Analyte sensor using multiple enzymes and related methods

Abstract

This invention provides an analyte sensor configured for the detection of glucose and lactate. [Solution] The analyte sensor includes at least first and second working electrodes; a lactate-responsive active region and a glucose-responsive active region positioned above each working electrode; and a mass transfer limiting membrane positioned above each active region. The mass transfer limiting membrane has different permeability values, resulting in variable analyte permeability in the lactate-responsive active region and the glucose-responsive active region. The analyte sensor is configured to be partially inserted into the user's skin, so that lactate and glucose are detected in vivo when the distal portion of the analyte sensor comes into contact with interstitial fluid.

Description

[Background technology]

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

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

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

[0004] Regular in vitro analyte monitoring may be sufficient to determine the physiological state of many individuals. However, in vitro analyte monitoring can be inconvenient or painful for some. Furthermore, if analyte measurements are not taken at the appropriate time, there is no way to recover the lost data.

[0005] Continuous analyte monitoring using implanted sensors may be a more desirable approach for individuals with severe analyte dysregulation and / or rapidly fluctuating analyte levels, but it may also be beneficial for other individuals. While continuous analyte monitoring with implanted sensors can be advantageous, there are challenges associated with these types of measurements. Intravenous analyte sensors have the advantage of providing analyte concentrations directly from the blood, but they are invasive and can be painful for individuals to wear, especially over long periods. Subcutaneous, interstitial, or cutaneous analyte sensors are often less painful for individuals 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, 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 can be inconvenient because the individual needs to wear multiple analyte sensors. Furthermore, multiple analyte sensors can represent an unacceptable cost burden for the individual or insurance company. Additionally, there is a higher probability that one of the independent analyte sensors will fail during such a detection protocol.

[0007] In vivo analyte sensors may also include a membrane placed over at least the embedded portion of the analyte sensor. In one embodiment, the membrane can improve the biocompatibility of the analyte sensor. In another embodiment, the membrane may be permeable or semipermeable to the analyte of interest and restrict the overall analyte flux to the active region of the analyte sensor. That is, the membrane can function as a mass transfer limiting membrane. By using a mass transfer limiting membrane to restrict analyte access to the active region of the sensor, sensor overload (saturation) can be avoided, thereby improving detection performance and accuracy. Such membranes are highly specialized in restricting the mass transfer of a particular analyte and may allow other substances to pass through the membrane at significantly different rates. The different membrane permeability of various potential analytes is a major obstacle to developing analyte sensors configured for the analysis of multiple analytes. In other words, different membrane permeability values ​​can lead to significantly different sensitivities to multiple analytes, thereby complicating the analysis. Different sensitivities to multiple analytes can sometimes be partially offset by using active regions of different sizes (e.g., a smaller active region for highly sensitive / permeable analytes and a larger active region for less sensitive / permeable analytes). However, this approach presents significant manufacturing challenges and is not always applicable. [Brief explanation of the drawing]

[0008] The accompanying drawings are included to illustrate specific aspects of the disclosure and should not be considered exclusive embodiments. The disclosed subject matter can be substantially modified, altered, combined, and replaced with equivalents in form and function without departing from the scope of the disclosure. [Figure 1] An example of a detection system that may incorporate the analyte sensor of this disclosure is shown in the diagram. [Figure 2A] A diagram shows an exemplary two-electrode analyte sensor configuration having a single working electrode, suitable for use in some embodiments disclosed herein. [Figure 2B]A diagram shows an exemplary three-electrode analyte sensor configuration having a single working electrode, suitable for use in some embodiments disclosed herein. [Figure 2C] Same as above. [Figure 3] A diagram shows an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode, suitable for use in some embodiments disclosed herein. [Figure 4] An exemplary analyte sensor configuration suitable for use in some embodiments disclosed herein is shown, in which two different active regions are arranged on the surface of a single working electrode. [Figure 5A] The present disclosure illustrates coordinated enzymatic reaction cycles related to ethanol detection using alcohol oxidase and xanthine oxidase directly placed on a working electrode, according to various embodiments of this disclosure. [Figure 5B] The present disclosure illustrates a coordinated enzyme reaction cycle related to ketone detection using β-hydroxybutyrate dehydrogenase, nicotinamide adenine dinucleotide, and diaphorase directly placed on a working electrode, according to various embodiments of this disclosure. [Figure 5C] The present disclosure illustrates a coordinated enzymatic reaction cycle related to ketone detection using β-hydroxybutyrate dehydrogenase, nicotinamide adenine dinucleotide, NADH oxidase, and superoxide dismutase directly placed on a working electrode, according to various embodiments of this disclosure. [Figure 5D] The present disclosure illustrates a coordinated enzymatic reaction cycle related to ketone detection using β-hydroxybutyrate dehydrogenase, nicotinamide adenine dinucleotide, and poly-1,10-phenanthroline-5,6-dione, all directly positioned on a working electrode, according to various embodiments of this disclosure. [Figure 5E] Various embodiments of this disclosure illustrate coordinated enzymatic reaction cycles related to ethanol detection using glucose oxidase, catalase, and xanthine oxidase, where glucose oxidase is located away from the working electrode and xanthine oxidase is located directly on the working electrode. [Figure 6A]A diagram of an exemplary working electrode is shown, suitable for use in some embodiments disclosed herein, in which a first active region is located directly on the surface of the working electrode and a second active region is separated from the working electrode by a film. [Figure 6B] Same as above. [Figure 6C] The diagram shows an exemplary working electrode in which a first active region and a second active region are arranged on the working electrode with a lateral spacing between them, and one of the active regions is separated from the working electrode by a membrane. [Figure 7] An exemplary schematic diagram of a portion of an analyte sensor suitable for use in some embodiments disclosed herein is shown, which has two working electrodes and features a bilayer film covering one of the two working electrodes. [Figure 8] An exemplary cyclic voltammogram obtained with an analyte-free buffer using a working electrode containing two different osmium complexes as electron transfer mediators is shown. [Figure 9] Figure 8 shows four replicas of the electrode response in 5 mM glucose / 5 mM lactate buffer when the electrode was circulated between the E1 and E2 potentials. [Figure 10] Three replicates of the response of an electrode containing both alcohol oxidase and xanthine oxidase in its active region upon exposure to various ethanol concentrations are shown. [Figure 11A] Figure 10 shows an illustrative plot of the average current response of the electrodes versus the ethanol concentration. [Figure 11B] An illustrative plot of the current response versus ethanol concentration at a single electrode is shown. [Figure 12A] Two replicates of the responses of electrodes containing glucose oxidase and xanthine oxidase, layered in separate active regions and separated by a membrane, to exposure to various ethanol concentrations are shown, with catalase present in the active region containing glucose oxidase. [Figure 12B]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 are shown, with catalase being present separately in the active regions. [Figure 13] An exemplary plot of the average current response of the electrode of FIG. 12 versus ethanol concentration is shown. [Figure 14] An exemplary plot of the response of electrodes covered with polymers 1A and 1B to a 5 mM lactate solution is shown. [Figure 15] An exemplary plot of the response of an electrode covered with polymer 2 to a 5 mM lactate solution is shown. [Figure 16] An exemplary plot of the response of an electrode covered with polymer 3 to a 5 mM lactate solution is shown. [Figure 17] An exemplary plot of the response of electrodes covered with polymer 3 to lactate solutions having various lactate concentrations is shown. [Figure 18] An exemplary plot of the response of an electrode covered with polymer 4 to a 5 mM lactate solution is shown. [Figure 19] An exemplary plot of the response of electrodes covered with polymer 4 to lactate solutions having various lactate concentrations is shown. [Figure 20] An exemplary plot of the response of an electrode covered with a two-layer film comprising a lower layer of cross-linked PVP (polymer 2) and an upper layer of cross-linked polymer 1B to a 5 mM lactate solution is shown. [Figure 21] An exemplary plot of the response of an electrode covered with a two-layer film comprising a lower layer of cross-linked PVP (polymer 2) and an upper layer of cross-linked polymer 1A to a 5 mM lactate solution is shown, where the electrode was dip-coated various times with formulations 1 and 3. [Figure 22] An exemplary plot of the response of an electrode covered with a mixed film comprising cross-linked PVP (polymer 2) and cross-linked polymer 1B to a 5 mM lactate solution is shown. [Figure 23] An exemplary plot of the response of electrodes covered with a mixed film comprising cross-linked PVP (polymer 2) and cross-linked polymer 1B to various lactate concentrations is shown. [Figure 24] Exemplary plots of the response of electrodes covered with mixed films containing crosslinked PVP (polymer 2) and crosslinked polymer 1B in various ratios are shown. [Figure 25] An exemplary plot of the response of a sensor containing two working electrodes to a 30 mM glucose / 5 mM lactate solution is shown, with the lactate-responsive working electrode covered by a bilayer membrane and the glucose-responsive working electrode covered by a homogeneous membrane. [Figure 26] Exemplary plots of the response of a sensor containing two working electrodes to solutions with varying concentrations of glucose and lactate are shown, with the lactate-responsive working electrode covered by a bilayer membrane and the glucose-responsive working electrode covered by a homogeneous membrane. [Figure 27] Four replicas of the electrode responses, including diaphorase, NAD+, and β-hydroxybutyrate dehydrogenase, when exposed to various β-hydroxybutyrate concentrations are shown. [Figure 28] Figure 27 shows an exemplary plot of the average current response of the electrodes versus the β-hydroxybutyrate concentration. [Figure 29] Figure 27 shows an exemplary plot of the current response of the electrode after exposure to 8 mM β-hydroxybutyrate in 100 mM PBS at 33°C for 2 weeks. [Figure 30] The following are illustrative plots of the sensor performance of groups 1 to 4 in Example 6. [Figure 31] Exemplary plots of the responses of Group 1 analytes from Example 6 to lactate solutions with various lactate concentrations are shown. [Figure 32] This is a schematic diagram of an exemplary analyte monitoring and vehicle control system according to one or more embodiments of the present disclosure. [Modes for carrying out the invention]

[0009] Detailed explanation This disclosure generally describes analytic sensors and methods that use multiple enzymes for detection, more specifically, analytic sensors and methods in which multiple enzymes function independently or in coordination to detect one or more analytics.

[0010] As mentioned above, analyte sensors are typically used to detect a single analyte. If the detection of multiple analytes is desired, a corresponding number of analyte sensors may be used. This approach may be undesirable, among other things, due to cost concerns, the need for individuals to wear multiple analyte sensors, and the increased possibility of individual sensor failure.

[0011] Some analyte sensors utilize enzymatic reactions as the basis for detecting the target analyte. Since enzymes often exhibit reaction specificity for specific substrates or related classes of substrates, they can provide analyte sensors with detection chemistry configured to analyze a single target analyte. Therefore, analyte sensors for analyzing a single analyte typically incorporate only the corresponding single enzyme to facilitate the appropriate enzymatic reaction for detection. Currently, incorporating multiple enzymes into a single analyte sensor to provide detection capabilities for multiple analytes can be quite challenging. This is due to differences in analyte sensitivity and the potential incompatibility of one or more enzymes for a given set of analytical conditions.

[0012] In contrast to analyte sensors featuring a single enzyme, this disclosure describes analyte sensors in which multiple enzymes are present in one or more active regions of the sensor. Many advantages can be realized by incorporating multiple enzymes into analyte sensors in the various ways described herein. In some sensor configurations of this disclosure, multiple enzymes can facilitate the independent detection of multiple analytes, such as glucose and lactate. Membranes configured to provide permeability tailored to multiple analytes are also described herein, facilitating analyte detection by a single analyte sensor by leveling the sensor's sensitivity to each analyte. In other sensor configurations of this disclosure, multiple enzymes are selected to function in coordination, facilitating the detection of a single target analyte that would have been 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. Furthermore, this disclosure can provide sensors that are smaller in size and reduce the complexity of the measuring electronics. Thus, analyte sensors using multiple enzymes in various configurations can facilitate the efficient detection of one or more analytes as disclosed herein.

[0013] Analyte sensors containing multiple enzymes can function with greater stability in the presence of appropriate stabilizers, regardless of whether the enzymes act independently or in coordination. Suitable stabilizers 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 the biological environment. Albumin, on the other hand, is not thought to exhibit the ability to remove reactive species, and consequently, their ability to stabilize the response of the analyte sensors of this disclosure is remarkable.

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

[0015] Figure 1 shows a diagram of an exemplary detection system into which the analyte sensor of this disclosure may be incorporated. As shown, the detection system 100 includes a sensor control device 102 and a reader device 120 configured to communicate with each other via a wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted local communication path or link. The reader device 120 may, according to some embodiments, constitute an output medium for displaying the analyte concentration and a warning or notification determined by the sensor 104 or its associated processor, and may allow one or more user inputs. The reader device 120 may be a multipurpose smartphone or a dedicated electronic reader device. Although only one reader device 120 is shown, in certain cases, there may be multiple reader devices 120. The reader device 120 may also communicate with remote terminals 170 and / or trusted computer systems 180, respectively, via a communication path / link 141 and / or 142, which is also wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. In addition to or instead of the above, the reader device 120 may communicate with a network 150 (e.g., a cellular network, the Internet, or a cloud server) via a communication path / link 151. The network 150 may be further communicated to a remote terminal 170 via a communication path / link 152 and / or to a trusted computer system 180 via a 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 the intervening reader device 120. For example, according to some embodiments, 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, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is incorporated entirely herein by reference.Any suitable electronic communication protocol, such as Near Field Communication (NFC), Radio Frequency Identification (RFID), Bluetooth® or Bluetooth® Low Energy Protocol, or Wi-Fi®, can be used for each of the communication paths or links. The remote terminal 170 and / or trusted computer system 180 are accessible, according to some embodiments, to individuals other than the primary user who are interested in the user's analysis level. The reader device 120 may include a display 122 and an optional input component 121. The display 122 may include a touchscreen interface, according to some embodiments.

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

[0017] The sensor 104 is adapted to be at least partially inserted into the tissue of interest, such as within the dermis or subcutaneous layer of the skin. The sensor 104 may include a sensor tail of sufficient length to be inserted to a desired depth in a given tissue. The sensor tail may include at least one working electrode and one or more active regions (detection regions / spots or detection layers) positioned on at least one working electrode and active to detect one or more analytes of interest. In general, according to one or more embodiments of the present disclosure, one or more active regions may include multiple enzymes. According to some embodiments, the active regions may include a polymer material to which at least some of the enzymes are covalently bonded. In various embodiments of the present disclosure, the analyte may be monitored in any biological fluid of interest, such as dermal fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, or amniotic fluid. 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, once data is acquired, it may be stored in memory until it is transmitted (e.g., every minute, every 5 minutes, or at other predetermined intervals) and communicated automatically and periodically at a specific frequency or after a specific period of time 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 may be communicated from the sensor 104 using RFID technology when the sensor electronics enter the communication range of the reader device 120. The data may remain stored in the sensor 104's memory until it is communicated to the reader device 120. Thus, the patient does not need to be in constant proximity to the reader device 120, but can instead upload data at a convenient time. In yet another embodiment, a combination of automatic and non-automatic data transfer can be implemented. For example, data transfer may continue automatically until the reader device 120 leaves the communication range of the sensor 104.

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

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

[0021] The analyte sensors described herein may feature multiple enzymes on the active region of a single working electrode or on two or more separate working electrodes. According to various embodiments of this 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. Subsequently, sensor configurations featuring multiple working electrodes are described separately with reference to Figure 3. Multiple enzymes can 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. A related two-electrode detection motif may include a working electrode and a second electrode, the second electrode functioning as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). In both two-electrode and three-electrode detection motifs, one or more active regions of the analyte sensor may be in contact with the working electrode. One or more active regions may include multiple enzymes according to embodiments of the present disclosure, the multiple enzymes residing in a single active region and / or multiple active regions. In some embodiments, the various electrodes may be stacked at least partially on top of each other (layered), as will be described in more detail below. In some or other embodiments, the various electrodes may be separated laterally from each other on the sensor tail. Similarly, the related active regions on each electrode may be stacked vertically on top of each other or separated laterally. In any case, the various electrodes may be electrically insulated from each other by a dielectric material or similar insulator.

[0023] Figure 2A shows a diagram of an exemplary two-electrode analyte sensor configuration having a single working electrode, suitable for use in some embodiments of the disclosure herein. As shown, the analyte sensor 200 includes a substrate 212 positioned between a working electrode 214 and a counter / reference electrode 216. Alternatively, the working electrode 214 and the counter / reference electrode 216 may be positioned on the same side of the substrate 212 with a dielectric material inserted between them (configuration not shown). The active region 218 is positioned as at least one layer on at least a portion of the working electrode 214. In various embodiments, the active region 218 may include multiple spots or a single spot configured for the detection of one or more analytes of interest. In general, multiple enzymes may be present in the active region 218 (i.e., in a single spot or multiple spots).

[0024] Continuing to refer to Figure 2A, according to some embodiments, the membrane 220 may cover at least the active region 218 and optionally cover part or all of the working electrode 214 and / or the counter / reference electrode 216, or the entire analyte sensor 200. One or both sides of the analyte sensor 200 may be covered with the membrane 220. The membrane 220 may comprise one or more polymer membrane materials having the ability to restrict the analyte flux to the active region 218. The composition of the membrane 220 may vary depending on what the analyte is, as will be further described herein. The analyte sensor 200 may be operable to assay one or more analytes by any of the electrochemical detection techniques by coulometry, amperometry, voltammetry, or potentiometry.

[0025] Figures 2B and 2C illustrate exemplary three-electrode analyte sensor configurations having a single working electrode, suitable for use in some embodiments of the disclosure herein. Three-electrode analyte sensor configurations using a single working electrode can be similar to those shown for analyte sensor 200 in Figure 2A, except that analyte sensors 201 and 202 (Figures 2B and 2C) include an additional electrode 217. With the additional electrode 217, the counter / reference electrode 216 can function as either the counter electrode or the reference electrode, while the additional electrode 217 performs the other electrode function. The working electrode 214 continues to perform its original function. The additional electrode 217 can be positioned either on the working electrode 210 or on electrode 216, with a dielectric isolation layer in between. For example, as shown in Figure 2B, dielectric layers 219a, 219b, and 219c isolate electrodes 214, 216, and 217 from each other, providing electrical insulation. Alternatively, at least one of electrodes 214, 216, and 217 may be positioned on the opposite side of the substrate 212, as shown in Figure 2C. Thus, in some embodiments, electrodes 214 (working electrode) and 216 (counter electrode) may be positioned on the opposite side of the substrate 212, and electrode 217 (reference electrode) may be positioned on one of electrodes 214 or 216 and separated therefrom by a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, and the position of the reference material layer 230 is not limited to those shown in Figures 2B and 2C. Similar to the sensor 200 shown in Figure 2A, the active regions 218 of analyte sensors 201 and 202 may include multiple spots or a single spot configured for the detection of one or more analytes of interest. In general, multiple enzymes may be present in the active regions 218 of analyte sensors 201 and 202. Furthermore, the analyte sensors 201 and 202 may be operable to assay one or more analytes by any of the following electrochemical detection techniques: coulometry, amperometry, voltammetry, or potentiometry.

[0026] Similar to the analyte sensor 200, the film 220 can also cover the active region 218 and other sensor components in the analyte sensors 201 and 202. In some embodiments, an additional electrode 217 may be covered by the film 220. In Figures 2B and 2C, electrodes 214, 216 and 217 are depicted as all covered by the film 220, but it should be recognized that in some embodiments, only the working electrode 214 may be covered. Furthermore, the thickness of the film 220 on each of electrodes 214, 216 and 217 may be the same or different. In the sensor configurations of Figures 2B and 2C, as in the two-electrode analyte sensor configuration (Figure 2A), one or both sides of the analyte sensors 201 and 202 may be covered by the film 220, or the entirety of the analyte sensors 201 and 202 may be covered. Therefore, the three-electrode sensor configuration shown in Figures 2B and 2C should be understood as a non-limiting example of the embodiments disclosed herein, comprising alternative electrode and / or layer configurations within the scope of this disclosure.

[0027] Next, we will describe in more detail analyte sensor configurations having multiple working electrodes. While the following description is primarily directed towards analyte sensor configurations having two working electrodes, it should be understood that by extending the disclosure herein, three or more working electrodes can also be incorporated without issue. Additional working electrodes may allow for additional active regions and corresponding detection capabilities to be provided to analyte sensors having such features.

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

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

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

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

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

[0033] According to various embodiments of this disclosure, electron transfer agents can be covalently bonded to the polymer in each active region. The manner of covalent bonding is not considered to be particularly limited. Covalent bonding of the electron transfer agent to the polymer can occur by polymerizing monomer units having covalently bonded electron transfer agents, or, if the polymer has already been synthesized, by reacting the electron transfer agent separately with the polymer. According to some embodiments, a bifunctional spacer can covalently bond an electron transfer agent to the polymer in the active region, where the first functional group is reactive with the polymer (e.g., a functional group that can quaternize a pyridine nitrogen atom or an imidazole nitrogen atom), and the second functional group is reactive with the electron transfer agent (e.g., a functional group that reacts with a ligand that coordinates a metal ion).

[0034] Similarly, according to some or other various embodiments of this disclosure, enzymes within one or more active regions may be covalently bonded to a polymer. When multiple enzymes are present in a single active region, in some embodiments, all of the multiple enzymes may be covalently bonded to the polymer, while in other embodiments, only some of the multiple enzymes may be covalently bonded to the polymer. For example, a first enzyme may be covalently bonded to the polymer, while a second enzyme may not be covalently bonded to the polymer. According to more specific embodiments, covalent bonding of an enzyme to a polymer may occur via a crosslinking agent introduced with a suitable crosslinking agent. Suitable crosslinking agents for reactions 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, imide esters, epichlorohydrin, or derivatized variants thereof. Suitable crosslinking agents for reactions with free carboxylic acid groups in the enzyme may include, for example, carbodiimide. Crosslinking is generally intermolecular, but in some embodiments it may be intramolecular.

[0035] Electron transfer agents and / or enzymes can bind to the polymer within the active region by means other than covalent bonding. In some embodiments, electron transfer agents and / or enzymes may bind to the polymer ionically or coordinationally. For example, a charged polymer may ionically bond with a conversely charged electron transfer agent or enzyme. In yet another embodiment, electron transfer agents and / or enzymes may be physically encombined into the polymer without binding to it.

[0036] Next, various configurations suitable for arranging multiple enzymes in the analyte sensor of this disclosure will be described in more detail. Multiple enzymes can be deposited within one or more active regions of the sensor. The active regions have a size of approximately 0.01 mm. 2 ~about 1mm 2 The active region may be within this range, but larger or smaller active regions are also intended herein.

[0037] In some embodiments, multiple enzymes may be arranged within separate active regions on a single working electrode. When multiple enzymes are arranged in this manner, each active region can facilitate the detection of separate analytes, as described below. At least one of the active regions can generate a signal independently of the others.

[0038] According to some embodiments, an analytic sensor of the present disclosure having multiple active regions on a single working electrode may include a sensor tail comprising at least a working electrode, and at least two active regions disposed on the surface of the working electrode. Each active region comprises an analytic responsive enzyme and a polymer, the analytic responsive enzymes in each active region being different. Each active region has a redox potential, and the redox potential of the first active region is sufficiently far from the redox potential of the second active region, allowing for the generation of a signal from the first active region independently of the signal from the second active region. In a more specific embodiment, such an analytic sensor may include a single working electrode having at least two active regions. To facilitate electron transfer, an electron transfer agent may be incorporated into each active region.

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

[0040] In more specific embodiments, the sensor tail may be configured for insertion into tissue. Suitable tissues are not considered particularly limited and are described in more detail above. Similarly, considerations for positioning the sensor tail at a specific location within the tissue are described 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, at least about 150 mV, or at least about 200 mV. The upper limit of the separation between redox potentials is determined in vivo by the working electrochemical window. By sufficiently separating the magnitudes of the redox potentials of the active regions, electrochemical reactions can occur in the first active region without substantially inducing electrochemical reactions in the second active region. Thus, signals from the first active region can be generated independently above their corresponding redox potential. In contrast, above the redox potential of the second active region, electrochemical reactions can occur in both active regions. Therefore, signals obtained above the redox potential of the second active region may include signal contributions from both the first and second active regions, and the signal is a composite signal. Next, the signal contribution from the second active region above its redox potential can be determined by subtracting the signal obtained only from the first active region above its corresponding redox potential from the composite signal. Similar considerations apply to the analysis of the signal contribution 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 sufficiently differentiate their redox potentials 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, which is different from the first electron transfer agent and the second electron transfer agent. According to various embodiments of this disclosure, the redox potentials of the first and second active regions can be sufficiently differentiated by changing the metal centers and / or ligands present in a given electron transfer agent. According to even more specific embodiments, the first electron transfer agent may be covalently bonded to the polymer in the first active region, and the second electron transfer agent may be covalently bonded to the polymer in the second active region. The covalent bonding of the first and second electron transfer agents may be the same or different. Similar considerations apply when selecting electron transfer agents 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 this disclosure, the analyte-responsive enzyme in each active region may be covalently bonded (or otherwise immobilized) to the polymer within each active region. In even more specific embodiments, the analyte-responsive enzyme and electron transfer agent within each active region may be covalently bonded to the polymer within each active region. When contained in a single active region, the first analyte-responsive enzyme and the first electron transfer agent may be covalently bonded to a first portion of the polymer, and the second analyte-responsive enzyme and the second electron transfer agent may be covalently bonded to a second portion of the polymer. The polymers of the first and second portions may be the same or different.

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

[0045] It should also be understood that the sensitivity (output current) of the analyte sensor to each analyte can be changed by changing the coverage (area or size) of the active region, the area ratio of the active regions to each other, the properties and thickness of the mass transfer limiting film covering the active region, and any combination thereof. These parameter changes can be readily implemented by those skilled in the art who are given the benefit of the disclosure herein.

[0046] While the above description is directed primarily towards analytic sensors configured to detect two different analytic compounds, it should be understood that the above concept can be extended to the detection of three or more analytic compounds using a corresponding number of active regions arranged on a single working electrode. Specifically, in further embodiments of this disclosure, the same number of different analytic compounds can be detected using an analytic sensor that uses three or more active regions and a corresponding number of different enzymes (and electron transfer agents) within them. If the redox potential of each active region is sufficiently far from the redox potential of the other active regions, the signal contribution from each active region can be analyzed in a manner related to the above method to provide the concentration of each analytic compound.

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

[0048] Similarly, it should be understood that some analytic sensors of this disclosure having two or more active regions positioned on a given working electrode may include two or more analytic enzymes in at least one active region. According to a more specific embodiment, two or more analytic enzymes within a given active region may interact cooperatively to produce a signal proportional to the concentration of a single analytic. Thus, the analytic enzymes do not necessarily have to be present in a 1:1 ratio for the selection of a particular analytic. Analytic sensors containing cooperatively interacting enzymes are described in further detail below.

[0049] Accordingly, a method for detecting multiple analytes using an analyte sensor characterized by multiple enzymes arranged on a single working electrode is also described herein. In various embodiments, such a method may include exposing the analyte sensor to a fluid containing at least one analyte. The analyte sensor includes at least a working electrode, in particular a single working electrode, and a sensor tail comprising at least two active regions arranged on the surface of the working electrode. Each active region comprises an analyte-responsive enzyme and a polymer, wherein the analyte-responsive enzymes in each active region are different. Each active region has a redox potential, and the redox potential of the first active region is sufficiently far from the redox potential of the second active region, allowing for the generation of a signal from the first active region independent of the generation of a signal from the second active region. This method further includes: acquiring a first signal at a redox potential above that of the first active region such that the first signal is proportional to the concentration of the first analyte; acquiring a second signal at a redox potential above that of the second active region such that the second signal is a composite signal including signal contributions from the first active region and signal contributions from the second active region; and subtracting the first signal from the second signal to obtain a differential signal proportional to the concentration of the second analyte.

[0050] In a more specific embodiment, the redox potential associated with the first active region may be set at least about 100 mV, or at least about 150 mV, or at least about 200 mV, from the redox potential of the second active region in order to set it sufficiently apart for independent signal generation from the first active region.

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

[0052] In some embodiments, the signals associated with each active region can be correlated to the corresponding analyte concentration by referring to a lookup table or calibration curve for each analyte. 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 curve for the analyte sensor of the present disclosure may be linear.

[0053] The processor can determine which sensor response value in the lookup table is closest to the one measured for a sample with an unknown analyte concentration and report the analyte concentration accordingly. In some or other embodiments, if the sensor response value for a sample with an unknown analyte concentration falls between the values ​​recorded in the lookup table, the processor can estimate the analyte concentration by interpolating between the two lookup table values. Interpolation may assume a linear concentration variation between the two values ​​reported in the lookup table. Interpolation can be used when the sensor response differs sufficiently from a given value in the lookup table (e.g., variation of about 10% or more).

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

[0055] Embodiments of an analyte sensor having two different active regions arranged on a given working electrode can use the sensor configurations related to those shown in Figures 2A-2C and above. However, it should be understood that a suitable analyte sensor 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 different active regions. It should also be understood that other analyte sensor configurations having two or more different active regions arranged on the surface of a given working electrode are also within the scope of this disclosure. For example, the position, orientation, or function of the working electrode and the counter electrode and / or reference electrode may differ from those shown in the drawings of this application.

[0056] Figure 4 shows an exemplary analyte sensor configuration suitable for use in several embodiments of the disclosure herein, in which two different active regions are arranged on the surface of a single working electrode. The analyte sensor configuration of Figure 4 is most similar to that of Figure 2C and can be better understood by referring to Figure 2C. Where appropriate, common reference numerals with Figure 2C are used in Figure 4 for clarity, and for the sake of brevity, features having a common structure and / or function are not described in further detail. In this case as well, it should be understood that other analyte sensor configurations can similarly incorporate the features described below with respect to Figure 4.

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

[0058] The redox potentials of the first active region 218a and the second active region 218b can be sufficiently separated from each other to allow signal generation from the first active region 218a independently of signal generation from the second active region 218b. Therefore, the analyte sensor 400 can operate at a first potential where the redox reaction occurs within the first active region 218a but not within the second active region 218b. Thus, the first analyte (e.g., glucose) can be selectively detected at or above the redox potential of the first active region 218a, unless the applied potential is sufficiently high to facilitate the redox reaction with the second active region 218b. The concentration of the first analyte can be determined from the sensor response of the first active region 218a by referring to a lookup table or calibration curve.

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

[0060] As mentioned above, similar considerations also apply to separating the first and second signals from a single active region containing two different analyte-responsive enzymes in order to determine the concentrations of the first and second analytes, which are distinct from each other.

[0061] In some or other embodiments of this disclosure, multiple enzymes may reside in a single active region. Unlike sensor configurations in which multiple enzymes function independently to detect different analytes, particularly those in which they are spaced apart in separate active regions on the surface of the working electrode, according to some embodiments of this disclosure, multiple enzymes arranged in a single active region function cooperatively, particularly in the presence of a stabilizer, to facilitate the detection of a single analyte. As used herein, the term “cooperatively” and its grammatical variations refer to a combined enzymatic reaction in which the product of a first enzymatic reaction becomes a substrate for a second enzymatic reaction, and the second enzymatic reaction serves as the basis for measuring the concentration of the substrate (analyte) that reacted during the first enzymatic reaction. When a single enzyme cannot facilitate detection, it may be desirable to utilize two enzymes that act cooperatively to detect a given analyte of interest. Situations in which a single enzyme may not be effective in facilitating the detection of an analyte include, for example, situations in which the enzyme is inhibited by one or more products of an enzymatic reaction or, when placed in an analyte sensor, cannot cycle between oxidized and reduced states.

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

[0063] In a more specific embodiment, an analyte sensor featuring at least two enzymes that interact cooperatively may include a sensor tail containing at least a working electrode; and at least one active region located on the surface of the working electrode. The at least one active region comprises a first enzyme, a second enzyme, and a polymer. The first and second enzymes can interact cooperatively, the first enzyme can convert the analyte to a first product, and the second enzyme can convert the first product to a second product, thereby generating a signal at the working electrode. The second enzyme does not react with the analyte. At least the second enzyme is covalently bonded to the polymer in at least one active region. As will be described in more detail below, analysis of the signal resulting from the reaction of the first product to the second product (e.g., a current measured at a fixed input voltage) can provide a basis for detecting the analyte and measuring its concentration.

[0064] More specifically, the active region may include an electron transfer agent as described above, and only the second enzyme can exchange electrons with the electron transfer agent, in which case the first enzyme can indirectly transfer electrons to the second enzyme, as described below. Thus, more specific embodiments of this disclosure may feature a first enzyme that is not covalently bonded to the polymer (and therefore less likely to exchange electrons with the electron transfer agent) and a second enzyme that is covalently bonded to the polymer (to facilitate electron exchange with the electron transfer agent). In either case, the electron transfer agent may be covalently bonded to the polymer in the active region. Coordinate bonds are also included within the scope of covalent bonding as disclosed herein.

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

[0066] According to some embodiments, the sensor tail may be configured to be inserted into the tissue of interest. Thus, according to some embodiments, an analytic sensor comprising enzymes that can interact cooperatively with each other in a given active region may be adapted to analyze the concentration of an analytic in a biological fluid within a living organism. Again, the nature of the biological fluid is not particularly limited.

[0067] As described above, a sensor configuration incorporating two enzymes capable of cooperative interaction may include one in which at least one active region contains an electron transfer agent covalently bonded to the polymer. In this case as well, according to the disclosure herein, coordination bonds are also included within the scope of covalent bonding. In such embodiments, at least a second enzyme may also be covalently bonded to the polymer. In some embodiments, the first enzyme is not covalently bonded to the polymer. In other embodiments, both the first and second enzymes may be covalently bonded to the polymer in at least one active region. Covalent bonding of the first enzyme to the polymer may be desirable, for example, to reduce the possibility of the first enzyme leaching from at least one active region.

[0068] An analytic sensor configuration suitable for incorporating two enzymes that interact cooperatively in one or more active regions on a working electrode is shown in Figures 2A-2C and may be similar to those described in more detail above. Enzymes capable of cooperative interaction (i.e., cooperative enzymes or cooperative enzyme pairs) can also be incorporated into an analytic sensor configuration having multiple working electrodes (Figure 3) or multiple active regions (Figure 4) arranged on a given working electrode. Any analytic sensor disclosed herein having a cooperative enzyme pair directly arranged on the surface of a working electrode can employ any of the aforementioned analytic sensor configurations. An analytic sensor configuration having two or more enzymes that interact cooperatively in multiple active regions, where one of the active regions is away from the working electrode, will be described further below with reference to Figures 6A and 6B.

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

[0070] Another pair of synergistic enzymes suitable for use in this disclosure, in which both enzymes are directly positioned on the surface of the working electrode, is β-hydroxybutyrate dehydrogenase and diaphorase. This synergistic enzyme pair can be used for the detection of ketone bodies, with β-hydroxybutyrate being a representative molecule indicating the presence of ketones. In a sensor configuration including this synergistic enzyme pair, β-hydroxybutyrate dehydrogenase is used to detect β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD). + ) can be converted to acetoacetate and reduced nicotinamide adenine dinucleotide (NADH). Next, NADH is reduced under the mediated influence of diaphorase, and the electrons transferred during this process provide the basis for ketone detection at the working electrode. β-hydroxybutyrate and diaphorase (NADH) for ketone detection + The synergistic reaction between the two enzymes (mediated by a cofactor) is shown in Figure 5B. Along with the synergistic enzyme pair, albumin may be present as a stabilizer.

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

[0072] Creatine amidohydrolase and sarcosine oxidase are another pair of synergistic enzymes that may be suitable for use in the disclosure herein, provided that both enzymes are directly positioned 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, the detection of hydrogen peroxide at the working electrode may serve as a basis for quantifying creatine and / or sarcosine.

[0073] Further details will be provided regarding the detection of ethanol and other alcohols using alcohol oxidase and xanthine oxidase via coordinated enzymatic reactions. Alcohol oxidase interacts with ethanol to form acetaldehyde and hydrogen peroxide. Other alcohols react to form aldehydes with corresponding higher or lower carbon number atoms. Advantageously, alcohol oxidase catalyzes only the forward conversion from ethanol to acetaldehyde (in contrast to alcohol dehydrogenase, which carries out the reaction reversibly), which can be advantageous for using this enzyme in analyte sensors. Furthermore, since alcohol oxidase contains a strongly bound flavin cofactor, it is not always necessary to combine the alcohol oxidase with an exogenous cofactor to activate the enzyme and promote alcohol oxidation.

[0074] In principle, alcohol oxidase alone can be used for ethanol detection in analyte sensors by assaying either acetaldehyde or hydrogen peroxide products generated by the enzymatic reaction. However, this approach has two problems. First, both acetaldehyde and hydrogen peroxide inhibit alcohol oxidase. Therefore, if these compounds are not removed from the sensor environment, alcohol oxidase becomes inactive to promote ethanol oxidation, thereby rendering the analyte sensor inoperable for assaying ethanol. Furthermore, if acetaldehyde and hydrogen peroxide are segregated or quenched with other agents, there are no species available for electrochemical detection. Second, alcohol oxidase does not freely exchange electrons with redox mediators other than oxygen molecules. Therefore, electron transfer agents associated with polymers within the active region of the analyte sensor, such as osmium and other transition metal complexes described herein, are ineffective in cycling alcohol oxidase from an inactive reducing state to an oxidizing state that is reactive with ethanol. Thus, alcohol oxidase can be covalently bonded to a polymer of any choice, but there is no particular advantage to the electron transfer process by doing so. In other words, the covalent bonding of alcohol oxidase to the polymer does not facilitate electron transfer by electron transfer agents.

[0075] The coordinated combination of alcohol oxidase and xanthine oxidase, directly and especially together in a given active region on the working electrode, can overcome at least some of the aforementioned challenges associated with ethanol detection using analytes sensors that utilize alcohol oxidase. Acetaldehyde and other aldehydes can function as substrates for xanthine oxidase, and acetaldehyde is enzymatically converted to acetic acid. Thus, xanthine oxidase can remove acetaldehyde from the sensor environment, thereby preventing the inactivation of the acetaldehyde base of alcohol oxidase. To remove hydrogen peroxide, catalase may be present in the active region (e.g., as a catalase-hydrogen peroxide complex), thereby preventing the 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 polymers within the active region of the analytes sensor. In this way, xanthine oxidase can cycle between its oxidized and reduced forms, thereby enabling the analytes sensor to maintain an active sensing state. Therefore, the detection of ethanol in the aforementioned analyte sensor is based on the enzymatic reaction between xanthine oxidase and acetaldehyde, the enzymatic reaction product of ethanol. Furthermore, by configuring the enzyme in the analyte sensor in the manner described above, alcohol oxidase may maintain its activity through re-oxidation with oxygen molecules.

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

[0077] Continuing with Figure 5A, the acetaldehyde enzymatically formed from ethanol then undergoes a subsequent reaction with oxidized xanthine oxidase in the presence of a flavin cofactor naturally present with the enzyme. This process produces acetic acid and reduces the xanthine oxidase. The reduced xanthine oxidase then reacts with a transition metal electron transfer agent bound to the polymer to transfer electrons to the working electrode, thereby generating an electric current and regenerating oxidized xanthine oxidase. Although not shown in Figure 5A, hydrogen peroxide is separately removed from the sensor environment by catalase present in the active region.

[0078] As can be 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 present, and thus to the amount of ethanol. The correlation between the working electrode current and the ethanol concentration can be determined by referring to a lookup table of currents at known ethanol concentrations or by using a calibration curve. These concepts are explained in more detail above.

[0079] Similarly, the current generated at the working electrode when analyzing ketones may be proportional to the amount of β-hydroxybutyrate that is oxidized to form acetoacetone (Figures 5B-5D). Therefore, the correlation of the current at the working electrode can be performed in the same manner as provided above for ethanol (e.g., using a calibration curve or lookup table).

[0080] Therefore, 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 a sensor tail comprising at least a working electrode and at least one active region disposed on the surface of the working electrode, wherein the at least one active region comprises alcohol oxidase, xanthine oxidase, catalase, a polymer, and an electron transfer agent. According to a particular embodiment, the electron transfer agent and xanthine oxidase may be covalently bonded to the polymer, but the alcohol oxidase is not covalently bonded to the polymer. The alcohol oxidase and xanthine oxidase 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 placed directly on the working electrode to achieve the foregoing.

[0081] According to a more specific embodiment, the catalase in at least one active region of the alcohol sensor is not covalently bonded to the polymer. The catalase may be present in an amount ranging from about 1% to about 50% by weight of the polymer, more 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] Accordingly, the disclosure also provides a detection method based on a cooperative enzyme reaction, wherein a cooperative enzyme pair is directly positioned on the surface of a working electrode. According to various embodiments, the detection method may include exposing an analyte sensor to a fluid containing an analyte, the analyte sensor including at least a working electrode and a sensor tail including at least one active region positioned on the surface of the working electrode, the at least one active region including a first enzyme, a second enzyme, and a polymer. The first and second enzymes can interact cooperatively, and the second enzyme is covalently bonded to the polymer and is nonreactive 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 with the concentration of the analyte in the fluid.

[0083] In more specific embodiments, when the method described above is carried out, the electron transfer agent may also be covalently bonded to the polymer. Suitable electron transfer agents are described in more detail above. In some or other embodiments, particularly in the presence of a covalently bonded electron transfer agent, the first enzyme is not covalently bonded to the polymer in at least one active region.

[0084] In more specific embodiments, the ethanol detection method of the present disclosure may include exposing an analyte sensor to a fluid containing ethanol, particularly a biological fluid, the analyte sensor comprising at least a working electrode and a sensor tail disposed on the surface of the working electrode and comprising at least one active region containing alcohol oxidase, xanthine oxidase, catalase, a polymer, and an electron transfer agent. The electron transfer agent and xanthine oxidase are covalently bonded to the polymer, while the alcohol oxidase is not covalently bonded to the polymer. The alcohol oxidase and xanthine oxidase can interact cooperatively. The method further includes reacting ethanol with alcohol oxidase to form acetaldehyde; reacting acetaldehyde with xanthine oxidase to form acetic acid to 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 within a single active region of an analyte sensor may interact cooperatively to determine the analyte concentration, the enzymes may also function independently of each other to detect alternative analytes in other embodiments. For example, if the second enzyme in the above analyte sensor is xanthine oxidase, instead of using the analyte sensor to detect ethanol, the analyte sensor can be used to detect any of the 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 way, if no alcohol is present, alcohol oxidase remains unused and / or these species can be inactivated by acetaldehyde / hydrogen peroxide if they are not removed by xanthine oxidase or other species. Thus, a sensor containing cooperative enzymes can also be considered capable of detecting multiple analytes, one analyte from a cooperative enzyme pair, and at least a second analyte from one of the members of the cooperative 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 can also interact cooperatively to facilitate the detection of a single analyte. In some cases, both of the enzymes may be located directly on the surface of the working electrode, as described in more detail above. In alternative analyte sensor configurations containing multiple enzymes in separate active regions, one of the active regions can be isolated from the working electrode so that electron transfer to the working electrode occurs from only one active region. That is, as described in more detail below, the active region isolated from the working electrode may facilitate the enzymatic reaction of the analyte of interest, producing a reaction product (substrate) that reacts with the enzyme in the active region in direct contact with the working electrode. The 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. The correlation between the signal and the analyte concentration can be achieved in a manner similar to that described in more detail above.

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

[0088] Before further describing the cooperative enzyme reaction shown in Figure 5E, we will first describe in more detail exemplary analyte sensor configurations featuring at least one active region isolated from the working electrode. As previously mentioned, all analyte sensor configurations shown in Figures 2A–4 feature one or more working electrodes, each having one or more active regions directly located on the surface of the working electrode. In contrast, Figures 6A, 6B, and 6C show working electrodes in which the first active region is directly located on the surface of the working electrode, and the second active region is separated from the working electrode (at a distance or remotely) by a membrane. The working electrode configurations shown in Figures 6A, 6B, and 6C can substitute for any of the specific working electrode configurations shown in Figures 2A–4. That is, the working electrode configurations shown in Figures 6A, 6B, and 6C can be combined in any suitable way with counter electrodes and / or reference electrodes, membranes, substrates, and similar structures within the analyte sensor.

[0089] As shown in Figure 6A, the working electrode 400 has an active region 402 directly located on its surface. The active region 402 contains a first enzyme covalently bonded to a first polymer. Typically, an electron transfer agent is also present in the active region 402, and the electron transfer agent is also covalently bonded to the polymer. The active region 402 is covered by a membrane 404. The membrane 404 can also cover the surface of the working electrode 400, as well as other parts of the analyte sensor on which the working electrode 400 resides, as shown in the figure. The membrane 404 isolates the active region 406 from the working electrode 400, thereby preventing electron exchange between the two. The active region 406 contains a second enzyme covalently bonded to a second polymer, but without a separate electron transfer agent. Figure 6A shows the active region 406 located directly above the active region 402, and it should be understood that in alternative configurations compatible with this disclosure, they may be laterally separated from each other. The film 408 provides mass transfer limiting properties by covering the active region 406 and, optionally, other sensor components. Similarly, as shown in Figure 6B, the film 404 does not necessarily have to extend the same lateral distance over the working electrode 400 as the film 408 does. In fact, the film 404 in Figure 6B covers the active region 402 but only a portion of the surface of the working electrode 400, while the film 408 covers the active region 406, the surface of the film 404, and the rest of the surface of the working electrode 400 not covered by the film 404. The active regions 402 and 406 can also be laterally offset from each other in some embodiments, as shown in Figure 6C, where the active region 406 is again isolated from the working electrode 400 by the film 404.

[0090] Membrane 408 is permeable to the analyte and any additional components required to facilitate the enzymatic reaction in the active region 406. In contrast, membrane 404 is permeable to the product formed in the active region 406. That is, the analyte reacts in the active region 406 to form a first product, which then diffuses through membrane 404 and subsequently reacts further in the active region 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, the lead 410 may be extended between the active regions 402 and 406. In an alcohol sensor characterized by detection based on a coordinated enzymatic reaction between glucose oxidase and xanthine oxidase, glucose oxidase is located in the active region 406 and xanthine oxidase is located in the active region 402. Referring again to Figure 5E, and continuing to refer to Figures 6A, 6B, and 6C, glucose oxidase is located in the active region 406 and converts exogenous glucose to D-gluconolactone-1,5-dione and hydrogen peroxide. Unlike the alcohol sensor characterized by detection based on a coordinated enzymatic reaction between alcohol oxidase and xanthine oxide (Figure 5A), catalase plays a more active role in the coordinated enzymatic reaction shown in Figure 5E. Specifically, catalase reacts with hydrogen peroxide to form a catalase-hydrogen peroxide complex (the same peroxide removal function that catalase exhibits in the coordinated enzymatic reaction between alcohol oxidase and xanthine oxidase), and the complex subsequently reacts with ethanol to form acetaldehyde. When ethanol is reacted with the catalase-hydrogen peroxide complex, the acetaldehyde formed in the active region 406 diffuses through membrane 404, separating the active region 406 from the active region 402. Alternatively, catalase may be present in the active region 402, in which case the hydrogen peroxide formed in the active region 406 diffuses through membrane 404 to the active region 402, where it forms the catalase-hydrogen peroxide complex and oxidizes ethanol to acetaldehyde. Once acetaldehyde is formed in the active region 402, the coordinated enzymatic reaction may continue as shown in Figure 5E. Membrane 404 may contain cross-linked polyvinylpyridine that is permeable to acetaldehyde. Next, the acetaldehyde reacts with xanthine oxidase in the active region 402 to form acetic acid in the same manner as described above for Figure 5A.

[0092] Accordingly, the alcohol sensor of this disclosure may include: a sensor tail comprising at least a working electrode; a first active region disposed on the surface of the working electrode, comprising xanthine oxidase, catalase, a first polymer, and an electron transfer agent, wherein the xanthine oxidase and electron transfer agent are covalently bonded to the first polymer; a first membrane covering the first active region, comprising a first membrane polymer and permeable to acetaldehyde; a second active region disposed on the first membrane, comprising glucose oxidase, catalase, and a second polymer, wherein the glucose oxidase is covalently bonded to the second polymer; and a second membrane covering the second active region, comprising a second membrane polymer and permeable to glucose and alcohol. Here, the glucose oxidase and xanthine oxidase interact cooperatively to generate a signal at the working electrode that is proportional to the alcohol concentration. In a more specific embodiment, the alcohol may be ethanol.

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

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

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

[0096] In yet another embodiment of this disclosure, multiple enzymes may be placed within the active regions of separate working electrodes. Therefore, signals associated with the enzymatic reactions occurring within each active region can be measured separately by having each working electrode respond simultaneously or at different times. The signals associated with each active region can then be correlated with the concentrations of separate analytes.

[0097] As described above, a membrane (i.e., a mass transfer limiting membrane) can cover one or more active regions of an analyte sensor to enhance biocompatibility and alter the analyte flux to the active region. Such membranes may be present in any of the analyte sensors disclosed herein. Since different analytes may exhibit different permeability values ​​within a given membrane, an analyte sensor configured to analyze multiple analytes may exhibit different sensitivities to each analyte. One approach to address these different sensitivities may involve utilizing different membrane thicknesses across each active region. While this approach is feasible, it can be difficult to implement from a manufacturing standpoint; that is, varying the membrane thickness at different locations using typical dip-coating techniques used for membrane deposition can be challenging. Another possible approach is to use active regions of different sizes for each analyte.

[0098] Analyte sensors having active regions configured to assay different analytes on separate working electrodes can address the aforementioned problems associated with different analyte sensitivities. Specifically, the following disclosures provide various methods for modifying membrane permeability on each working electrode and leveling analyte membrane permeability at each location. In other words, the disclosures herein enable independent variation of analyte permeability and sensitivity at each working electrode. According to the disclosures herein, mass transfer limiting membranes comprising two or more different membrane polymers can provide more leveled analyte permeability at each working electrode. Specific membrane configurations suitable for leveling 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 containing membrane polymers individually unsuitable for promoting permeability of a given analyte can 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 region on each working electrode to provide equivalent sensitivity values ​​for each analyte.

[0099] Accordingly, in some embodiments, an analytic sensor characterized by two or more enzymes arranged on separate working electrodes may include a sensor tail comprising at least a first working electrode and a second working electrode, a first active region located on the surface of the first working electrode, a second active region located on the surface of the second working electrode, a multi-component membrane covering the first active region, and a homogeneous membrane covering the second active region. The first active region comprises a first polymer and a first analytic enzyme that reacts with a first analytic, and the second active region comprises a second polymer and a second analytic enzyme that reacts with a second analytic. The first and second analytic enzymes are different and reactive with different analytics. The multi-component membrane comprises at least a first and second membrane polymer that are different from each other. The homogeneous membrane comprises one of the first and second membrane polymers.

[0100] The specific 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 will be described in more detail below, bilayer membranes and mixed membranes may function to equalize the permeability of the analyte.

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

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

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

[0104] Figure 7 shows an exemplary schematic diagram of a subset of analyte sensors having two working electrodes and featuring a bilayer film covering one of the two working electrodes, which is suitable for use in some embodiments of the disclosure herein. As shown in Figure 7, the analyte sensor features a sensor tail 600 having working electrodes 614a and 614b positioned on opposite sides of the substrate 612. An active region 618a is positioned on the working electrode 614a, and an active region 618b is positioned on the working electrode 614b. The active regions 618a and 618b contain different analyte-responsive enzymes and are configured to assay for different analytes according to the disclosure herein. Although Figure 7 shows that the active regions 618a and 618b are positioned approximately opposite each other with respect to the substrate 612, it should be understood that the active regions 618a and 618b may be laterally separated (shifted) from each other on the opposite side of the substrate 612. The lateral separation of the active regions 618a and 618b may be particularly advantageous for covering each active region 618a and 618b with a mass transfer limiting film, as described below.

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

[0106] According to one or more embodiments, an analytic sensor having multiple active regions on separate working electrodes, one of which is covered with a bilayer film, can exhibit leveled or independently variable analytic permeability. That is, the analytic sensor can have closer sensitivity to two different analytics than in the absence of the bilayer film. In such an analytic sensor configuration, the active region covered with a homogeneous film (e.g., film layer 620 in Figure 7) can exhibit analytic permeability to a first analytic, characteristic of its particular film polymer. Surprisingly, the bilayer film (e.g., bilayer film 621 in Figure 7) may contain a film polymer that does not adversely affect the permeability of the second analytic (i.e., a film polymer with neutral permeability), thereby allowing the other film polymer, including the bilayer film, to exhibit its characteristic permeability to the second analytic as if the first film polymer were not present. Thus, according to various embodiments, the film polymer with neutral permeability and the film polymer including the homogeneous film can constitute the same polymer.

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

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

[0109] In other embodiments of the present disclosure, the multicomponent membrane may comprise 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 Figure 7, except that the bilayer membrane 621 is replaced with a mixed membrane comprising two different membrane polymers of a homogeneous blend. Similar to an analyte sensor comprising a bilayer membrane positioned over one of the active regions, a homogeneous membrane comprising either the first or second membrane polymer of the mixed membrane may cover the other active region on the second working electrode.

[0110] Similar to a bilayer membrane, a mixed membrane containing 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 quite characteristic of the other membrane polymer in the mixture. Thus, according to various embodiments of the present disclosure, one of the membrane polymers of the homogeneous membrane and the mixed membrane may be selected such that the permeability of the second analyte through the mixed membrane is substantially altered by its membrane polymer. In certain embodiments, the first active region may contain a glucose-responsive enzyme such as glucose oxidase, and the second active region may contain a lactate-responsive enzyme such as lactate oxidase. Thus, according to such embodiments, the first active region containing the glucose-responsive enzyme may be covered with a mixed membrane, and the second active region containing the lactate-responsive enzyme may be overacted by a homogeneous (single-component membrane polymer) membrane. In more specific embodiments, the second active region may contain a polymer, albumin, and a lactate-responsive enzyme covalently bonded to the polymer. In more specific embodiments, the homogeneous film covering the second active region may include at least a crosslinked polyvinylpyridine homopolymer or copolymer, and the mixed film covering the first active region may also include a polyvinylpyridine homopolymer or copolymer.

[0111] As referenced above, the bilayer and mixed films can equalize analytic permeability in the analytic sensor of the present disclosure, where two or more active regions are spatially separated from each other and covered with different mass transfer limiting films. Specifically, the bilayer and mixed films of the present disclosure can equalize analytic permeability in an analytic sensor having separate working electrodes and comprising two or more active regions having different enzymes, with at least one active region positioned at each working electrode. Thus, such films can advantageously enable independent variation of sensor sensitivity for each analytic. The film thickness and / or the relative ratio of the first film polymer to the second film polymer represent other parameters that can be modified to adjust the characteristic permeability of the analytic at each working electrode.

[0112] Therefore, a method for using an analyte sensor comprising two working electrodes may involve exposing the analyte sensor to a fluid containing at least one analyte. The analyte sensor comprises a sensor tail comprising at least a first working electrode and a second working electrode. A first active region is located on the surface of the first working electrode, and a second active region is located on the surface of the second working electrode. The first active region comprises a first analyte-responsive enzyme that reacts with a first polymer and a first analyte, and the second active region comprises a second analyte-responsive enzyme that reacts with a second polymer and a second analyte. The first and second analyte-responsive enzymes are different. A multi-component membrane covers the first active region, and a homogeneous membrane covers the second active region. The multi-component membrane comprises at least a first and a second membrane polymer that are different from each other, and the homogeneous membrane comprises either the first or the second membrane polymer. This method further includes obtaining a first signal above the redox potential of a first active region, obtaining a second signal above the redox potential of a second active region, and correlating the first signal with the concentration of a first analyte in the fluid and the second signal with the concentration of a second analyte in the fluid. The first signal is proportional to the concentration of the first analyte in the fluid, and the second signal is proportional to the concentration of the second analyte in the fluid.

[0113] In a more specific embodiment, the first signal and the second signal may be measured at different times. In such an embodiment, the potential may be applied alternately to the first working electrode and the second working electrode. In another embodiment, the first signal and the second signal may be measured simultaneously through the first channel and the second channel, in which case the potential may be applied to both electrodes simultaneously.

[0114] Embodiments disclosed herein include the following: A. An analytic sensor comprising two active regions having different analytic enzymes. The analytic sensor comprises 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 comprising an analytic enzyme and a polymer, wherein the analytic enzymes in each active region are different. Here, each active region has a redox potential, and the redox potential of the first active region is sufficiently far from the redox potential of the second active region, so that a signal can be generated from the first active region independently of the signal generation from the second active region.

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

[0116] C. An analyte sensor comprising two or more enzymes capable of cooperatively interacting with each other. The analyte sensor comprises a sensor tail including at least a working electrode; and at least one active region disposed on the surface of the working electrode, the at least one active region comprising a first enzyme, a second enzyme, and a polymer, wherein the first and second enzymes are capable of cooperatively interacting with each other; the first enzyme can convert the analyte into a first product; the second enzyme can convert the first product into a second product to generate a signal at the working electrode; the second enzyme is covalently bonded to the polymer and is nonreactive with the analyte.

[0117] D. A method for assaying an analyte using two or more enzymes that can interact cooperatively with each other. The method comprises: exposing an analyte sensor to a fluid containing the analyte; the analyte sensor comprising a sensor tail including at least a working electrode and at least one active region located on the surface of the working electrode, wherein the at least one active region comprises a first enzyme, a second enzyme, and a polymer; the first enzyme and the second enzyme can interact cooperatively, the second enzyme being covalently bonded to the polymer and nonreactive with the analyte; reacting the analyte with the first enzyme to form a first product; reacting the first product with the second enzyme to form a second product, thereby generating a signal at the working electrode; and correlating the signal with the concentration of the analyte in the fluid.

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

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

[0120] G. An analyte sensor comprising two or more working electrodes covered with different mass transfer limiting membranes. The analyte sensor comprises a sensor tail comprising at least a first working electrode and a second working electrode; a first active region disposed on the surface of the first working electrode, comprising a first polymer and a first analyte-responsive enzyme that reacts with a first analyte; a second active region disposed on the surface of the second working electrode, comprising a second polymer and a second analyte-responsive enzyme that reacts with a second analyte; wherein the first and second analyte-responsive enzymes are different; a multi-component membrane covering the first active region, comprising at least a first and second membrane polymer that are different from each other; and a homogeneous membrane covering the second active region, having a different composition from the multi-component membrane, comprising one of the first and second membrane polymers.

[0121] H. A method for assaying two or more analytes using two working electrodes covered with different mass transfer limiting membranes. The method comprises: exposing an analyte sensor to a fluid containing at least one analyte; the analyte sensor comprising a sensor tail containing at least a first working electrode and a second working electrode; a first active region located on the surface of the first working electrode, comprising a first analyte-responsive enzyme that reacts with a first polymer and a first analyte; a second active region located on the surface of the second working electrode, comprising a second analyte-responsive enzyme that reacts with a second polymer and a second analyte; the first and second analyte-responsive enzymes being different; and a multicomponent membrane covering the first active region. The method includes: a homogeneous film covering a second active region, the multicomponent film comprising at least one first film polymer and a second film polymer that are different from each other, the homogeneous film comprising either the first or second film polymer and having a different composition from the multicomponent film; obtaining a first signal proportional to the concentration of a first analyte in the fluid at an oxidation-reduction potential above that of the first active region; obtaining a second signal proportional to the concentration of a second analyte in the fluid at an oxidation-reduction potential above that of the second active region; and correlating the first signal with the concentration of a first analyte in the fluid and the second signal with the concentration of a second analyte in the fluid.

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

[0123] J. A method for detecting alcohol using the cooperative interaction between glucose oxidase and xanthine oxidase. The method involves exposing an analyte sensor to a fluid containing ethanol and glucose; the analyte sensor comprises a sensor tail comprising: at least a working electrode; a first active region disposed on the surface of the working electrode, comprising xanthine oxidase, catalase, a first polymer, and an electron transfer agent, wherein the xanthine oxidase and electron transfer agent are covalently bonded to the first polymer; a first membrane covering the first active region, comprising a first membrane polymer, and being permeable to acetaldehyde; a second active region disposed on the first membrane, comprising glucose oxidase, catalase, and a second polymer, wherein the glucose oxidase is covalently bonded to the second polymer. A second active region covalently bonded to the polymer; and a second membrane covering the second active region, comprising a second membrane polymer and permeable to glucose and alcohol; the ability of glucose oxidase and xanthine oxidase to interact cooperatively; the production of hydrogen peroxide by oxidizing glucose with glucose oxidase; the formation of a catalase-hydrogen peroxide complex; the production of acetaldehyde by oxidizing alcohol with the catalase-hydrogen peroxide complex; the formation of acetic acid by reacting acetaldehyde with xanthine oxidase to generate a signal at the working electrode; and the correlation of the signal with the alcohol concentration in the liquid.

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

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

[0126] Element 4: The first electron transfer agent is covalently bonded to the polymer in the first active region, and the second electron transfer agent is covalently bonded to the polymer in the second active region. Element 5: The analyte-responsive enzymes in each active region are covalently bonded to the polymer.

[0127] Element 6: The analyte sensor further includes a mass transfer limiting membrane covering at least two active regions. Element 7: At least one of the two active regions contains two or more analyte-responsive enzymes, which interact cooperatively to produce 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 within the body. Element 9: The mass transfer limiting membrane covers at least two of the above-mentioned active regions.

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

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

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

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

[0133] Element 18: The analyte sensor further includes a mass transfer limiting membrane covering at least one of the above active regions. Element 19: The fluid is a biological fluid, and the analyte sensor is exposed to the biological fluid within the body.

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

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

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

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

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

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

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

[0141] Element 33: The analyte-responsive enzymes in each active region are covalently bonded to the polymer. Element 34: The fluid is a biological fluid, and the analyte sensor is exposed to the biological fluid within the body.

[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 through the first channel and the second channel.

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

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

[0145] Element 41: The fluid is a biological fluid, and the analyte sensor is exposed to the biological fluid within the living organism. As a non-restrictive example, the following are exemplary combinations applicable to A-J:

[0146] An analyte sensor of A combined with elements 1 and 2; 1 and 3; 1, 3, and 4; 1 and 5; 1 and 6; 1 and 7; 2 and 3; 2-4; 2 and 5; 2 and 6; 2 and 7; 3-5; 3 and 4; 3 and 5; 3 and 6; 3 and 7; 5 and 6; 6 and 7; 2, 3, and 5; 2, 3, and 6; 2-5; and 2, 5, and 6. Method B, combined with elements 2 and 3; 2-4; 2 and 5; 2 and 6; 2 and 7; 2 and 8; 3-5; 3 and 4; 3 and 5; 3 and 6; 3 and 7; 3 and 8; 5 and 6; 5 and 8; 6 and 7; 6 and 8; 7 and 8; 2, 3 and 5; 2, 3, 5 and 8; 2, 3 and 6; 2, 3, 6 and 8; 2-5; 2-5 and 8; 2, 5 and 6; 2, 5, 6 and 8; 2, 3 and 8; 3, 5 and 8; 2, 6 and 8; 2, 5 and 8; any one of 2-8 and 9; and any one of 2-8 and 10.

[0147] Elements 11 and 12; 11, 12 and 13; 12 and 13; 11 and 14; 11 and 15; 11 and 17; 11 and 18; 12 and 13; 12 and 14; 12 and 15; 12 and 16; 12 and 17; 12 and 18; 15 and 16; 15 and 17; 15 and 18; 16 and 17; 16 and 18; 17 and 18; 11, 12 and 13; 11, 12 and 14; 11-14; 11-14 and 16; 11-14 and 17; 11-14 and 18; 15-17; 15-18; and C analyte sensor in combination with 15, 17 and 18. Elements 11 and 12; 11, 12 and 13; 12 and 13; 11 and 14; 11 and 15; 11 and 17; 11 and 18; 11 and 19; 11 and 20; 12 and 13; 12 and 14; 12 and 15; 12 and 16; 12 and 17; 12 and 18; 12 and 19; 12 and 20; 15 and 16; 15 and 17; 15 and 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, combined with 8 and 20; 15-17; 15-17 and 19; 15-17 and 20; 15-18; 15-18 and 19; 15-18 and 20; 15, 17 and 18; 15, 17, 18 and 19; 15, 17, 18 and 20; any one of 11-16 and 19; and any one of 11-16 and 20.

[0148] Analyte sensor E combined with elements 21 and 22; 21 and 23; 22 and 23; and 21-23. Method F combined with elements 21 and 22; 21 and 23; 22 and 23; 21-23; 21 and 24; 21 and 25.

[0149] An analyte sensor of G combined 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 combined 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] Analyte sensor I in combination with elements 37 and 38; 37 and 39; 37 and 40; 38 and 39; 38 and 40; and 39 and 40. Method 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: A1: Analyte sensor with a multi-component membrane. The analyte sensor comprises a sensor tail configured to be inserted into tissue, the sensor tail including at least a working electrode; and first and second active regions disposed on the sensor tail, each containing at least two different enzymes for measuring the concentration of at least one analyte; the first active region is covered with a first membrane polymer and a second membrane polymer, which are different from each other.

[0153] B1: An analytic sensor having two active regions on a working electrode and configured to detect different analytic compounds. The analytic sensor is configured to be inserted into tissue and includes a sensor tail containing at least a working electrode; and at least two active regions positioned on the sensor tail, each active region containing an enzyme, an electron transfer agent, and a polymer, the enzymes in each active region being different and responding to different analytic compounds; each active region having a redox potential, the redox potential of the first active region being sufficiently far from the redox potential of the second active region, and the first active region being able to generate a signal independently of the signal generation from the second active region.

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

[0155] Element 2': The first active region is covered by a bilayer film containing the first membrane polymer placed on the second membrane polymer, and the second membrane polymer covers the second active region as a homogeneous film. Element 3': The first active region contains a first enzyme from at least two different enzymes, and the second active region contains a second enzyme from at least two different enzymes.

[0156] Element 4': The first enzyme is nonreactive with at least one analyte, and the first and second enzymes can interact cooperatively to produce a signal proportional to the concentration of the at least one analyte.

[0157] Element 5': The second enzyme can convert at least one analyte into a product that reacts with the first enzyme, and as a result, the first enzyme can react 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 and second active regions further comprises catalase.

[0159] Element 7': Catalase is located in the first active region. Element 8': The first active region is positioned directly on the working electrode and further includes an electron transfer agent. Element 9': The first membrane polymer is directly positioned on the first active region, the second active region is directly positioned on the first membrane polymer, and the second membrane polymer is directly positioned on the second active region.

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

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

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

[0163] Element 14': The first active region contains a first electron transfer agent, and the second active region contains a second electron transfer agent different from the first electron transfer agent. Embodiment B1 may 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 approximately 100 mV away from the redox potential of the second active region. Element 16': The first active region contains a first electron transfer agent, and the second active region contains a second electron transfer agent different from the first electron transfer agent.

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

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

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

[0168] [ka]

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

[0170] [Table 1]

[0171] [Table 2]

[0172] To deposit each active region, approximately 20 nL of each solution was deposited onto the carbon working electrode, resulting in each region being approximately 0.1 mm thick. 2 Two separate spots with a certain area were formed. One spot contained a glucose oxidase formulation, and the other spot contained a lactose oxidase formulation. After deposition, the working electrode was cured overnight at 25°C.

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

[0174] After preparation, the electrodes were analyzed by cyclic voltammetry in a buffer solution free of glucose and lactate. The resulting cyclic voltammograms are shown in Figure 8. Since neither glucose nor lactate contributed to the current, Figure 8 shows the characteristic anode and cathode peaks of the two osmium complexes. The redox potentials reported above were calculated from the average of the cathode and anode peaks of each osmium complex.

[0175] To analyze glucose and lactate, the electrode was positioned at a potential exceeding the average redox potential of the first polymer, specifically +40 mV (E1 in Figure 8). At this potential, oxidation of the osmium complex in the first polymer and glucose may occur, but oxidation of the osmium complex in the second polymer or lactate does not. To oxidize both osmium complexes, and both glucose and lactate, the electrode was positioned at a potential exceeding the average redox potential of the second polymer, specifically +250 mV (E2 in Figure 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 replicas of the electrode response in 5 mM glucose / 5 mM lactate buffer as it cycled between E1 and E2. As shown, the current at E1 is approximately 5 nA, which is due to glucose oxidation, and the current at E2 is approximately 10.5 nA, which is due to the oxidation of both glucose and lactate. Taking the difference between the currents measured at E1 and E2, we obtain a contribution of approximately 5.5 nA at E2 due to lactate oxidation. Unknown glucose and lactate concentrations can be similarly analyzed by comparison with a lookup table or calibration curve.

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

[0178] [Table 3]

[0179] Approximately 15 nL of solution, approximately 0.05 mm 2 The carbon was deposited on the working electrode as a single spot with a certain surface area. After deposition, the working electrode was cured overnight at 25°C. 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 dipping 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 two days. Film deposition can also be achieved using spray coating, screen printing, or a similar process alternately.

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

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

[0182] [Table 4]

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

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

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

[0186] [Table 5]

[0187] Approximately 15 nL of the second spotting solution, approximately 0.05 mm 2 It was deposited onto the PVP film as a single spot (GOX spot) with a certain area. After deposition, it was cured overnight at 25°C.

[0188] After curing, a second film was deposited on the GOX spots and PVP film. In this case, the film polymer was a crosslinked polyvinylpyridine-co-styrene polymer, with some pyridine nitrogen atoms functionalized with non-crosslinked poly(ethylene glycol) tails and some pyridine nitrogen atoms functionalized with alkyl sulfonic acid groups. The film at this location was deposited from a coating solution containing 35 mg / mL of crosslinked polyvinylpyridine-co-styrene polymer and 100 mg / mL of PEGDE400. Film deposition was achieved by dipping the electrode into the coating solution three times. Film deposition can also be achieved by spray coating, screen printing, or by alternating similar processes. After deposition, the electrode was cured overnight at 25°C, and then further cured in a dry vial at 56°C for two days.

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

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

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

[0192] [Table 6]

[0193] The deposition and curing of the active region were carried out as described in Example 1. However, the 0.1 mm of Example 1 was used. 2 Instead of a single spot with an area of ​​0.01 mm, each one is 0.01 mm 2Six spots with the specified area were deposited. Unless otherwise noted below, film deposition was performed by dip coating (1 to 5 dips of the electrode with a waiting time of approximately 10 minutes between dips). After dip coating was complete, the film was cured at 25°C for 24 hours, followed by curing in a dry vial at 56°C for 48 hours.

[0194] The electrode response was measured by placing the active region of the electrode in a beaker containing 100 mM, pH=7.5 phosphate-buffered saline at 37°C. The potential was raised to +40 mV relative to Ag / AgCl, and the current was continuously monitored thereafter. To measure the response at various lactate concentrations, sodium lactate was added to the buffer in 1 mM increments up to a maximum of 5 mM. To determine the stability of the response, the current was measured over a long period, such as two weeks, in 5 mM sodium lactate.

[0195] Membrane Polymers 1A and 1B: The first membrane polymer tested was a crosslinked polyvinylpyridine-co-styrene polymer, in which some of the pyridine nitrogen atoms were functionalized with non-crosslinked poly(ethylene glycol) tails and some of the pyridine nitrogen atoms were functionalized with alkyl sulfonic acid groups. Two different crosslinking agents were used to influence the crosslinking 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 crosslinked polymers are shown herein as polymer 1A and 1B, respectively.

[0196] Figure 14 shows exemplary plots of the responses of electrodes coated with polymers 1A and 1B to a 5 mM lactate solution. As shown, neither formulation yielded a stable sensor response over time. The sensor current provided by polymer 1A (formulation 1) decreased slowly over a two-week measurement period, while the sensor current provided by polymer 1B (formulation 2) initially increased in the first week of lactate exposure and then decreased. In contrast, both of these membranes provided a stable response in the presence of a 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] Figure 15 shows an exemplary plot of the response of an electrode coated with polymer 2 (composition 3) to a 5 mM lactate solution. Similar to polymers 1A and 1B, polymer 2 did not provide a stable current response over time. The response decreased significantly during the first 48 hours, after which it remained relatively stable. Furthermore, the sensitivity was far below the target value of approximately 1 nA / mM. Similar to polymers 1A and 1B, polymer 2 provided a stable current response in the presence of glucose analytes (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 (composition 4) to a 5 mM lactate solution. Unlike polymer 2, which was crosslinked with a high molecular weight variant of the same crosslinking agent, polymer 3 surprisingly yielded a stable current response over time. Furthermore, the current responded rapidly and achieved a stable current as the amount of added lactate was increased in 1 mM increments (Figure 17).

[0201] Membrane Polymer 4: The fourth membrane polymer tested was a PVP containing 3-4% by weight of uncrosslinked PEG side chains, which was then crosslinked with PEGDGE1000. Thus, the tested membrane polymer contained both uncrosslinked PEG chains 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 (composition 5) to a 5 mM lactate solution. Unlike polymers 2 and 3, polymer 4 surprisingly provided a stable current response over time. Furthermore, the current responded rapidly and achieved stable values ​​as the amount of added lactate was increased in 1 mM increments (Figure 19).

[0203] Two-layer films containing polymers 2 and 1B or polymers 2 and 1A: Formulation 3 (polymer 2) was coated onto the electrode surface by repeated dip coating operations. The film could be deposited by alternately using spray coating, screen printing, or similar processes. Next, Formulation 2 (polymer 1B) was coated onto the deposited cross-linked PVP layer by repeated dip coating operations. There was a 10-minute waiting period between consecutive dips. After all dips were completed, the sensor was cured at 25°C for 24 hours, followed by curing in a dry vial at 56°C for 48 hours. As shown above, none of these film polymers provided satisfactory performance when used alone.

[0204] Figure 20 shows an exemplary plot of the response of an electrode covered with a bilayer film containing a lower layer of crosslinked PVP (polymer 2) and an upper layer of crosslinked polymer 1B to a 5 mM lactate solution. Despite the fact that neither polymer alone provides acceptable performance, unlike polymer 1B or PVP crosslinked with the same crosslinking agent (polymer 2), the bilayer film containing these film polymers surprisingly yielded a stable current response over time with an acceptable level of sensitivity. The response data in Figure 20 were for electrodes immersed twice in formulation 3 (polymer 2) and four times in formulation 2 (polymer 1B).

[0205] The amount (thickness) of each film polymer in the bilayer film can change the sensor performance for polymer 2 and polymer 1A, as shown below. Therefore, the Gly3 crosslinked variant of polymer 1B (i.e., polymer 1A) may provide similarly acceptable performance when combined with polymer 2 in a bilayer film, even if neither polymer alone provides acceptable performance.

[0206] Figure 21 shows an exemplary plot of the response of an electrode covered with a bilayer film containing a lower layer of crosslinked PVP (polymer 2) and an upper layer of crosslinked polymer 1A to a 5 mM lactate solution, with the electrode dip-coated with formulation 1 (polymer 1A) and formulation 3 (polymer 2) a number of times. In this case, the crosslinking agent for PVP (polymer 2) remained PEGDGE1000, while the crosslinking agent for polymer 1A was Gly3. This indicates that this crosslinking agent is also suitable for use in bilayer film configurations. As shown in Figure 21, a good balance between sensitivity and stable current response was obtained by dip-coating the electrode twice with formulation 3 and four times with formulation 1. Changing the number of dip-coating operations changed the thickness of each component of the bilayer film and the relative mass ratios of the film polymers. As shown in Figure 21, if the PVP layer is too thin (0 or 1 dipping of polymer 2), sensitivity is high, but response stability is low. On the other hand, if the electrode is too thick (more than three immersions), it may become less sensitive and have poor response stability.

[0207] A mixed membrane containing membrane polymers 1B and 3: The mixed membrane formulation (formulation 6) was prepared by mixing 1.5 mL of PVP (100 mg / mL) in 80:20 ethanol:HEPES buffer, 2.5 mL of the copolymer 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. Therefore, after crosslinking, formulation 6 contains polymer 1B and polymer 3, which are each crosslinked with PEG400.

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

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

[0210] Example 4: Performance of a sensor containing two working electrodes covered with a two-layer mass transfer limiting film. In this example, a first working electrode containing glucose oxidase and a second working electrode containing lactate oxidase were covered with a two-layer film. The active region containing glucose oxidase was deposited using a glucose oxidase formulation as described in Example 1 (Table 1). The active region containing lactate oxidase was deposited using a lactate oxidase formulation as described in Example 3 (Table 6). The deposition and curing of the active regions were carried out as described in Example 1, except that the 0.1 mm film used in Example 1 was used. 2 Instead of a single spot with an area of ​​0.01 mm, each one is 0.01 mm2 Five spots with the specified area 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 film in this example. That is, polymer 3 was deposited on a second working electrode characterized by 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. Subsequently, the entire assembly (i.e., both working electrodes, the PVP coating on the second working electrode, and the counter electrode and reference electrode) was dip-coated with formulation 2. Curing was carried out again at 25°C for 24 hours, followed by baking at 56°C for 48 hours in a dry environment. Thus, a homogeneous film was deposited on the first working electrode (glucose-responsive), and a bilayer film was deposited on the second working electrode (lactic acid-responsive). The crosslinked PVP (polymer 3) was in contact with the lactate-responsive active region on the second working electrode.

[0211] This sensor was used to simultaneously assay glucose and lactate in 100 mM PBS at 37°C. In the first experiment, the sensor was exposed to a 100 mM PBS solution containing 30 mM glucose and 5 mM lactate for two weeks at 37°C. In this test, the sensor maintained a voltage of +40 mV relative to Ag / AgCl. Figure 25 shows exemplary plots of the sensor response for each working electrode upon exposure to 30 mM glucose and 5 mM lactate. As shown, the sensor response remained remarkably stable throughout the observation period.

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

[0213] Example 5: Detection of ketones using an analyte sensor having cooperatively interacting diaphorase and β-hydroxybutyrate dehydrogenase. In this example, the membrane formulations shown in Table 7 below were coated onto a carbon working electrode. Approximately 0.01 mm of each was applied to the working electrode. 2 Deposition was carried out to place six spots with a given area. After deposition, the working electrode was cured overnight at 25°C. Subsequently, the PVP film was applied to the working electrode via dip coating using a coating solution containing 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 carried out at 25°C for 24 hours, followed by 56°C for 48 hours in a dry vial.

[0214] [Table 7]

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

[0216] Example 6: Comparison of Lactate Sensor Responses in Various Sensor Configurations. To assay the performance of lactate-responsive sensors characterized by various formulation arrangements, two different lactate oxidase / polymer formulations for active region deposition and two different membrane polymer formulations for mass transfer limiting membrane deposition were prepared. Details of the formulations and the processes used to prepare the analyte sensors are described below. In general, the analyte sensors were prepared in the same manner as described above.

[0217] [Table 8]

[0218] [Table 9]

[0219] To deposit each active region, approximately 20 nL of each solution was deposited onto the carbon working electrode, resulting in each region being approximately 0.01 mm thick. 2 Six individual spots with the specified area were formed. The spots were formed by applying formulation A four times and formulation B six times. After deposition, the working electrode was cured overnight at 25°C. Formulation A is equivalent to that used to deposit the active region of the glucose-responsive analyte sensor, except that lactate oxidase was used instead of glucose oxidase.

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

[0221] [Table 10]

[0222] [Table 11]

[0223] Using dip coating, mass transfer limiting films were deposited on each active region prepared as described above. Formulation C was deposited using 4 dips, and formulation D was deposited using 44 dips. A waiting time of approximately 10 minutes was used between dips. After completion of dip coating, the films were cured at 25°C for 24 hours, followed by curing in a dry vial at 56°C for 48 hours. Mass transfer limiting films can also be deposited using spray coating, screen printing, or similar processes alternately. Formulation C corresponds to the one used to deposit a mass transfer limiting film within a glucose-responsive analyte sensor.

[0224] Lactate-responsive analyte sensors were prepared using the deposition conditions specified above. All possible combinations of active region and mass transfer limiting membrane 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 to maintain an action potential of +40 mV relative to Ag / AgCl. The tested combinations of active region and mass transfer limiting membrane are shown in Table 12. The test results are shown in Figure 30.

[0225] [Table 12]

[0226] As shown in Figure 30, a lactate-responsive analyte sensor with an active region and mass transfer limiting membrane formulated similarly to those used without issue in the glucose-responsive analyte sensor (Group 1) exhibited insufficient performance when exposed to lactate. As shown, the signal intensity was less than 0.5 nA for all samples tested. This is an undesirable low value for a viable lactate-responsive sensor. In formulation C (Group 2), the signal intensity was even lower when polyvinylpyridine and a different crosslinking agent were used instead of polyvinylpyridine-co-styrene and the Gly3 crosslinking agent.

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

[0228] Surprisingly, the combination of an active region containing human serum albumin and a mass transfer limiting membrane containing a cross-linked polyvinylpyridine homopolymer (Group 4) yielded an acceptable combination of high signal intensity and extended signal stability over time. As shown in Figure 30, all replicated sensors in Group 4 had initial signal intensities clustered within 1 nA of each other between 4 nA and 5 nA. This level of signal intensity and variability is within the range in which a commercially viable lactate-responsive analyte sensor could be developed. Furthermore, the signal intensity changed by only a fraction of an nA over 190 hours of signal observation. This, too, is within the range suitable for the development of a commercially viable sensor.

[0229] As shown in Figure 31, the current observed for the sensors in group 4 responded quickly and achieved stable values ​​when lactic acid was added in increasing amounts of 1 mM increments to a PBS solution that initially did not contain lactic acid.

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

[0231] Analyte sensor ignition lock Vehicle failsafes such as ignition locks may be used to prevent an operator from operating a vehicle in the event of a malfunction or when the vehicle is not in a safe operating condition. Operating a vehicle in a malfunctioning state can pose a significant risk to the operator and the general public. One common type of ignition lock is designed to prevent drunk driving, and more specifically, to prevent an individual from operating a vehicle while intoxicated by the use of alcohol. Such a locking device connects a breath alcohol analyzer or an optical sensor to the vehicle's ignition system, and the driver is required 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, since other impairments and conditions can also afflict an operator, it is necessary to closely monitor to ensure that an operator does not operate a vehicle when impaired. For example, an operator driving while experiencing hypoglycemia due to diabetes may experience dizziness, confusion, headache, loss of consciousness, seizures, and delayed reflexes, any of which can endanger the life of the operator and those within or near the vehicle.

[0233] Analyte monitoring systems have been developed to facilitate the 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 is useful in treating diabetic conditions. However, other analyte monitoring systems are designed to detect and monitor other analytes present in the operator's bodily fluids, and abnormal analyte levels detected in the operator may indicate that the operator is not currently fit to safely operate the vehicle.

[0234] The following description concerns an analytic monitoring and vehicle control system used to prevent vehicle operation when the operator's analytic levels exceed a predetermined threshold. By appropriately deploying the sensor control device 102 (Figure 1), the user can rationally track and monitor the levels and trends of bodily fluid analytics. When certain analytic levels exceed a specific threshold, 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 return the analytic levels to a safe range before attempting to operate the vehicle. However, in some cases, the user may feel no problem operating the vehicle, yet still have an unsafe analytic level that could suddenly cause a dangerous physical injury. In such cases, it may be beneficial to install a fail-safe system that prevents or warns the user of the possibility of endangering themselves or others by operating the vehicle.

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

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

[0237] As shown in the figure, the system 3200 may further include a reader device 120, and the sensor control device 102 may communicate with the reader device 120 via a local communication path or link to 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 the vehicle's electrical system and is powered by the vehicle battery or, otherwise, by a separate battery. In such embodiments, data transmitted from the sensor control device 102 to the reader device 120 may then be transmitted by the reader device 120 to the control module 3204 for processing. However, in other embodiments, the sensor control device 102 may communicate directly with the control module 3204 via any wireless communication protocol such as BLUETOOTH®. In such embodiments, 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 in a broad sense and means all kinds of transportable vehicles that can be operated by a human user or “operator,” but may also include autonomous vehicles used to transport people. Examples of vehicle 3206 include, but are not limited to, any kind of automobile, truck, sports utility vehicle, aircraft, ship, spacecraft, and / or any other means of transport, or combinations thereof.

[0239] The control module 3204 may include a communication interface for communicating information with the sensor control device 102 and / or the reader device 120. In the case of exemplary Bluetooth®-enabled sensor control device 102 and / or reader device 120, the sensor control device 102 can enter pairing mode when it approaches the vehicle 3206. 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 with them. For example, when operator 3202 approaches or enters the vehicle 3206, the control module 3204 can automatically detect the presence of the sensor control device 102 and enable communication between them 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 touchscreen display, or an information display. In such embodiments, the control module 3204 can communicate visually with the operator 3202 via the vehicle user interface 3208, and can also communicate audibly with the operator 3202 via an audio speaker included in the vehicle 3206. However, in other embodiments, the control module 3204 may be configured to communicate with a reader device 120 so that it can communicate with the operator 3202.

[0241] As shown in the figure, 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 level of operator 3202 obtained by the sensor control device 102. The operation of the vehicle 3206 may be controlled, disabled, or modified by disabling one or more critical systems of the vehicle 3206 or by activating warning systems within the vehicle 3206. If the real-time measured analyte level of operator 3202 is within a predetermined safety range, it may be considered safe for operator 3202 to operate the vehicle 3206. However, if the real-time measured analyte level of operator 3202 is outside a predetermined safety range or exceeds a predetermined threshold, the computer system 3210 may be programmed to control, disable, or modify 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 detected analyte level of operator 3202 is outside a predetermined range or exceeds a predetermined threshold, and thus, if operator 3202 is identified as being unable to safely operate vehicle 3206, the operation of the vehicle is gradually and safely disabled. Critical vehicle systems of vehicle 3206 that may be disabled include the ignition system (e.g., energy switching / control system), transmission system (or gearbox), fuel system, and energy supply system (e.g., battery, capacitor, conversion / reaction battery, etc.). When an elevated or degraded (unsafe) analyte level is detected, the computer system 3210 may prevent critical vehicle systems from functioning or operating. As a result, operator 3202 will not be able to start or operate vehicle 3206, thereby preventing 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 level of operator 3202 exceeds or surpasses a predetermined threshold. Non-critical vehicle systems that can be activated include, for example, a vehicle horn, vehicle lights, or an audible warning system installed in vehicle 3206. In such embodiments, the activation of a non-critical vehicle system can alert law enforcement and others (e.g., operators of adjacent vehicles, bystanders, pedestrians, etc.) of operator 3202 who may be driving under impaired conditions, enabling legal action to be taken to promptly address the related problem and notifying others of a potentially dangerous situation.

[0244] In further embodiments, or in addition, the computer system 3210 may be configured to automatically call one or more emergency contacts when the operator's analyte level is outside a predetermined safe operating range or exceeds a predetermined threshold. In such embodiments, the computer system 3210 may operate via a reader device 120 (e.g., a mobile phone) or a cellular or satellite communication system (e.g., OnStar®) incorporated in the vehicle 3206. In further 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 analyte level is outside a predetermined safe operating range or exceeds a predetermined threshold. Examples of emergency contacts include, but are not limited to, a spouse, parent, medical professional (e.g., doctor), 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 an operator 3202, more specifically, a sensor control device 102. In such embodiments, the proximity sensor 3212 may be configured to monitor the entire area of ​​the driver's seat 3214 within 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 indicator that an operator 3202 is in the driver's seat 3214 and may be attempting to operate the vehicle 3206. In such cases, a signal may be sent to the control module 3204 to alert the computer system 3210 that an operator 3202 is in the vehicle 3206 and may be attempting to operate the vehicle 3206. If the operator 3202's real-time measured analyte level is within a predetermined safety range or below a predetermined level, the computer system 3210 may allow the operator 3202 to operate the vehicle 3206. However, if the operator 3202's real-time measured analyte level is outside a predetermined safety range or exceeds a predetermined threshold, the computer system 3210 can control, disable, or modify the operation of the vehicle 3206, as outlined above. As understood, the proximity sensor 3212 may be advantageous in preventing the operation of the vehicle 3206 only if the disabled operator 3202 is in the driver's seat 3214 and ready to operate the vehicle 3206. As a result, a user equipped with the sensor control device 102 can ride in the vehicle 3206 as a passenger at any time 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 the current state of the vehicle 3206, including whether the vehicle 3206 is currently moving or stationary. Furthermore, the vehicle state detection module 3216 may be configured to determine whether the motors in the vehicle 3206 are currently operating or stopped. In one or more embodiments, the vehicle state detection module 3216 may provide a state signal to the control module 3204, which can use the state signal to determine which vehicle operation to activate or disable when the operator's 3202's real-time measured analyte level is outside a predetermined safety range or exceeds 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, transmission system, ignition system, or any combination thereof. In contrast, if the status signal indicates that vehicle 3206 is moving, the control module 3204 may activate the vehicle horn, flash the vehicle lights, or emit a warning sound to operator 3202 and / or people around operator 3202 that operator 3202 is not functioning correctly.

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

[0248] If not done automatically, operator 3202 may be prompted to obtain the current analyte level when pairing the sensor control device 102 with the control module 3204. In some cases, operation of the vehicle 3206 may be prevented until the current analyte level is obtained. If the current analyte level is within the 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 operator 3202 to obtain additional current analyte levels after operating the vehicle 3206 over a predetermined period of time (e.g., 1 hour, 2 hours, 5 hours, etc.).

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

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

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

[0252] In some embodiments, if an unsafe analyte level is measured while operator 3202 is operating vehicle 3206, the control module 3204 may be configured to issue one or more warnings (visual or audible) to alert operator 3202 about the unsafe analyte level. In some cases, the volume of the stereo in vehicle 3206 may be automatically lowered so that 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 operator 3202. Examples of corrective actions include, but are not limited to, slowing down and stopping vehicle 3206, locating and driving to a nearby convenience store or pharmacy, and locating a nearby hospital or medical facility. If vehicle 3206 is an autonomous vehicle and the current analyte level puts operator 3202 in a potentially dangerous situation, the control module 3204 may automatically direct vehicle 3206 to a medical facility for treatment. Alternatively, or in addition, the control module 3204 may gradually reduce or limit the speed of the vehicle 3206 when an unsafe level of analyte is detected, thereby stopping the operator 3202 and addressing the problem before continuing to operate the vehicle 3206.

[0253] System 3200 may be useful in several different scenarios to protect the operator 3202 and / or people around operator 3202 while driving. In some applications, system 3200 may be voluntarily incorporated by the operator to detect faults in real time. In other applications, system 3200 may be required by the owner of vehicle 3206 to detect faults of operator 3202. In such applications, the owner of vehicle 3206 may be a transport company or a trucking company. In yet another application, system 3200 may be legally required of operator 3202 to detect faults.

[0254] Embodiments disclosed herein include the following: An analyte monitoring and vehicle control system including a sensor control device having sensors to detect and monitor one or more analytes present in an operator's body, and a control module that communicates 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, the operation of the vehicle being controlled or disabled by the computer system if the operator's real-time measured analyte level exceeds a predetermined safety threshold.

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

[0256] Embodiments K and L may each 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 positioned percutaneously under the operator's skin to detect and monitor analytes present in the operator's bodily fluids. Element 2: The sensor control device includes an in vitro analyte sensor. Element 3: Further comprises a reader device that receives data from the sensor control device and transmits the data to a control module. Element 4: The vehicle includes a transport body selected from the group consisting of automobiles, autonomous vehicles, trucks, sports utility vehicles, aircraft, ships, spacecraft, or any combination thereof. Element 5: The sensor control device pairs with a control module for communication when an operator approaches the vehicle. Element 6: Further comprises a vehicle user interface included in the vehicle that communicates 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: Vehicle operation is controlled by at least one of activating one or more non-critical systems of the vehicle, calling or messaging one or more emergency contacts, and gradually reducing the vehicle's speed. Element 9: Further comprising one or more proximity sensors installed in the vehicle to monitor the driver's seat area of ​​the vehicle and detect the presence of an operator. Element 10: The control module further includes a vehicle state detection module that detects the current state of the vehicle. Element 11: The control module generates a visual or auditory warning that the operator can perceive when the operator's real-time measured analyte level is outside a predetermined safety threshold. Element 12: The visual or auditory warning is generated at specific time increments before an unsafe analyte level is reached. Element 13: The visual or auditory warning includes one or more proposed corrective actions 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 includes receiving data from a sensor control device and transmitting the data to a control module using a reader device that communicates with the sensor control device and the control module. Element 16: Disabling the operation of the vehicle includes disabling one or more critical systems of the vehicle, the critical systems being selected from the group consisting of the ignition system, transmission system, fuel system, and energy supply system. Element 17: Controlling the operation of the vehicle includes activating one or more non-critical systems of the vehicle, calling or messaging one or more emergency contacts, and gradually reducing the speed of the vehicle. Element 18: Further includes monitoring the driver's seat area of ​​the vehicle using one or more proximity sensors mounted on the vehicle and detecting the presence of an operator. Element 19: Further includes detecting the current state of the vehicle using a vehicle state detection module included in the control module. Element 20: Further includes using the control module to generate a visual or auditory warning perceptible to the operator when the operator's real-time measured analyte level 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 herein. It is understood that in developing a physical embodiment incorporating an embodiment of the present invention, a number of implementation-specific decisions must be made to achieve the developer's objectives, such as compliance with system-related, business-related, government-related, and other constraints. These will vary depending on the implementation and may vary from case to case. While the developer's efforts may be time-consuming, such efforts are routine for those skilled in the art and will be of interest to the present disclosure.

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

[0260] As used herein, the phrase “at least one” that precedes a set of items and separates any of the items, accompanied by the terms “and” or “or,” applies to the entire list, rather than to each member of the list (i.e., each item). The phrase “at least one of ~” may mean at least one of any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. For example, the phrase “at least one of A, B, and C” or “at least one of A, B, or C” refers to A only, B only, or C only; any combination of A, B, and C; and / or at least one of each of A, B, and C, respectively.

[0261] Accordingly, the disclosed systems, tools, and methods are well-suited to achieving the stated objectives and benefits, as well as those inherent therein. The teachings of this disclosure can be modified and implemented in different equivalent ways, which will be obvious to those skilled in the art who are interested in the teachings herein; therefore, the specific embodiments disclosed above are merely illustrative. Furthermore, except as described in the appended claims, it is not intended to limit the scope to the structural or design details shown herein. Accordingly, it is obvious that the specific exemplary embodiments disclosed above can be modified, combined, or altered, and all such variations are considered to be within the scope of this disclosure. The systems, tools, and methods disclosed exemplary herein can be adequately implemented without elements not specifically disclosed herein and / or any optional elements disclosed herein. While systems, tools, and methods are described in terms of "including" various components or steps, systems, tools, and methods can also "essentially consist of" or "consist of" various components and steps. All numbers and scopes disclosed above are subject to some variation. Whenever a numerical range with lower and upper limits is disclosed, any number within that range and any range included therein are specifically disclosed. In particular, all range values ​​disclosed herein (in the form of "about a to about b," or equivalently "about a to b," or equivalently "about a~b") should be understood to describe all numbers and ranges included in the broader range of values. Furthermore, unless explicitly and clearly defined by the patentee, the terms in the claims have their original, ordinary meanings. In the event of any inconsistency in the use of a word or term between this specification and one or more patent documents or other documents that may be incorporated herein by reference, the definitions in accordance with this specification shall prevail.

Claims

1. An electrochemical analyte sensor for detecting glucose and lactate in living organisms, At least a first working electrode and a second working electrode, A lactate-responsive active region disposed on the first working electrode, the lactate-responsive active region containing a lactate-responsive enzyme, A glucose-responsive active region disposed on the second working electrode, the glucose-responsive active region containing a glucose-responsive enzyme, and A mass transfer limiting membrane, A first membrane disposed on the lactic acid-responsive active region, A second membrane disposed on the first membrane and the glucose-responsive active region, The mass transfer limiting membrane including The first membrane and the second membrane have different permeability values ​​that result in variable analyte permeability in the lactate-responsive active region and the glucose-responsive active region, The analyte sensor is configured to be partially inserted into the user's skin, so that lactic acid and glucose are detected in vivo when the distal portion of the analyte sensor comes into contact with interstitial fluid.

2. The analyte sensor according to claim 1, wherein the lactic acid-responsive active region further comprises a first polymer and albumin.

3. The analyte sensor according to claim 2, wherein the lactic acid-responsive enzyme is covalently bonded to the first polymer.

4. The analyte sensor according to claim 2, wherein the lactic acid-responsive active region further comprises a first electron transfer agent.

5. The analyte sensor according to claim 4, wherein the first electron transfer agent is covalently bonded to the first polymer.

6. The analyte sensor according to claim 1, wherein the glucose-responsive active region further comprises a second polymer.

7. The analyte sensor according to claim 6, wherein the glucose-responsive enzyme is covalently bonded to the second polymer.

8. The analyte sensor according to claim 6, wherein the glucose-responsive active region further comprises a second electron transfer agent.

9. The analyte sensor according to claim 8, wherein the second electron transfer agent is covalently bonded to the second polymer.

10. The analyte sensor according to claim 1, wherein the lactic acid-responsive enzyme is lactate oxidase.

11. The analyte sensor according to claim 10, wherein the glucose-responsive enzyme is glucose oxidase.

12. The analyte sensor according to claim 1, wherein the first membrane comprises a crosslinked polyvinylpyridine homopolymer or copolymer.

13. The analyte sensor according to claim 12, wherein the second membrane comprises a cross-linked polyvinylpyridine-co-styrene polymer.

14. The analyte sensor according to claim 2, wherein the first polymer comprises polyvinylpyridine, polyvinylimidazole, or a copolymer thereof.

15. The analyte sensor according to claim 6, wherein the second polymer comprises polyvinylpyridine, polyvinylimidazole, or a copolymer thereof.

16. The analyte sensor according to claim 1, wherein the first working electrode is configured to generate a first signal indicating lactic acid concentration, and the second working electrode is configured to generate a second signal indicating glucose concentration.

17. A sensor control device, (i) The analyte sensor according to any one of claims 1 to 16, and (ii) A sensor electronic device configured to determine the lactic acid concentration and glucose concentration for each analyte using a calibration curve based on the first and second signals. Sensor control devices, including those mentioned above.

18. An analyte detection system comprising the sensor control device according to claim 17 for detecting glucose and lactate in a living organism.

19. The analyte detection system according to claim 18, further comprising a reader device for displaying lactic acid concentration and glucose concentration.

20. A method for detecting glucose and lactate in vivo, Exposing the analyte sensor according to any one of claims 1 to 16 to a fluid containing glucose and lactic acid, Applying a potential to the first working electrode and the second working electrode, The first signal is obtained at an oxidation-reduction potential above the first active region, wherein the first signal is proportional to the concentration of lactic acid in the fluid. The method involves obtaining a second signal at a redox potential above the second active region, wherein the second signal is proportional to the concentration of glucose in the fluid. Correlating the first signal with the concentration of lactic acid in the fluid, and The second signal is correlated with the concentration of glucose in the fluid. A method that includes this.

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