Methods for monitoring levels of diaphorase inhibitors

Abstract

To provide analyte sensors featuring an enzyme system comprising diaphorase and a NAD-dependent dehydrogenase.SOLUTION: Analyte sensors featuring an enzyme system comprising diaphorase and a NAD-dependent dehydrogenase may be utilized to detect inhibitors of diaphorase, provided that the transfer of electrons to a working electrode is rate-limiting with respect to the diaphorase. Such analyte sensors may comprise a sensor tail comprising at least a first working electrode, a first active area disposed upon a surface of the first working electrode, and an analyte-permeable membrane overcoating at least the first active area. The enzyme system comprises: NAD, reduced NAD, or any combination thereof; a NAD-dependent dehydrogenase, such as NAD-dependent glucose dehydrogenase; and diaphorase. Inhibitors of diaphorase that may be detected include, for example, warfarin, dicoumarol, and similar compounds. A second active area may be present to facilitate detection of an analyte differing from the inhibitor of diaphorase.SELECTED DRAWING: None

Description

[Background technology]

[0001] Detecting various analytes within an individual can sometimes be essential for monitoring a person's health and well-being. Some analytes are biomolecules produced in the body, and their concentrations can fluctuate due to underlying physiological conditions or exposure to specific environmental factors. Drug or drug metabolite concentrations can similarly be analyzed as a measure of an individual's health and can assist medical professionals in making medication and treatment decisions. Deviations from normal analyte levels can often indicate a worsening metabolic state, disease, exposure to specific environmental conditions, or an ineffective treatment regimen. While a particular pathological source can dysregulate a single analyte alone, it is common for multiple analytes to be dysregulated simultaneously, either due to the same pathological source or coexisting (related) conditions. When multiple analytes are dysregulated, the degree of dysregulation can vary for each analyte. To achieve a complete assessment of an individual's health, each analyte may need to be monitored.

[0002] Coumarin-based drugs, such as warfarin and dicoumarol, are commonly used anticoagulants in patients with cardiovascular disease. Their mechanism of action involves competitive inhibition of vitamin K epoxide reductase, which depletes vitamin K in the blood and results in reduced blood clotting. Despite their usefulness, maintaining therapeutically effective doses of coumarin-based drugs in vivo can be extremely difficult. Patients taking coumarin-based drugs must carefully adjust their diet to avoid foods rich in vitamin K, such as green leafy vegetables, to avoid reactivating the blood clotting cycle and displacing enzyme-bound coumarin. Furthermore, patients may respond differently to coumarin-based drugs or metabolize them at widely different rates. If plasma levels become too high as a result of excessive or frequent coumarin administration, dangerous bleeding events can occur. Similarly, the therapeutic window for inhibiting blood clotting can easily be exceeded. Another drawback is that coumarin-based drugs, such as warfarin, can potentiate the effects of some diabetes medications, causing extreme hypoglycemia. Therefore, healthcare professionals who prescribe coumarin-based drugs must usually carefully titrate a particular patient to a therapeutically effective dose and then monitor for adverse side effects after prescribing, particularly in diabetic patients.

[0003] Periodic ex vivo analyte monitoring using collected bodily fluids is often sufficient for monitoring the health of many individuals. Indeed, when titrating a coumarin-based drug to a therapeutically effective dose, multiple blood draws may be required during subsequent post-prescription maintenance monitoring. Because coumarin-based drug dosages may change frequently, a significant number of blood draws may be required for a patient over time. Not only can multiple blood draws be painful, but they are often performed in a doctor's office at set collection times, which can be inconvenient for a patient's work or personal schedule. Furthermore, the periodic nature of blood draws may provide healthcare professionals with only a limited view of the in vivo profile of the coumarin-based drug and other analytes.

[0004] In vivo analyte sensors, particularly those that use enzyme-based detection to provide detection specificity, have partially addressed the aforementioned problems for certain analytes and are seeing increasing use. Indeed, in vivo analyte sensors that utilize glucose-responsive enzymes to monitor blood glucose levels are now commonly used among diabetics. Other types of analytes can be monitored using other enzymes or enzyme systems that include multiple enzymes acting in concert. However, there are currently relatively few in vivo analyte sensors featuring enzyme-based detection that can satisfactorily analyze drugs or drug metabolites, such as coumarin-based drugs. [Brief explanation of the drawings]

[0005] The following figures are included to illustrate certain aspects of the present disclosure and should not be considered exclusive embodiments. The disclosed subject matter is capable of considerable modification, alteration, combination, and equivalents in form and function without departing from the scope of the present disclosure. [Figure 1] FIG. 1 shows a diagram of an exemplary sensing system that can incorporate the analyte sensors of the present disclosure. [Figure 2A] 2A-2C show an enzyme system configured to detect ketones. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 3A] FIG. 3A shows how the enzyme system of FIG. 2A can be modified to detect glucose. [Figure 3B] Figure 3B shows how the enzyme system of Figure 3A can be further modified to detect inhibitors of diaphorase. Thus, Figure 3B shows an enzyme system configured to detect inhibitors of diaphorase. [Figure 4A] 4A-4C show cross-sectional views of exemplary analyte sensors having active regions suitable for detecting inhibitors of diaphorase. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 5A] 5A-5C show cross-sectional views of an exemplary analyte sensor having a single working electrode and multiple active areas suitable for detecting an inhibitor of diaphorase and another analyte. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 6] FIG. 6 shows a cross-sectional view of an exemplary analyte sensor having two working electrodes and multiple active areas suitable for detecting an inhibitor of diaphorase and another analyte. [Figure 7A] 7A-7C are perspective views of an exemplary analyte sensor featuring electrodes arranged concentrically relative to one another and including an active area suitable for detecting an inhibitor of diaphorase and another analyte. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 8A] 8A and 8B show an enzyme system configured to detect glucose. [Figure 8B] Same as above. [Figure 9] FIG. 9 shows an enzyme system configured to detect creatinine. [Figure 10] FIG. 10 is a graph showing the results of titrating NADH and dicoumarol (DCM) into a PBS solution exposed to the analyte sensors of Example 1 (Sensors 1-4). [Figure 11A] 11A and 11B are graphs showing the results of titrating NADH and dicoumarol (DCM) into a PBS solution exposed to an analyte sensor having varying amounts of electron transfer agent in the active area of ​​the analyte sensor. [Figure 11B] Same as above. [Figure 12] FIG. 12 is a graph of sensor response as a function of NADH concentration for analyte sensors having varying amounts of electron transfer agent in the active area of ​​the analyte sensor. [Figure 13]FIG. 13 is a graph of normalized sensor response as a function of dicoumarol concentration for analyte sensors having varying amounts of electron transfer agent in the active area of ​​the analyte sensor. [Figure 14A] 14A and 14B are graphs showing the results of titrations of glucose and dicoumarol (DCM) in PBS solution exposed to the analyte sensors of Example 2 (sensors 5-8). [Figure 14B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0006] The present disclosure generally describes analyte sensors that use multiple enzymes for the detection of one or more analytes, and more specifically, analyte sensors that employ multiple enzymes acting in concert to detect inhibitors of diaphorase, such as coumarin-based drugs, and corresponding methods for their use. Additional analytes may be simultaneously detected using another enzyme or enzyme system disposed on the same analyte sensor.

[0007] As mentioned above, due to the specificity often found in enzymes for particular substrates or classes of substrates, analyte sensors using enzyme-based detection are typically used to assay a single analyte, such as glucose or a related analyte. For this purpose, analyte sensors that combine single enzymes and enzyme systems containing multiple enzymes acting in concert can be used. As used herein, the term "in concert" refers to coupled enzyme reactions in which the product of a first enzyme reaction becomes the substrate for a second enzyme reaction, and the second or subsequent enzyme reaction serves as the basis for measuring the concentration of the analyte. To facilitate detection, the analyte may react during or otherwise influence at least one of the enzyme reactions in the enzyme system. Using in vivo analyte sensors featuring enzymes or enzyme systems to facilitate detection can be particularly advantageous for avoiding the frequent withdrawal of bodily fluids that may be required for analyte monitoring. Monitoring drugs and drug metabolites using in vivo analyte sensors can be particularly problematic because it is rare to identify an appropriate enzyme system to facilitate the specific detection of a particular drug or drug metabolite.

[0008] Coumarin-based drugs, such as warfarin and dicoumarol, are a class of drugs for which in vivo monitoring is highly desirable due to the difficulty of titrating and maintaining therapeutically effective levels. Currently, it is believed that there are no effective methods for detecting and quantifying coumarin-based drugs or their metabolites in vivo, especially methods that use enzymes or enzyme systems to facilitate detection. Vitamin K epoxide reductase, the target enzyme of some coumarin-based drugs, has not yet been utilized for a viable enzyme-based detection scheme for coumarin-based drugs.

[0009] Coumarin-based drugs, as well as some other types of compounds, are highly effective inhibitors of the enzyme diaphorase. This disclosure demonstrates that analyte sensors featuring enzyme systems containing diaphorase can be configured to effectively detect coumarin-based drugs as well as other inhibitors of the diaphorase enzyme. The enzyme systems can be electrically coupled to a working electrode to facilitate electrochemical analyte detection. Enzyme systems configured to detect coumarin-based drugs and similar inhibitors are characterized by diaphorase and at least one additional enzyme acting in concert to generate an electrochemical signal at the working electrode. To facilitate the detection of coumarin-based drugs and other diaphorase inhibitors, the enzyme system is made rate-limiting with respect to diaphorase so that the electrochemical signal (e.g., current) received at the working electrode can be correlated with the amount of coumarin-based drug or other diaphorase inhibitor present. Suitable enzyme systems that include diaphorase and are rate-limiting with respect to the diaphorase are described in further detail below. Advantageously, such enzyme systems can take advantage of the high natural concentrations of glucose or other chemical species in biological fluids to initiate the enzyme cascade, ultimately resulting in electron transfer to the working electrode, as also described below, such that no additional reagents other than those contained within the analyte sensor itself are required to facilitate detection.

[0010] In addition to detecting coumarin-based drugs and other diaphorase inhibitors, the analyte sensors disclosed herein can be further configured to detect one or more additional analytes. Examples of other analytes that can be analyzed using additional detection chemistries housed within the same analyte sensor include, for example, glucose, ketones, creatinine, lactate, A1c, pH, etc. As discussed above, in vivo analyte sensors featuring enzyme-based detection of glucose are currently widely used among diabetic patients. Detection systems for other analytes, one or more of which may also be dysregulated in diabetic patients, are also known. A detection system for glucose or any one or more of the other aforementioned analytes may be incorporated into the analyte sensors disclosed herein in combination with an enzyme system configured to detect diaphorase inhibitors. Further details regarding how additional sensing chemistries can be incorporated into the analyte sensors of the present disclosure are provided below.

[0011] The ability to monitor coumarin-based drugs in vivo represents a significant and advantageous clinical advance provided by the present disclosure. Furthermore, because coumarin-based drugs tend to potentiate the effects of diabetes medications, which can lead to further dosing dysregulation of the coumarin-based drug, it may be even more advantageous to monitor glucose levels in vivo in conjunction with the analysis of the coumarin-based drug. Simultaneous monitoring of glucose and coumarin-based drug concentrations using an analyte sensor configured to detect both analytes may provide a wealth of information to medical professionals and potentially provide improved patient outcomes. Similarly, it may be desirable to monitor other commonly dysregulated analytes in conjunction with monitoring the concentrations of coumarin-based drugs and other diaphorase inhibitors.

[0012] Before describing the analyte sensors of the present disclosure in further detail, an overview of a suitable in vivo analyte sensor configuration and a sensor system using the analyte sensor will first be provided so that embodiments of the present disclosure can be better understood. FIG. 1 shows a diagram of an exemplary sensing system that can incorporate the analyte sensors of the present disclosure, specifically, analyte sensors including an active region responsive to an inhibitor of diaphorase. As shown, sensing system 100 includes a sensor control device 102 and a reader device 120 configured to communicate with each other via a local communication path or link 140, which may be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. According to some embodiments, reader device 120 can configure an output medium for viewing analyte concentrations and alerts or notifications determined by sensor 104 or its associated processor, as well as for enabling one or more user inputs. Reader device 120 may be a general-purpose smartphone or a dedicated electronic reader instrument. While only one reader device 120 is shown, multiple reader devices 120 may be present in certain instances. Reader device 120 can communicate with remote terminal 170 and / or trusted computer system 180 via communication paths / links 141 and / or 142, respectively, which may also be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. Additionally or alternatively, reader device 120 may be in communication with network 150 (e.g., a cellular network, the Internet, or a cloud server) via communication path / link 151. Network 150 may further be communicatively coupled to remote terminal 170 via communication path / link 152 and / or to trusted computer system 180 via communication path / link 153. Alternatively, sensor 104 may communicate directly with remote terminal 170 and / or trusted computer system 180 without the presence of an intervening reader device 120.For example, sensor 104 may communicate with remote terminal 170 and / or trusted computer system 180 via a direct communication link to network 150, according to some embodiments, as described in U.S. Patent Application Publication No. 2011 / 0213225, the entire contents of which are incorporated herein by reference. Any suitable electronic communication protocol may be used for each communication path or link, such as near field communication (NFC), radio frequency identification (RFID), BLUETOOTH® or BLUETOOTH® Low Energy protocol, WiFi, etc. Remote terminal 170 and / or trusted computer system 180, according to some embodiments, may be accessible by individuals other than the primary user who are interested in the user's analyte levels. Reader device 120 may include a display 122 and optional input components 121. Display 122, according to some embodiments, may include a touchscreen interface.

[0013] The sensor control unit 102 includes a sensor housing 103 that may house circuitry and a power source for operating the sensor 104. Optionally, the power source and / or active circuitry may be omitted. A processor (not shown) may be communicatively coupled to the sensor 104, the processor being physically located within the sensor housing 103 or the reader device 120. The sensor 104 protrudes from an underside of the sensor housing 103 and extends through an adhesive layer 105, which, according to some embodiments, is adapted to adhere the sensor housing 103 to a tissue surface, such as skin.

[0014] The sensor 104 is adapted to be at least partially inserted into a target tissue, such as into the dermal or subcutaneous layer of the skin. The sensor 104 may include a sensor tail of sufficient length for insertion to a desired depth in a given tissue. The sensor tail includes at least one working electrode and an active region containing an enzyme system responsive to an inhibitor of diaphorase to facilitate detection of coumarin-based drugs and other inhibitors of diaphorase. Additional active regions may also be present to facilitate detection of one or more additional analytes, as specified in more detail herein. A counter electrode may be present in combination with the at least one working electrode, and optionally in further combination with a reference electrode. Specific electrode configurations on the sensor tail are described in more detail below with reference to Figures 3A-7C.

[0015] Active regions responsive to additional analytes may similarly feature an appropriate enzyme or enzyme system to facilitate detection of the additional analyte. When the active region responsive to another analyte is a glucose-responsive active region, for example, the glucose-responsive active region may include a glucose-responsive enzyme. Active regions responsive to other analytes may include regions responsive to, for example, ketone, lactate, creatinine, pH, etc., and may feature other enzymes or enzyme systems suitable for assaying these analytes. Suitable enzyme systems for detecting these analytes are further described below, particularly with reference to Figures 2A-2C, 8A, 8B, and 9. According to various embodiments, one or more enzymes in the active region may be covalently attached to a polymer comprising the active region. Inhibitors of diaphorase and any additional analytes may be monitored in any biological fluid of interest, such as dermal fluid, interstitial fluid, plasma, blood, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, etc. In certain embodiments, the analyte sensors of the present disclosure may be adapted to assay dermal or interstitial fluid to measure the concentration of an inhibitor of diaphorase and / or an additional analyte in vivo.

[0016] One or more mass transport limiting membranes can cover the active area responsive to the diaphorase inhibitor and, if present, the active area responsive to another analyte. Analyte sensors often use membranes covering the active area to limit mass transport or improve biocompatibility. The mass transport limiting membrane may also be referred to herein as an analyte-permeable membrane. Restricting analyte access to the active area using a mass transport limiting membrane can help avoid sensor overload (saturation), thereby improving detection performance and accuracy. When a single analyte sensor is used to assay multiple analytes, different permeability values ​​may be exhibited by different analytes crossing a given mass transport limiting membrane, potentially resulting in different sensitivities for each analyte. Advantageously, the sensor structure can be used to incorporate different mass transport limiting membranes on each active area as needed to facilitate detection of multiple analytes. If a single mass transport limiting membrane provides sufficient permeability for both analytes, a simpler sensor structure can be used.

[0017] Referring again to FIG. 1 , the sensor 104 can automatically transfer data to the reader device 120. For example, analyte concentration data (i.e., coumarin-based drug concentration and / or glucose concentration, ketone concentration, lactate concentration, or creatinine concentration, or pH value) may be automatically and periodically communicated, such as at a specific frequency when the data is acquired or after a specific period of time has passed, with the data stored in memory until transmission (e.g., every minute, every five minutes, or other predetermined time period). In other embodiments, the sensor 104 can communicate with the reader device 120 in a non-automatic manner, not according to a set schedule. For example, data may be communicated from the sensor 104 using RFID technology when the sensor electronics are brought within communication range of the reader device 120. The data may remain stored in the memory of the sensor 104 until communicated to the reader device 120. Thus, a user need not maintain constant proximity to the reader device 120 but can instead upload data at a convenient time. In still other embodiments, a combination of automatic and non-automatic data transfer can be implemented. For example, data transfer may continue automatically until the reader device 120 is no longer within communication range of the sensor 104 .

[0018] An introducer may be temporarily present to facilitate the introduction of the sensor 104 into tissue. In an exemplary embodiment, the introducer may comprise a needle or similar sharp. It should be appreciated that other types of introducers, such as a sheath or blade, may be present in alternative embodiments. More specifically, the needle or other introducer may be temporarily present in proximity to the sensor 104 prior to tissue insertion and then withdrawn. While present, the needle or other introducer may facilitate the insertion of the sensor 104 into tissue by opening an access path for the sensor 104 to follow. For example, the needle, according to one or more embodiments, may facilitate penetration of the epidermis as an access path to the dermis to allow implantation of the sensor 104 to occur. After opening the access path, the needle or other introducer may be withdrawn so that it does not present a sharps hazard. In an exemplary embodiment, a suitable needle may be solid or hollow, beveled or non-beveled, and / or circular or non-circular in cross section. In more particular embodiments, suitable needles may be equivalent in cross-sectional diameter and / or tip design to acupuncture needles, which may have a cross-sectional diameter of about 250 microns. However, it should be recognized that suitable needles may have larger or smaller cross-sectional diameters as needed for a particular application.

[0019] In some embodiments, the tip of the needle (while present) may be angled beyond the end of the sensor 104 so that the needle penetrates the tissue first, opening an access path for the sensor 104. In other exemplary embodiments, the sensor 104 may reside within a lumen or groove in the needle, which similarly opens an access path for the sensor 104. In either case, the needle may be subsequently withdrawn after facilitating sensor insertion.

[0020] FIG. 2A shows an enzyme system configured for detecting ketones. Additional enzyme systems suitable for detecting ketones are shown in FIGS. 2B and 2C, which are further described below. In the enzyme system shown in FIG. 2A, β-hydroxybutyrate serves as a surrogate for ketones formed in vivo, which undergoes a reaction with an enzyme system including β-hydroxybutyrate dehydrogenase (HBDH) and diaphorase to facilitate ketone detection within a ketone-responsive active area disposed on the surface of at least one working electrode, as further described herein. Within the ketone-responsive active area, β-hydroxybutyrate dehydrogenase can convert β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD) to acetoacetate and reduced nicotinamide adenine dinucleotide (NADH), respectively. It should be understood that the term "nicotinamide adenine dinucleotide (NAD)" includes the phosphate-bound form of the aforementioned enzyme cofactor. That is, the use of the term "NAD" herein refers to both NAD+ phosphate and NADH phosphate, specifically the diphosphate linking two nucleotides, one containing an adenine nucleobase and the other containing a nicotinamide nucleobase. NAD+ and NADH enzyme cofactors help facilitate the cooperative enzymatic reactions disclosed herein. Once formed, NADH can undergo diaphorase-mediated oxidation, and the electrons transferred during this process provide the basis for ketone detection at the working electrode. Thus, there is a 1:1 molar correspondence between the amount of electrons transferred to the working electrode and the amount of converted β-hydroxybutyrate, thereby providing the basis for ketone detection and quantification based on the amount of current measured at the working electrode. Electron transfer to the working electrode can occur with the additional mediation of an electron transfer agent, such as an osmium (Os) compound or a similar transition metal complex, as described in more detail below. Albumin may also be present within the active region as a stabilizer. β-hydroxybutyrate dehydrogenase and diaphorase can be covalently attached to a polymer comprising a ketone-responsive active region.The NAD+ may or may not be covalently bound to a polymer, but if the NAD+ is not covalently bound, the NAD+ may be physically retained within the ketone-responsive active region, for example, by having a mass transport limiting membrane covering the ketone-responsive active region that is also permeable to ketones.

[0021] This disclosure demonstrates how the enzyme system shown in Figure 2A can be modified to respond to other analytes. Figures 3A and 3B show how the enzyme system of Figure 2A can be sequentially modified to become responsive to inhibitors of glucose and diaphorase, respectively. By replacing β-hydroxybutyrate dehydrogenase with NAD-dependent glucose dehydrogenase in the active region, as shown in Figure 3A, the analyte sensor becomes responsive to glucose, where gluconolactone is formed as a product of glucose oxidation. Diaphorase can facilitate the transfer of electrons between NAD and the electron transfer agent. An even simpler enzyme-based detection scheme for glucose is shown below in Figures 8A and 8B, in which glucose oxidase or FAD-dependent glucose dehydrogenase can transfer electrons to the electron transfer agent without the presence of additional enzymes.

[0022] The enzyme system shown in Figure 3A can provide a linear response to glucose, provided there is sufficient turnover of downstream enzymes and cofactors (diaphorase and NAD+ / NADH, respectively) to facilitate the transfer of all electrons generated during glucose oxidation to the working electrode. In the present disclosure, the enzyme system of Figure 3A can be further modified to make diaphorase rate-limiting for electron transfer to the working electrode. By making diaphorase rate-limiting, diaphorase acts as a "valve" to control the flow of electrons to the working electrode. If diaphorase is rate-limiting, a constant signal will be generated regardless of the glucose concentration upstream of diaphorase. However, if diaphorase is rate-limiting, inhibitors of diaphorase, such as coumarin-based drugs and other diaphorase inhibitors, can alter the flow of electrons to the working electrode and serve as a basis for inhibitor detection. More specifically, a decrease in electron flow to the working electrode can be correlated with the amount of inhibitor present. Furthermore, because glucose is ubiquitous in biological fluids, glucose can serve as a "fuel" to provide a constant flow of electrons to the rate-limiting diaphorase enzyme. Thus, Figure 3B shows an enzyme system configured to detect inhibitors of diaphorase that keeps these considerations in mind. Other NAD-dependent dehydrogenases can be used in place of the NAD-dependent glucose dehydrogenase in Figure 3B, provided that a ready source of substrate for the other NAD-dependent dehydrogenase is present in the biological fluid being analyzed.

[0023] Thus, the analyte sensor of the present disclosure may include a sensor tail comprising at least a first working electrode, a first active region disposed on the surface of the first working electrode, and an analyte-permeable membrane covering at least the first active region. The enzyme system includes nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), or any combination thereof; an NAD-dependent dehydrogenase; and diaphorase; and electron transfer from the first active region to the first working electrode is rate-limiting for diaphorase, such that the first active region responds to a diaphorase inhibitor, as described above. Optionally, the analyte-permeable membrane covering the first active region may be omitted if NAD or NADH can be appropriately retained within the first active region, for example, via physical attraction or covalent bonding to a polymer comprising the first active region.

[0024] In a particular embodiment, the NAD-dependent dehydrogenase may be an NAD-dependent glucose dehydrogenase, and glucose present in the fluid under analysis may provide the source of electrons to the working electrode. Other NAD-dependent dehydrogenases may be similarly utilized, provided that a ready source of substrate for the other NAD-dependent dehydrogenase is present in the fluid under analysis, particularly a biological fluid.

[0025] To make the analyte sensor responsive to a diaphorase inhibitor, the first active region may include a rate-limiting amount of diaphorase with respect to transferring electrons to the first working electrode, the diaphorase may be modified to be rate-limiting with respect to transferring electrons to the first working electrode, or any combination thereof. For example, wild-type diaphorase may be modified to have reduced activity.

[0026] As mentioned above, inhibitors of diaphorase that can be monitored using the analyte sensors disclosed herein include warfarin and dicoumarol. Other diaphorase inhibitors can be assayed using the analyte sensors disclosed herein, including, for example, N-methylmaleimide, diphenyleneiodonium, 5,6-dimethylxanthenone-4-acetic acid, flavone-8-acetic acid, dimethylbenzylalkammonium chloride, 7,8-dihydroxyflavone, chrysin, and any combination thereof.

[0027] The analyte sensors disclosed herein feature at least one active area responsive to an inhibitor of diaphorase on a working electrode in combination with at least one additional electrode, which may be a counter electrode, a reference electrode, and / or a counter / reference electrode. In some cases, additional working electrodes may be present. Analyte sensors featuring an active area responsive to an inhibitor of diaphorase in combination with an active area responsive to another analyte, such as glucose, are also contemplated by the present disclosure and are further discussed herein. Exemplary analyte sensor configurations adapted to assay one or more analytes are further discussed below.

[0028] Sensor configurations featuring an active region responsive to an inhibitor of diaphorase but not an active region responsive to another analyte may use two-electrode or three-electrode detection motifs, as further described herein with reference to Figures 4A-4C. Sensor configurations featuring both an active region responsive to an inhibitor of diaphorase and an active region responsive to another analyte, either on separate working electrodes or on the same working electrode, are described separately below with reference to Figures 5A-7C. Sensor configurations with multiple working electrodes may be particularly advantageous for analyte sensors that include active regions configured to monitor two or more different analytes within the same sensor tail, because the signal contribution from each active region can be more easily determined.

[0029] When a single working electrode is present in the analyte sensor, a three-electrode sensor configuration may include a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration may include a working electrode and a second electrode, where the second electrode can function as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). The various electrodes may be at least partially stacked (layered) on one another and / or laterally spaced apart on the sensor tail. Suitable sensor configurations may be substantially planar or substantially cylindrical in shape. In any of the sensor configurations disclosed herein, the various electrodes may be electrically insulated from one another by a dielectric material or similar insulator. An analyte sensor including an active region responsive to an inhibitor of diaphorase and an active region responsive to another analyte, such as glucose, may feature the active regions laterally spaced apart on the working electrode.

[0030] Analyte sensors featuring multiple working electrodes may also include at least one additional electrode. If one additional electrode is present, the one additional electrode may function as a counter / reference electrode for each of the multiple working electrodes. If two additional electrodes are present, one of the additional electrodes may function as a counter electrode for each of the multiple working electrodes, and the other of the additional electrodes may function as a reference electrode for each of the multiple working electrodes.

[0031] 4A shows a diagram of an exemplary two-electrode analyte sensor configuration suitable for use in the present disclosure. As shown, analyte sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter / reference electrode 216. Alternatively, working electrode 214 and counter / reference electrode 216 may be disposed on the same side of substrate 212 with a dielectric material between them (configuration not shown). An active region 218 responsive to a diaphorase inhibitor is disposed as at least one layer on at least a portion of working electrode 214. Active region 218 may include multiple spots or a single spot configured for detection of a diaphorase inhibitor, as discussed further herein.

[0032] 4A , according to some embodiments, membrane 220 can cover at least active area 218 and, optionally, part or all of working electrode 214 and / or counter / reference electrode 216, or the entire analyte sensor 200. One or both sides of analyte sensor 200 may be covered with membrane 220. Membrane 220 can include one or more polymeric membrane materials capable of limiting analyte flux to active area 218 (i.e., membrane 220 is a mass-transport-limiting membrane with some permeability to inhibitors of diaphorase). The composition and thickness of membrane 220 can be varied to promote a desired flux of diaphorase inhibitor to active area 218. In a non-limiting example, membrane 220 can be coated onto active area 218 by one or more of spray coating, dip coating, printing, and / or similar deposition techniques. The thickness of the membrane can be selected so that the current generated at working electrode 214 remains correlable to the amount of diaphorase inhibitor present. Analyte sensor 200 may be operable to assay inhibitors of diaphorase by any of the following electrochemical detection techniques: coulometric, amperometric, electrolytic, or potentiometric.

[0033] 4B and 4C show diagrams of exemplary three-electrode analyte sensor configurations, also suitable for use in the present disclosure. The three-electrode analyte sensor configuration can be similar to that shown for analyte sensor 200 in FIG. 4A, except for the inclusion of an additional electrode 217 within analyte sensors 201 and 202 (FIGS. 4B and 4C). With the additional electrode 217, counter / reference electrode 216 may then function as either a counter electrode or a reference electrode, with the additional electrode 217 performing other electrode functions not otherwise considered. Working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed on either working electrode 214 or electrode 216, with a separating layer of dielectric material sandwiched between them. For example, as shown in FIG. 4B, dielectric layers 219a, 219b, and 219c separate electrodes 214, 216, and 217 from one another and provide electrical insulation. Alternatively, at least one of electrodes 214, 216, and 217 may be disposed on opposite sides of substrate 212, as shown in FIG. 4C. Thus, in some embodiments, electrode 214 (working electrode) and electrode 216 (counter electrode) may be located on opposite sides of substrate 212, and electrode 217 (reference electrode) is located on one of electrodes 214 or 216 and spaced therefrom using a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, and the location of reference material layer 230 is not limited to the locations depicted in FIGS. 4B and 4C. Similar to sensor 200 shown in FIG. 4A, active area 218 in analyte sensors 201 and 202 may include multiple spots or a single spot. Analyte sensors 201 and 202 may similarly be operable to assay inhibitors of diaphorase by any of coulometric, amperometric, electrolytic, or potentiometric electrochemical detection techniques.

[0034] Similar to analyte sensor 200, membrane 220 may also cover active area 218 and other sensor components in analyte sensors 201 and 202, thereby functioning as a mass transport limiting membrane. Additional electrode 217 may be covered with membrane 220 in some embodiments. While FIGS. 4B and 4C show all of electrodes 214, 216, and 217 as being covered with membrane 220, it should be appreciated that in some embodiments, only working electrode 214 may be covered. Furthermore, the thickness of membrane 220 on each of electrodes 214, 216, and 217 may be the same or different. In non-limiting examples, membrane 220 may be coated onto active area 218 by one or more of spray coating, dip coating, printing, and / or similar deposition techniques. As in the two-electrode analyte sensor configuration (FIG. 4A), one or both faces of analyte sensors 201 and 202 may be covered with membrane 220 in the sensor configurations of FIGS. 4B and 4C, or the entire analyte sensors 201 and 202 may be covered. Thus, the three-electrode sensor configurations shown in FIGS. 4B and 4C should be understood as non-limiting of the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of the present disclosure.

[0035] Analyte sensors having both an active area responsive to an inhibitor of diaphorase and an active area responsive to another analyte, each located on a single working electrode or on multiple working electrodes, are described in further detail with reference to FIGS. 5A-7C.

[0036] FIG. 5A shows an exemplary configuration of a sensor 203 comprising a single working electrode having both an active region responsive to an inhibitor of diaphorase and an active region responsive to another analyte disposed thereon. FIG. 5A is similar to FIG. 4A except for the presence of two active regions on the working electrode 214: active region 218a (responsive to an inhibitor of diaphorase) and active region 218b (responsive to another analyte), which are laterally spaced apart from each other on the surface of the working electrode 214. Active regions 218a and 218b may include multiple spots or a single spot configured for detection of each analyte. The composition of membrane 220 may be different in active regions 218a and 218b or may be compositionally the same. For example, if the membrane 220 is compositionally different between the active areas 218a and 218b, a single membrane polymer may be present in one of the active areas (e.g., active area 218b) and a bilayer of membrane polymers or a mixture of membrane polymers may be present in the other active area (e.g., active area 218a). One or more of spray coating, dip coating, printing, and / or similar deposition techniques may be used to deposit compositionally homogeneous or compositionally different membranes 220 in the active areas 218a and 218b. The first active area 218a and the second active area 218b may be configured to detect their corresponding analytes at different working electrode potentials, as discussed further below.

[0037] Figures 5B and 5C show cross-sectional views of exemplary three-electrode sensor configurations for sensors 204 and 205, respectively, each featuring a single working electrode with both active area 218a (responsive to an inhibitor of diaphorase) and active area 218b (responsive to another analyte) disposed thereon. Figures 5B and 5C are otherwise similar to and may be better understood by reference to Figures 4B and 4C, respectively. As with Figure 5A, the composition of membrane 220 may be compositionally the same or different in active areas 218a and 218b.

[0038] Exemplary sensor configurations including multiple working electrodes, specifically two working electrodes, are described in further detail with reference to Figures 6-7C. While the following description is primarily directed to sensor configurations including two working electrodes, it should be understood that more than two working electrodes may be incorporated through extensions of the disclosure herein. Additional working electrodes can be used to provide the analyte sensor with additional sensing capabilities beyond just sensing an inhibitor of diaphorase and one additional analyte. That is, an analyte sensor including more than two working electrodes may be suitable for detecting a corresponding number of additional analytes.

[0039] 6 shows a cross-sectional view of an exemplary analyte sensor configuration including two working electrodes, a reference electrode, and a counter electrode suitable for use in the present disclosure. As shown, analyte sensor 300 includes working electrodes 304 and 306 disposed on opposite sides of substrate 302. Active area 310a (responsive to an inhibitor of diaphorase) is disposed on the surface of working electrode 304, and active area 310b (responsive to another analyte) is disposed on the surface of working electrode 306. Counter electrode 320 is electrically insulated from working electrode 304 by dielectric layer 322, and reference electrode 321 is electrically insulated from working electrode 306 by dielectric layer 323. Outer dielectric layers 330 and 332 are disposed on reference electrode 321 and counter electrode 320, respectively. Membrane 340, according to various embodiments, can cover at least active areas 310a and 310b, and optionally other components of analyte sensor 300 or the entire analyte sensor 300 as well. Again, membrane 340 can be compositionally the same or compositionally different in active areas 310a and 310b, as needed, to regulate analyte flux at each location. Compositional differences can include, for example, a mixture of multiple membrane polymers or bilayers of multiple membrane polymers.

[0040] Alternative sensor configurations having multiple working electrodes and differing from the configuration shown in Figure 6 may feature counter / reference electrodes instead of separate counter and reference electrodes 320, 321, and / or may feature layer and / or film arrangements different from those explicitly depicted. For example, the positioning of counter electrode 320 and reference electrode 321 may be reversed from that shown in Figure 6. Furthermore, working electrodes 304 and 306 do not necessarily need to be on opposite sides of substrate 302 in the manner shown in Figure 6.

[0041] While preferred sensor configurations may feature electrodes that are substantially planar in nature, it should be understood that sensor configurations featuring non-planar electrodes may be advantageous and particularly suitable for use in the present disclosure. In particular, substantially cylindrical electrodes arranged concentrically relative to one another may facilitate the deposition of mass transport limiting films with different compositions in two distinct active regions, as described below. Figures 7A-7C show perspective views of an analyte sensor featuring two working electrodes arranged concentrically relative to one another. It should be understood that sensor configurations with a concentric electrode arrangement but lacking a second working electrode are also possible in the present disclosure.

[0042] 7A shows a perspective view of an exemplary sensor configuration in which multiple electrodes are substantially cylindrical and concentrically arranged around a central substrate. As shown, the analyte sensor 400 includes a central substrate 402 around which all of the electrodes and dielectric layers are concentrically arranged. In particular, a working electrode 410 is disposed on a surface of the central substrate 402, with a dielectric layer 412 disposed on a portion of the working electrode 410 distal to the sensor tip 404. A working electrode 420 is disposed on the dielectric layer 412, with a dielectric layer 422 disposed on a portion of the working electrode 420 distal to the sensor tip 404. A counter electrode 430 is disposed on the dielectric layer 422, with a dielectric layer 432 disposed on a portion of the counter electrode 430 distal to the sensor tip 404. A reference electrode 440 is disposed on the dielectric layer 432, with a dielectric layer 442 disposed on a portion of the reference electrode 440 distal to the sensor tip 404. Thus, the exposed surfaces of the working electrode 410, working electrode 420, counter electrode 430, and reference electrode 440 are spaced apart from one another along the longitudinal axis B of the analyte sensor 400. The surface areas of the working electrode 410, working electrode 420, counter electrode 430, and reference electrode 440 gradually increase in size with distance from the sensor tip 404.

[0043] 7A , active area 414a (responsive to an inhibitor of diaphorase) and active area 414b (responsive to another analyte) are disposed on the exposed surfaces of working electrodes 410 and 420, respectively, so that they can come into contact with a fluid for sensing both analytes. While active areas 414a and 414b are shown as three separate spots in FIG. 7A , it should be understood that in alternative sensor configurations, there may be fewer or more than three spots. Furthermore, the positioning of active area 414a and active area 414b may be reversed from that shown in FIG. 7A .

[0044] In Figure 7A, sensor 400 is partially coated with a film 450 over working electrodes 410 and 420 and active areas 414a and 414b disposed thereon. Figure 7B shows an alternative sensor configuration in which substantially the entire sensor 401 is covered with film 450. Film 450 may be compositionally the same or different in active areas 414a and 414b. Dip-coating techniques may be particularly desirable for applying films in substantially cylindrical sensor configurations.

[0045] It should further be understood that the arrangement of the various electrodes in FIGS. 7A and 7B may differ from that explicitly shown. For example, the positions of the counter electrode 430 and reference electrode 440 may be reversed from the configuration shown in FIGS. 7A and 7B. Similarly, the positions of the working electrodes 410 and 420 are not limited to those explicitly depicted in FIGS. 7A and 7B. FIG. 7C illustrates an alternative sensor configuration to that shown in FIG. 7B, in which the sensor 405 includes the counter electrode 430 and reference electrode 440 located more proximally to the sensor tip 404 and the working electrodes 410 and 420 located more distally to the sensor tip 404. A sensor configuration in which the working electrodes 410 and 420 are located more distally to the sensor tip 404 may be advantageous by providing a larger surface area for deposition of active regions 414a and 414b (five separate sensing spots illustratively shown for each in FIG. 7C), thereby facilitating increased signal strength in some cases.

[0046] 7A-7C each show a sensor configuration supported on a central substrate 402, it should be understood that alternative sensor configurations may instead be electrode-supported or may lack a central substrate 402 (configurations not shown). In particular, the innermost concentric electrode may be utilized to support the other electrodes and dielectric layers. For example, the counter electrode 430 may be the innermost concentric electrode and may be used to position the reference electrode 440, the working electrodes 410 and 420, and the dielectric layers 432, 442, 412, and 422 thereon. It should again be understood that other electrode and dielectric layer configurations may be employed in sensor configurations lacking a central substrate 402, in light of the disclosure herein.

[0047] Thus, the analyte sensors of the present disclosure may further include an active region responsive to an analyte other than the diaphorase inhibitor, also disposed on the sensor tail. Thus, the analyte sensors of the present disclosure may be configured to analyze multiple analytes in certain embodiments. Other analytes that may be monitored in addition to the diaphorase inhibitor include, for example, glucose, ketones, lactate, creatinine, pH, or any combination thereof. Suitable enzymes, enzyme systems, or similar detection protocols for assaying these additional analytes in the analyte sensors are discussed further below.

[0048] In some embodiments, the analyte sensor may further include a glucose-responsive active region comprising a glucose-responsive enzyme disposed on the sensor tail. Suitable glucose-responsive enzymes include, for example, glucose oxidase or glucose dehydrogenase (e.g., pyrroloquinoline quinone (PQQ) or cofactor-dependent glucose dehydrogenases, such as flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase or nicotinamide adenine dinucleotide (NAD)-dependent glucose dehydrogenase). Glucose oxidase and glucose dehydrogenase are distinguished by their ability to utilize oxygen as an electron acceptor in oxidizing glucose; glucose oxidase can utilize oxygen as an electron acceptor, while glucose dehydrogenase transfers electrons to a natural or artificial electron acceptor, such as an enzyme cofactor. Exemplary enzyme-based detection schemes for analyzing glucose are further illustrated in Figures 3A, 8A, and 8B, which utilize glucose oxidase or glucose dehydrogenase to facilitate detection. Both glucose oxidase and glucose dehydrogenase can be covalently attached to a polymer containing a glucose-responsive active region and can exchange electrons with an electron transfer agent (e.g., an osmium (Os) complex or similar transition metal complex), which can also be covalently attached to the polymer. Suitable electron transfer agents are described in more detail below. Simultaneous detection of diaphorase inhibitors and glucose may be particularly desirable because dicoumarol and other coumarin-based drugs tend to affect the activity of certain diabetes medications. Therefore, an analyte sensor capable of analyzing both diaphorase inhibitors and glucose could facilitate treatment decisions and potentially improve patient outcomes. Considerations for detecting a second analyte, such as glucose, in combination with an inhibitor or diaphorase are provided below.

[0049] In some embodiments, the analyte sensor can further comprise a ketone-responsive active region including an enzyme system that works in concert to facilitate the detection of ketones. Suitable enzyme systems for facilitating the detection of ketones are described above with reference to FIG. 2A. Additional enzyme systems that may work in concert to facilitate the detection of ketones are shown in FIGS. 2B and 2C. In FIGS. 2B and 2C, there is also a 1:1 molar correspondence between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted, thereby providing the basis for ketone detection. Further details regarding ketone-responsive enzyme systems can be found in commonly owned U.S. Patent Application No. 16 / 774,835, entitled "Analyte Sensors and Sensing Methods Features Dual Detection of Glucose and Ketones," filed January 28, 2020, and published as U.S. Patent Application Publication No. 2020 / 0237275, which is incorporated herein by reference in its entirety.

[0050] As shown in Figure 2B, β-hydroxybutyrate dehydrogenase (HBDH) can again convert β-hydroxybutyrate and NAD+ to acetoacetate and NADH, respectively. Instead of electron transfer to the working electrode being completed by diaphorase (see Figure 2A) and a transition metal electron transfer agent, the reduced form of NADH oxidase (NADHOx(Red)) undergoes a reaction to form the corresponding oxidized form (NADHOx(Ox)). NADHOx(Red) can then be reformed through reaction with molecular oxygen to produce superoxide, which can undergo subsequent conversion to hydrogen peroxide under superoxide dismutase (SOD)-mediated conditions. The hydrogen peroxide can then undergo oxidation at the working electrode, providing a signal that can be correlated to the amount of ketone originally present. SOD can be covalently attached to a polymer in the ketone-responsive active region according to various embodiments. Similar to the enzyme system shown in Figure 2A, β-hydroxybutyrate dehydrogenase and NADH oxidase may be covalently bound to a polymer in the ketone-responsive active region, and NAD / NADH may or may not be covalently bound to a polymer in the ketone-responsive active region. If NAD is not covalently bound, it may be physically retained within the ketone-responsive active region, for example, using a membrane polymer covering the ketone-responsive active region.

[0051] As shown in Figure 2C, another enzymatic detection chemistry for ketones utilizes β-hydroxybutyrate dehydrogenase (HBDH) to convert β-hydroxybutyrate and NAD+ to acetoacetate and NADH, respectively. The electron transfer cycle in this case is completed by oxidation of NADH by 1,10-phenanthroline-5,6-dione to reform NAD+, which then transfers the electrons to the working electrode. The 1,10-phenanthroline-5,6-dione may or may not be covalently bound to a polymer within the ketone-responsive active region. Similar to the enzyme system shown in Figure 2A, β-hydroxybutyrate dehydrogenase may be covalently bound to a polymer within the ketone-responsive active region, and NAD+ / NADH may or may not be covalently bound to the polymer. The inclusion of albumin in the ketone-responsive active region can provide a surprising improvement in response stability. An appropriate membrane polymer can promote the retention of NAD+ within the ketone-responsive active region.

[0052] Simultaneous detection of diaphorase inhibitors and ketones may be particularly desirable due to the prevalence of diabetic patients experiencing ketoacidosis. Therefore, an analyte sensor capable of analyzing both diaphorase inhibitors and ketones could facilitate treatment decisions and potentially improve outcomes for such individuals. In addition to providing health benefits to diabetic patients, an analyte sensor featuring detection capabilities for both diaphorase inhibitors and ketones could be beneficial for other individuals who wish to monitor ketone levels, such as those following a ketogenic diet. A ketogenic diet may be beneficial for promoting weight loss and helping epilepsy patients manage their condition. Coumarin-based drugs may sometimes be used by such individuals in response to heart health concerns.

[0053] In some embodiments, the analyte sensor can further include a creatinine-responsive active region that includes an enzyme system that operates in concert to facilitate the detection of creatinine. A suitable enzyme system that can be used to detect creatinine in the analyte sensors disclosed herein is shown in FIG. 9 and described in further detail below. Additional details regarding creatinine-responsive enzyme systems can be found in commonly owned U.S. Patent Application No. 16 / 582,583, entitled "Analyte Sensors and Sensing Methods for Detecting Creatine," filed September 25, 2019, and published as U.S. Patent Application Publication No. 2020 / 0241015, which is incorporated herein by reference in its entirety.

[0054] As shown in Figure 9, creatinine can react reversibly hydrolytically in the presence of creatinine amidohydrolase (CNH) to form creatine. Creatine can then undergo catalytic hydrolysis in the presence of creatine amidohydrolase (CRH) to form sarcosine. Neither of these reactions produces an electron flow (e.g., oxidation or reduction) that provides the basis for electrochemical detection of creatinine.

[0055] Continuing with FIG. 9 , sarcosine produced via creatine hydrolysis can undergo oxidation in the presence of oxidized sarcosine oxidase (SOX(Ox)) to form glycine and formaldehyde, thereby generating reduced sarcosine oxidase (SOX(Red)) in the process. Hydrogen peroxide can also be generated in the presence of oxygen. The reduced form of sarcosine oxidase can then undergo reoxidation in the presence of oxidized form of an electron transfer agent (e.g., an Os(III) complex), thereby generating the corresponding reduced form of the electron transfer agent (e.g., an Os(II) complex) and delivering electron flow to the working electrode.

[0056] Oxygen can inhibit the concerted reaction sequence used to detect creatinine according to the above disclosure. Specifically, the reduced form of sarcosine oxidase can undergo a reaction with oxygen to reform the corresponding oxidized form of the enzyme, but without exchanging electrons with the electron transfer agent. When the reaction with oxygen occurs, all of the enzymes remain activated, but electrons do not flow to the working electrode. Without being bound by theory or mechanism, it is believed that the competitive reaction with oxygen results from a kinetic effect. That is, it is believed that the oxidation of the reduced form of sarcosine oxidase by oxygen occurs faster than the oxidation promoted by the electron transfer agent. Hydrogen peroxide is also formed in the presence of oxygen.

[0057] The desired reaction pathway for facilitating creatinine detection, as shown in Figure 9, can be facilitated by including an oxygen scavenger in close proximity to the enzyme system. Various oxygen scavengers and their placement may be suitable, including oxidase enzymes such as glucose oxidase. Small molecule oxygen scavengers may also be suitable, but they may be completely consumed before the sensor's lifetime is fully exhausted. In contrast, enzymes can undergo reversible oxidation and reduction, thereby resulting in longer sensor lifetime. By inhibiting the oxidation of the reduced form of sarcosine oxidase by oxygen, a slower electron exchange reaction with the electron transfer agent can occur, thereby enabling the generation of a current at the working electrode. The magnitude of the current generated is proportional to the amount of creatinine initially reacted.

[0058] The oxygen scavenger used to promote the desired reaction pathway in Figure 9 can be an oxidase enzyme in any embodiment of the present disclosure. Any oxidase enzyme can be used to promote oxygen removal in proximity to the enzyme system, provided that a suitable substrate for the enzyme is also present, thereby providing a reagent for reacting with oxygen in the presence of the oxidase enzyme. Oxidase enzymes that may be suitable for oxygen removal in the present disclosure include, but are not limited to, glucose oxidase, lactate oxidase, xanthine oxidase, and the like. Glucose oxidase may be a particularly desirable oxidase enzyme for promoting oxygen removal due to the ready availability of glucose in various biological fluids. Reaction 1 below illustrates the enzymatic reaction promoted by glucose oxidase to result in oxygen removal.

[0059]

number

[0060] Although the concentration of available lactate in vivo is lower than that of glucose, it is still sufficient to facilitate oxygen removal. An oxidase enzyme, such as glucose oxidase, may be located in any location suitable for facilitating oxygen removal in the analyte sensors disclosed herein. For example, glucose oxidase may be located on the sensor tail such that the glucose oxidase is functional and / or non-functional to facilitate glucose detection. When non-functional to facilitate glucose detection, the glucose oxidase may be located on the sensor tail such that electrons generated during glucose oxidation are unable to reach the working electrode, such as by electrically isolating the glucose oxidase from the working electrode.

[0061] Simultaneous detection of diaphorase and creatinine inhibitors may be particularly desirable due to the prevalence of diabetic patients experiencing diabetic neuropathy. For example, diabetic neuropathy can result from high blood glucose levels and lead to end-stage renal failure. Diabetic neuropathy is the leading cause of renal failure in the United States and is experienced by a significant number of diabetic patients within 10 to 20 years of the onset of the disease. Creatinine levels may be a particularly interesting analyte for monitoring an individual's susceptibility to renal failure, especially due to diabetic neuropathy. Therefore, an analyte sensor capable of analyzing both diaphorase inhibitors and creatinine could facilitate treatment decisions and potentially improve treatment outcomes for such individuals. Coumarin-based drugs may sometimes be used by individuals who are also concerned about potential renal failure.

[0062] In some embodiments, the analyte sensor may further comprise a lactate-responsive active region comprising a lactate-responsive enzyme disposed on the sensor tail. Suitable lactate-responsive enzymes may include, for example, lactate oxidase. Lactate oxidase or other lactate-responsive enzymes may be covalently attached to a polymer comprising the lactate-responsive active region and exchange electrons with an electron transfer agent (e.g., an osmium (Os) complex or similar transition metal complex), which may also be covalently attached to the polymer. Suitable electron transfer agents are described in further detail below. As described in further detail in commonly owned U.S. Patent Application Publication No. 2019 / 0320947, which is incorporated herein by reference in its entirety, albumin, such as human serum albumin, may be present in the lactate-responsive active region to stabilize the sensor response. Lactate levels may change in response to numerous environmental or physiological factors, including, for example, feeding, stress, exercise, sepsis or septic shock, infection, hypoxia, the presence of cancerous tissue, etc.

[0063] In some embodiments, the analyte sensor may further comprise an active area responsive to pH. A suitable analyte sensor configured to determine pH is described in commonly assigned U.S. Patent Application Publication No. 2020 / 0060592, incorporated herein by reference in its entirety. Such an analyte sensor may comprise a sensor tail comprising a first working electrode and a second working electrode, wherein the first active area located on the first working electrode comprises a material having a pH-dependent redox chemistry, and the second active area located on the second working electrode comprises a material having a redox chemistry that is substantially invariant to pH. By obtaining the difference between the first signal and the second signal, this difference can be correlated to the pH of the fluid to which the analyte sensor is exposed.

[0064] Thus, some embodiments of the analyte sensors disclosed herein can include a sensor tail including at least a first working electrode, a first active region containing an enzyme system responsive to a diaphorase inhibitor, and a second active region responsive to another analyte, such as a glucose-responsive active region, a lactate-responsive active region, a ketone-responsive active region, a creatinine-responsive active region, or a pH-responsive active region. The first active region responsive to the diaphorase inhibitor and the other active regions can be disposed on the surface of the first working electrode and spaced apart from one another. Each active region can have a redox potential, and the redox potential of the first active region responsive to the diaphorase inhibitor can be sufficiently separated from the redox potential of the second active region to allow independent generation of a signal from one of the active regions. As non-limiting examples, the redox potentials can differ by at least about 100 mV, or at least about 150 mV, or at least about 200 mV. The upper limit of the separation between the redox potentials is determined by the in vivo operating electrochemical window. The redox potentials of the two active regions are sufficiently separated from one another that an electrochemical reaction can occur in one of the two active regions (i.e., the first active region or the second active region) without substantially inducing an electrochemical reaction in the other active region. Thus, a signal from one of the first or second active regions can be independently generated at or above its corresponding redox potential (the lower redox potential) but below the redox potential of the other active region. The differential signal can allow the signal contribution from each analyte to be resolved.

[0065] Some or other embodiments of the analyte sensors disclosed herein may feature an active region responsive to a diaphorase inhibitor and an active region responsive to a different analyte located on the surface of a different working electrode. Such analyte sensors may include at least a first working electrode and a second working electrode, and a sensor tail including an active region responsive to a diaphorase inhibitor located on the surface of the first working electrode and a second active region responsive to a different analyte located on the surface of the second working electrode. A membrane may cover at least one of the first and second active regions. The membrane may be a mass-transport-limiting membrane or may include a multi-component membrane in which the membrane covers at least one of the active regions. The multi-component membrane may include a bilayer of two different membrane polymers or a mixture of two different membrane polymers, with one membrane polymer covering the other active region.

[0066] Electron transfer agents may be present in any of the active regions disclosed herein, particularly in the active region responsive to a diaphorase inhibitor and, if present, in the active region responsive to another analyte. A suitable electron transfer agent can facilitate the transport of electrons to an adjacent working electrode after one or more analytes undergo an enzymatic redox reaction in the corresponding active region, thereby generating an electron current indicative of the presence of that particular analyte. The amount of current generated is proportional to the amount of analyte present. Depending on the sensor configuration used, the electron transfer agents in the active region responsive to a diaphorase inhibitor and the active region responsive to another analyte may be the same or different. For example, if two different active regions are disposed on the same working electrode, the electron transfer agents in each active region may be different (e.g., chemically distinct so that the electron transfer agents exhibit different redox potentials). If multiple working electrodes are present, the electron transfer agents in each active region may be the same or different, since each working electrode can be interrogated separately.

[0067] Suitable electron transfer agents may include electroreducible and electrooxidizable ions, complexes, or molecules (e.g., quinones) with redox potentials several hundred millivolts higher or lower than the redox potential of a standard calomel electrode (SCE). According to some embodiments, suitable electron transfer agents may include low-potential osmium complexes, such as those described in U.S. Pat. Nos. 6,134,461 and 6,605,220, the disclosures of which are incorporated herein by reference in their entireties. Further examples of suitable electron transfer agents include those described in U.S. Pat. Nos. 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are incorporated herein by reference in their entireties. Other suitable electron transfer agents include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt, including, for example, metallocene compounds thereof. Suitable ligands for metal complexes can also include bidentate or higher dentate ligands, such as, for example, bipyridine, biimidazole, phenanthroline, or pyridyl (imidazole). Other suitable bidentate ligands can include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher dentate ligands can be present in the metal complex to achieve a complete coordination sphere.

[0068] Active regions suitable for detecting any of the analytes disclosed herein can include a polymer to which an electron transfer agent is covalently attached. Any of the electron transfer agents disclosed herein may include suitable functional groups to facilitate covalent attachment to the polymer in the active region. Suitable examples of polymer-bound electron transfer agents include those described in U.S. Pat. Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are incorporated herein by reference in their entireties. Suitable polymers for inclusion in the active region include, but are not limited to, polyvinylpyridine (e.g., poly(4-vinylpyridine)), polyvinylimidazole (e.g., poly(1-vinylimidazole)), or any copolymers thereof. Exemplary copolymers that may be suitable for inclusion in the active region include those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile. The polymers in each active region can be the same or different.

[0069] In certain embodiments of the present disclosure, the mass transport limiting membrane covering at least one of the active regions can include a cross-linked polyvinylpyridine homopolymer or copolymer. When a mass transport limiting membrane covers each active region, the mass transport limiting membranes can have the same or different compositions. When the membrane compositions are different, the membrane can include a bilayer membrane or a homogeneous mixture of two different membrane polymers, one of which can be a cross-linked polyvinylpyridine homopolymer or copolymer. Suitable techniques for depositing the mass transport limiting membrane on the active region can include, for example, spray coating, painting, inkjet printing, stenciling, roller coating, dip coating, etc., and any combination thereof.

[0070] Covalent attachment of the electron transfer agent to the polymer comprising the active region can occur by polymerizing a monomer unit having a covalently attached electron transfer agent, or the electron transfer agent can be reacted separately with the polymer after the polymer has already been synthesized. A bifunctional spacer can covalently attach the electron transfer agent to the polymer within the active region, with one functional group reactive with the polymer (e.g., a functional group capable of quaternizing the pyridine nitrogen atom or imidazole nitrogen atom) and a second functional group reactive with the electron transfer agent (e.g., a functional group reactive with a ligand that coordinates to a metal ion).

[0071] Similarly, one or more enzymes within an active region may be covalently bound to the polymer comprising the active region. When an enzyme system comprising multiple enzymes is present in a given active region, in some embodiments, all of the enzymes may be covalently bound to the polymer, while in other embodiments, only a portion of the enzymes may be covalently bound to the polymer. For example, one or more enzymes comprising the enzyme system may be covalently bound to the polymer, or at least one enzyme may be noncovalently bound to the polymer, such that the noncovalently bound enzyme is physically entrapped within the polymer. Covalent binding of an enzyme to a polymer in a given active region may occur via a cross-linker introduced with an appropriate cross-linking agent. Suitable cross-linkers for reaction with free amino groups in enzymes (e.g., free side-chain amines in lysines) may include, for example, polyethylene glycol diglycidyl ether (PEFDGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatized variants thereof. Suitable cross-linkers for reaction with free carboxylic acid groups in enzymes may include, for example, carbodiimides. Cross-linking of the enzyme to the polymer is usually intermolecular, but in some embodiments can be intramolecular, hi certain embodiments, all of the enzymes within a given active region can be covalently bound to the polymer.

[0072] The electron transfer agent and / or enzyme may also be associated with the polymer in the active region through means other than covalent bonding. In some embodiments, the electron transfer agent and / or enzyme may be ionically or coordinately bonded to the polymer. For example, a charged polymer may be ionically associated with an oppositely charged electron transfer agent or enzyme. In yet other embodiments, the electron transfer agent and / or enzyme may be physically entrapped within the polymer without being bound to the polymer. The physically entrapped electron transfer agent and / or enzyme may still adequately interact with the fluid to facilitate analyte detection without substantially leaching from the active region.

[0073] The polymer in the active region can be selected to limit the outward diffusion of NAD+ or another cofactor that is not covalently bound to the polymer. The limited outward diffusion of the cofactor can facilitate a reasonable sensor lifetime (days to weeks) while still allowing sufficient inward analyte diffusion to facilitate detection.

[0074] The active area in the analyte sensors disclosed herein may comprise one or more discrete spots (e.g., 1 to about 10 spots, or even more discrete spots), which may range in size from about 0.01 mm to about 1 mm, although larger or smaller individual spots within the active area are also contemplated herein. Active areas defined as a continuous band around a cylindrical electrode are also possible in the present disclosure. When an active area responsive to an inhibitor of diaphorase and an active area responsive to a different analyte are present, the number and / or size of the individual spots may be the same or different.

[0075] It should also be understood that the sensitivity (output current) of the analyte sensor to each analyte can be altered by varying the coverage (area or size) of the active regions, the area ratio between the active regions, and the identity, thickness, and / or composition of the mass transport limiting membrane covering the active regions. Once given the benefit of the disclosure herein, alterations to these parameters can be readily made by one of ordinary skill in the art.

[0076] In more specific embodiments, the analyte sensors of the present disclosure may include a sensor tail configured for insertion into tissue. Suitable tissues are not considered to be particularly limited and are discussed in more detail above. Similarly, considerations for deploying the sensor tail to a specific location within a given tissue, such as the dermis layer of skin, are addressed above.

[0077] A detection method for assaying an inhibitor of diaphorase may include exposing an analyte sensor to a fluid containing a substrate for an NAD-dependent dehydrogenase and an inhibitor of diaphorase, the analyte sensor including at least a first working electrode and a first active region disposed on a surface of the first working electrode, the first active region including an electron transfer agent and an enzyme system including NAD+, NADH, or any combination thereof; an NAD-dependent dehydrogenase; and diaphorase; and a sensor tail including an analyte-permeable membrane covering at least the first active region, wherein electron transfer from the first active region to the first working electrode is rate-limiting for diaphorase such that the first active region is responsive to the inhibitor; applying a potential to the first working electrode; obtaining a first signal at or above the redox potential of the first active region that is proportional to the concentration of the inhibitor in the fluid; and correlating the first signal to the concentration of the inhibitor in the fluid. Optionally, the analyte-permeable membrane covering the first active region can be omitted if NAD or NADH can be adequately retained within the first active region, for example, via physical attraction or covalent bonding to the polymer comprising the first active region. Any inhibitor of diaphorase can be assayed using the analyte sensors disclosed herein, including those identified above. Electron transfer to the first working electrode can be rate-limiting for diaphorase in any suitable manner described above.

[0078] In certain instances, the NAD-dependent dehydrogenase may be an NAD-dependent glucose dehydrogenase and the substrate is glucose. Because glucose is readily present in biological fluids, this substrate / dehydrogenase combination may be particularly advantageous for providing a supply of electrons to facilitate the detection of inhibitors of diaphorase.

[0079] In some embodiments, the first signal can be correlated to the corresponding concentration of the diaphorase inhibitor by referencing a lookup table or calibration curve. A lookup table for a particular inhibitor can be entered by assaying multiple samples with known inhibitor concentrations and recording the sensor response at each concentration. Similarly, a calibration curve for an inhibitor can be determined by plotting the analyte sensor response as a function of inhibitor concentration and determining a suitable calibration function (e.g., by regression, particularly linear regression) over the calibration range.

[0080] The processor can determine which sensor response value in the lookup table is closest to that measured for the sample with the unknown analyte concentration and then report the analyte concentration accordingly. In some or other embodiments, if the sensor response value for the sample with the unknown analyte concentration falls between the recorded values ​​in the lookup table, the processor can estimate the analyte concentration by interpolating between the two lookup table values. The interpolation can assume a linear concentration variation between the two values ​​reported in the lookup table. Interpolation can be used if the sensor response differs by a sufficient amount from a given value in the lookup table, such as by a variation of about 10% or more.

[0081] Similarly, according to some or various other embodiments, the processor can input the sensor response values ​​for samples with unknown analyte concentrations into a corresponding calibration function, and the processor can then report the analyte concentration accordingly.

[0082] The sensor tail can further include a second working electrode having an active region thereon that responds to a different analyte than the inhibitor, such as a glucose-responsive active region. Thus, the method can further include obtaining a second signal at or above the redox potential of the glucose-responsive active region that is proportional to the glucose concentration in the fluid, and correlating the second signal to the glucose concentration in the fluid. Other analytes can be similarly analyzed by using appropriate active regions and applied potentials.

[0083] According to more specific embodiments, the first signal and the second signal may be measured at different times. Thus, in such embodiments, a potential may be applied alternately to the first and second working electrodes. In other specific embodiments, the first and second signals may be measured simultaneously via the first and second channels, in which case a potential may be applied simultaneously to both working electrodes. In either case, the signal associated with each active region may then be correlated with the concentration of an inhibitor of diaphorase and another analyte, such as glucose or a similar analyte, using a lookup table or calibration function in a manner similar to that described above.

[0084] Embodiments disclosed herein include: A. Analyte Sensor Responsive to Diaphorase Inhibitors The analyte sensor includes a sensor tail including at least a first working electrode and a first active region disposed on the surface of the first working electrode, the first active region including an electron transfer agent and an enzyme system including nicotinamide adenine dinucleotide (NAD), reduced NAD, or any combination thereof, an NAD-dependent dehydrogenase, and diaphorase, wherein electron transfer from the first active region to the first working electrode is rate-limiting for diaphorase such that the first active region responds to diaphorase inhibitors.

[0085] B. A method for assaying an inhibitor of diaphorase, the method comprising: exposing an analyte sensor to a fluid containing a substrate for nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase and an inhibitor of diaphorase, the analyte sensor comprising a sensor tail including at least a first working electrode and a first active region disposed on a surface of the first working electrode, the first active region comprising an electron transfer agent and an enzyme system including NAD, reduced NAD, or any combination thereof, an NAD-dependent dehydrogenase, wherein electron transfer from the first active region to the first working electrode is rate-limiting for diaphorase such that the first active region is responsive to the inhibitor; applying a potential to the first working electrode; obtaining a first signal at or above the redox potential of the first active region that is proportional to the concentration of the inhibitor in the fluid; and correlating the first signal to the concentration of the inhibitor in the fluid.

[0086] Embodiment A may have one or more of the following additional elements in any combination: Element 1: The NAD-dependent dehydrogenase, which is an NAD-dependent glucose dehydrogenase.

[0087] Element 2: the first active region comprising a rate-limiting amount of diaphorase with respect to electron transfer to the first working electrode, the diaphorase modified to be rate-limiting with respect to electron transfer to the first working electrode, or any combination thereof.

[0088] Element 3: The diaphorase inhibitor, comprising at least one compound selected from the group consisting of warfarin, dicoumarol, N-methylmaleimide, diphenyleneiodonium, 5,6-dimethylxanthenone-4-acetic acid, flavone-8-acetic acid, dimethylbenzyl(alk)ammonium chloride, 7,8-dihydroxyflavone, chrysin, and any combination thereof.

[0089] Element 4: The analyte sensor further comprising an analyte-permeable membrane covering at least the first active area, the analyte-permeable membrane being permeable to the inhibitor. Element 5: The analyte sensor further comprising a second active region that responds to a different analyte than the inhibitor.

[0090] Element 6: The second active region, which is a glucose-responsive active region comprising a glucose-responsive enzyme disposed on the sensor tail. Element 6A: An analyte-permeable membrane that is permeable to glucose, covering the second active area.

[0091] Element 7: The analyte sensor further comprising a second working electrode, wherein a second active area is disposed on a surface of the second working electrode, and an analyte-permeable membrane covering the second active area.

[0092] Element 8: The sensor tail configured to be inserted into tissue. Element 9: at least the electron transfer agent, the diaphorase, and the NAD-dependent dehydrogenase covalently attached to a polymer comprising a first active region.

[0093] Element 10: The first active region further comprises albumin. By way of non-limiting example, exemplary combinations applicable to A include, but are not limited to: 1 and 2; 1 and 3; 1 and 4; 1 and 5; 1 and 6; 1, 6 and 6A; 1, 6, 6A and 7; 1 and 7; 1 and 8; 1 and 9; 1 and 10; 1, 2 and 3; 1, 2 and 4; 1, 2, 4 and 5; 1, 4, 5 and 7; 1, 2 and 9; 1, 2 and 10; 2 and 3; 2-4; 2 and 4; 2 and 5; 2, 4 and 5; 2 and 6; 2, 6 and 6A; 2, 6, and 7; 2, 6, 6A and 7; 2 and 7; 2 and 8; 2 and 9; 2 and 10, 2, 4, 5 and 7; 2, 3 and 4; 2, 3, 5 and 6; 2, 4, 5, 6 and 6A; 2, 4, 5 and 7; 3 and 4; 3 and 5; and 3, 4 and 5; 3, 4, 5 and 6; 3, 4, 5, 6 and 6A; 3 and 7; 3 and 8; 3 and 9; 3 and 10; 4 and 5; 4, 5 and 6; 4, 5, 6 and 6A; 4 and 7; 4 and 8; 4 and 9; 4 and 10; 8 and 9; 8 and 10; and 9 and 10.

[0094] Embodiment B may have one or more of the following additional elements in any combination: Element 11: The NAD-dependent glucose dehydrogenase, wherein the substrate is glucose.

[0095] Element 12: The first active region comprises a rate-limiting amount of diaphorase with respect to electron transfer to the first working electrode, the diaphorase is modified to be rate-limiting with respect to electron transfer to the first working electrode, or any combination thereof.

[0096] Element 13: The inhibitor comprising at least one compound selected from the group consisting of warfarin, dicoumarol, N-methylmaleimide, diphenyleneiodonium, 5,6-dimethylxanthenone-4-acetic acid, flavone-8-acetic acid, dimethylbenzyl(alk)ammonium chloride, 7,8-dihydroxyflavone, chrysin, and any combination thereof.

[0097] Element 14: The analyte-permeable membrane covering at least the first active area, the analyte-permeable membrane being permeable to an inhibitor. Element 15: The sensor tail further comprising a second active region that responds to a different analyte than the inhibitor.

[0098] Element 16: A glucose-responsive active region comprising a glucose-responsive enzyme disposed on the sensor tail, the method comprising obtaining a second signal equal to or greater than the redox potential of the glucose-responsive active region, the second signal being proportional to the glucose concentration in the fluid, the second active region further comprising correlating the second signal to the glucose concentration in the fluid.

[0099] Element 16A: The substrate, which is glucose. Element 16B: An analyte-permeable membrane that is permeable to glucose and covers the second active area.

[0100] Element 17: A second active region disposed on a surface of a second working electrode, wherein a second potential is applied to the second working electrode and a second signal equal to or greater than the redox potential of the second active region is obtained.

[0101] Element 18: An analyte-permeable membrane covering the second active area. Element 19: the first signal and the second signal are obtained at different times. Element 20: said first signal and said second signal simultaneously acquired via a first channel and a second channel.

[0102] Element 21: At least the electron transfer agent, the diaphorase, and the NAD-dependent dehydrogenase covalently attached to a polymer comprising the first active region. Element 22: The first active region further comprises albumin.

[0103] Element 23: The fluid, wherein the fluid is a biological fluid and the analyte sensor is exposed to the biological fluid in vivo. By way of non-limiting example, exemplary combinations applicable to B include, but are not limited to, the following: 11 and 12; 11 and 13; 11-13; 11 and 14; 11, 12 and 14; 11, 13 and 14; 11-14; 11 and 15, 11, 15 and 16; 11, 15, 16 and 16A; 11, 15, 16, 16B; 11, 15 and 17; 11, 15, 17 and 18; 11, 15, 1 7, 18 and 19;11, 15, 17, 18 and 20;11 and 21;11 and 22;11 and 23;11, 12 and 21;11, 12, 13 and 21;11, 12 and 23;11, 12, 13 and 23;12 and 13;12 and 14;12-14;12 and 15;12, 15 and 16;12, 15, 16 and 16A;12, 15, 16 and 16B;12, 15 and 17;12, 15, 17 and 18;12, 13, 14 15 and 17;12, 13, 14, 15, 17 and 18;12, 15, 17 and 19;12, 15, 17 and 20;12 and 21;12 and 22;12 and 23;13 and 14;13 and 15;13, 15 and 16;13, 15, 16 and 16A;13, 15, 16 and 16B;13 and 17;13, 17 and 18;13, 17 and 19;13, 17 and 20;13 and 21;13, 17 and 18;13, 17 and 19;13, 17 and 20;13 and 21;13 and 22;13 and 23;14 and 1 5;14-16;14, 15, 16 and 16A;14, 15, 16 and 16B;14, 15 and 17;14, 15, 17 and 18;14, 15, 17 and 19;14, 15, 17 and 20;14 and 21;14 and 22;14 and 23;15 and 16;15, 16 and 16A;15, 16 and 16B;15 and 17;15 and 18;15, 17 and 18;15, 17 and 19;15, 17 and 20;15 and 21;15 and 22;15 and 23;21 and 22;21 and 23; and 22 and 23.

[0104] To facilitate a better understanding of the embodiments described herein, examples of various representative embodiments are provided below, which should not be read as limiting or defining the scope of the present invention.

[0105] Example A poly(vinylpyridine)-bound transition metal complex was prepared having the structure shown in Formula 1. Further details regarding this transition metal complex and electron transfer therewith are provided in commonly owned U.S. Pat. No. 6,605,200, incorporated by reference above. The subscripts for each monomer represent exemplary atomic ratios and do not indicate a particular monomer order.

[0106] [ka]

[0107] Example 1 Inhibition of diaphorase by dicoumarol. For this example, the spotting formulations shown in Table 1 below were coated onto separate carbon working electrodes. Using a microsyringe, 35 nL of each formulation was deposited onto a separate carbon working electrode as a single spot with an area of ​​approximately 0.2 mm. After deposition, the working electrodes were cured overnight at 25°C.

[0108] [Table 1]

[0109] The electrodes were exposed to fresh phosphate-buffered saline (PBS) solution, and then various amounts of NADH and dicoumarol were titrated into the buffered solution. To "power" the sensors and complete the enzyme system identified above (Figure 3B), no glucose or GDH was added. NADH was titrated up to 30 μM. Dicoumarol was then titrated up to a concentration of 100 μM in a buffered solution containing 30 μM NADH. After titrating the dicoumarol concentration to 100 μM, the NADH concentration was finally titrated up to 40 μM. Figure 10 is a graph showing the results of titrating NADH and dicoumarol (DCM) into a PBS solution exposed to the analyte sensors (sensors 1–4) of Example 1. As shown, all sensors were responsive to increasing NADH concentrations, but only for sensors 2 and 4, each containing a low concentration of electron transfer agent, there was a sharp and sensitive decrease in signal as the dicoumarol concentration increased. The higher concentration of diaphorase in sensor 2 resulted in a higher signal in sensor 2. Given the goal of making the sensor response diaphorase-limited, further optimization efforts focused on optimizing the sensor response at low diaphorase loading concentrations.

[0110] Next, several electrodes were fabricated as described above using 10 mM MES buffer containing 0.2 mg / mL diaphorase, 4 mg / mL PEGDGE400, and varying amounts of electron transfer agent (0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 4, and 8 mg / mL). NADH and dicoumarol were then titrated in PBS solution exposed to the analyte sensor. In this case, NADH was titrated up to 160 μM, and dicoumarol was titrated up to 80 μM. Figures 11A and 11B are graphs showing the results of titrating NADH and dicoumarol (DCM) in PBS solution exposed to electrodes with varying amounts of electron transfer agent in the active area above the electrode. Figure 11A shows the raw current response, and Figure 11B shows the normalized current response. Figure 12 is a graph of sensor response as a function of NADH concentration for electrodes with varying amounts of electron transfer agent on the active area above the electrode, and Figure 13 is a graph of normalized sensor response as a function of dicoumarol concentration for electrodes with varying amounts of electron transfer agent on the active area above the electrode. As shown, a concentration of 0.4 mg / mL of electron transfer agent provided the optimal combination of strong inhibition and good sensitivity for the detection of diaphorase inhibitors.

[0111] Example 2 Detection of dicoumarol using an analyte sensor with an active region involving glucose dehydrogenase and rate-limiting electron transfer to a working electrode. For this example, the spotting formulations shown in Table 2 below were coated onto separate carbon working electrodes. Using a microsyringe, 35 nL of each formulation was deposited onto a separate carbon working electrode as a single spot with an area of ​​approximately 0.2 mm. After deposition, the working electrodes were cured overnight at 25°C.

[0112] [Table 2]

[0113] Glucose and dicoumarol were then gradually increased in the PBS solution in which the sensor was immersed. A 500 μM amount of NAD was first added to the buffer, followed by three additions of glucose up to 50 μM. Dicoumarol (DCM) was then gradually increased to 160 μM. Figures 14A and 14B are graphs showing the results of gradually increasing glucose and dicoumarol (DCM) in a PBS solution exposed to an electrode with varying amounts of NAD-dependent glucose dehydrogenase in the active area of ​​the electrode. Figure 14A shows the raw current response, and Figure 14B shows the normalized current response. As shown, increasing the amount of glucose dehydrogenase increased the signal response during glucose and DCM addition. Overall, the DCM response decreased as the amount of DCM added increased.

[0114] Unless otherwise indicated, all numbers expressing quantities and the like in the specification and the related claims should be understood to be modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by 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 scope of the claims, each numerical parameter should, at the very least, be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0115] One or more exemplary embodiments incorporating various features are presented herein. For the sake of clarity, not all features of a physical implementation are described or illustrated in this application. It should be understood that in developing a physical embodiment incorporating embodiments of the present invention, numerous implementation-specific decisions must be made to achieve the developer's goals, such as compliance with system-related, business-related, government-related, and other constraints, which vary from implementation to implementation and from time to time. While the developer's efforts may be time-consuming, such efforts would nevertheless be routine for one of ordinary skill in the art and would have the benefit of this disclosure.

[0116] Although various systems, tools, and methods are described herein in terms of "comprising" various components or steps, the systems, tools, and methods may also be described herein in terms of "consisting essentially of" various components and steps. It can also be "essentially of" or "consist of."

[0117] As used herein, the phrase "at least one of" a series of preceding items, along with the term "and" or "or" separating any of the items, modifies the list as a whole, rather than each member (i.e., each item) of the list. The phrase "at least one of" allows for the meaning to include at least one of any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. By way of example, the phrase "at least one of A, B, and C" or "at least one of A, B, or C" refers, respectively, to A only, B only, or C only; any combination of A, B, and C; and / or at least one of each of A, B, and C.

[0118] Thus, the disclosed systems, tools, and methods are well adapted to achieve the objects and advantages mentioned, as well as those inherent therein. The specific embodiments disclosed above are illustrative only, as the teachings of the present disclosure may be modified and implemented in different but similar manners apparent to those skilled in the art having the benefit of the teachings herein. Furthermore, no limitations are intended to the details of construction or design herein shown, other than as described in the following claims. It is therefore evident that the specific exemplary embodiments disclosed above may be altered, combined, or modified, and all such variations are contemplated within the scope of the present disclosure. The systems, tools, and methods illustratively disclosed herein may suitably be practiced in the absence of any element not specifically disclosed herein and / or optional element disclosed herein. While systems, tools, and methods will be described in terms of "comprising," "containing," or "including" various components or steps, the systems, tools, and methods may also "consist essentially of" or "consist of" various components and steps. All numbers and ranges disclosed above may vary to some extent. When a numerical range with a lower and upper limit is disclosed, any number falling within that range and any included range is specifically disclosed. In particular, any range of values ​​disclosed herein in the form of "about a to about b," or equivalently, "approximately a to b," or equivalently, "approximately a to b," should be understood to represent any number or range encompassed within the broader range of values. Additionally, terms in the claims have their plain and ordinary meaning unless expressly and unambiguously defined otherwise by the patent owner. Furthermore, the indefinite article "a" or "an," as used in the claims, is defined herein to mean one or more of the element it introduces.In the event of any discrepancy in the use of a word or term in this specification and one or more patents or other documents that may be incorporated herein by reference, the definition consistent with this specification shall prevail.

[0119] The following supplementary notes correspond to the claims as filed. [Appendix 1] In the analyte sensor: a sensor tail comprising at least a first working electrode; and a first active region disposed on a surface of the first working electrode, the first active region comprising an electron transfer agent; Nicotinamide adenine dinucleotide (NAD), reduced NAD, or any combination thereof; NAD-dependent dehydrogenase, and an enzyme system including diaphorase, The analyte sensor, wherein electron transfer from the first active region to the first working electrode is rate-limiting for the diaphorase such that the first active region responds to an inhibitor of diaphorase. [Appendix 2] The analyte sensor according to Appendix 1, wherein the NAD-dependent dehydrogenase is an NAD-dependent glucose dehydrogenase. [Appendix 3] The analyte sensor of Appendix 1, wherein the first active region comprises the diaphorase in a rate-limiting amount with respect to electron transfer to the first working electrode, the diaphorase is modified to be rate-limiting with respect to electron transfer to the first working electrode, or any combination thereof. [Appendix 4] The analyte sensor of Appendix 1, wherein the diaphorase inhibitor comprises at least one compound selected from the group consisting of warfarin, dicoumarol, N-methylmaleimide, diphenyleneiodonium, 5,6-dimethylxanthenone-4-acetic acid, flavone-8-acetic acid, dimethylbenzyl(alk)ammonium chloride, 7,8-dihydroxyflavone, chrysin, and any combination thereof. [Supplementary Note 5] further comprising an analyte-permeable membrane covering at least the first active area; 10. The analyte sensor of claim 1, wherein the analyte-permeable membrane is permeable to the inhibitor. [Appendix 6] The analyte sensor of Appendix 1, further comprising a second active region that responds to a different analyte than the inhibitor. [Appendix 7] The analyte sensor of Appendix 6, wherein the second active region is a glucose-responsive active region comprising a glucose-responsive enzyme disposed on the sensor tail. [Supplementary Note 8] A second working electrode, the second active region being disposed on a surface of the second working electrode; 7. The analyte sensor of claim 6, further comprising an analyte-permeable membrane covering the second active area. [Appendix 9] The analyte sensor of Appendix 1, wherein the sensor tail is configured to be inserted into tissue. [Appendix 10] The analyte sensor of Appendix 1, wherein at least the electron transfer agent, the diaphorase, and the NAD-dependent dehydrogenase are covalently bonded to a polymer comprising the first active region. [Appendix 11] The analyte sensor of Appendix 1, wherein the first active region further comprises albumin. [Appendix 12] Exposing the analyte sensor to a fluid containing a substrate for nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase and an inhibitor of diaphorase; the analyte sensor comprises a sensor tail including at least a first working electrode and a first active area disposed on a surface of the first working electrode, the first active area including an electron transfer agent and an enzyme system including NAD, reduced NAD, or any combination thereof; and the NAD-dependent dehydrogenase; exposing the analyte sensor, wherein electron transfer from the first active area to the first working electrode is rate-limiting to the diaphorase, such that the first active area responds to the inhibitor; applying a potential to the first working electrode; obtaining a first signal equal to or greater than the redox potential of the first active region, the first signal being proportional to a concentration of the inhibitor in the fluid; and correlating the first signal to a concentration of the inhibitor in the fluid. [Appendix 13] The method described in Appendix 12, wherein the NAD-dependent dehydrogenase is an NAD-dependent glucose dehydrogenase and the substrate is glucose. [Appendix 14] The method of Appendices 12, wherein the first active region comprises the diaphorase in a rate-limiting amount with respect to electron transfer to the first working electrode, the diaphorase has been modified to be rate-limiting with respect to electron transfer to the first working electrode, or any combination thereof. [Appendix 15] The method of Appendices 12, wherein the inhibitor comprises at least one compound selected from the group consisting of warfarin, dicoumarol, N-methylmaleimide, diphenyleneiodonium, 5,6-dimethylxanthenone-4-acetic acid, flavone-8-acetic acid, dimethylbenzyl(alk)ammonium chloride, 7,8-dihydroxyflavone, chrysin, and any combination thereof. [Appendix 16] The method of Appendices 12, wherein an analyte-permeable membrane covers at least the first active area, and the analyte-permeable membrane is permeable to the inhibitor. [Appendix 17] The method of Appendices 12, wherein the sensor tail further comprises a second active region that responds to a different analyte than the inhibitor. [Appendix 18] The second active region is a glucose-responsive active region comprising a glucose-responsive enzyme disposed on the sensor tail, and the method further comprises: obtaining a second signal equal to or greater than the redox potential of the glucose-responsive active region, the second signal being proportional to the glucose concentration in the fluid; 18. The method of claim 17, further comprising correlating the second signal to a glucose concentration in the fluid. [Appendix 19] The method of Appendices 17, wherein the second active region is disposed on a surface of a second working electrode, and a second potential is applied to the second working electrode to obtain a second signal equal to or greater than the redox potential of the second active region. [Appendix 20] The method of Appendices 19, wherein an analyte-permeable membrane covers the second active area. [Appendix 21] The method of Appendices 19, wherein the first signal and the second signal are acquired at different times. [Appendix 22] The method of Appendices 19, wherein the first signal and the second signal are acquired simultaneously via a first channel and a second channel. [Appendix 23] The method of Appendices 12, wherein at least the electron transfer agent, the diaphorase, and the NAD-dependent dehydrogenase are covalently bound to a polymer comprising the first active region. [Appendix 24] The method of Appendices 12, wherein the first active region further comprises albumin. [Appendix 25] The method of Appendices 12, wherein the fluid is a biological fluid and the analyte sensor is exposed to the biological fluid in vivo.

Claims

1. 1. A method for monitoring the level of a diaphorase inhibitor in a subject using an analyte sensor already inserted into the biological fluid of the subject, comprising: the analyte sensor is exposed to the biological fluid containing a substrate for nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase and a diaphorase inhibitor, the analyte sensor comprising: a first working electrode and a first active region disposed on a surface of the first working electrode, the first active region comprising an electron transfer agent and an enzyme system, the enzyme system comprising NAD as a cofactor, reduced NAD, or any combination thereof; an NAD-dependent dehydrogenase; diaphorase; and a polymer, wherein one or more of the NAD, the reduced NAD, the NAD-dependent dehydrogenase, and the diaphorase are covalently bound to the polymer, and electron transfer from the first active region to the first working electrode is rate-limiting with respect to the diaphorase; applying a potential to the first working electrode; obtaining a first signal at or above the redox potential of the first active region, the first signal being proportional to the concentration of a diaphorase inhibitor in the biological fluid; correlating the first signal to the concentration of the diaphorase inhibitor in the biological fluid. The method comprising:

2. 2. The method of claim 1, wherein the NAD-dependent dehydrogenase is an NAD-dependent glucose dehydrogenase.

3. The method of claim 1 , wherein the electron transfer agent comprises a transition metal complex.

4. The method of claim 3 , wherein the transition metal complex comprises ruthenium or osmium.

5. 10. The method of claim 1, wherein the polymer is polyvinylpyridine, polyvinylimidazole, or any copolymer thereof.

6. The method of claim 1 , wherein the electron transfer agent comprises an osmium complex bound to a poly(vinylpyridine) polymer.

7. 2. The method of claim 1, wherein the diaphorase inhibitor comprises at least one compound selected from the group consisting of warfarin, dicoumarol, N-methylmaleimide, diphenyleneiodonium, 5,6-dimethylxanthenone-4-acetic acid, flavone-8-acetic acid, dimethylbenzyl(alk)ammonium chloride, 7,8-dihydroxyflavone, chrysin, and any combination thereof.

8. The method of claim 1 , wherein the substrate is glucose.

9. 2. The method of claim 1, wherein the first working electrode comprises the diaphorase in a rate-limiting amount with respect to electron transfer to the first working electrode, or the diaphorase is modified to be rate-limiting with respect to electron transfer to the first working electrode, or any combination thereof.

10. 10. The method of claim 1, wherein an analyte-permeable membrane covers at least the first active area, the analyte-permeable membrane being permeable to the diaphorase inhibitor.

11. 10. The method of claim 1, further comprising a second active area disposed on a second working electrode, the second active area responsive to a second analyte.

12. the second active region is a glucose-responsive active region comprising a glucose-responsive enzyme disposed on the analyte sensor; The method comprises: obtaining a second signal at or above the redox potential of the glucose-responsive active region, the second signal being proportional to the concentration of glucose in the biological fluid; correlating the second signal to the concentration of glucose in the biological fluid. The method of claim 11 , comprising:

13. the second active region is disposed on a surface of a second working electrode; 12. The method of claim 11, wherein a second potential is applied to the second working electrode to obtain a second signal at or above the redox potential of the second active region.

14. The method of claim 11 , wherein an analyte-permeable membrane covers the second active area.

15. 14. The method of claim 13, wherein the first signal and the second signal are obtained at different times.

16. The method of claim 13 , wherein the first signal and the second signal are obtained simultaneously via a first channel and a second channel.

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