensor systems using the analyte sensors is first provided so that embodiments of the present disclosure can be better understood. 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] FIG. 1 illustrates a diagram of an exemplary sensing system that may incorporate the analyte sensors of the present disclosure. As shown, the sensing system 100 includes a sensor control device 102 and a reader device 120 configured to communicate with each other via a local communication path or link that may be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. The reader device 120 may, according to some embodiments, configure an output medium for displaying the analyte concentration and an alert or notification determined by the sensor 104 or its associated processor, as well as allowing for one or more user inputs. The reader device 120 may be a general-purpose smartphone or a dedicated electronic reader instrument. Although only one reader device 120 is shown, in certain cases, there may be multiple reader devices 120. The reader device 120 may also communicate with a remote terminal 170 and / or a trusted computer system 180, respectively, via communication paths / links 141 and / or 142, also wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. Additionally or alternatively, the reader device 120 may communicate with a network 150 (e.g., a cellular network, the Internet, or a cloud server) via communication path / link 151. The network 150 may be further communicatively connected to a remote terminal 170 via communication path / link 152 and / or to a trusted computer system 180 via communication path / link 153. Alternatively, the sensor 104 may 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, the sensor 104 may communicate with the remote terminal 170 and / or the trusted computer system 180 via a direct communication link to the network 150 according to some embodiments, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is incorporated herein by reference in its entirety.Any suitable electronic communication protocol may be used for each of the communication paths or links, such as Near Field Communication (NFC), Radio Frequency Identification (RFID), BLUETOOTH or BLUETOOTH low energy protocols, WiFi, etc. The remote terminal 170 and / or the trusted computer system 180, according to some embodiments, are accessible by individuals other than the primary user who are interested in the user's analyte levels. The reader device 120 may include a display 122 and an optional input component 121. The display 122, according to some embodiments, may include a touch screen interface.

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

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

[0018] In some embodiments, the sensor 104 can automatically transfer data to the reader device 120. For example, analyte concentration data can be stored in memory as the data is acquired until it is transmitted (e.g., every minute, every five minutes, or at other predefined time periods) and communicated automatically and periodically, such as at a specific frequency or after a specific time period has elapsed. In other embodiments, the sensor 104 can communicate with the reader device 120 in a non-automatic manner rather than according to a set schedule. For example, data can be communicated from the sensor 104 using RFID technology when the sensor electronics come within communication range of the reader device 120. The data can remain stored in the memory of the sensor 104 until communicated to the reader device 120. Thus, the patient does not need to be in constant proximity to the reader device 120, but can instead upload data at a convenient time. In still other embodiments, a combination of automatic and non-automatic data transfer can be implemented. For example, data transfer can continue automatically until the reader device 120 is no longer within communication range of the sensor 104.

[0019] An introducer may be temporarily present to facilitate introduction of the sensor 104 into the tissue. In an exemplary embodiment, the introducer may include a needle or similar sharp. It should be appreciated that in alternative embodiments, other types of introducers, such as a sheath or blade, may be present. More specifically, the needle or other introducer may be temporarily present in the vicinity of the sensor 104 prior to tissue insertion and then withdrawn. While present, the needle or other introducer may facilitate insertion of the sensor 104 into the tissue by opening an access path for the sensor 104 to follow. For example, according to one or more embodiments, the needle may facilitate penetration of the epidermis as an access path to the dermis to allow implantation of the sensor 104 to occur. After opening the access path, the needle or other introducer may be withdrawn so as not to present a sharps hazard. In an exemplary embodiment, a suitable needle may be solid or hollow, beveled or non-beveled, and / or circular or non-circular in cross section. In more particular embodiments, suitable needles may be comparable in cross-sectional diameter and / or tip design to acupuncture needles, which may have a cross-sectional diameter of about 250 microns. However, it should be recognized that suitable needles may have larger or smaller cross-sectional diameters as required for a particular application.

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

[0021] The analyte sensors described herein may feature multiple enzymes on the active area of ​​a single working electrode or on two or more separate working electrodes. According to various embodiments of the present disclosure, the single working electrode configuration of the analyte sensor may use a two-electrode or three-electrode detection motif. Sensor configurations featuring a single working electrode are described below with reference to Figures 2A-2C. Sensor configurations featuring multiple working electrodes are then described separately with reference to Figure 3. Multiple enzymes may be incorporated into any of the sensor configurations described below, and specific configurations suitable for incorporating multiple enzymes are described in further detail below.

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

[0023] 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 present disclosure. 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 may be disposed on the same side of the substrate 212 with a dielectric material interposed therebetween (configuration not shown). An active area 218 is disposed as at least one layer on at least a portion of the working electrode 214. In various embodiments, the active area 218 may include multiple spots or a single spot configured for detection of one or more analytes of interest. In general, multiple enzymes may be present in the active area 218 (i.e., in a single spot or in multiple spots).

[0024] 2A , the membrane 220, according to some embodiments, covers at least the active area 218 and can optionally cover some or all of the working electrode 214 and / or counter / reference electrode 216, or the entire analyte sensor 200. One or both sides of the analyte sensor 200 may be covered with the membrane 220. The membrane 220 may include one or more polymeric membrane materials capable of restricting analyte flux to the active area 218. As described further herein, the composition of the membrane 220 may vary depending on the analyte. The analyte sensor 200 may be operable to assay one or more analytes by any of the following electrochemical detection techniques: coulometric, amperometric, voltammetric, or potentiometric.

[0025] 2B and 2C show diagrams of an exemplary three-electrode analyte sensor configuration with a single working electrode suitable for use in some embodiments of the present disclosure. The three-electrode analyte sensor configuration using a single working electrode can be similar to that shown for the analyte sensor 200 of FIG. 2A, except for the inclusion of an additional electrode 217 in the analyte sensors 201 and 202 (FIGS. 2B and 2C). With the additional electrode 217, the counter / reference electrode 216 can function as either a counter electrode or a reference electrode, and the additional electrode 217 performs the other electrode function. The working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed on either the working electrode 210 or on the electrode 216, with a dielectric separation layer between them. For example, as shown in FIG. 2B, the 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 may be disposed on the opposite side of the substrate 212, as shown in FIG. 2C. Thus, in some embodiments, the electrode 214 (working electrode) and the electrode 216 (counter electrode) may be disposed on the opposite side of the substrate 212, and the electrode 217 (reference electrode) may be disposed on one of the electrodes 214 or 216 and separated therefrom by a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) may be present on the electrode 217, and the location of the reference material layer 230 is not limited to that shown in FIGS. 2B and 2C. Similar to the sensor 200 shown in FIG. 2A, the active area 218 of the analyte sensors 201 and 202 may include multiple spots or a single spot configured for detection of one or more analytes of interest. In general, multiple enzymes may be present in the active area 218 of the analyte sensors 201 and 202. Additionally, analyte sensors 201 and 202 may be operable to assay one or more analytes by any of the following electrochemical detection techniques: coulometric, amperometric, voltammetric, or potentiometric.

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

[0027] Analyte sensor configurations having multiple working electrodes will now be described in more detail. While the following description is primarily directed to analyte sensor configurations having two working electrodes, it should be understood that the disclosure herein may be expanded to incorporate three or more working electrodes without issue. Additional working electrodes may enable the provision of additional active areas and corresponding sensing capabilities to analyte sensors having such features.

[0028] FIG. 3 shows a diagram of an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode suitable for use in some embodiments of the present disclosure. As shown in FIG. 3, the analyte sensor 300 includes working electrodes 304 and 306, respectively, disposed on opposing sides of a substrate 302. An active area 310 is disposed on the surface of the working electrode 304, and an active area 312 is disposed on the surface of the working electrode 306. In general, multiple enzymes may be present in the active areas 310 and 312, with each active area 310, 312 including one or more enzymes. For example, in certain embodiments, a glucose-responsive enzyme may be present in the active area 310 and a lactate-responsive enzyme may be present in the active area 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. Membrane 340 may cover at least active areas 310 and 312, according to various embodiments. Other components of analyte sensor 300 may be covered with membrane 340 as well, and as noted above, one or both sides of analyte sensor 300, or portions thereof, may be covered with membrane 340. Similar to analyte sensors 200, 201, and 202, analyte sensor 300 may be operable to assay one or more analytes by any of the following electrochemical detection techniques: coulometric, amperometric, voltammetric, or potentiometric.

[0029] Alternative analyte sensor configurations having multiple working electrodes and different from those shown in Figure 3 may feature counter / reference electrodes instead of separate counter and reference electrodes 320, 321, and / or different layer and / or membrane arrangements than those explicitly shown. For example, the arrangement of counter electrode 320 and reference electrode 321 can be reversed from that shown in Figure 3. Furthermore, working electrodes 304 and 306 do not necessarily have to be on opposing sides of substrate 302 in the manner shown in Figure 3.

[0030] An analyte sensor configuration featuring a working electrode having a separate active area is shown in Figures 6A and 6B and described further below. According to various embodiments of the present disclosure, an electron transfer agent may be present in one or more active areas of any of the analyte sensors or analyte sensor configurations disclosed herein. A suitable electron transfer agent may facilitate the transport of electrons to the working electrode when the analyte (enzyme substrate) undergoes an oxidation-reduction reaction. The selection of the electron transfer agent in each active area may determine the oxidation-reduction potential observed for each. When multiple active areas are present, the electron transfer agent in each active area may be the same or different.

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

[0032] According to various embodiments of the present disclosure, a polymer may be present in each active area of ​​any of the analyte sensors or analyte sensor configurations disclosed herein. Polymers suitable for inclusion in the active area may include, but are not limited to, polyvinylpyridine (e.g., poly(4-vinylpyridine)), polyvinylimidazole (e.g., poly(1-vinylimidazole)), or any copolymers thereof. Exemplary copolymers that may be suitable for inclusion in the active area include those that include monomer units such as, for example, styrene, acrylamide, methacrylamide, or acrylonitrile. When multiple active areas are present, the polymers in each active area may be the same or different.

[0033] According to various embodiments of the present disclosure, the electron transfer agent can be covalently bonded to the polymer in each active region. The manner 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 a monomer unit having a covalently bonded electron transfer agent, or the electron transfer agent can be reacted separately with the polymer after the polymer has already been synthesized. According to some embodiments, a bifunctional spacer can covalently bond the electron transfer agent to the polymer in the active region, where a first functional group is reactive with the polymer (e.g., a functional group that can quaternize the pyridine nitrogen atom or the imidazole nitrogen atom) and a second functional group is reactive with the electron transfer agent (e.g., a functional group that reacts with a ligand that coordinates a metal ion).

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

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

[0036] Various configurations suitable for disposing multiple enzymes on the analyte sensors of the present disclosure will now be described in further detail. The multiple enzymes can be deposited within one or more active areas of the sensor. The active areas can be on the order of about 0.01 mm in size. 2 ~about 1mm 2 , although larger or smaller active areas are also contemplated herein.

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

[0038] According to some embodiments, the disclosed analyte sensor having multiple active regions on a single working electrode may include 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 includes an analyte-responsive enzyme and a polymer, and the analyte-responsive enzyme in each active region is different. Each active region has an oxidation-reduction potential, and the oxidation-reduction potential of the first active region is sufficiently separated from the oxidation-reduction potential of the second active region to allow for the generation of a signal from the first active region independent of the signal from the second active region. In more specific embodiments, such an analyte sensor may include a single working electrode having at least two active regions. An electron transfer agent may be incorporated within each active region to facilitate electron transfer.

[0039] An alternative sensor configuration may include a single active area that includes both a first analyte-responsive enzyme and a second analyte-responsive enzyme along with an electron transfer agent. Each enzyme may be covalently attached to a separate portion of the polymer within the single active area. If the sensing chemicals for facilitating electron transfer for each analyte are not overly diluted in the single active area, the single active area may facilitate detection of the analytes in a similar manner as described below for separate active areas. Such a sensor configuration may 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 may be configured for insertion into tissue. Suitable tissues are not considered to be particularly limited and are described in more detail above. Similarly, considerations for placing the sensor tail at a particular location within tissue are discussed above.

[0041] In more specific embodiments, the redox potential associated with the first active region may be separated from the redox potential of the second active region by at least about 100 mV, or at least about 150 mV, or at least about 200 mV. The upper limit of separation between the redox potentials is determined in vivo by the working electrochemical window. By separating the redox potentials of the active regions sufficiently in magnitude from each other, electrochemical reactions may occur in the first active region without substantially inducing electrochemical reactions in the second active region. Thus, a signal from a first active region may be independently generated above its corresponding redox potential. In contrast, above the redox potential of the second active region, electrochemical reactions may occur in both active regions. Thus, a signal obtained above the redox potential of the second active region may include signal contributions from both the first active region and the second active region, and the signal is a composite signal. The signal contribution from a second active region above that redox potential can then be determined by subtracting from the composite signal the signal obtained from only the first active region above the corresponding redox potential. Similar considerations 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 contain different electron transfer agents to separate the redox potentials sufficiently in magnitude when the active regions are located on the same working electrode. More specifically, the first active region may contain a first electron transfer agent and the second active region may contain a second electron transfer agent, the first and second electron transfer agents being different. According to various embodiments of the present disclosure, the metal center and / or ligands present in a given electron transfer agent may be varied to separate the redox potentials of the first and second active regions sufficiently. According to even more specific embodiments, the first electron transfer agent may be covalently attached to a polymer in the first active region and the second electron transfer agent may be covalently attached to a polymer in the second active region. The covalent attachment aspects of the first and second electron transfer agents may be the same or different. Similar considerations apply when selecting an electron transfer agent suitable for use in combination with a first analyte responsive enzyme and a second analyte responsive enzyme contained within a single active region, in accordance with the above disclosure.

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

[0044] Ideally, the first and second active regions disposed on a single working electrode can be configured to rapidly achieve a steady-state current upon operation of the analyte sensor at a given potential. Rapid achievement of a steady-state current can be promoted by selecting an electron transfer agent for each active region that rapidly changes its oxidation state upon exposure to a potential equal to or greater than its redox potential. Making the active regions as thin as possible can also facilitate rapid achievement of a steady-state current. For example, a suitable thickness for the first and second active regions can range from about 0.1 microns to about 10 microns. In some or other embodiments, combining conductive materials, such as, for example, carbon nanotubes, graphene, or metal nanoparticles, within one or more active regions can facilitate rapid achievement of a steady-state current. A suitable amount of conductive particles can range from about 0.1% to about 50% by weight, or from about 1% to about 50% by weight, or from about 0.1% to about 10% by weight, or from about 1% to about 10% by weight of the active region. Stabilizers can also be used to promote 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 areas, the area ratio of the active areas to each other, the nature and thickness of the mass transport limiting membrane covering the active areas, and any combination thereof. Modification of these parameters can be readily implemented by one of ordinary skill in the art given the benefit of the disclosure herein.

[0046] While the above description is directed primarily to an analyte sensor configured to detect two different analytes, it should be understood that the above concepts can be extended to detect three or more analytes using a corresponding number of active areas disposed on a single working electrode. Specifically, in further embodiments of the present disclosure, an analyte sensor using three or more active areas and a corresponding number of different enzymes (and electron transfer agents) therein can be used to detect the same number of different analytes. If the redox potential of each active area is sufficiently far from the redox potential of the other active areas, the signal contribution from each active area can be analyzed in a manner related to the above method to provide the concentration of each analyte.

[0047] In more specific 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, in addition to suitable electron transfer agents and polymers as described in more detail above. According to certain embodiments, an analyte sensor suitable for detecting glucose and lactate may include a working electrode on which a first active region and a second active region are disposed, and a mass transport 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 bound to the polymer, and the first active region includes a glucose-responsive enzyme (e.g., glucose oxidase) covalently bound to the polymer. Different first and second electron transfer agents may be present in each active region. In more specific embodiments, the mass transport limiting membrane may include at least a cross-linked polyvinylpyridine homopolymer or copolymer. The composition of the mass transport limiting membrane may be the same or different when the mass transport limiting membrane covers each active region. In certain embodiments, the mass transport limiting membrane covering the first active area can be single component (comprising a single membrane polymer) and the mass transport limiting membrane covering the second active area can be multicomponent (comprising two or more different membrane polymers, one of which is a polyvinylpyridine homopolymer or copolymer), either a bilayer or a homogeneous mixture.

[0048] Similarly, it should also be understood that some analyte sensors of the present disclosure having two or more active areas disposed on a given working electrode may include two or more analyte-responsive enzymes in at least one active area. According to more specific embodiments, two or more analyte-responsive enzymes in a given active area may interact in a coordinated manner to generate a signal proportional to the concentration of a single analyte. Thus, the analyte-responsive enzymes do not necessarily have to be present in a 1:1 ratio for a particular analyte selection. Analyte sensors including enzymes that interact in a coordinated manner are described in further detail below.

[0049] Thus, also described herein are methods of multi-analyte detection using an analyte sensor featuring multiple enzymes disposed on a single working electrode. In various embodiments, such methods 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 areas disposed on a surface of the working electrode. Each active area includes an analyte-responsive enzyme and a polymer, and the analyte-responsive enzyme in each active area is different. Each active area has an oxidation-reduction potential, and the oxidation-reduction potential of a first active area is sufficiently separated from the oxidation-reduction potential of a second active area to allow generation of a signal from the first active area independent of generation of a signal from the second active area. The method further includes obtaining a first signal at or above the redox potential of the first active area, such that the first signal is proportional to the concentration of the first analyte; obtaining a second signal at or above the redox potential of the second active area, such that the second signal is a composite signal including a signal contribution from the first active area and a signal contribution from the second active area; and subtracting the first signal from the second signal to obtain a differential signal proportional to the concentration of the second analyte.

[0050] In more specific embodiments, the redox potential associated with the first active region may be separated from the redox potential of the second active region by at least about 100 mV, or at least about 150 mV, or at least about 200 mV, to provide sufficient separation for independent generation of a signal from the first active region.

[0051] In some or other more specific embodiments, the fluid is a biological fluid and the analyte sensor is exposed to the biological fluid in vivo in an individual. Biological fluids suitable for analysis with an analyte sensor having at least two distinct active areas disposed on a given working electrode may include any of the biological fluids described in more detail above.

[0052] In some embodiments, the signal associated with each active area can be correlated to a corresponding analyte concentration by referencing a lookup table or calibration curve for each analyte. A lookup table for each analyte can be created by analyzing multiple samples with known analyte concentrations and recording the sensor response at each concentration of each analyte. Similarly, a 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 curves of the analyte sensors 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 the sample with unknown analyte concentration and report the analyte concentration accordingly. In some or other embodiments, if the sensor response value of the sample with 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 sensor response is sufficiently different from a given value in the lookup table (e.g., a variation of about 10% or more).

[0054] Similarly, according to some or various other embodiments, the processor can input the sensor response values ​​of samples having unknown analyte concentrations into a corresponding calibration curve, and the sensor can then report the analyte concentration accordingly.

[0055] Analyte sensor embodiments having two distinct active areas disposed on a given working electrode may use sensor configurations related to those shown in Figures 2A-2C and above. However, it should be understood that suitable analyte sensors may also feature multiple working electrodes, such as the sensor configuration shown in Figure 3, where at least one of the working electrodes has at least two active areas that are different from each other. It should be understood that other analyte sensor configurations having two or more distinct active areas disposed on a 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 and / or reference electrodes may differ from those shown in the drawings of this application.

[0056] Figure 4 illustrates an exemplary analyte sensor configuration suitable for use in some embodiments of the present disclosure, in which two distinct active regions are disposed on a surface of a single working electrode. The analyte sensor configuration of Figure 4 is most similar to that of Figure 2C and may be better understood with reference to Figure 2C. Where appropriate, common reference numbers with Figure 2C have been used in Figure 4 for purposes of clarity, and for purposes of brevity, features having common structure and / or function have not been described in further detail. Again, it should be understood that other analyte sensor configurations may similarly incorporate the features described below with respect to Figure 4.

[0057] 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, which may be covalently attached to the polymer that includes the active region 218a. The active region 218b includes a second electron transfer agent and a second analyte responsive enzyme, which may be covalently attached to the polymer that includes the active region 218b. The first and second electron transfer agents may be compositionally different to provide separation of the redox potentials of the first and second active regions 218a and 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 area 218a and the second active area 218b may be sufficiently separated from each other to allow for generation of a signal from the first active area 218a independent of signal generation from the second active area 218b. Thus, the analyte sensor 400 may be operated at a first potential where a redox reaction occurs in the first active area 218a but not in the second active area 218b. Thus, a first analyte (e.g., glucose) may be selectively detected at or above the redox potential of the first active area 218a as long as the applied potential is not high enough to promote a redox reaction with the second active area 218b. The concentration of the first analyte may be determined from the sensor response of the first active area 218a by reference to a look-up table or calibration curve.

[0059] At or above the redox potential of the second active area 218b, separate redox reactions may occur simultaneously or nearly simultaneously in both the first active area 218a and the second active area 218b. As a result, a signal generated at or above the redox potential of the second active area 218b may include a composite signal having signal contributions from both the first active area 218a and the second active area 218b. To determine the concentration of a second analyte (e.g., lactate) from the composite signal, the signal from the first active area 218a at or above the corresponding redox potential may be subtracted from the composite signal to provide a differential signal associated only with the second active area 218b. Once the differential signal is determined, the concentration of the second analyte may be determined by referencing a lookup table or calibration curve.

[0060] As discussed above, similar considerations apply to isolating a first signal and a second signal from a single active area containing two different analyte-responsive enzymes to determine the concentrations of first and second analytes that are different from one another.

[0061] In some or other embodiments of the present disclosure, multiple enzymes may be present in a single active area. Unlike sensor configurations in which multiple enzymes function independently to detect different analytes, particularly those spaced apart in separate active areas on the surface of the working electrode, according to some embodiments of the present disclosure, multiple enzymes located in a single active area function in concert to facilitate detection of a single analyte, particularly in the presence of a stabilizer. As used herein, the term "in concert" and grammatical variations thereof refer to a combined enzymatic reaction in which the product of a first enzymatic reaction is a substrate for a second enzymatic reaction, which serves as the basis for measuring the concentration of the substrate (analyte) reacted during the first enzymatic reaction. When a single enzyme cannot facilitate detection, it may be desirable to utilize two enzymes acting in concert with each other to detect a given analyte of interest. Situations in which a single enzyme may be ineffective in facilitating detection of an analyte include, for example, situations in which the enzyme is inhibited by one or more products of the enzymatic reaction or is unable to cycle between oxidized and reduced states when placed in the analyte sensor.

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

[0063] In a more specific embodiment, an analyte sensor featuring at least two enzymes that interact with each other in a coordinated manner may include a sensor tail including at least a working electrode; and at least one active area disposed on the surface of the working electrode. The at least one active area includes a first enzyme, a second enzyme, and a polymer. The first and second enzymes may interact in a coordinated manner, the first enzyme may convert the analyte to a first product, and the second enzyme may convert the first product to a second product to generate a signal at the working electrode. The second enzyme does not react with the analyte. The at least second enzyme is covalently attached to the polymer in the at least one active area. As described in more detail below, analysis of a signal (e.g., a current measured at a fixed input voltage) resulting from the reaction of the first product to the second product may provide a basis for detecting and measuring the concentration of the analyte.

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

[0065] According to some embodiments, stabilizers may be present in the active region. Stabilizers that are particularly suitable for analyte sensors that include cooperatively interacting enzymes include, for example, catalase and albumin.

[0066] According to some embodiments, the sensor tail can be configured to be inserted into a tissue of interest, such that, according to some embodiments, an analyte sensor including enzymes capable of interacting with each other in a coordinated manner at a given active area can be adapted to analyze the concentration of an analyte in a biological fluid in vivo, again without any particular limitation on what the biological fluid may be.

[0067] As described above, sensor configurations incorporating two enzymes capable of cooperative interaction may include those in which at least one active region includes an electron transfer agent covalently bound to a polymer. Again, in accordance with the disclosure herein, coordinate bonds are also included within the scope of covalent bonds. In such embodiments, at least a second enzyme may also be covalently bound to a polymer. In some embodiments, the first enzyme is not covalently bound to a polymer. In other embodiments, both the first enzyme and the second enzyme may be covalently bound to a polymer in at least one active region. Covalent binding of the first enzyme to a polymer may be desirable, for example, to reduce the possibility of leaching of the first enzyme from at least one active region.

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

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

[0070] Another pair of co-enzymes that may be suitable for use in the present disclosure, where both enzymes are directly disposed on the surface of the working electrode, is β-hydroxybutyrate dehydrogenase and diaphorase. This co-enzyme pair can be used to detect ketone bodies, with β-hydroxybutyrate being a representative molecule that indicates the presence of ketones. In a sensor configuration that includes this co-enzyme pair, β-hydroxybutyrate dehydrogenase catalyzes the synthesis of β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD + ) can be converted to acetoacetate and reduced nicotinamide adenine dinucleotide (NADH). NADH then undergoes diaphorase-mediated reduction, and the electrons transferred during this process provide the basis for ketone detection at the working electrode. + The concerted reaction between the cofactor-mediated enzymes is shown in Figure 5B. Along with the concerted enzyme pair, albumin may be present as a stabilizer.

[0071] Yet another alternative enzymatic detection chemistry for ketones is shown in Figures 5C and 5D. As shown in Figure 5C, β-hydroxybutyrate dehydrogenase reacts with β-hydroxybutyrate and NAD + Instead of the electron transfer to the working electrode being completed by diaphorase (see FIG. 5B), the reduced form of NADH oxidase (NADHOx(red)) undergoes a reaction to form the corresponding oxidized form (NADHOx(Ox)). NADHOx(red) is then reformed by reaction with molecular oxygen to generate superoxide, which can then be converted to hydrogen peroxide via superoxide dismutase (SOD). According to various embodiments, SOD can be covalently attached to a polymer in the active region. The hydrogen peroxide can then undergo a reaction at the working electrode to provide a signal that can be correlated to the amount of ketone present. FIG. 5D shows that β-hydroxybutyrate dehydrogenase again converts β-hydroxybutyrate and NAD + This paper presents another alternative enzyme detection chemistry that can convert albumin to acetoacetate and NADH. The detection cycle in this case is completed by oxidation of poly-1,10-phenanthroline-5,6-dione at the working electrode. As with the other detection chemistries disclosed herein, the inclusion of albumin in the active region can provide a surprising improvement in response stability.

[0072] Creatine amidohydrolase and sarcosine oxidase are another pair of cooperative enzymes that may be suitable for use in the present disclosure when both enzymes are directly disposed on the working electrode. Creatine amidohydrolase produces sarcosine and urea from creatine. Sarcosine oxidase can then catalyze the reaction of sarcosine to form glycine, formaldehyde, and hydrogen peroxide. Thus, detection of hydrogen peroxide at the working electrode can serve as the basis for quantifying creatine and / or sarcosine.

[0073] The detection of ethanol and other alcohols using alcohol oxidase and xanthine oxidase via a concerted enzymatic reaction is described in further detail. Alcohol oxidase interacts with ethanol to form acetaldehyde and hydrogen peroxide. Other alcohols react to form corresponding higher or lower carbon number aldehydes. Advantageously, alcohol oxidase only catalyzes the forward conversion of ethanol to acetaldehyde (as opposed to performing the reaction reversibly as in the case of alcohol dehydrogenase), which may be advantageous for the use of this enzyme in an analyte sensor. Furthermore, because alcohol oxidase contains a tightly bound flavin cofactor, exogenous cofactors do not necessarily need to be combined with alcohol oxidase to activate the enzyme to facilitate alcohol oxidation.

[0074] In principle, alcohol oxidase alone can be used for ethanol detection in an analyte sensor by assaying either the acetaldehyde or hydrogen peroxide products generated in the enzymatic reaction. However, there are two problems with this approach. First, both acetaldehyde and hydrogen peroxide inhibit alcohol oxidase. Thus, if these compounds are not removed from the sensor environment, alcohol oxidase will become inactive to promote ethanol oxidation, thereby rendering the analyte sensor non-functional to assay ethanol. Furthermore, if acetaldehyde and hydrogen peroxide are sequestered or quenched with other agents, no species will be available for electrochemical detection. Second, alcohol oxidase does not freely exchange electrons with redox mediators other than molecular oxygen. Therefore, electron transfer agents associated with the polymer in the active region of the analyte sensor, such as the 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. Thus, although alcohol oxidase can optionally be covalently attached to the polymer, doing so offers no particular advantage to the electron transfer process. That is, the covalent attachment of alcohol oxidase to the polymer does not aid in facilitating electron transfer by the electron transfer agent.

[0075] The concerted combination of alcohol oxidase and xanthine oxidase directly on the working electrode, particularly together in a given active area, may overcome at least some of the aforementioned challenges associated with ethanol detection using an analyte sensor using alcohol oxidase. Acetaldehyde and other aldehydes can serve as substrates for xanthine oxidase, which 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 area (e.g., as a catalase-hydrogen peroxide complex), thereby preventing inactivation of alcohol oxidase by this species. Furthermore, unlike alcohol oxidase, xanthine oxidase can exchange electrons with osmium and other transition metal complexes associated with the polymer in the active area of ​​the analyte sensor. In this way, xanthine oxidase can cycle between its oxidized and reduced forms, thereby allowing the analyte sensor to maintain an active sensing state. Therefore, the detection of ethanol in the above-mentioned analyte sensor is based on the enzymatic reaction between xanthine oxidase and acetaldehyde, which is the enzymatic reaction product of ethanol. Furthermore, by configuring the enzyme in the analyte sensor in the above-mentioned manner, alcohol oxidase may be reoxidized with molecular oxygen to maintain its activity.

[0076] FIG. 5A shows a concerted enzyme reaction cycle associated with ethanol detection using alcohol oxidase and xanthine oxidase directly disposed on the working electrode according to various embodiments of the present disclosure. Xanthine oxidase is covalently bound to a polymer at the active area of ​​the analyte sensor, and alcohol oxidase is non-covalently bound to the polymer at the active area. In addition to xanthine oxidase, an osmium complex or other transition metal complex capable of exchanging electrons with the enzyme is also covalently bound to the polymer. As shown in FIG. 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 reoxidized with molecular oxygen as shown to return alcohol oxidase to its catalytically active oxidized form.

[0077] With continued reference to FIG. 5A, acetaldehyde enzymatically formed from ethanol then undergoes a subsequent reaction with oxidized xanthine oxidase in the presence of flavin cofactors naturally present with the enzyme. This process produces acetic acid and converts xanthine oxidase to a reduced state. The reduced xanthine oxidase can then 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 form of xanthine oxidase. Although not shown in FIG. 5A, hydrogen peroxide is separately removed from the sensor environment by catalase present in the active region.

[0078] As can be seen from Figure 5A, the amount of acetaldehyde enzymatically formed is proportional to the amount of ethanol originally present. Therefore, the current generated at the working electrode during xanthine oxidase oxidation of acetaldehyde can be proportional to the amount of acetaldehyde and therefore the amount of ethanol present. Correlation of the working electrode current to ethanol concentration can be done by referencing a look-up table of currents at known ethanol concentrations or by utilizing a calibration curve. These concepts are explained in more detail above.

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

[0080] Thus, in a more specific embodiment, the present disclosure provides an alcohol sensor based on the cooperative enzymatic reaction of alcohol oxidase and xanthine oxidase. More specifically, the alcohol sensor may include at least a working electrode and a sensor tail including at least one active area disposed on the surface of the working electrode, where the at least one active area includes alcohol oxidase, xanthine oxidase, catalase, a polymer, and an electron transfer agent. According to a specific embodiment, the electron transfer agent and xanthine oxidase may be covalently attached to the polymer, but alcohol oxidase is not covalently attached to the polymer. The alcohol oxidase and xanthine oxidase can interact cooperatively to generate a signal at the working electrode that is proportional to the alcohol concentration. More specifically, both alcohol oxidase and xanthine oxidase are disposed directly on the working electrode to achieve the foregoing.

[0081] According to more specific embodiments, the catalase in at least one active area of ​​the alcohol sensor is not covalently bound to the polymer, and the catalase may be present in an amount ranging from about 1% to about 50% by weight of the polymer, more specifically, from about 1% to about 10% by weight of the polymer, or from about 1% to about 5% by weight of the polymer.

[0082] Thus, the present disclosure also provides a detection method based on a cooperative enzyme reaction, in which the cooperative enzyme pair is disposed directly on the surface of the working electrode. According to various embodiments, the detection method may include exposing an analyte sensor to a fluid containing an analyte, the analyte sensor includes at least a working electrode and a sensor tail including at least one active area disposed on the surface of the working electrode, the at least one active area including a first enzyme, a second enzyme, and a polymer. The first enzyme and the second enzyme can interact cooperatively, and the second enzyme is covalently attached 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 generating 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 carrying out the aforementioned method, an electron transfer agent may also be covalently attached to the polymer. Suitable electron transfer agents are described in more detail above. In some or other embodiments, particularly when a covalently attached electron transfer agent is present, the first enzyme is not covalently attached to the polymer in at least one active region.

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

[0085] It should be understood that while two different enzymes in a single active area of ​​an analyte sensor may interact cooperatively with each other to determine an analyte concentration, the enzymes may 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 instead be used to detect any of a wide variety of substrates that have affinity for xanthine oxidase. Alternative substrates for xanthine oxidase may include, for example, hypoxanthine, xanthine, uric acid, purines, pterins, and similar compounds. When the analyte sensor is used in this manner, in the absence of alcohol, alcohol oxidase remains unused and / or may be inactivated by acetaldehyde / hydrogen peroxide if these species are not removed by xanthine oxidase or other species. Thus, a sensor containing a coordinated enzyme may also be considered to be capable of detecting multiple analytes, one analyte from the coordinated enzyme pair, and at least a second analyte from one of the members of the coordinated enzyme pair acting independently. 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 referenced above, multiple enzymes located in separate active regions may also interact in a coordinated manner to facilitate the detection of a single analyte. In some cases, multiple enzymes may both be located 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 may be isolated from the working electrode such that electron transfer to the working electrode occurs only from one active region. That is, as described in more detail below, the active region isolated from the working electrode may facilitate an enzymatic reaction of the analyte of interest to produce a reaction product (substrate) that reacts with the enzyme in the active region in direct contact with the working electrode. A signal associated with the enzymatic reaction occurring in the active region in direct contact with the working electrode then provides the basis for detecting the analyte. Correlation of the signal to the analyte concentration may be achieved in a similar manner as described in more detail above.

[0087] More specifically, FIG. 5E illustrates a concerted enzymatic reaction cycle associated with ethanol detection using glucose oxidase and xanthine oxidase according to various embodiments of the present disclosure, which is further mediated by catalase when only xanthine oxidase or xanthine oxidase and catalase are disposed on the surface of the working electrode. The concerted enzymatic reaction cycle illustrated in FIG. 5E relies on glucose and ethanol coexisting with each other in the fluid during analysis, as described below. Glucose is a ubiquitous biological nutrient, and therefore often coexists with other analytes when assaying biological fluids. However, if a particular fluid under analysis lacks glucose, certain embodiments of the present disclosure may feature the addition of glucose to the fluid and the use of the concerted enzymatic reaction of glucose oxidase and xanthine oxidase to facilitate the detection of ethanol or another alcohol.

[0088] Before further describing the concerted enzymatic reaction shown in FIG. 5E, an exemplary analyte sensor configuration featuring at least one active area isolated from the working electrode will first be described in more detail. As previously mentioned, the analyte sensor configurations shown in FIGS. 2A-4 all feature one or more working electrodes with one or more active areas 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 area is disposed directly on the surface of the working electrode and a second active area is separated (at a distance or remote) from the working electrode by a membrane. The working electrode configurations shown in FIGS. 6A, 6B, and 6C can be substituted 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 and / or reference electrodes, membranes, substrates, and similar structures in the analyte sensor.

[0089] As shown in FIG. 6A, the working electrode 400 has an active area 402 disposed directly on its surface. The active area 402 includes a first enzyme covalently bonded to a first polymer. Typically, an electron transfer agent is also present in the active area 402, also covalently bonded to the polymer. The active area 402 is covered with a membrane 404. The membrane 404 can also cover the surface of the working electrode 400, as shown, as well as other portions of the analyte sensor where the working electrode 400 resides. The membrane 404 isolates the active area 406 from the working electrode 400, such that electron exchange between the two is prevented. The active area 406 includes a second enzyme covalently bonded to a second polymer, but no separate electron transfer agent is present. While FIG. 6A shows the active area 406 disposed directly above the active area 402, it should be understood that they can be laterally spaced apart from one another in alternative configurations compatible with the present disclosure. The membrane 408 provides mass transport limiting properties by covering the active area 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 area 402, but only a portion of the surface of the working electrode 400, while the membrane 408 covers the active area 406, the surface of the membrane 404, and the remaining portions of the surface of the working electrode 400 that are not covered by the membrane 404. The active areas 402 and 406 may also be laterally offset from one another in some embodiments, as shown in FIG. 6C, where the active area 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 area 406. In contrast, the membrane 404 is permeable to the product formed in the active area 406. That is, the analyte reacts in the active area 406 to form a first product, which then diffuses through the membrane 404 and subsequently reacts further in the active area 402 to form a second product. The second product is then detectable based on electron exchange with the working electrode 400.

[0091] Optionally, if glucose detection is desired, a lead 410 may extend between active areas 402 and 406. In the alcohol sensor characterized by detection based on the concerted enzymatic reaction between glucose oxidase and xanthine oxidase, glucose oxidase is present in the active region 406 and xanthine oxidase is present in the active region 402. With continued reference to Figures 6A, 6B and 6C, and again with reference to Figure 5E, glucose oxidase is present in the active region 406 and converts exogenous glucose into D-gluconolactone-1,5-dione and hydrogen peroxide. Unlike the alcohol sensor characterized by detection based on the concerted enzymatic reaction between alcohol oxidase and xanthine oxide (Figure 5A), catalase plays a more active role in the concerted enzymatic reaction shown in Figure 5E. That is, catalase reacts with hydrogen peroxide to form a catalase-hydrogen peroxide complex (the same peroxide scavenging function that catalase exhibits in the concerted enzymatic reaction between alcohol oxidase and xanthine oxidase), and the complex then reacts with ethanol to form acetaldehyde. Acetaldehyde formed in active region 406 upon reacting ethanol with catalase-hydrogen peroxide complex diffuses through membrane 404, which separates active region 406 from active region 402. Alternatively, catalase may be present in active region 402, in which case hydrogen peroxide formed in active region 406 may diffuse through membrane 404 to active region 402, forming catalase-hydrogen peroxide complex in active region 402 and oxidizing ethanol to acetaldehyde in active region 402. Once acetaldehyde is formed in active region 402, the concerted enzymatic reaction may continue as shown in FIG. 5E. Membrane 404 may include cross-linked polyvinylpyridine that is permeable to acetaldehyde. Acetaldehyde then reacts with xanthine oxidase in active region 402 to form acetic acid in a manner similar to that described above for FIG. 5A.

[0092] Thus, the alcohol sensor of the present disclosure may include a sensor tail including at least a working electrode; a first active area disposed on the surface of the working electrode, the first active area 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 area, the first membrane including a first membrane polymer and permeable to acetaldehyde; a second active area disposed on the first membrane, the second active area including glucose oxidase, catalase, and a second polymer, the glucose oxidase being covalently bonded to the second polymer; and a second membrane covering the second active area, the second membrane including a second membrane polymer and permeable to glucose and alcohol. Here, the glucose oxidase and the xanthine oxidase can interact cooperatively to generate a signal at the working electrode that is proportional to the alcohol concentration. 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 may be a cross-linked polyvinylpyridine. In an embodiment of the present disclosure, the cross-linked polyvinylpyridine is easily permeable to acetaldehyde. The second membrane polymer may be a cross-linked polyvinylpyridine-co-styrene polymer, in which some of the pyridine nitrogen atoms are functionalized with non-cross-linked poly(ethylene glycol) tails and some of the pyridine nitrogen atoms are functionalized with alkylsulfonic acid groups. Such a second membrane polymer is easily permeable to both glucose and ethanol.

[0094] According to some embodiments, the catalase is not covalently bound to the first polymer or the second polymer in the first active region or the second active region. The catalase may be physically constrained within the first active region and the second active region by either 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 including glucose oxidase and xanthine oxidase interacting in concert may include 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 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 may include a biological fluid. Correlation of the signal with the alcohol concentration in the fluid may be performed using any suitable correlation technique as outlined in more detail above.

[0096] In yet other embodiments of the present disclosure, multiple enzymes may be disposed within separate working electrode active areas, such that the signal associated with the enzymatic reaction occurring within each active area can be measured separately by responding to each working electrode simultaneously or at different times, and the signal associated with each active area can then be correlated with the concentration of a separate analyte.

[0097] As discussed above, a membrane (i.e., a mass transport limiting membrane) can cover one or more active areas of an analyte sensor to enhance biocompatibility and modify analyte flux to the active area. Such a membrane can be present in any of the analyte sensors disclosed herein. Because different analytes may exhibit different permeability values ​​within a given membrane, an analyte sensor configured to analyze multiple analytes may exhibit different sensitivity to each analyte. One approach to address the different sensitivity values ​​may include utilizing different membrane thicknesses across each active area. While this approach is feasible, it may be difficult to implement from a manufacturing perspective; i.e., it may 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 different sized active areas for each analyte.

[0098] Analyte sensors having active areas configured to assay different analytes on separate working electrodes may address the aforementioned problems associated with different analyte sensitivity. That is, the disclosure below provides various ways in which the membrane permeability can be altered on each working electrode to equalize the analyte membrane permeability at each location. That is, the disclosure herein allows for independent variation of the analyte permeability and sensitivity at each working electrode. According to the disclosure herein, a mass-transfer limiting membrane comprising two or more different membrane polymers may provide a more equalized analyte permeability at each working electrode. Particular membrane configurations that may be suitable for equalizing the analyte permeability on one or more working electrodes include bilayer and mixed membranes, each comprising two or more different membrane polymers. Surprisingly, bilayer and mixed membranes comprising membrane polymers that are individually inappropriate to promote the permeability of a given analyte may provide satisfactory performance when arranged in a bilayer or mixed membrane, as described below. This approach may be advantageous compared to varying the size of the active area on each working electrode to provide comparable sensitivity values ​​for each analyte.

[0099] Thus, in some embodiments, an analyte sensor featuring two or more enzymes disposed on separate working electrodes may include a sensor tail including at least a first working electrode and a second working electrode, a first active area located on the surface of the first working electrode, a second active area located on the surface of the second working electrode, a multi-component membrane covering the first active area, and a homogeneous membrane covering the second active area. The first active area includes a first polymer and a first analyte-responsive enzyme that reacts with a first analyte, and the second active area includes a second polymer and a second analyte-responsive enzyme that reacts with a second analyte. The first analyte-responsive enzyme and the second analyte-responsive enzyme are different and 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 particular configuration of the multi-component membrane described above may include a bilayer membrane in some embodiments, or a mixture of membrane polymers in other embodiments. Surprisingly, as described in more detail below, bilayer and mixed membranes may function to equalize the permeability of analytes.

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

[0102] According to more specific embodiments of the present disclosure, an analyte sensor having multiple working electrodes may include active regions in which an electron transfer agent is covalently attached to a polymer at each active region. In some or other embodiments, such an analyte sensor may feature a first analyte responsive enzyme covalently attached to a polymer at a first active region and a second analyte responsive enzyme covalently attached to a polymer at a second active region. Again, in certain embodiments, the first analyte responsive enzyme may be a glucose responsive enzyme, such as glucose oxidase, and the second analyte responsive enzyme may be a lactate responsive enzyme, such as lactate oxidase.

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

[0104] FIG. 7 illustrates an exemplary schematic diagram of a portion of an analyte sensor having two working electrodes and featuring a bilayer membrane covering one of the two working electrodes, which is adapted 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 sides of a substrate 612. An active area 618a is disposed on the working electrode 614a, and an active area 618b is disposed on the working electrode 614b. The active areas 618a and 618b include different analyte-responsive enzymes and are configured to assay for different analytes according to the disclosure herein. Although FIG. 7 illustrates that the active areas 618a and 618b are disposed approximately opposite one another with respect to the substrate 612, it should be understood that the active areas 618a and 618b may be laterally spaced apart (offset) from one another on opposite sides of the substrate 612. The laterally spaced apart configuration of active areas 618a and 618b may be particularly advantageous for covering each active area 618a and 618b with a mass transport limiting membrane, as described below.

[0105] As further shown in Figure 7, active area 618a is covered with membrane layer 620. Membrane layer 620 is a homogenous membrane comprising a single membrane polymer. Active area 618b is covered with a bilayer membrane 621 comprising membrane layer 621a in direct contact with active area 618b and membrane layer 621b covering membrane layer 621a. Membrane layers 621a and 621b comprise different membrane polymers. As noted above, in certain embodiments, membrane layer 620 and membrane layer 621b may comprise the same membrane polymer.

[0106] According to one or more embodiments, an analyte sensor having multiple active areas on separate working electrodes, one of the active areas covered with a bilayer membrane, may exhibit a leveled or independently variable analyte permeability. That is, the analyte sensor may have sensitivity to two different analytes that are closer to each other than they would be if the bilayer membrane were not present. In such an analyte sensor configuration, the active area covered with a homogeneous membrane (e.g., membrane layer 620 of FIG. 7) may exhibit an analyte permeability characteristic of that particular membrane polymer for a first analyte. Surprisingly, the bilayer membrane (e.g., bilayer membrane 621 of FIG. 7) may include a membrane polymer that does not adversely affect the permeability of a second analyte (i.e., a membrane polymer with a neutral permeability effect), thereby allowing the other membrane polymer that comprises 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, the membrane polymer with a neutral permeability effect and the membrane polymer that comprises the homogeneous membrane may constitute the same polymer.

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

[0108] In certain embodiments, the first active area may include a glucose-responsive enzyme, such as glucose oxidase, and the second active area may include a lactate-responsive enzyme, such as lactate oxidase. Thus, according to such embodiments, the first active area including the glucose-responsive enzyme may be covered with a bilayer membrane, and the second active area including the lactate-responsive enzyme may be overacted by a homogeneous (single component membrane polymer) membrane. In even more specific embodiments, the second active area may include a polymer, albumin, and a lactate-responsive enzyme covalently bound to the polymer. In even more specific embodiments, the homogeneous membrane covering the second active area may include at least a cross-linked polyvinylpyridine homopolymer or copolymer, and the bilayer membrane covering the first active area 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 that includes two different membrane polymers of a homogeneous blend. Similar to the analyte sensor including a bilayer membrane disposed on one of the active areas, a homogeneous membrane including either the first membrane polymer or the second membrane polymer of the mixed membrane may cover the other active area on the second working electrode.

[0110] Similar to bilayer membranes, a mixed membrane that includes a membrane polymer that neutrally affects the permeability of a second analyte may allow the mixed membrane to exhibit a permeability to the second analyte that is fairly characteristic of the other membrane polymer in the mixture. Thus, according to various embodiments of the present disclosure, the membrane polymer of the homogeneous membrane and one of the membrane polymers of the mixed membrane may be selected such that the permeability of the second analyte through the mixed membrane is not substantially altered by the membrane polymer. 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 including the glucose-responsive enzyme may be covered with a mixed membrane, and the second active region including the lactate-responsive enzyme may be overacted by a homogeneous (single component membrane polymer) membrane. In even more specific embodiments, the second active region may include a polymer, albumin, and a lactate-responsive enzyme covalently bound to the polymer. In a more specific embodiment, the homogeneous film covering the second active area may include at least a cross-linked polyvinylpyridine homopolymer or copolymer, and the mixed film covering the first active area may also include a polyvinylpyridine homopolymer or copolymer.

[0111] As referenced above, bilayer and mixed membranes can equalize analyte permeability in analyte sensors of the present disclosure, where two or more active regions can be spatially separated from one another and covered with different mass transport limiting membranes. Specifically, bilayer and mixed membranes of the present disclosure can equalize analyte permeability in analyte sensors having separate working electrodes and including two or more active regions with different enzymes, with at least one active region disposed at each working electrode. Thus, such membranes can advantageously allow the sensor sensitivity to be independently varied 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 altered to tune the characteristic permeability of the analyte at each working electrode.

[0112] Thus, a method for using an analyte sensor including two working electrodes may include exposing the analyte sensor to a fluid including 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 area is disposed on a surface of the first working electrode, and a second active area is disposed on a surface of the second working electrode. The first active area includes a first polymer and a first analyte-responsive enzyme that reacts with the first analyte, and the second active area includes a second polymer and a second analyte-responsive enzyme that reacts with the second analyte. The first analyte-responsive enzyme and the second analyte-responsive enzyme are different. A multi-component membrane covers the first active area, and a homogeneous membrane covers the second active area. The multi-component membrane includes at least a first membrane polymer and a second membrane polymer that are different from each other, and the homogeneous membrane includes one of the first membrane polymer or the second membrane polymer. The method further includes acquiring a first signal at or above the redox potential of the first active area, acquiring a second signal at or above the redox potential of the second active area, and correlating the first signal to a concentration of a first analyte in the fluid and the second signal to a concentration of a second analyte in the fluid, where 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 the first channel and the second channel, in which case the potential can be applied simultaneously to both electrodes.

[0114] Embodiments disclosed herein include the following: A. An analyte sensor comprising two active regions having different analyte responsive enzymes. The analyte sensor comprises a sensor tail including at least a working electrode; and at least two active regions disposed on a surface of the working electrode, each active region comprising an analyte responsive enzyme and a polymer, and the analyte responsive enzyme in each active region is different, wherein each active region has an oxidation-reduction potential, and the oxidation-reduction potential of a first active region is sufficiently separated from the oxidation-reduction potential of a second active region to allow generation of a signal from the first active region independent of generation of a signal from the second active region.

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

[0116] C. An analyte sensor comprising two or more enzymes capable of interacting with each other in a coordinated manner. The analyte sensor comprises a sensor tail including at least a working electrode; and at least one active area disposed on a surface of the working electrode, the at least one active area comprising a first enzyme, a second enzyme, and a polymer, the first enzyme and the second enzyme capable of interacting in a coordinated manner; the first enzyme capable of converting an analyte to a first product, and the second enzyme capable of converting the first product to a second product to generate a signal at the working electrode; the second enzyme is covalently bound to the polymer and is non-reactive with the analyte.

[0117] D. A method for assaying an analyte using two or more enzymes capable of interacting with each other in a coordinated manner, the method including exposing an analyte sensor to a fluid containing an analyte; the analyte sensor includes a sensor tail including at least a working electrode and at least one active area disposed on a surface of the working electrode, the at least one active area including a first enzyme, a second enzyme, and a polymer; the first enzyme and the second enzyme are capable of interacting in a coordinated manner, the second enzyme being covalently attached 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 to generate a signal at the working electrode; and correlating the signal to a concentration of the analyte in the fluid.

[0118] E. Alcohol Sensor. The alcohol sensor comprises a sensor tail including at least a working electrode; and at least one active area disposed on a surface of the working electrode, the at least one active area comprising alcohol oxidase, xanthine oxidase, catalase, a polymer, and an electron transfer agent; the electron transfer agent and the xanthine oxidase are covalently bound to the polymer, and the alcohol oxidase is not covalently bound to the polymer; and the alcohol oxidase and the xanthine oxidase can interact in a coordinated manner to generate a signal at the working electrode that is proportional to the alcohol concentration.

[0119] F. A method for detecting alcohol, the method including exposing an analyte sensor to a fluid including ethanol; the analyte sensor includes at least a working electrode and a sensor tail including at least one active region disposed on a 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 the xanthine oxidase are covalently attached to the polymer, and the alcohol oxidase is not covalently attached to the polymer; and the alcohol oxidase and the xanthine oxidase are capable of interacting in a coordinated manner; reacting the ethanol with the alcohol oxidase to form acetaldehyde; reacting the acetaldehyde with the xanthine oxidase to form acetic acid to generate a signal at the working electrode; and correlating the signal with the ethanol concentration in the fluid.

[0120] G. An analyte sensor comprising two or more working electrodes covered with different mass transport limiting membranes, the analyte sensor comprising a sensor tail comprising at least a first working electrode and a second working electrode; a first active area disposed on the first working electrode, the first active area comprising a first polymer and a first analyte responsive enzyme reactive with a first analyte; a second active area disposed on the second working electrode, the second active area comprising a second polymer and a second analyte responsive enzyme reactive with a second analyte, wherein the first analyte responsive enzyme and the second analyte responsive enzyme are different; a multicomponent membrane covering the first active area, the multicomponent membrane comprising 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 area, the homogeneous membrane having a composition different from the multicomponent membrane, the homogeneous membrane comprising one of the first membrane polymer and the second membrane polymer.

[0121] H. A method of assaying two or more analytes using two working electrodes covered with different mass transport limiting membranes, the method comprising: 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 disposed on a surface of the first working electrode, the first active region including a first polymer and a first analyte responsive enzyme reactive with the first analyte, and a second active region disposed on a surface of the second working electrode, the second active region including a second polymer and a second analyte responsive enzyme reactive with the second analyte; the first analyte responsive enzyme and the second analyte responsive enzyme are different; and a multi-component membrane covering the first active region. the homogenous membrane covering the second active area, the multi-component membrane comprising at least a first membrane polymer and a second membrane polymer that are different from each other, the homogenous membrane comprising one of the first membrane polymer or the second membrane polymer and different in composition from the multi-component membrane; obtaining a first signal at or above the redox potential of the first active area, the first signal being proportional to a concentration of a first analyte in the fluid; obtaining a second signal at or above the redox potential of the second active area, the second signal being proportional to a concentration of a second analyte in the fluid; and correlating the first signal to the concentration of the first analyte in the fluid and correlating the second signal to the concentration of the second analyte in the fluid.

[0122] I. An alcohol sensor comprising glucose oxidase and xanthine oxidase interacting in a coordinated manner. The alcohol sensor comprises a sensor tail including at least a working electrode; a first active area disposed on a surface of the working electrode, the first active area comprising xanthine oxidase, catalase, a first polymer, and an electron transfer agent; the xanthine oxidase and the electron transfer agent being covalently bonded to the first polymer; a first membrane covering the first active area, the first membrane comprising a first membrane polymer and permeable to acetaldehyde; a second active area disposed on the first membrane, the second active area comprising glucose oxidase, catalase, and a second polymer; the glucose oxidase being covalently bonded to the second polymer; a second membrane covering the second active area, the second membrane comprising a second membrane polymer and permeable to glucose and alcohol; the glucose oxidase and the xanthine oxidase can interact in a coordinated manner to generate a signal at the working electrode that is proportional to an alcohol concentration.

[0123] J. A method for detecting alcohol using cooperative interactions between glucose oxidase and xanthine oxidase, the method comprising exposing an analyte sensor to a fluid containing ethanol and glucose; the analyte sensor comprising a sensor tail comprising: at least a working electrode; a first active area disposed on a surface of the working electrode, the first active area comprising xanthine oxidase, catalase, a first polymer, and an electron transfer agent, the xanthine oxidase and the electron transfer agent being covalently bonded to the first polymer; a first membrane covering the first active area, the first membrane comprising a first membrane polymer and permeable to acetaldehyde; a second active area disposed on the first membrane, the second active area comprising glucose oxidase, catalase, and a second polymer, the glucose oxidase being covalently bonded to the second polymer; a second active area covalently bonded to the polymer of said first active area; and a second membrane covering the second active area, said second membrane comprising a second membrane polymer, said second membrane being permeable to glucose and alcohol; said glucose oxidase and said xanthine oxidase being capable of interacting in a coordinated manner; oxidizing glucose with said glucose oxidase to produce hydrogen peroxide; forming a catalase-hydrogen peroxide complex; oxidizing alcohol with said catalase-hydrogen peroxide complex to form acetaldehyde; reacting said acetaldehyde with said xanthine oxidase to form acetic acid and produce a signal at a working electrode; and correlating said signal with the concentration of alcohol in said 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 and the second active region includes a second electron transfer agent, and the first and second electron transfer agents are different.

[0126] Element 4: A first electron transfer agent is covalently attached to the polymer at a first active region, and a second electron transfer agent is covalently attached to the polymer at a second active region. Element 5: The analyte responsive enzyme of each active region is covalently attached to a polymer.

[0127] Element 6: The analyte sensor further comprises a mass transport limiting membrane covering at least the at least two active areas. Element 7: At least one of the at least two active regions comprises two or more analyte responsive enzymes, which interact in a coordinated manner 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: A mass transport limiting membrane covers at least the at least two active areas.

[0129] Element 10: The analyte sensor includes glucose oxidase as a first enzyme and lactate oxidase as a 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 comprises catalase.

[0131] Element 13: The catalase is not covalently attached to the polymer. Element 14: The alcohol oxidase is not covalently attached to a polymer. Element 15: The first enzyme is not covalently attached to the polymer.

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

[0133] Element 18: The analyte sensor further comprises a mass transport limiting membrane covering at least said at least one active area. Element 19: The fluid is a biological fluid and the analyte sensor is exposed to the biological fluid in vivo.

[0134] Element 20: A mass transport limiting membrane covers at least one active area. Each of embodiments E and F may have one or more of the following additional elements in any combination.

[0135] Element 21: The catalase is not covalently attached to the polymer. Element 22: The sensor tail is configured for insertion into tissue. Element 23: The alcohol sensor further includes a mass transport limiting membrane covering at least the at least one active area.

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

[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 comprises a bilayer membrane.

[0138] Element 27: A first membrane polymer is disposed directly on the first active area, and a second membrane polymer is disposed on the first membrane polymer to define a bilayer membrane, the second membrane polymer also being present in the homogenous membrane.

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

[0140] Element 31: A first analyte responsive enzyme is covalently attached to the polymer at a first active area and a second analyte responsive enzyme is covalently attached to the polymer at a second active area. Element 32: An electron transfer agent is covalently attached to the polymer in each active region.

[0141] Element 33: The analyte responsive enzyme of each active region is covalently attached to a 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: The catalase is not covalently attached 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: Wherein the first membrane polymer comprises crosslinked polyvinylpyridine.

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

[0146] Analyte sensor A in combination 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; and 2, 5 and 6. Method B in combination 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; and any one of 2 to 8 and 10.

[0147] Analyte sensors of C in combination with elements 11 and 12; 11, 12 and 13; 12 and 13; 11 and 14; 11 and 15; 11 and 17; 11 and 18; 12 and 13; 12 and 14; 12 and 15; 12 and 16; 12 and 17; 12 and 18; 15 and 16; 15 and 17; 15 and 18; 16 and 17; 16 and 18; 17 and 18; 11, 12 and 13; 11, 12 and 14; 11-14; 11-14 and 16; 11-14 and 17; 11-14 and 18; 15-17; 15-18; and 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 1 8;15 and 19;15 and 20;16 and 17;16 and 18;16 and 19;16 and 20;17 and 18;17 and 19;17 and 20;18 and 19;18 and 20;19 and 20;11, 12 and 13;11, 12, 13 and 19;11, 12, 13 and 20;11, 12 and 14;11, 12, 14 and 1 9;11, 12, 14 and 20;11-14;11-14 and 19;11-14 and 20;11-14 and 16;11-14, 16 and 19;11-14, 16 and 20;11-14 and 17;11-14, 17 and 19;11-14, 17 and 20;11-14 and 18;11-14, 18 and 19;11-14, 1 Method D in combination with any one of 11 to 16 and 19; and any one of 11 to 16 and 20; 15 to 17; 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 any one of 11 to 16 and 20.

[0148] The analyte sensor of E in combination with elements 21 and 22; 21 and 23; 22 and 23; and 21-23. The method of F in combination with elements 21 and 22; 21 and 23; 22 and 23; 21-23; 21 and 24; 21 and 25.

[0149] Analyte sensor 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] Method 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] The 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. The 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: Analyte sensor with a multi-component membrane, the analyte sensor comprising: a sensor tail configured for insertion into tissue, the sensor tail including at least a working electrode; and first and second active regions including at least two different enzymes for measuring the concentration of at least one analyte disposed on the sensor tail; the first active region is covered with a first membrane polymer and a second membrane polymer that are different from each other.

[0153] B1: An analyte sensor having two active regions on a working electrode and configured to sense different analytes. The analyte sensor includes a sensor tail configured for insertion into tissue and including 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 in each active region being different and responsive to different analytes; each active region having an oxidation-reduction potential, the oxidation-reduction potential of a first active region being sufficiently separated from the oxidation-reduction potential of a second active region to allow signal generation from the first active region independent 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': A first active area is covered with a mixture of a first membrane polymer and a second membrane polymer, one of the first membrane polymer and the second membrane polymer covering the second active area as a homogenous membrane.

[0155] Element 2': A first active area is covered with a bilayer membrane comprising a first membrane polymer disposed on a second membrane polymer, the second membrane polymer covering the second active area as a homogenous membrane. Element 3': The first active region comprises a first enzyme of at least two different enzymes and the second active region comprises 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 interact in a coordinated manner to generate a signal proportional to the concentration of said at least one analyte.

[0157] Element 5': The second enzyme is capable of converting 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 comprises 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 comprises an electron transfer agent. Element 9': a first membrane polymer is disposed directly on the first active area, a second active area is disposed directly on the first membrane polymer, and a second membrane polymer is disposed directly on the second active area.

[0160] Element 10': The sensor tail includes a first working electrode and a second working electrode, a first active area disposed on the surface of the first working electrode, a second active area disposed on the surface of the second working electrode, a first enzyme reacting with a first analyte to generate a signal proportional to the concentration of the first analyte, and a second enzyme reacting with a second analyte to generate a signal proportional to the concentration of the second analyte.

[0161] Element 11': The first and second active regions each have an oxidation-reduction potential, and the oxidation-reduction potential of the first active region is sufficiently separated from the oxidation-reduction potential of the second active region to allow generation of a signal from the first active region independent of generation of a signal from the second active region.

[0162] Element 12': 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 13': the signal from the first active area corresponds to a first analyte concentration and the signal from the second active area corresponds to a second analyte concentration.

[0163] Element 14': The first active region includes a first electron transfer agent and the second active region includes a second electron transfer agent that is 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 includes a first electron transfer agent and the second active region includes a second electron transfer agent that is different from the first electron transfer agent.

[0165] Element 17': The first and second active areas are covered with mass transport limiting membranes, the first active area being covered with a single membrane polymer and the second active area being 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 given and should not be construed as limiting or defining the scope of the invention.

[0167] Working Example Example 1: Detection of glucose and lactate using an analyte sensor with two different active areas on a single working electrode. Two solutions containing different poly(vinylpyridine)-bound transition metal complexes were prepared. The 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] [ka]

[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 (270 mV separation, see FIG. 8). In addition to the transition metal complexes that act as the respective electron transfer agents, the polymer of formula 1 contained glucose oxidase (GOX) covalently bound thereto, and the polymer of formula 2 contained lactose oxidase (LOX) covalently bound 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 formulated as specified in Tables 1 and 2 below.

[0170] [Table 1]

[0171] [Table 2]

[0172] To deposit each active area, approximately 20 nL of each solution was deposited onto the carbon working electrode, each measuring approximately 0.1 mm 2 Two separate, distinct spots with an area of ​​0.01 μm were formed. One spot contained the glucose oxidase formulation and the other spot contained the lactose oxidase formulation. After deposition, the working electrode was cured overnight at 25° C.

[0173] After curing, the membrane was deposited on the working electrode. The membrane polymer was disclosed in a patent application filed on June 13, 2018, entitled "Temperature-Insensitive Membrane Materials and Analyte Sensors Including the Same." The electrode was a polyvinylpyridine copolymer with amine-free polyether side chain functional groups, as described in U.S. Provisional Patent Application No. 62 / 684,438, entitled "Sensors Containing the Same." Film deposition was achieved by dip-coating the electrode three times into a solution containing 4 mL of the film polymer (120 mg / mL) and 0.35 mL PEG1000 (200 mg / mL). Alternate spray coating, screen printing, or similar processes can be used to deposit the film. After deposition, the electrode was allowed to cure overnight at 25°C and then further cured in a dry vial at 56°C for 2 days.

[0174] After preparation, the electrodes were analyzed by cyclic voltammetry in buffer containing neither glucose nor lactate. The resulting cyclic voltammograms are shown in Figure 8. Since there was no current contribution from either glucose or lactate, Figure 8 shows the characteristic anodic and cathodic peaks for the two osmium complexes. The redox potentials reported above were calculated from the average of the cathodic and anodic peaks for 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 FIG. 8). At this potential, oxidation of the osmium complexes in the first polymer and glucose can occur, but not in the second polymer or lactate. To oxidize both osmium complexes and 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 FIG. 8).

[0176] Glucose and lactate analysis was performed by immersing the electrode in a buffer containing 5 mM glucose and 5 mM lactate, and E1 and E2 potentials were applied sequentially. Figure 9 shows four replicates of the electrode response in 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. Unknown glucose and lactate concentrations can be similarly analyzed by comparison to a lookup table or calibration curve.

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

[0178] [Table 3]

[0179] Approximately 15 nL of solution was applied to a 2 The working electrode was cured at 25° C. overnight after deposition. After curing, a poly(4-vinylpyridine) (PVP) film was deposited onto the working electrode from a coating solution containing 100 mg / mL PVP and 100 mg / mL PEGDE400. Film deposition was achieved by dip-coating the electrode three times into the coating solution. After deposition, the electrode was cured overnight at 25° C. and then further cured in a dry vial at 56° C. for 2 days. Spray coating, screen printing, or similar processes can be alternately used to deposit the film.

[0180] Ethanol analysis was performed by immersing the electrode in ethanol-containing PBS solutions, each containing various concentrations of ethanol. FIG. 10 shows three replicates of the response of an electrode containing alcohol oxidase and xanthine oxidase together in the sensing spot upon exposure to various ethanol concentrations. As shown, the current response increased for 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 with two different enzymes (XOX / GOX) working in concert on a single working electrode. A first spotting solution was prepared with the formulation shown in Table 4. All components were dissolved in 10 mM HEPES buffer at pH 8. Cross-linking was achieved using polyethylene glycol diglycidyl ether.

[0182] [Table 4]

[0183] Approximately 15 nL of the first spotting solution was placed at a depth of approximately 0.05 mm. 2 The solution was deposited on a carbon working electrode as a single spot (XOX spot) with an area of ​​100 nm to 100 nm. After deposition, the working electrode was cured at 25° C. overnight.

[0184] After curing, poly(4-vinylpyridine) (PVP) films were deposited on the working electrodes and XOX spots from a coating solution containing 100 mg / mL PVP and 100 mg / mL PEGDE400. Film deposition was achieved by dip-coating the electrodes three times into the coating solution. Alternately, spray coating, screen printing, or similar processes can be used to deposit the films. After deposition, the electrodes were cured overnight at 25°C and then further cured in a dry vial at 56°C for 2 days.

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

[0186] [Table 5]

[0187] Approximately 15 nL of the second spotting solution was placed at a depth of approximately 0.05 mm. 2 The mixture was deposited on the PVP film from above as a single spot (GOX spot) having an area of ​​100 μm. After deposition, the mixture was cured overnight at 25° C.

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

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

[0190] FIG. 12B shows comparative response data upon exposure 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 a separate active region. As shown, the sensor response was greater when catalase was included in the active region containing xanthine oxidase.

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

[0192] [Table 6]

[0193] Deposition and curing of the active area was performed as described in Example 1, except that the 0.1 mm 2 Instead of a single spot with an area of ​​0.01 mm each 2Six spots with an area of ​​100 nm were deposited. Unless otherwise noted below, film deposition was performed by dip coating (1-5 dippings of the electrode with approximately 10 min wait time between dippings). After completion of dip coating, the films were cured at 25 °C for 24 h, followed by curing at 56 °C for 48 h 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 increased to +40 mV vs. Ag / AgCl and the current was then continuously monitored. To measure the response at different lactate concentrations, sodium lactate was added to the buffer in 1 mM increments up to 5 mM. To determine the response stability, the current was measured over an extended period of time, such as 2 weeks in 5 mM sodium lactate.

[0195] Membrane Polymers 1A and 1B: The first membrane polymer tested was a cross-linked polyvinylpyridine-co-styrene polymer in which 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. Two different cross-linkers were used to affect cross-linking of this membrane polymer: glycerol triglycidyl ether (Gly3-formulation 1) and polyethylene glycol diglycidyl ether 400 (PEGDGE400-formulation 2). Formulation 1 contained 4 mL of membrane polymer (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 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 referred to herein as Polymers 1A and 1B, respectively.

[0196] FIG. 14 shows an exemplary plot of the response of electrodes coated 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) slowly decreased over the two-week measurement period, whereas the sensor current provided by polymer 1B (formulation 2) initially increased during the first week of lactate exposure and then decreased. In contrast, both of these films provided a stable response in the presence of 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 referred to herein as Polymer 2. 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] FIG. 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. There was a significant decrease in response over the first 48 hours, followed by relatively stable operation. Furthermore, the sensitivity was well below the target value of about 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 referred to herein as Polymer 3. 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. Unlike polymer 2 crosslinked with a higher molecular weight variant of the same crosslinker, polymer 3 surprisingly provided a stable current response over time. Moreover, the current responded quickly and achieved a stable current as the amount of lactate added 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 membrane polymer tested contained both non-crosslinked PEG and crosslinked PEG1000 chains. This membrane polymer is referred to herein as Polymer 4. 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. Moreover, the current responded quickly and achieved a stable value as the amount of lactate added was increased in 1 mM increments (Figure 19).

[0203] Bilayer membranes containing polymers 2 and 1B or polymers 2 and 1A: Formulation 3 (polymer 2) was coated on the electrode surface by repeated dip-coating operations. Spray coating, screen printing, or similar processes can be used alternately to deposit the membrane. Formulation 2 (polymer 1B) was then coated on the deposited cross-linked PVP layer by repeated dip-coating operations. There was a 10 minute waiting time between successive immersions. After all immersion operations were completed, the sensor was cured at 25°C for 24 hours, followed by curing at 56°C for 48 hours in a dry 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 bottom layer of crosslinked PVP (Polymer 2) and a top layer of crosslinked Polymer 1B to a 5 mM lactate solution. Unlike Polymer 1B or PVP crosslinked with the same crosslinker (Polymer 2), bilayer membranes containing these membrane polymers surprisingly provided a stable current response over time with an acceptable level of sensitivity, even though neither polymer alone provided acceptable performance. The response data in Figure 20 was for an electrode 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 can change the sensor performance, as shown below for polymer 2 and polymer 1 A. Thus, the Gly3-bridged variant of polymer 1B (i.e., polymer 1A) may provide similarly acceptable performance when combined in a bilayer with polymer 2, even though neither polymer provided acceptable performance alone.

[0206] FIG. 21 shows an exemplary plot of the response to 5 mM lactate solution of an electrode covered with a bilayer membrane including a bottom layer of crosslinked PVP (polymer 2) and a top layer of crosslinked polymer 1A, where the electrode was dip-coated various times with formulation 1 (polymer 1A) and formulation 3 (polymer 2). The crosslinker for PVP (polymer 2) in this case remained PEGDGE1000, while the crosslinker for polymer 1A was Gly3. This indicates that this crosslinker is also suitable for use in bilayer membrane configurations. As shown in FIG. 21, a good balance of sensitivity and stable current response was obtained by dip-coating the electrode twice with formulation 3 and four times with formulation 1. Varying the number of dip-coating operations changed the thickness of each component of the bilayer membrane and the mass ratio of the membrane polymers to each other. As shown in FIG. 21, a PVP layer that is too thin (zero or one polymer 2 dip) results in high sensitivity but low response stability. On the other hand, if it is too thick (more than three immersions), the electrode may have low sensitivity and poor response stability.

[0207] Mixed membranes containing membrane polymers 1B and 3: A 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 used to prepare formulations 1A and 1B (140 mg / mL) 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, each of which has been crosslinked with PEG400.

[0208] Figure 22 shows an exemplary plot of the response of an electrode covered with a blended membrane containing cross-linked PVP (polymer 3) and cross-linked polymer 1B to a 5 mM lactate solution. Similar to the bilayer containing one of the same components (polymer 1B), the blended membrane provided a stable current response over time and an acceptable level of sensitivity. Moreover, the current responded quickly and achieved a stable value as the amount of lactate added was increased in 1 mM increments (Figure 23).

[0209] Figure 24 shows an exemplary plot of the response of a sensor coated with a blend film containing various ratios of cross-linked PVP (polymer 3) and cross-linked polymer 1 B. As shown in Figure 24, a higher amount of polymer 1 B increases the sensitivity but reduces the response stability.

[0210] Example 4: Performance of a sensor containing two working electrodes covered with a bilayer mass transport limiting membrane. In this example, a first working electrode containing glucose oxidase and a second working electrode containing lactate oxidase were covered with a bilayer membrane. The active area containing glucose oxidase was deposited using a glucose oxidase formulation as described in Example 1 (Table 1). The active area containing lactate oxidase was deposited using a lactate oxidase formulation as described in Example 3 (Table 6). The active areas were deposited and cured as described in Example 1, except that the 0.1 mm mass transport limiting membrane of Example 1 was used. 2 Instead of a single spot with an area of ​​0.01 mm each2 Five spots with an area of ​​100 nm 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 membrane in this example. That is, polymer 3 was deposited on the second working electrode featuring lactate oxidase. Selective deposition on the second working electrode was achieved by a modified slot coating procedure. Curing was then carried out at 25° C. for 24 hours. The entire assembly (i.e., both working electrodes, the PVP coating on the second working electrode, as well as the counter and reference electrodes) was then dip-coated in formulation 2. Curing was again carried out at 25° C. for 24 hours, followed by baking at 56° C. for 48 hours in a dry environment. Thus, a homogenous membrane was deposited on the first working electrode (glucose-responsive) and a bilayer membrane was deposited on the second working electrode (lactate-responsive). The crosslinked PVP (polymer 3) was in contact with the lactate-responsive active area 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 for 2 weeks at 37° C. In this test, the sensor was held at +40 mV vs. Ag / AgCl. FIG. 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 sensor response remained very stable over the observation period.

[0212] Glucose and lactate were then added stepwise to 100 mM PBS at 37°C to measure the sensor's response to each analyte. In this test, the sensor was again held at +40 mV vs. Ag / AgCl. Glucose was added in a concentration range of 0-30 mM and lactate was added in a 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 to both analytes and remained stable at a given analyte concentration.

[0213] Example 5: Detection of ketones using an analyte sensor having diaphorase and β-hydroxybutyrate dehydrogenase interacting in concert. In this example, the membrane formulations shown in Table 7 below were coated onto a carbon working electrode. 2 Deposition was performed to place six spots with an area of ​​100 μm. After deposition, the working electrode was cured at 25 °C overnight. A PVP film was then applied to the working electrode via dip coating using a coating solution with the following formulation: 4 mL of 100 mg / mL PVP, 0.2 mL of 100 mg / mL PEGDGE400, and 0.0132 mL of 100 mg / mL PDMS. Film curing was performed at 25 °C for 24 h, followed by 56 °C for 48 h in a dry vial.

[0214] [Table 7]

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

[0216] Example 6: Comparison of lactate sensor response with various sensor configurations. To assay the performance of lactate-responsive sensors featuring various permutations of the formulations, two different lactate oxidase / polymer formulations for active area deposition and two different membrane polymer formulations for mass transport limiting membrane deposition were prepared. Details of the formulations and the process used to prepare the analyte sensors are provided below. In general, the analyte sensors were prepared in a similar manner as described above.

[0217] [Table 8]

[0218] [Table 9]

[0219] To deposit each active area, approximately 20 nL of each solution was deposited onto the carbon working electrode, each measuring approximately 0.01 mm 2 Six individual spots with an area of ​​100 nm were formed. Four applications of Formulation A and six applications of Formulation B were used to form the spots. After deposition, the working electrodes were cured overnight at 25° C. Formulation A corresponds to that used to deposit the active area of ​​the glucose-responsive analyte sensor, except that lactate oxidase was used instead of glucose oxidase.

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

[0221] [Table 10]

[0222] [Table 11]

[0223] A mass transport limiting membrane was deposited on each active area prepared as above using dip coating. Formulation C was deposited using 4 dips and Formulation D was deposited using 44 dips. A wait time of about 10 minutes was used between dips. After completion of dip coating, the membrane was cured at 25° C. for 24 hours, followed by curing at 56° C. for 48 hours in a dry vial. Alternately, spray coating, screen printing, or similar processes can be used to deposit the mass transport limiting membrane. Formulation C corresponds to that used to deposit the mass transport limiting membrane in the glucose-responsive analyte sensor.

[0224] Lactate-responsive analyte sensors were prepared using the deposition conditions specified above. All possible combinations of active regions and mass transport limiting membranes were prepared, and eight 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 vs. Ag / AgCl. The tested combinations of active regions and mass transport limiting membranes are shown in Table 12. The test results are shown in FIG. 30.

[0225] [Table 12]

[0226] As shown in Figure 30, lactate-responsive analyte sensors with active areas and mass transport limiting membranes formulated similarly to those successfully used in glucose-responsive analyte sensors (Group 1) yielded poor performance when exposed to lactate. As shown, signal strength was less than 0.5 nA for all samples tested, an undesirably low value for a viable lactate-responsive sensor. When polyvinylpyridine and a different crosslinker were used in place of polyvinylpyridine-co-styrene and the Gly3 crosslinker in Formulation C (Group 2), the signal strength was even lower.

[0227] Furthermore, as shown in Figure 30, the incorporation of human serum albumin significantly improved the sensor performance. For example, sample group 3 exhibited significantly higher signal intensity than that achieved with either group 1 or group 2 samples. However, there was considerable variability in initial signal intensity between samples in this group (>4 nA variability). Furthermore, there was a steady decrease in signal intensity from the maximum signal intensity initially observed. The variability in response and poor signal stability over time make this sample group combination similarly unlikely to be suitable for a viable lactate-responsive analyte sensor.

[0228] Surprisingly, the combination of an active region containing human serum albumin and a mass transport limiting membrane containing cross-linked 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 between 4 and 5 nA, clustered within 1 nA of each other. This level of signal intensity and variability is within the range in which a commercially viable lactate-responsive analyte sensor may be developed. Furthermore, the signal intensity changed by no more than a few tenths of a nA over 190 hours of signal observation. This is also within the range that may be suitable for the development of a commercially viable sensor.

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

[0230] Unless otherwise noted, all numbers expressing quantities and the like in the specification and the associated claims should be understood in all instances to be modified by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by embodiments of the invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed at least in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0231] Analyte Sensor Ignition Lock Vehicle fail-safes, such as ignition locks, are sometimes used to prevent an operator from operating a vehicle when impaired or otherwise in an unsafe condition to operate the vehicle. Operating a vehicle while impaired can pose serious danger to the operator and the public. One common type of ignition lock is designed to prevent drunk driving, and more specifically, to prevent an individual from operating a vehicle while intoxicated by the use of alcohol. Such locking devices connect a breath alcohol analyzer or optical sensor to the vehicle's ignition system and require the driver to 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, other impairments and conditions may also afflict the operator and must be closely monitored to ensure that the operator does not operate a vehicle while impaired. For example, an operator who is diabetic and has low blood sugar while driving may experience lightheadedness, confusion, headaches, loss of consciousness, seizures, and slowed reflexes, any of which may endanger their own life and the lives of others in or near the vehicle.

[0233] Analyte monitoring systems have been developed to facilitate long-term monitoring of analytes in bodily fluids (e.g., blood). Some analyte monitoring systems are designed to detect and monitor blood glucose levels, which aid in the treatment of diabetic conditions. However, other analyte monitoring systems are designed to detect and monitor other analytes present in an operator's bodily fluids, and abnormal analyte levels detected in the operator may indicate that the operator is currently unfit to safely operate a vehicle.

[0234] The following description describes an analyte monitoring and vehicle control system used to prevent operation of a vehicle when an operator's analyte levels exceed a predetermined threshold. By properly deploying the sensor control device 102 (FIG. 1), a user can intelligently track and monitor bodily fluid analyte levels and trends. When some analyte levels exceed a certain threshold, a physical or cognitive impairment may occur that prevents the user from safely operating the vehicle. In such cases, the user must take appropriate measures to bring the analyte levels back into a safe range before attempting to operate the vehicle. However, in some cases, the user may feel perfectly fine to operate the vehicle, yet have unsafe analyte levels that may suddenly cause the onset of a dangerous physical impairment. In such cases, it may be advantageous to install a fail-safe system that prevents or warns the user from operating the vehicle and potentially endangering themselves or others.

[0235] 32 is a schematic diagram of an exemplary analyte monitoring and vehicle control system 3200 in accordance with 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 that can be deployed on a user or "operator" 3202 or delivered to a target monitoring location on the operator's 3202 body, such as the back of the arm. As described above, the sensor control device 102 includes a sensor 104 (FIG. 1) that, when properly deployed, is transdermally placed within the skin to detect and monitor analytes present within the bodily fluids of the operator 3202. An adhesive patch 105 (FIG. 1) applied to the bottom of the sensor control device 102 adheres to the skin to secure the sensor control device 102 in place during operation.

[0236] Although the system 3200 is described herein as including an on-body sensor control device 102 for detecting and reporting analyte levels, the system 3200 could instead incorporate an in vitro analyte sensor (e.g., a self-monitoring blood glucose "SMBG" meter) without departing from the scope of the present disclosure. Thus, the term "sensor control device" should be construed herein to include not only on-body sensor systems as primarily described above, but also traditional handheld sensor systems.

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

[0238] In the illustrated embodiment, vehicle 3206 is depicted as an automobile. However, as used herein, the term "vehicle" is used broadly and is meant to include any type of transportation that can be operated by a human user or "operator," but may also include autonomous vehicles used to transport humans. Examples of vehicle 3206 include, but are not limited to, any type of automobile, truck, sport utility vehicle, aircraft, watercraft, spacecraft, and / or any other mode of transportation, or combinations thereof.

[0239] The control module 3204 may include a communication interface for communicating information between the sensor control device 102 and / or the reader device 120. In the case of an exemplary BLUETOOTH-enabled sensor control device 102 and / or reader device 120, when the sensor control device 102 approaches the vehicle 3206, it may enter a pairing mode. During pairing, the control module 3204 may be programmed and configured to automatically detect the presence of the sensor control device 102 and / or the reader device 120 and establish communication therewith. For example, when the operator 3202 approaches or enters the vehicle 3206, the control module 3204 may automatically detect the presence of the sensor control device 102 and / or the reader device 120 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 visually communicate with the operator 3202 via the vehicle user interface 3208 and can also audibly communicate 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 such that it can communicate with the operator 3202.

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

[0242] In some embodiments, for example, the computer system 3210 may be configured to disable various critical vehicle systems when the operator 3202's detected analyte levels are outside of a predetermined range or exceed a predetermined threshold, thus gradually and safely disabling operation of the vehicle upon identifying the operator 3202 as impaired from safely operating the vehicle 3206. Critical vehicle systems of the vehicle 3206 that may be disabled include the ignition system (e.g., energy switching / control system), transmission system (or gearbox), fuel system, energy supply system (e.g., battery, capacitor, conversion / reaction cell, etc.). When elevated or depressed (unsafe) analyte levels are detected, the computer system 3210 may prevent critical vehicle systems from functioning or operating. As a result, the operator 3202 is unable to start or operate the vehicle 3206, thereby preventing the operator 3202 from endangering himself and / or others.

[0243] In other embodiments, or in addition, the computer system 3210 may be configured to activate various non-critical vehicle systems when the detected analyte levels of the operator 3202 rise above or exceed a predetermined threshold. Non-critical vehicle systems that may be activated include, for example, a vehicle horn, vehicle lights, or an audible warning system installed on the vehicle 3206. In such embodiments, activation of a non-critical vehicle system may alert law enforcement and others (e.g., operators of adjacent vehicles, bystanders, pedestrians, etc.) of the operator 3202 that may be operating in an impaired condition, thus enabling law enforcement to quickly address the problem associated therewith and notifying others of a potentially dangerous situation.

[0244] In yet other embodiments, or in addition, the computer system 3210 may be configured to automatically call one or more emergency contacts when the operator's 3202 analyte levels are outside of a predetermined safe operating range or exceed a predetermined threshold. In such embodiments, the computer system 3210 may operate via the reader device 120 (e.g., a cell phone) or a cellular or satellite communication system (e.g., OnStar®) integrated into the vehicle 3206. In other embodiments, or in addition, the computer system 3210 may be configured to automatically send a message (e.g., a text or SMS message, email, etc.) to an emergency contact when the operator's 3202 analyte levels are outside of a predetermined safe operating range or exceed a predetermined threshold. Examples of emergency contacts include, but are not limited to, a spouse, a parent, a medical professional (e.g., a doctor), a hospital, 911, or any combination thereof.

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

[0246] In some embodiments, the control module 3204 may further include a vehicle state detection module 3216 configured to detect a current state of the vehicle 3206, including whether the vehicle 3206 is currently moving or stationary. Additionally, the vehicle state detection module 3216 may be configured to determine whether a motor in the vehicle 3206 is currently running or stopped. In one or more embodiments, the vehicle state detection module 3216 may provide a state signal to the control module 3204, and the control module 3204 may use the state signal to determine which vehicle operations to activate or disable when the real-time measured analyte levels of the operator 3202 are outside of a predetermined safe range or exceed a predetermined threshold. For example, if the state signal indicates that the vehicle 3206 is stationary, the control module 3204 may disable the vehicle fuel system, the transmission system, the ignition system, or any combination thereof. In contrast, if the status signal indicates that the vehicle 3206 is moving, the control module 3204 may activate the vehicle horn, flash the vehicle lights, or sound an alarm to the operator 3202 and / or those around the operator 3202 that the operator 3202 is not normal.

[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 vehicle user interface 3208. A digital dashboard may then be displayed on the reader device 120 and / or vehicle user interface 3208 showing current analyte levels, trends, historical data, and predicted analyte levels. However, if the current analyte levels are outside of 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 an embodiment, a visual or audio warning may be issued by the control module 3204 to inform the operator 3202 why the vehicle 3206 will not start. More specifically, a visual alert (e.g., a written message) may be generated and displayed on the reader device 120 or vehicle user interface 3208, or an audible alert (e.g., a voice message) may be communicated through a speaker in the reader device or vehicle 3206.

[0248] If not done automatically, the operator 3202 may be prompted to obtain a current analyte level upon pairing the sensor control device 102 with the control module 3204. In some cases, operation of the vehicle 3206 may be prevented until a current analyte level is obtained. If the current analyte level is within safety limits, the computer system 3210 may allow operation of the vehicle 3206. In some embodiments, and unless done automatically, the control module 3204 may prompt the operator 3202 to obtain an additional current analyte level after operating the vehicle 3206 for a predetermined period of time (e.g., after 1 hour, 2 hours, 5 hours, etc.).

[0249] In some embodiments, the control module 3204 may be configured to issue visual or audio recommendations or instructions to the operator 3202 that may aid in returning the measured analyte levels to a safe range. In such embodiments, such visual or audio recommendations may prompt the user to take some action that may result in returning the analyte levels to a safe range. Additionally, in some embodiments, the operator 3202 may be able to verbally communicate with the control module 3204 by issuing verbal responses or commands. This may prove advantageous in preventing distracted operation of the vehicle 3206.

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

[0251] In some embodiments, the computer system 3210 may be configured or programmed to calculate a predictive timeline when the operator's 3202 analyte levels are likely to deviate from a predefined safe range or exceed a predefined threshold. In such embodiments, the control module 3204 may be configured to issue a visual or audio warning to the operator 3202 indicating approximately how much time the operator 3202 has before reaching an unsafe analyte level and a condition in which a potentially dangerous medical condition may result. Multiple warnings may be provided to indicate that the operator has a certain increment of time left before reaching an unsafe analyte level. For example, a visual or audio warning may be issued when an unsafe analyte level is about to be reached within 1 hour, 30 minutes, 10 minutes, 5 minutes, 1 minute, and any time increment therebetween. Additionally, a visual or audio warning may be issued when the operator's analyte levels reach an unsafe level or exceed a predefined threshold.

[0252] In some embodiments, if 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 alert the operator 3202 of the unsafe analyte level. In some cases, the volume of a stereo in the vehicle 3206 may be automatically lowered so that the operator 3202 can hear the audible warning. In such embodiments, the control module 3204 may be configured to suggest one or more corrective actions to the operator 3202. Examples of corrective actions include, but are not limited to, slowing and stopping the vehicle 3206, locating and driving to a nearby convenience store or pharmacy, and locating a nearby hospital or medical facility. If the vehicle 3206 is an autonomous vehicle and the current analyte level places the operator 3202 in a potentially unsafe condition, the control module 3204 may automatically direct the vehicle 3206 to a medical facility for treatment. Alternatively, or in addition, the control module 3204 can gradually slow or limit the speed of the vehicle 3206 when an unsafe analyte level is detected, thus stopping the operator 3202 and addressing the problem before continuing to operate the vehicle 3206.

[0253] The system 3200 may be useful in several different scenarios to protect the operator 3202 and / or people around the operator 3202 while driving. In some applications, the system 3200 may be voluntarily installed by the operator to detect impairments in real time. In other applications, the 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 may be a transportation or trucking company. In still other applications, the system 3200 may be legally mandated on the operator 3202 to detect impairments.

[0254] Embodiments disclosed herein include the following: K. 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 the vehicle's electrical system, the control module including a computer system programmed to receive and process data provided by the sensor control device, wherein operation of the vehicle is controlled or disabled by the computer system when the operator's real-time measured analyte levels exceed a predetermined safety threshold.

[0255] L. A method comprising: 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 the vehicle; and controlling or disabling operation of the vehicle using a computer system of the control module when a real-time measured analyte level in the operator exceeds a predetermined safety threshold.

[0256] Each of the embodiments K and L may have one or more of the following additional elements in any combination: Element 1: A sensor control device is coupled to an operator, and a sensor is transdermally placed under the skin of the operator for detecting and monitoring an analyte present in the operator's bodily fluid. Element 2: The sensor control device includes an in vitro analyte sensor. Element 3: The sensor control device further includes a reader device that receives data from the sensor control device and transmits the data to the control module. Element 4: The vehicle includes a vehicle selected from the group consisting of an automobile, an autonomous vehicle, a truck, a sports utility vehicle, an aircraft, a watercraft, 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 includes a vehicle user interface included in the vehicle and in communication with the control module. Element 7: The operation of the vehicle is disabled by disabling one or more critical systems of the vehicle, the critical systems being 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 driver's area of ​​the vehicle and detect the presence of an operator. Element 10: The control module further comprises a vehicle state detection module that detects a current state of the vehicle. Element 11: The control module generates a visual or audible warning perceptible to the operator when the operator's real-time measured analyte level is outside of a predetermined safety threshold. Element 12: The visual or audible warning is generated at a specific time increment before an unsafe analyte level is reached. Element 13: The visual or audible warning includes one or more suggested corrective actions that are communicated to the operator. Element 14: The control module includes a bypass function that allows the operator to operate the vehicle when the operator's real-time measured analyte level exceeds a predetermined threshold.

[0257] Element 15: further comprising receiving data from the sensor control device and transmitting the data to the control module using a reader device that communicates with the sensor control device and the control module. Element 16: disabling the operation of the vehicle includes disabling one or more critical systems of the vehicle, the critical systems being 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 comprising monitoring a driver's area of ​​the vehicle using one or more proximity sensors mounted on the vehicle to detect the presence of an operator. Element 19: further comprising detecting a current state of the vehicle using a vehicle state detection module included in the control module. Element 20: further comprising generating a visual or audio alert perceivable by 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 embodiment incorporating an embodiment of the present invention, it is understood that numerous implementation-specific decisions must be made to achieve the developer's goals, such as compliance with system-related, business-related, government-related and other constraints. These will vary from implementation to implementation and from case to case. While the developer's efforts may be time consuming, such efforts are nevertheless routine for those of ordinary skill in the art who would have 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 may also "consist essentially of" or "consist of" various components and steps.

[0260] As used herein, the phrase "at least one of" with the term "and" or "or" preceding a list of items and separating any of the items modifies the entire list, not each member (i.e., each item) of the list. The phrase "at least one of" allows for the meaning of including at least one of any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. By way of example, the phrase "at least one of A, B, and C" or "at least one of A, B, or C" refers, respectively, to A only, B only, or C only; any combination of A, B, and C; and / or at least one of each of A, B, and C.

[0261] Thus, the disclosed systems, tools, and methods are well adapted to achieve the objects and advantages mentioned, as well as those inherent therein. The specific embodiments disclosed above are illustrative only, as the teachings of the disclosure may be modified and implemented in different and equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Moreover, no limitations are intended to the details of construction or design shown herein, except as set forth in the appended claims. Thus, it will be apparent that the specific exemplary embodiments disclosed above may be altered, combined, or modified, and all such variations are deemed to be within the scope of the present disclosure. The systems, tools, and methods illustratively disclosed herein may suitably be practiced in the absence of elements not specifically disclosed herein and / or optional elements disclosed herein. Although the systems, tools, and methods are described in terms of "including" various components or steps, the systems, tools, and methods may also "consist essentially of" or "consist of" various components and steps. All of the numbers and ranges disclosed above may vary somewhat. Whenever a numerical range with a lower limit and an upper limit is disclosed, any number within that range and any included range is 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 to b") should be understood to describe all numbers and ranges within the broader range of values. In addition, unless expressly and unambiguously defined by the patent owner, the claim terms have their ordinary meaning. In the event of a discrepancy in the usage of a word or term between this specification and one or more patent documents or other documents that may be incorporated by reference herein, the definition in accordance with this specification shall prevail.

Claims

1. 1. An analyte sensor comprising: a) a working electrode; b) an ethanol-responsive active region, i. a first active area disposed directly on a surface of the working electrode, the first active area comprising xanthine oxidase and an electron transfer agent in an amount sufficient to react with acetaldehyde to produce a signal at the working electrode proportional to the alcohol concentration; and ii. the ethanol-responsive active region comprising a second active region isolated from the working electrode, the second active region comprising glucose oxidase and catalase in sufficient amounts to form acetaldehyde upon exposure to alcohol; c) a first membrane disposed on the first active area, the first membrane being permeable to acetaldehyde; wherein the second active area is vertically stacked above the first active area separated by the first membrane.

2. The analyte sensor of claim 1, further comprising a second membrane disposed on the second active area, the second membrane being permeable to glucose and alcohol.

3. The analyte sensor of claim 1 , wherein the first active region further comprises catalase.

4. The analyte sensor of claim 2 , wherein the first membrane is disposed directly over the first active area and the second membrane is disposed directly over the second active area.

5. 10. The analyte sensor of claim 1, wherein the first membrane is a cross-linked polyvinylpyridine homopolymer or copolymer.

6. 3. The analyte sensor of claim 2, wherein the second membrane is a cross-linked polyvinylpyridine-co-styrene polymer.

7. The analyte sensor of claim 1 , wherein the first active area comprises a first polymer.

8. The analyte sensor of claim 7 , wherein the xanthine oxidase is covalently attached to the first polymer.

9. The analyte sensor of claim 7 , wherein the electron transfer agent is covalently attached to the first polymer.

10. The analyte sensor of claim 1 , wherein the second active area comprises a second polymer.

11. The analyte sensor of claim 10 , wherein the glucose oxidase is covalently bonded to the second polymer.

12. 1. An analyte sensor comprising: a) a working electrode; b) an ethanol-responsive active region, i. a first active area disposed directly on a surface of the working electrode, the first active area comprising xanthine oxidase, an electron transfer agent, and catalase in an amount sufficient to react with acetaldehyde to produce a signal at the working electrode proportional to the alcohol concentration; and ii. the ethanol-responsive active region comprising a second active region isolated from the working electrode, the second active region comprising glucose oxidase and catalase in sufficient amounts to form acetaldehyde upon exposure to alcohol; c) a first membrane, permeable to acetaldehyde, disposed on the first active area; d) a second membrane disposed on the second active area, the second membrane being permeable to glucose and alcohol; wherein the second active area is vertically stacked above the first active area separated by the first membrane.

13. 13. The analyte sensor of claim 12, wherein the first membrane is a cross-linked polyvinylpyridine homopolymer or copolymer.

14. 13. The analyte sensor of claim 12, wherein the second membrane is a cross-linked polyvinylpyridine-co-styrene polymer.

15. The analyte sensor of claim 12 , wherein the first active area comprises a first polymer.

16. 16. The analyte sensor of claim 15, wherein the xanthine oxidase is covalently attached to the first polymer.

17. The analyte sensor of claim 15 , wherein the electron transfer agent is covalently attached to the first polymer.

18. The analyte sensor of claim 12 , wherein the second active area comprises a second polymer.

19. 20. The analyte sensor of claim 18, wherein the glucose oxidase is covalently bonded to the second polymer.

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