Analyte sensors and sensing methods for detecting monoamine oxidase inhibitors

Abstract

The invention relates to analyte sensors and sensing methods for detecting warfarin inhibitors. Analyte sensors featuring an enzyme system comprising a warfarin enzyme and an NAD-dependent dehydrogenase can be used to detect warfarin inhibitors, provided that the transfer of electrons to the working electrode is rate limiting with respect to the warfarin enzyme. The analyte sensor can include a sensor tail comprising at least a first working electrode, a first active area disposed on the surface of the first working electrode, and an analyte permeable membrane that coats at least the first active area. The enzyme system comprises NAD, reduced NAD, or any combination thereof; an NAD-dependent dehydrogenase such as an NAD-dependent glucose dehydrogenase; and a warfarin enzyme. Warfarin inhibitors that can be detected include, for example, warfarin, dicumarol, and similar compounds. A second active area can be present to facilitate detection of an analyte different from a warfarin inhibitor.

Description

[0001] This application is a divisional application of Chinese Invention Patent Application No. 202180038475.8.

[0002] Cross-reference with related applications

[0003] not applicable. Background Technology

[0004] Detecting various analytes in an individual can sometimes be crucial for monitoring their health and well-being. Some analytes are internally produced biomolecules, and their concentrations can vary due to baseline physiological conditions or exposure to specific environmental factors. Similarly, the concentrations of drugs or drug metabolites can be analyzed as a measure of individual health, aiding healthcare professionals in making decisions regarding medication and treatment. Deviations from normal analyte levels often indicate a deteriorating metabolic condition, disease, exposure to specific environmental conditions, or ineffective treatment regimens. While a specific pathological source can cause dysregulation of a single analyte on its own, it is common for multiple analytes to be dysregulated simultaneously due to the same pathological source or comorbid (related) conditions. In cases of multiple analyte dysregulation, the degree of dysregulation can vary among the analytes. To achieve a comprehensive assessment of an individual's health, it may be necessary to monitor every single analyte.

[0005] Coumarin-based drugs, such as warfarin and dicumarol, 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 thus reduces blood clotting. Despite their effectiveness, it is very difficult to maintain therapeutically effective levels of coumarin-based drugs in the body. Patients taking coumarin-based drugs must carefully regulate their diet to avoid vitamin K-rich foods such as leafy green vegetables, thereby avoiding reactivation of the blood clotting cycle and replacement of the enzyme-bound coumarin. Furthermore, patients may respond differently to coumarin-based drugs and / or metabolize them at significantly different rates. If plasma levels of coumarin-based drugs become excessively high due to overdosing or overly frequent administration, it can lead to dangerous bleeding events. Similarly, they are also prone to dropping below the therapeutic window of inhibition of blood clotting. As another difficulty, coumarin-based drugs, such as warfarin, can enhance the effects of certain diabetes medications and cause extremely low blood glucose levels. Correspondingly, healthcare professionals prescribing coumarin-based medications often have to carefully and gradually increase the effective dose for a particular patient and subsequently monitor for the development of adverse side effects, especially for diabetic patients.

[0006] Periodic in vitro analyte monitoring using extracted bodily fluids is often sufficient for monitoring the health of many individuals. In fact, multiple blood draws may be required as coumarin-based drugs are gradually increased to a therapeutically effective dose, followed by continuous maintenance monitoring. Because the dosage of coumarin-based drugs can vary frequently, patients may require a significant number of blood draws over time. These multiple blood draws can be painful, and the frequent occurrence of them at fixed collection times in the physician's workplace can be inconvenient for patients' work or personal schedules. Furthermore, the periodic nature of the blood draws may only provide medical personnel with limited observation of the in vivo characteristics of coumarin-based drugs and other analytes.

[0007] In vivo analyte sensors (especially those using enzyme-based detection to provide detection specificity) address some of the aforementioned difficulties with certain analytes and are being used increasingly frequently. In fact, in vivo analyte sensors that monitor blood glucose levels using glucose-responsive enzymes are currently widely used in diabetic patients. Other types of analytes can be monitored using other enzymes or enzyme systems containing multiple enzymes that work synergistically. However, relatively few in vivo analyte sensors currently exist that feature enzyme-based detection and can satisfactorily analyze drugs or drug metabolites, such as coumarin-based drugs. Attached Figure Description

[0008] The following figures are included to illustrate certain aspects of this disclosure and should not be considered as exclusive embodiments. The disclosed subject matter can be considerably modified, altered, combined, and equivalent in form and function without departing from the scope of this disclosure.

[0009] Figure 1 A diagram of an exemplary sensing system is shown, which can be incorporated into the analyte sensor of this disclosure.

[0010] Figure 2A-2C This shows the configuration of the enzyme system used to detect ketones.

[0011] Figure 3A How can the display be modified? Figure 2A An enzyme system is used to detect glucose. Figure 3B How can the display be further modified? Figure 3A The enzyme system is used to detect flavotransferase inhibitors. Therefore, Figure 3B This shows the enzyme system configuration used to detect flavoprotein inhibitors.

[0012] Figures 4A-4C This diagram shows a cross-sectional view of an exemplary analyte sensor with an active region suitable for detecting a flavoprotein inhibitor.

[0013] Figures 5A-5CThis diagram shows a cross-sectional view of an exemplary analyte sensor with a single working electrode and an active region suitable for detecting a flavoprotein inhibitor and another analyte.

[0014] Figure 6 This diagram shows a cross-sectional view of an exemplary analyte sensor with two working electrodes and an active region suitable for detecting a flavoprotein inhibitor and another analyte.

[0015] Figures 7A-7C This diagram shows a perspective view of an exemplary analyte sensor, characterized by electrodes arranged concentrically and containing an active region suitable for detecting a flavozyme inhibitor and another analyte.

[0016] Figure 8A and 8B This shows the configuration of the enzyme system used to detect glucose.

[0017] Figure 9 This shows the configuration of the enzyme system used to detect creatinine.

[0018] Figure 10 The figure shows the results of titrating NADH and dicoumarin (DCM) in PBS solution exposed to the analyte sensors (sensors 1-4) of Example 1.

[0019] Figure 11A and 11B The figure shows the results of titrating NADH and dicoumarin (DCM) in a PBS solution exposed to an analyte sensor, on which a varying amount of electron transfer agent is present in the active region.

[0020] Figure 12 The diagram shows the sensor response of the analyte sensor as a function of NADH concentration, with varying amounts of electron transfer agent in the active region of the sensor.

[0021] Figure 13 The figure shows the normalized sensor response of the analyte sensor as a function of the concentration of dicumarol, with varying amounts of electron transfer agent in the active region of the sensor.

[0022] Figure 14A and 14B The figure shows the results of titrating glucose and dicoumarin (DCM) in PBS solution exposed to the analyte sensors (sensors 5-8) of Example 2. Summary of the Invention

[0023] This disclosure generally describes analyte sensors that use multiple enzymes to detect one or more analytes, and more particularly describes analyte sensors that use multiple enzymes working synergistically to detect flavoxel inhibitors such as coumarin-based drugs, and corresponding methods of use. Other analytes can be detected simultaneously using separate enzymes or enzyme systems located on the same analyte sensor.

[0024] As discussed above, analyte sensors using enzyme-based detection are generally used to test single analytes, such as glucose or related analytes, due to the specificity of common enzymes for specific substrates or substrate classes. For this purpose, analyte sensors can be used that employ a single enzyme or an enzyme system comprising multiple enzymes acting synergistically. As used herein, the term "synergistic" refers to coupled enzyme reactions in which the product of a first enzyme reaction becomes the substrate of a second enzyme reaction, and the second or subsequent enzyme reaction serves as the basis for measuring analyte concentration. To facilitate detection, the analyte can react or influence said reaction during at least one of the enzyme reactions in the enzyme system. Using in vivo analyte sensors characterized by enzymes or enzyme systems to facilitate detection can particularly advantageously avoid frequent aspirations of bodily fluids, which might otherwise be necessary for analyte monitoring. Monitoring drugs and drug metabolites with in vivo analyte sensors can be particularly problematic because finding suitable enzyme systems is crucial for facilitating the specific detection of a particular drug or drug metabolite.

[0025] Coumarin-based drugs, such as warfarin and dicumarol, are a class of drugs for which in vivo monitoring is highly desirable due to the difficulty of gradually increasing and maintaining these drugs at therapeutically effective levels. Currently, there is no believed to be an efficient method for the in vivo detection and quantification of coumarin-based drugs and their metabolites, particularly using enzymes or enzyme systems to facilitate detection. Vitamin K epoxide reductase (a target enzyme for some coumarin-based drugs) has not yet been used in feasible enzymatic detection protocols for coumarin-based drugs.

[0026] Coumarin-based drugs and several other types of compounds are also potent inhibitors of flavoxelases. This disclosure demonstrates that an analyte sensor characterized by an enzyme system comprising a flavoxelase can be configured to efficiently detect coumarin-based drugs and other inhibitors of flavoxelases. The enzyme system can be electrically coupled to a working electrode to facilitate electrochemical analyte detection. An enzyme system configured to detect coumarin-based drugs and similar inhibitors is characterized by the synergistic action of the flavoxelase and at least one additional enzyme to generate an electrochemical signal at the working electrode. To facilitate the detection of coumarin-based drugs and other flavoxelase inhibitors, the enzyme system is rate-limited with respect to the flavoxelase, 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 flavoxelase inhibitor present. Suitable enzyme systems comprising flavoxelases and rate-limited with respect to the flavoxelase are described in further detail below. Advantageously, the enzyme system can utilize high natural concentrations of glucose or other species in biological fluids to initiate an enzyme cascade, ultimately causing electron transfer to the working electrode, as also explained below. Thus, apart from those encapsulated within the analyte sensor itself, no additional reagents are needed to facilitate detection.

[0027] In addition to detecting coumarin-based drugs and other flavoxant inhibitors, the analyte sensors disclosed herein can be further configured to detect one or more additional analytes. Exemplary examples of other analytes that can be encapsulated in the same analyte sensor for further detection include, for example, glucose, ketones, creatinine, lactate, A1c, pH, etc. As mentioned above, in vivo analyte sensors featuring glucose zymase detection are currently widely used in diabetic patients. Detection systems for other analytes (one or more of which may be simultaneously dysregulated in diabetic patients) are also known. Detection systems for glucose or any one or more of the aforementioned analytes can also be combined with enzyme systems for detecting flavoxant inhibitors and incorporated into the analyte sensors disclosed herein. Further details on how additional sensing chemistry can be incorporated into the analyte sensors of this disclosure are provided below.

[0028] The ability to monitor coumarin-based drugs in vivo represents a significant and advantageous clinical advancement provided by this disclosure. Furthermore, the combination of in vivo monitoring of glucose levels with the analysis of coumarin-based drugs can be further advantageous because coumarin-based drugs tend to enhance the effects of diabetes medications, potentially leading to additional dosing dysregulation of these drugs. Simultaneous monitoring of both concentrations using analyte sensors configured to detect glucose and coumarin-based drug analytes can provide healthcare professionals with a wealth of information and potentially improve patient outcomes. Similarly, it is desirable to combine the monitoring of other commonly dysregulated analytes with the monitoring of coumarin-based drugs and other flavoprotein inhibitors.

[0029] Before describing the analyte sensor of this disclosure in further detail, an overview of a suitable in vivo analyte sensor construction and a sensor system using the analyte sensor will be provided to better understand embodiments of this disclosure. Figure 1 A diagram illustrating an exemplary sensing system that can be incorporated into the analyte sensors of this disclosure (particularly analyte sensors comprising an active region responsive to a flavoxin inhibitor). 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, one-way or two-way, and encrypted or unencrypted). Reader device 120 may constitute an output medium for observing analyte concentrations determined by sensor 104 or its associated processor and providing prompts or notifications, and, according to some embodiments, allowing input from one or more users. Reader device 120 may be a multi-purpose smartphone or a dedicated electronic reader device. Although only one reader device 120 is shown, multiple reader devices 120 may be present in some cases. Reader device 120 may also communicate with remote terminal 170 and / or trusted computer system 180 via communication paths / links 141 and / or 142 (which may also be wired or wireless, one-way or two-way, and encrypted or unencrypted). The reader device 120 may also, or alternatively, communicate with a network 150 (e.g., a mobile phone network, the Internet, or a cloud server) via a communication path / link 151. The network 150 may further be communicatively coupled to a remote terminal 170 via a communication path / link 152 and / or to a trusted computer system 180 via a communication path / link 153. Alternatively, the sensor 104 may communicate directly with the remote terminal 170 and / or the trusted computer system 180 without the intermediary reader device 120. For example, according to some embodiments, the sensor 104 may communicate with the remote terminal 170 and / or the trusted computer system 180 via a direct communication link with the network 150, as described in US Patent Application Publication 2011 / 0213225, the entire description of which is incorporated herein by reference. Any suitable electronic communication scheme may be used for each communication path or link, such as Near Field Communication (NFC), Radio Frequency Identification (RFID), Bluetooth® or Bluetooth® Low Energy, WiFi, etc. According to some embodiments, the remote terminal 170 and / or the trusted computer system 180 may be accessible to individuals other than the primary user who are interested in the user's analytics level. The reader device 120 may include a display 122 and optional input components 121. According to some embodiments, the display 122 may include a touchscreen interface.

[0030] The sensor control device 102 includes a sensor housing 103 that can house circuitry and a power supply for operating the sensor 104. Optionally, the power supply and / or operating circuitry may be omitted. A processor (not shown) may be communicatively coupled to the sensor 104, wherein the processor is physically located within the sensor housing 103 or the reader device 120. According to some embodiments, the sensor 104 protrudes from the underside of the sensor housing 103 and extends through an adhesive layer 105 suitable for attaching the sensor housing 103 to a tissue surface such as skin.

[0031] Sensor 104 is adapted for at least partial insertion into the dermis or subcutaneous layer of a relevant tissue, such as skin. Sensor 104 may include a sensor tail of sufficient length for insertion to a desired depth in a given tissue. The sensor tail may include at least one working electrode and an active region comprising an enzyme system responsive to a flavoxylase inhibitor, facilitating the detection of coumarin-based drugs and other flavoxylase inhibitors. Additional active regions may also be present to facilitate the detection of one or more additional analytes, as described further herein. A counter electrode may be present in combination with the at least one working electrode, optionally further in combination with a reference electrode. Specific electrode configurations on the sensor tail are described below. Figure 3A-7C To describe in more detail.

[0032] Similarly, the active region responding to an additional analyte may be characterized by a suitable enzyme or enzyme system for facilitating the detection of that additional analyte. For example, if the active region responding to another analyte is a glucose-responsive active region, then the glucose-responsive active region may contain a glucose-responsive enzyme. Active regions responding to other analytes may include those responding to, for example, ketones, lactate, creatinine, pH, etc., and may be characterized by separate enzymes or enzyme systems suitable for testing these analytes. Suitable enzyme systems for detecting these analytes are further described below, particularly with reference to... Figure 2A-2C 8A, 8B, and 9. According to various embodiments, one or more enzymes in the active region can be covalently bonded to the polymer constituting the active region. The flavoprotein enzyme inhibitor and any additional analytes can be monitored in any relevant biological fluid, such as skin fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, etc. In certain embodiments, the analyte sensor of this disclosure can be adapted to test skin fluid or interstitial fluid to determine the in vivo concentration of the flavoprotein enzyme inhibitor and / or additional analytes.

[0033] One or more mass transport restriction membranes can encapsulate an active region responding to a flavoxin inhibitor and an active region responding to another analyte (if present). Analytical sensors typically use membranes encapsulating the active regions to restrict mass transport and / or improve biocompatibility. Mass transport restriction membranes may also be referred to herein as analyte permeability membranes. Restricting analytes in and out of the active region with a mass transport restriction membrane helps avoid sensor overload (saturation), thereby improving detection power and accuracy. In the case of testing multiple analytes with a single analyte sensor, the various analytes can exhibit different permeability values ​​across a given mass transport restriction membrane, potentially leading to different sensitivities to each analyte. Advantageously, sensor constructions can be used to incorporate different mass transport restriction membranes over the active regions as needed, thereby facilitating the detection of multiple analytes. If a single mass transport restriction membrane provides satisfactory permeability to both analytes, a simpler sensor construction can be used.

[0034] Refer again Figure 1 Sensor 104 can automatically transmit data to reader device 120. For example, analyte concentration data (i.e., drug concentrations based on coumarin and / or concentrations of glucose, ketones, lactate, or creatinine, or pH values) can be communicated automatically and periodically (e.g., at certain frequencies or after certain time intervals), with the data stored in memory until transmission (e.g., every minute, every five minutes, or other predetermined time intervals). In other embodiments, sensor 104 can communicate with reader device 120 in a non-automatic manner and without following a set schedule. For example, data can be communicated from sensor 104 using RFID technology when the sensor electronics are brought into the communication range of reader device 120. The data can remain stored in the memory of sensor 104 until communication with reader device 120 is reached. Thus, the user is not required to be in close proximity to reader device 120 at all times, but can upload data at convenient times. In other embodiments, a combination of automatic and non-automatic data transfer can be implemented. For example, data transfer can continue automatically until reader device 120 is no longer within the communication range of sensor 104.

[0035] An introducer may be briefly present to facilitate the introduction of sensor 104 into the tissue. In an exemplary embodiment, the introducer may comprise a needle or similar sharp object. It should be appreciated that other types of introducers, such as sheaths or paddles, may be present in alternative embodiments. More specifically, the needle or other introducer may be briefly located near sensor 104 before tissue insertion and then withdrawn. In its presence, the needle or other introducer may facilitate insertion of sensor 104 into the tissue by opening an inlet / outlet path that allows sensor 104 to follow. For example, according to one or more embodiments, the needle may facilitate penetration of the epidermis, which serves as an inlet / outlet path to the dermis, thereby allowing sensor 104 implantation. After opening the inlet / outlet path, the needle or other introducer may be withdrawn without posing a risk of sharpness. In an exemplary embodiment, a suitable needle may be solid or hollow, beveled or non-beveled, and / or have a circular or non-circular cross-section. In a more specific embodiment, a suitable needle may be comparable to an acupuncture needle in cross-sectional diameter and / or tip design, having a cross-sectional diameter of approximately 250 micrometers. However, it should be recognized that a suitable needle may have a larger or smaller cross-sectional diameter if required by a specific application.

[0036] In some embodiments, the tip of the needle (if present) may be angled above the end of the sensor 104, thereby allowing the needle to penetrate the tissue first and open the entry / exit path of the sensor 104. In other exemplary embodiments, the sensor 104 may be located within the lumen or groove of the needle, where the needle similarly opens the entry / exit path of the sensor 104. In either case, the needle may be retracted after facilitating sensor insertion.

[0037] Figure 2A The enzyme system configuration for ketone detection is shown. Additional enzyme systems suitable for ketone detection are shown. Figure 2B and 2C This is further described below. (In the example shown...) Figure 2A In the enzyme system, β-hydroxybutyrate acts as a substitute for ketones formed in vivo, reacting with an enzyme system comprising β-hydroxybutyrate dehydrogenase (HBDH) and a flavoprotein enzyme to facilitate ketone detection in a ketone-responsive active region disposed on at least one working electrode surface, as further described herein. Within the ketone-responsive active region, β-hydroxybutyrate dehydrogenase can react β-hydroxybutyrate with oxidized nicotinamide adenine dinucleotide (NAD). + The enzyme is converted into acetoacetic acid 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, as used herein, the term "NAD" refers to phosphate-bound NAD. + Both NADH and NAD are diphosphates that link two nucleotides, one containing an adenine nucleotide and the other a nicotinamide nucleotide. +The NADH enzyme cofactor helps facilitate the synergistic enzymatic reaction disclosed herein. Once formed, NADH can be oxidized under the mediation of the flavin transducer, where the electron transfer during this process provides 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 β-hydroxybutyrate converted, thereby providing the basis for ketone detection and quantification based on current measurements at the working electrode. The transfer of electrons to the working electrode can further occur under the mediation of electron transfer agents (such as osmium (Os) compounds or similar transition metal complexes), as described in further detail below. Albumin can further be present in the active region as a stabilizer. β-hydroxybutyrate dehydrogenase and flavin transducer can be covalently bonded to the polymer constituting the ketone-responsive active region. + It can or may not covalently bond to the polymer, but if NAD + If not covalently bonded, it can be physically retained within the ketone-responsive active region (e.g., using a mass transport restriction membrane covering the ketone-responsive active region), wherein the mass transport restriction membrane is also ketone-permeable.

[0038] This public display Figure 2A The enzyme system shown can be modified to become responsive to other analytes. Figure 3A and 3B show Figure 2A How can enzyme systems be sequentially modified to become responsive to glucose and flavoprotein inhibitors, respectively? For example... Figure 3A As shown, by replacing β-hydroxybutyrate dehydrogenase with an NAD-dependent glucose dehydrogenase in the active region, the analyte sensor can become glucose-responsive, in which case gluconolactone is formed as a product of glucose oxidation. The flavoprotein can facilitate the transfer of electrons between NAD and electron transfer agents. Even simpler enzyme-based detection schemes for glucose are shown below. Figure 8A and 8B In this process, glucose oxidase or FAD-dependent glucose dehydrogenase can transfer electrons to an electron transfer agent without the presence of an additional enzyme.

[0039] Figure 3A The enzyme system described herein can respond linearly to glucose, provided that there is a sufficient flow of downstream enzymes and cofactors (flavoprotein enzyme and NAD+, respectively). + (NADH) can be used to promote the transfer of all electrons generated during glucose oxidation to the working electrode. Further modifications are possible in this disclosure. Figure 3AThe enzyme system is designed such that the transfer of electrons from the flavoprotein to the working electrode is rate-limited. By making the flavoprotein rate-limited, it acts as a "valve" controlling the flow of electrons to the working electrode. In the case of rate-limited flavoprotein, a constant signal is resulted, regardless of the glucose concentration upstream of the flavoprotein. However, in the case of rate-limited flavoprotein, flavoprotein inhibitors, such as coumarin-based drugs and other flavoprotein inhibitors, can alter the flow of electrons to the working electrode and act as the basis for inhibitor detection. More specifically, the reduction in electron flow to the working electrode can be correlated with the amount of inhibitor present. Furthermore, since glucose is ubiquitous in biological fluids, it can act as "fuel" to provide a stable flow of electrons to the rate-limited flavoprotein. Accordingly, Figure 3B The enzyme system configuration for detecting flavin inhibitors is shown, taking these factors into account. Other NAD-dependent dehydrogenases can be used as... Figure 3B The alternative to NAD-dependent glucose dehydrogenase is provided that its substrate is readily available in the biological fluid being analyzed.

[0040] Accordingly, the analyte sensor of this 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 comprises nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), or any combination thereof; an NAD-dependent dehydrogenase; and a flavoxelase; wherein the transfer of electrons from the first active region to the first working electrode is rate-limited with respect to the flavoxelase, thereby the first active region is responsive to flavoxelase inhibitors, as explained above. Optionally, the analyte-permeable membrane covering the first active region may be omitted, provided that NAD or NADH can be sufficiently retained within the first active region (e.g., by physical entrainment or covalent bonding to the polymer constituting the first active region).

[0041] In specific instances, the NAD-dependent dehydrogenase can be an NAD-dependent glucose dehydrogenase, where glucose present in the analyte fluid can provide an electron source to the working electrode. Other NAD-dependent dehydrogenases can be used similarly, provided that their substrates are readily available in the analyte fluid (especially biological fluids).

[0042] To enable the analyte sensor to respond to a yellow translucency inhibitor, the first active region may contain a yellow translucency in an amount that is electron rate-limited with respect to transfer to the first working electrode. The yellow translucency may be modified to become electron rate-limited with respect to transfer to the first working electrode, or any combination thereof. For example, wild-type yellow translucency may be modified to become a modified yellow translucency with reduced activity.

[0043] As mentioned above, flavoxerase inhibitors that can be monitored using the analyte sensors disclosed herein include warfarin and dicumarol. Other flavoxerase inhibitors that can be tested using the analyte sensors disclosed herein include, for example, N-methylmaleimide, diphenyliodonium, 5,6-dimethylxanthonone-4-acetic acid, flavonoid-8-acetic acid, dimethylbenzylalkammonium chloride, 7,8-dihydroxyflavone, flavonoidin, and any combination thereof.

[0044] The analyte sensors disclosed herein are characterized at least by an active region on a working electrode that responds to a flavozyme inhibitor, and at least one additional electrode in combination therewith, which may be a counter electrode, a reference electrode, and / or a counter / reference electrode. An additional working electrode may be present in some cases. This disclosure also contemplates analyte sensors characterized by an active region responding to a flavozyme inhibitor and, in combination therewith, an active region responding to another analyte, such as glucose, and is further discussed herein. Exemplary analyte sensor constructions suitable for testing one or more analytes are further discussed below.

[0045] The sensor construction, characterized by responding to the active region of a flavoxin inhibitor but not to the active region of another analyte, can utilize two-electrode or three-electrode detection phantoms, as described herein. Figures 4A-4C Further description. The sensor configuration, characterized by both an active region responding to a flavoxin inhibitor and an active region responding to another analyte (either on separate working electrodes or on the same working electrode), is described below. Figure 5A-7C Described separately. For analyte sensors containing active regions configured to monitor two or more different analytes in the same sensor tail, a sensor configuration with multiple working electrodes can be particularly advantageous because it is easier to determine the signal contributed from each active region.

[0046] In the case where a single working electrode exists in the analyte sensor, a three-electrode sensor configuration may include a working electrode, a counter electrode, and a reference electrode. A corresponding two-electrode sensor configuration may include a working electrode and a second electrode, wherein the second electrode may serve as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). The electrodes may be at least partially stacked (layered) and / or laterally spaced from each other at the sensor tail. Suitable sensor configurations may be substantially flat or substantially cylindrical. In any sensor configuration disclosed herein, the electrodes may be electrically isolated from each other by a dielectric material or similar insulator. An analyte sensor containing an active region responsive to a flavoxin inhibitor and an active region responsive to another analyte, such as glucose, may be characterized by laterally spaced active regions on the working electrode.

[0047] The characteristic is that the analyte sensor with multiple working electrodes can similarly include at least one additional electrode. In the presence of one additional electrode, said additional electrode can serve as a counter / reference electrode for each of the multiple working electrodes. In the presence of two additional electrodes, one of said additional electrodes can serve as a counter electrode for each of the multiple working electrodes, while the other of said additional electrodes can serve as a reference electrode for each of the multiple working electrodes.

[0048] Figure 4A A diagram illustrating an exemplary two-electrode analyte sensor configuration that may be used in this disclosure. As shown, the analyte sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter / reference electrode 216. Alternatively, the working electrode 214 and the counter / reference electrode 216 may be located on the same side of the substrate 212, with a dielectric material (configuration not shown) interposed therebetween. An active region 218 responsive to a flavoxylase inhibitor is disposed as at least one layer on at least a portion of the working electrode 214. The active region 218 may include a plurality of sites or a single site configured for detecting the flavoxylase inhibitor, as further discussed herein.

[0049] Still refer to Figure 4A According to some embodiments, membrane 220 may cover at least the active region 218 and optionally cover part or all of the working electrode 214 and / or the counter / reference electrode 216, or the entire analyte sensor 200. One or both sides of the analyte sensor 200 may be covered by membrane 220. Membrane 220 may comprise one or more polymer membrane materials that have the ability to restrict the analyte flux reaching the active region 218 (i.e., membrane 220 is a mass transport restriction membrane with a certain degree of permeability to flavoxera inhibitors). The composition and thickness of membrane 220 may be varied to promote a desired flux of flavoxera inhibitors reaching the active region 218. In non-limiting examples, membrane 220 may be coated onto the active region 218 by one or more of spray coating, impregnation coating, printing, and / or similar deposition techniques. The membrane thickness may be selected such that the current generated at the working electrode 214 remains correlated with the amount of flavoxera inhibitor present. The analyte sensor 200 can be used to test flavoprotein inhibitors by any of the following electrochemical detection techniques: coulometric, amperometric, voltammetric, or potentiometric.

[0050] Figure 4B and 4C A diagram illustrating an exemplary three-electrode analyte sensor construction, which may also be used in this disclosure. The three-electrode analyte sensor construction can be similar to... Figure 4A The illustration shows the analyte sensor 200, with the exception that analyte sensors 201 and 202 include an additional electrode 217. Figure 4B and 4CWith the additional electrode 217 present, the counter / reference electrode 216 can act as either the counter electrode or the reference electrode, while the additional electrode 217 performs another electrode function not otherwise explained. The working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed on the working electrode 214 or the electrode 216, with a dielectric material spacer layer between them. For example, as... Figure 4B The dielectric layers 219a, 219b, and 219c space the electrodes 214, 216, and 217 apart from each other and provide electrical isolation. Alternatively, at least one of the electrodes 214, 216, and 217 may be located on the opposite surface of the substrate 212, as described above. Figure 4C As shown. Thus, in some embodiments, electrode 214 (working electrode) and electrode 216 (counter electrode) may be located on opposite surfaces of substrate 212, wherein electrode 217 (reference electrode) is located on one of electrodes 214 or 216 and spaced apart from it by a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, wherein the location of the reference material layer 230 is not limited to... Figure 4B and 4C The description. For those shown Figure 4A The active region 218 in the sensor 200, analyte sensors 201 and 202 can contain multiple sites or a single site. Analyte sensors 201 and 202 can similarly be used to test for flavoprotein inhibitors by any of the following electrochemical detection techniques: coulometric, galvanometric, voltammetric, or potentiometric.

[0051] Similar to analyte sensor 200, in analyte sensors 201 and 202, membrane 220 may also coat the active region 218 and other sensor components, thereby acting as a mass transport limiting membrane. In some embodiments, an additional electrode 217 may be coated with membrane 220. Although Figure 4B and 4C As described, all electrodes 214, 216, and 217 are covered with film 220. It should be understood that in some embodiments, only the working electrode 214 may be covered. Furthermore, the thickness of the film 220 on each of electrodes 214, 216, and 217 may be the same or different. In non-limiting examples, film 220 may be coated onto the active region 218 by one or more of spray coating, dip coating, printing, and / or similar deposition techniques. For example, in a two-electrode analyte sensor configuration (… Figure 4A ),exist Figure 4B and 4C In the sensor configuration, one or both sides of the analyte sensors 201 and 202 can be covered by the membrane 220, or all of the analyte sensors 201 and 202 can be covered. Accordingly, shown in Figure 4B and 4CThe three-electrode sensor configuration should be understood as not limiting the embodiments disclosed herein, wherein alternative electrode and / or layer configurations are still within the scope of this disclosure.

[0052] An analyte sensor reference having both an active region responding to a flavoxin inhibitor and an active region responding to another analyte (each located on a single working electrode or multiple working electrodes). Figure 5A-7C Further detailed description.

[0053] Figure 5A This illustrates an exemplary configuration of a sensor 203 with a single working electrode, on which active regions responsive to a flavozyme inhibitor and active regions responsive to another analyte are arranged. Figure 5A similar Figure 4A An exception is the presence of two active regions on the working electrode 214: active region 218a (responding to a flavozyme inhibitor) and active region 218b (responding to another analyte), which are laterally spaced from each other on the surface of the working electrode 214. Active regions 218a and 218b may contain multiple sites or a single site configured for detecting each analyte. The composition of the membrane 220 may vary or be identical in active regions 218a and 218b. For example, in cases where the composition of the membrane 220 varies in active regions 218a and 218b, a single membrane polymer may be present in one of the active regions (e.g., active region 218b), while a membrane polymer bilayer or a mixture of membrane polymers may be present in the other active region (e.g., active region 218a). One or more of spray coating, dip coating, printing, and / or similar deposition techniques may be used to deposit membranes 220 with uniform or different compositions in active regions 218a and 218b. The first active region 218a and the second active region 218b can be configured to detect their respective analytes at different working electrode potentials, as discussed further below.

[0054] Figure 5B and 5C Cross-sectional diagrams of exemplary three-electrode sensor configurations for sensors 204 and 205 are shown, each characterized by a single working electrode having an active region 218a (responding to a flavozyme inhibitor) and an active region 218b (responding to another analyte) disposed thereon. Figure 5B and 5C Other aspects are similar Figure 4B and 4C And you can refer to them for a better understanding. For Figure 5A The composition of membrane 220 in active regions 218a and 218b can be the same or different.

[0055] An exemplary sensor construction reference with multiple working electrodes, particularly two working electrodes. Figure 6-7CFurther detailed description follows. Although the following description primarily concerns a sensor construction with two working electrodes, it should be recognized that more than two working electrodes can be incorporated by expanding the disclosure herein. Additional working electrodes can be used to endow an analyte sensor with additional sensing capabilities beyond just a flavoxin inhibitor and one additional analyte. That is, an analyte sensor containing more than two working electrodes can be adapted to detect a proportionate number of additional analytes.

[0056] Figure 6 This illustration shows a cross-sectional schematic configuration of an exemplary analyte sensor having two working electrodes (a reference electrode and a counter electrode), which may be used in this disclosure. As shown, the analyte sensor 300 includes working electrodes 304 and 306 disposed on opposite surfaces of a substrate 302. An active region 310a (responding to a flavoxin inhibitor) is disposed on the surface of the working electrode 304, while an active region 310b (responding to another analyte) is disposed on the surface of the working electrode 306. A counter electrode 320 is electrically isolated from the working electrode 304 by a dielectric layer 322, while a reference electrode 321 is electrically isolated from the working electrode 306 by a dielectric layer 323. Outer dielectric layers 330 and 332 are located on the reference electrode 321 and the counter electrode 320, respectively. According to various embodiments, a membrane 340 may cover at least the active regions 310a and 310b, wherein other components or all of the analyte sensor 300 may optionally also be covered by the membrane 340. Furthermore, membrane 340 may have the same composition or a different composition (if desired) in active regions 310a and 310b to adjust the analyte flux at each location. Compositional variations may include, for example, a mixture of multiple membrane polymers or a bilayer of multiple membrane polymers.

[0057] It has multiple working electrodes and is different from Figure 6 The alternative sensor configuration shown may be characterized by counter / reference electrodes instead of separate counter and reference electrodes 320, 321, and / or by having a different layer and / or film arrangement than those explicitly described. For example, the positions of the counter electrode 320 and reference electrode 321 may be... Figure 6 The description in the text is the opposite. Furthermore, working electrodes 304 and 306 do not necessarily require... Figure 6 The arrangement shown is located on the opposite side of substrate 302.

[0058] While suitable sensor fabrications may feature substantially planar electrodes, it should be recognized that sensor fabrications featuring non-planar electrodes can be advantageous and particularly suitable for the purposes disclosed herein. In particular, substantially cylindrical electrodes arranged concentrically can facilitate the deposition of mass transport confinement films of different compositions in two distinct active regions, as described below. Figures 7A-7CThe diagram shows a perspective view of an analyte sensor, characterized by two working electrodes arranged concentrically. It should be recognized that a sensor configuration with a concentric electrode arrangement but lacking a second working electrode is also possible in this disclosure.

[0059] Figure 7A A perspective view showing an exemplary sensor configuration is displayed, in which a plurality of 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 electrodes and a dielectric layer are arranged concentrically. In particular, a working electrode 410 is disposed on the surface of the central substrate 402, and a dielectric layer 412 is 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, and a dielectric layer 422 is 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, and a dielectric layer 432 is 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, and a dielectric layer 442 is 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 each other along the longitudinal axis B of the analyte sensor 400. The surface area of ​​the working electrode 410, working electrode 420, counter electrode 430, and reference electrode 440 gradually increases as they move away from the sensor tip 404.

[0060] Still refer to Figure 7A Active regions 414a (responding to a flavoxin inhibitor) and 414b (responding to another analyte) are respectively arranged on the exposed surfaces of working electrodes 410 and 420, thereby allowing contact with the fluid for sensing of both analytes. Although active regions 414a and 414b are already... Figure 7A The description refers to three discrete points; however, it should be recognized that fewer or more than three points may exist in alternative sensor configurations. Furthermore, the locations of active regions 414a and 414b can be related to... Figure 7A The description is the opposite.

[0061] exist Figure 7A In the sensor 400, the upper portion of the working electrodes 410 and 420 and the active regions 414a and 414b disposed thereon are covered by a membrane 450. Figure 7B Alternative sensor configurations are shown, in which the entire sensor 401 is essentially covered by a membrane 450. The membrane 450 may be identical or varied in composition in the active regions 414a and 414b. For applying the membrane in a substantially cylindrical sensor configuration, an impregnation-coating technique may be particularly desirable.

[0062] It should also be recognized that various electrodes in Figure 7A and 7B The positions of the electrodes may differ from those explicitly depicted. For example, the positions of the counter electrode 430 and the reference electrode 440 may differ. Figure 7A and 7B The construction described herein is the opposite. Similarly, the positions of the working electrodes 410 and 420 are not limited to those described herein. Figure 7A and 7B Those explicitly described in the text. Figure 7C Showing Figure 7B The alternative sensor configurations shown include sensor 405 comprising a counter electrode 430 and a reference electrode 440 positioned closer to the sensor tip 404, and working electrodes 410 and 420 positioned further away from the sensor tip 404. The sensor configuration where the working electrodes 410 and 420 are positioned further away from the sensor tip 404 can be advantageous because it provides a larger surface area for depositing active regions 414a and 414b (in... Figure 7C (Each of the five discrete sensing points shown in the example) thereby facilitates increased signal strength in some cases.

[0063] although Figures 7A-7C Having described sensor configurations each mounted on a central substrate 402, it should be recognized that alternative sensor configurations can be electrode-loaded and lack a central substrate 402 (configuration not shown). In particular, the innermost concentric electrodes can be used to mount other electrodes and dielectric layers. For example, counter electrode 430 can be the innermost concentric electrode and is used to arrange reference electrode 440, working electrodes 410 and 420, and dielectric layers 432, 442, 412, and 422 thereon. Given this disclosure, it should be recognized again that alternative electrode and dielectric layer configurations can be used in sensor configurations lacking a central substrate 402.

[0064] Accordingly, the analyte sensor of this disclosure may also include an active region, also arranged at the tail of the sensor, that responds to an analyte different from the flavoxylase inhibitor. Accordingly, in a particular embodiment, the analyte sensor of this disclosure may be configured to analyze a variety of analytes. Other analytes that can be monitored besides flavoxylase inhibitors include, for example, glucose, ketones, lactate, creatinine, pH, or any combination thereof. Suitable enzymes, enzyme systems, or similar detection schemes for testing these additional analytes in the analyte sensor are further discussed below.

[0065] In some embodiments, the analyte sensor may further include a glucose-responsive active region containing a glucose-responsive enzyme disposed at the sensor tail. Suitable glucose-responsive enzymes may include, for example, glucose oxidases or glucose dehydrogenases (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 oxidases and glucose dehydrogenases are distinguished by their ability to use oxygen as an electron acceptor when oxidizing glucose: glucose oxidases can use oxygen as an electron acceptor, while glucose dehydrogenases transfer electrons to natural or artificial electron acceptors such as enzyme cofactors. Exemplary enzyme-based detection schemes for analyzing glucose are further shown in Figure 3A , 8A And 8B, which utilizes glucose oxidase or glucose dehydrogenase to facilitate detection. Both glucose oxidase and glucose dehydrogenase can be covalently bonded to polymers constituting the glucose-responsive active region and exchange electrons with electron transfer agents (e.g., osmium (Os) complexes or similar transition metal complexes) that can also be covalently bonded to the polymer. Suitable electron transfer agents are described in further detail below. Glucose oxidase can directly exchange electrons with electron transfer agents ( Figure 8A ), while glucose dehydrogenase can use cofactors to promote electron exchange with electron transfer agents ( Figure 3A and 8B FAD cofactors can directly exchange electrons with electron transfer agents, such as... Figure 8B As shown. In contrast, NAD cofactors can use flavotransferases to promote the transfer of electrons from cofactors to electron transfer agents, such as... Figure 3A As shown and described above. Further details concerning glucose-responsive active regions incorporated into glucose oxidase or glucose dehydrogenase and their use in glucose detection can be found, for example, in co-owned US Patent 8,268,143.

[0066] Simultaneous detection of flavoprotein inhibitors and glucose can be particularly desirable because dicumarol and other coumarin-based drugs tend to affect the activity of certain diabetes medications. Thus, analyte sensors capable of analyzing flavoprotein inhibitors and glucose could facilitate treatment decisions and potentially improve patient outcomes. Considerations regarding the combined detection of a second analyte, such as glucose, with a flavoprotein inhibitor are provided below.

[0067] In some embodiments, the analyte sensor may further include a ketone-responsive active region comprising a synergistic enzyme system to facilitate ketone detection. Suitable enzyme systems for facilitating ketone detection are referenced above. Figure 2A Description. Additional enzyme systems that can work synergistically to facilitate the detection of ketones are shown in... Figure 2B and2C .exist Figure 2B and 2C In this process, a 1:1 molar correspondence again exists between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted, thus providing the basis for ketone detection. Additional details concerning the enzyme system that responds to ketones can be found in the co-owned US patent application 16 / 774,835 entitled "Analyte Sensors and Sensing Methods Featuring Dual Detection of Glucose and Ketones," filed January 28, 2020, and published as US Patent Application Publication 2020 / 0237275, the entirety of which is incorporated herein by reference.

[0068] like Figure 2B As shown, β-hydroxybutyrate dehydrogenase (HBDH) can again separate β-hydroxybutyrate and NAD. + It is converted to acetoacetic acid and NADH. Electron transfer to the working electrode is facilitated by flavotransferase (see [link to flavotransferase]). Figure 2A Unlike transition metal electron transfer agents, the reduced form of NADH oxidase (NADHOx(Red)) reacts to form the corresponding oxidized form (NADHOx(Ox)). NADHOx(Red) can then be reformed by reacting with molecular oxygen to produce superoxide, which can subsequently be converted to hydrogen peroxide under the mediation of superoxide dismutase (SOD). Hydrogen peroxide can then be oxidized at the working electrode to provide a signal that can be correlated with the amount of ketone initially present. According to various embodiments, SOD can be covalently bonded to the polymer in the ketone-responsive active region. Figure 2A The enzyme systems shown are similar; β-hydroxybutyrate dehydrogenase and NADH oxidase can covalently bind to polymers in the ketone-responsive active region, while NAD... + / NADH may or may not be covalently bonded to polymers in ketone-responsive active regions. If NAD + If it is not covalently bonded, it can be physically retained in the ketone-responsive active region, for example, by coating the ketone-responsive active region with a membrane polymer.

[0069] like Figure 2C As shown, another enzymatic detection chemistry for ketones can use β-hydroxybutyrate dehydrogenase (HBDH) to separate β-hydroxybutyrate and NAD+. + They are converted to acetoacetic acid and NADH, respectively. The electron transfer cycle in this case is completed as follows: 1,10-phenanthroline-5,6-dione oxidizes NADH to reform NAD. +The 1,10-phenanthroline-5,6-dione then transfers electrons to the working electrode. The 1,10-phenanthroline-5,6-dione may or may not be covalently bonded to the polymer in the ketone-responsive active region. Figure 2A The enzyme systems shown are similar; β-hydroxybutyrate dehydrogenase can covalently bind to polymers in ketone-responsive active regions, while NAD... + NADH may or may not be covalently bonded to the polymer. The inclusion of albumin in the ketone-responsive active region can provide a surprising improvement in response stability. Suitable membrane polymers can promote NAD... + It remains in the ketone-responsive active region.

[0070] Simultaneous detection of flavoprotein inhibitors (FPIs) and ketones is particularly desirable due to the prevalence of ketoacidosis in diabetic patients. Thus, analyte sensors capable of analyzing both FPIs and ketones can facilitate treatment decisions and potentially improve treatment outcomes for these individuals. In addition to providing health benefits to diabetic patients, the ability to detect both FPIs and ketones by analyte sensors can also benefit other individuals wishing to monitor their ketone levels, such as those following a ketogenic diet. A ketogenic diet can help promote weight loss and assist individuals with epilepsy in managing their condition. These individuals may sometimes use coumarin-based medications to address heart health issues.

[0071] In some embodiments, the analyte sensor may further include a creatinine-responsive active region comprising a synergistic enzyme system to facilitate creatinine detection. Suitable enzyme systems that can be used in the analyte sensors disclosed herein for detecting creatinine are shown in... Figure 9 And further details are described below. Additional details relating to the enzyme system's response to creatinine can be found in co-owned US Patent Application 16 / 582,583 entitled "Analyte Sensors and Sensing Methods for Detecting Creatinine", filed September 25, 2019 and published as US Patent Application Publication 2020 / 0241015, which is incorporated herein by reference in its entirety.

[0072] like Figure 9 As shown, creatinine can reversibly and hydrolyze to form creatine in the presence of creatine amide hydrolase (CNH). Creatine can then undergo catalytic hydrolysis to form sarcosine in the presence of creatine amide hydrolase (CRH). These reactions do not generate electron flow (e.g., oxidation or reduction) to provide a basis for the electrochemical detection of creatinine.

[0073] Still refer to Figure 9Sarcosine, produced by creatine hydrolysis, can be oxidized in the presence of the oxidized form of sarcosine oxidase (SOx-ox) to form glycine and formaldehyde, thereby generating the reduced form of 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 be re-oxidized in the presence of an electron transfer agent in the oxidized form (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 an electron flow to the working electrode.

[0074] Oxygen can interfere with the concerted reaction sequence disclosed above for the detection of creatinine. Specifically, the reduced form of sarcosine oxidase can react with oxygen to reform the corresponding oxidized form of the enzyme, without exchanging electrons with an electron transfer agent. Although the enzyme remains fully active in the presence of oxygen, there is no electron flow to the working electrode. Regardless of theory or mechanism, the competitive reaction with oxygen is believed to be kinetic. That is, the oxidation of the reduced form of sarcosine oxidase with oxygen is believed to occur faster than the electron transfer agent-promoted oxidation. Hydrogen peroxide is also formed in the presence of oxygen.

[0075] like Figure 9 As shown, a desirable reaction pathway to facilitate creatinine detection can be promoted by including an oxygen scavenger near the enzyme system. Various oxygen scavengers and their properties can be suitable, including oxidases such as glucose oxidase. Small molecule oxygen scavengers can also be suitable, but they can be completely consumed before the sensor lifetime is fully exhausted. In contrast, the enzyme can undergo reversible oxidation and reduction, thereby providing a longer sensor lifetime. By hindering oxygen oxidation of the reduced form of sarcosine oxidase, a slower electron exchange reaction with an electron transfer agent can occur, thereby allowing a current to be generated at the working electrode. The intensity of the generated current is proportional to the amount of creatinine initially reacted.

[0076] In any embodiment of this disclosure, for promoting Figure 9 The oxygen scavenger in the desired reaction pathway can be an oxidase. Any oxidase can be used to promote oxygen scavenging from a neighboring enzyme system, provided that a suitable substrate for the enzyme is also present, thereby providing a reagent for the reaction with oxygen in the presence of the oxidase. Oxidases suitable for oxygen scavenging in this disclosure include, but are not limited to, glucose oxidase, lactate oxidase, xanthine oxidase, etc. Glucose oxidase is a particularly desirable oxidase for promoting oxygen scavenging because glucose is readily available in various body fluids. Reaction 1 below illustrates an enzymatic reaction facilitating oxygen scavenging promoted by glucose oxidase.

[0077] β-D-glucose + O2 -- D-gluconic acid-1,5-lactone + H2O2

[0078] Reaction 1

[0079] The concentration of lactic acid available in the body is lower than that of glucose, but still sufficient to promote oxygen capture.

[0080] Oxidases, such as glucose oxidase, can be located in the analyte sensors disclosed herein at any location suitable for promoting oxygen capture. For example, glucose oxidase can be located at the sensor tail so that it can and / or cannot be used to promote glucose detection. In cases where it cannot be used to promote glucose detection, glucose oxidase can be located at the sensor tail to prevent electrons generated during glucose oxidation from reaching the working electrode, for example, by electrically isolating the glucose oxidase from the working electrode.

[0081] Simultaneous detection of flavoprotein inhibitors (FPIs) and creatinine is particularly desirable because diabetic neuropathy is a common condition in diabetic patients. For example, diabetic neuropathy can arise from high blood sugar levels and lead to eventual kidney failure. Diabetic neuropathy is a leading cause of kidney failure in the United States, and a large number of diabetic patients experience it within the first 10-20 years of their diagnosis. Creatinine levels can be an analyte of particular interest for monitoring an individual's susceptibility to kidney failure, especially due to diabetic neuropathy. Thus, an analyte sensor capable of analyzing both FPIs and creatinine could facilitate treatment decisions and potentially improve treatment outcomes for such individuals. Individuals with underlying kidney failure problems may also benefit from coumarin-based medications.

[0082] In some embodiments, the analyte sensor may further include a lactate-responsive active region comprising a lactate-responsive enzyme disposed at the sensor tail. Suitable lactate-responsive enzymes may include, for example, lactate oxidase. Lactate oxidase or other lactate-responsive enzymes may be covalently bonded to the polymer constituting the lactate-responsive active region and exchange electrons with an electron transfer agent (e.g., an osmium (Os) complex or a similar transition metal complex) that may also be covalently bonded to the polymer. Suitable electron transfer agents are described in further detail below. Albumins, such as human serum albumin, may be present in the lactate-responsive active region to stabilize the sensor response, as further described in the commonly owned US Patent Application Publication 2019 / 0320947, the entirety of which is incorporated herein by reference. Lactate levels can vary in response to a number of environmental or physiological factors, including, for example, eating, stress, exercise, sepsis or septic shock, infection, hypoxia, the presence of cancerous tissue, etc.

[0083] In some embodiments, the analyte sensor may further include a pH-responsive active region. A suitable analyte sensor configured for determining pH is described in commonly owned US Patent Application Publication 2020 / 0060592, which is incorporated herein by reference in its entirety. The analyte sensor may include a sensor tail comprising a first working electrode and a second working electrode, wherein the first active region on the first working electrode contains a substance having pH-dependent redox chemistry, and the second active region on the second working electrode contains a substance having redox chemistry that is substantially independent of pH. By obtaining the difference between the first and second signals, this difference can be correlated with the pH of the fluid to which the analyte sensor is exposed.

[0084] Accordingly, certain embodiments of the analyte sensor disclosed herein may include a sensor tail comprising at least a first working electrode, and a first active region comprising an enzyme system responsive to a flavoxin 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 flavoxin inhibitor and the other active regions may be arranged on the surface of the first working electrode and spaced apart from each other. Each active region may have a redox potential, wherein the redox potential of the first active region responsive to the flavoxin inhibitor is sufficiently different from the redox potential of the second active region to allow for independent signal generation from one of the active regions. As a non-limiting example, the redox potential difference may be at least about 100 mV, or at least about 150 mV, or at least about 200 mV. The upper limit of the redox potential difference depends on the working electrochemical window in vivo. By sufficiently differentiating the redox potential intensities of the two active regions, an electrochemical reaction can occur in one of the two active regions (i.e., the first 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 the second active regions can be generated independently at a redox potential equal to or higher than its corresponding redox potential (the lower redox potential) but lower than that of the other active region. This differential signal allows for the decomposition of signal contributions from each analyte.

[0085] Some or other embodiments of the analyte sensor disclosed herein may be characterized by having an active region responsive to a flavozyme inhibitor and an active region responsive to another analyte located on different working electrode surfaces. The analyte sensor may include a sensor tail comprising at least a first and a second working electrode, an active region responsive to a flavozyme inhibitor disposed on the surface of the first working electrode, and a second active region responsive to a different analyte disposed 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 and may comprise multiple component membranes, wherein the membrane covers at least one active region. A multi-component membrane may comprise a bilayer of two different membrane polymers or a mixture of two different membrane polymers, wherein one membrane polymer covers another active region.

[0086] Electron transfer agents can be present in any of the active regions disclosed herein, particularly in active regions responding to flavozyme inhibitors and (if present) in active regions responding to another analyte. After one or more analytes undergo an enzymatic redox reaction in their respective active regions, a suitable electron transfer agent can facilitate electron transport to an adjacent working electrode, thereby generating an electron flow indicating the presence of a specific analyte. The amount of current generated is proportional to the amount of analyte present. Depending on the sensor construction used, the electron transfer agents in the active regions responding to flavozyme inhibitors and in the active regions responding to another analyte can be the same or different. For example, in the case where two different active regions are arranged on the same working electrode, the electron transfer agents in each active region can be different (e.g., chemically different, thus exhibiting different redox potentials). In the case of multiple working electrodes, the electron transfer agents in each active region can be the same or different because each working electrode can be queried separately.

[0087] Suitable electron transfer agents may include electroreducible and electrooxidizable ions, complexes, or molecules (e.g., quinones) having a redox potential of several hundred millivolts above or below the standard mercurous chloride electrode (SCE) redox potential. According to some embodiments, suitable electron transfer agents may include low-potential osmium complexes, such as those described in US Patents 6,134,461 and 6,605,200, all of which are incorporated herein by reference. Additional examples of suitable electron transfer agents include those described in US Patents 6,736,957, 7,501,053, and 7,754,093, all of which are incorporated herein by reference. Other suitable electron transfer agents may comprise metal compounds or complexes of, for example, ruthenium, osmium, iron (e.g., ferrocene or hexacyanoferrate), or cobalt, including their metallocene compounds. Suitable ligands for metal complexes may also include, for example, bidentate or higher-dentate ligands such as bipyridine, biimidazole, phenanthroline, or pyridyl (imidazole). Other suitable bidentate ligands may include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoaromatics. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher-dentate ligands may be present in metal complexes to achieve a fully coordinated sphere.

[0088] The active region suitable for detecting any analyte disclosed herein may comprise a polymer to which an electron transfer agent is covalently bonded. Any electron transfer agent disclosed herein may contain suitable functionality to facilitate covalent bonding with the polymer in the active region. Suitable examples of polymer-bonded electron transfer agents may include those described in US Patents 8,444,834, 8,268,143, and 6,605,201, all of which are incorporated herein by reference. Polymers suitable for inclusion in the active region may include, but are not limited to, polyvinylpyridine (e.g., poly(4-vinylpyridine)), polyvinylimidazole (e.g., poly(1-vinylimidazole)), or any copolymer thereof. Exemplary copolymers suitable for inclusion in the active region include those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile. The polymers in the various active regions may be the same or different.

[0089] In specific embodiments of this disclosure, the mass transport restriction film covering at least one of the active regions may comprise a crosslinked polyvinylpyridine homopolymer or copolymer. When the mass transport restriction film covers each active region, the composition of the mass transport restriction film may be the same or different. When the film composition is different, the film may comprise a bilayer or homogeneous mixture of two different membrane polymers, one of which may be a crosslinked polyvinylpyridine homopolymer or copolymer. Suitable techniques for depositing the mass transport restriction film on the active regions may include, for example, spray coating, coating, inkjet printing, stencil printing, roller coating, dip coating, and any combination thereof.

[0090] Covalent bonding of an electron transfer agent to the polymer constituting the active region can occur by polymerizing a monomer unit carrying the covalently bonded electron transfer agent, or by reacting the electron transfer agent separately with the polymer after its synthesis. Bifunctional spacers can covalently bond an electron transfer agent to the polymer in the active region, wherein the first functional group is reactive with the polymer (e.g., a functional group capable of quaternizing pyridine or imidazole nitrogen atoms) and the second functional group is reactive with the electron transfer agent (e.g., a functional group reactive with ligands of coordinating metal ions).

[0091] Similarly, one or more enzymes in the active region can be covalently bonded to the polymer constituting the active region. In the case of an enzyme system comprising multiple enzymes present in a given active region, in some embodiments all of the multiple enzymes can be covalently bonded to the polymer, while in other embodiments only some of the multiple enzymes can be covalently bonded to the polymer. For example, one or more enzymes constituting the enzyme system can be covalently bonded to the polymer and at least one enzyme can be non-covalently bound to the polymer, such that the non-covalently bonded enzyme is physically encased in the polymer. Covalent bonding of the enzyme to the polymer in a given active region can occur via cross-linking introduced by a suitable cross-linking agent. Suitable cross-linking agents for reacting with free amino groups in the enzyme (e.g., with free side-chain amines in lysine) can include cross-linking agents such as polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imine esters, epichlorohydrin, or derivatives thereof. Suitable cross-linking agents for reacting with free carboxylic acid groups in the enzyme can include, for example, carbodiimide. Crosslinking between enzymes and polymers is generally intermolecular, but in some embodiments it can be intramolecular. In a particular embodiment, the entire enzyme in a given active region can be covalently bonded to the polymer.

[0092] Electron transfer agents and / or enzymes can also bind to the polymer in the active region by means other than covalent bonding. In some embodiments, the electron transfer agent and / or enzyme can form ionic or coordination bonds with the polymer. For example, a charged polymer can form ionic bonds with an electron transfer agent or enzyme carrying an opposite charge. In other embodiments, the electron transfer agent and / or enzyme can be physically entrained in the polymer without being bonded to it. Physically entrained electron transfer agents and / or enzymes can still suitably interact with the fluid to facilitate analyte detection and do not substantially leach from the active region.

[0093] The polymer in the active region can be selected so that NAD not covalently bound to the polymer + Alternatively, the outward diffusion of another auxiliary factor may be restricted. Restricted outward diffusion of auxiliary factors can promote a reasonable sensor lifetime (days to weeks) while still allowing sufficient inward diffusion of analytes to facilitate detection.

[0094] The active region in the analyte sensor disclosed herein may comprise one or more discrete sites (e.g., one to about ten sites or even more discrete sites), and may have a diameter of about 0.01 mm. 2 approximately 1 mm 2 The size of the active region is considered, but this document also anticipates the presence of individual sites that may be larger or smaller within the active region. It is also possible, as disclosed herein, that the active region is defined as a continuous band surrounding a cylindrical electrode. In the presence of active regions responding to flavozyme inhibitors and active regions responding to different analytes, the number and / or size of individual sites may be the same or different.

[0095] It should also be recognized that the sensitivity (output current) of the analyte sensor to each analyte can be varied by changing the coverage (area or size) of the active region, the area ratio of the active regions to each other, and the characteristics, thickness, and / or composition of the mass transport limiting membrane covering the active region. These parameters can be readily modified by those skilled in the art once they have obtained the benefits disclosed herein.

[0096] In a more particular embodiment, the analyte sensor of this disclosure may include a sensor tail configured for insertion into tissue. Suitable tissues are not considered particularly limited and are described in more detail above. Similarly, considerations for configuring the sensor tail at a specific location in a given tissue, such as the dermis of the skin, have also been described above.

[0097] A detection method for testing flavoxylase inhibitors may include: exposing an analyte sensor to a fluid containing a substrate comprising an NAD-dependent dehydrogenase and a flavoxylase inhibitor; wherein the analyte sensor includes at least a sensor tail comprising a first working electrode, and a first active region disposed on the surface of the first working electrode, wherein the first active region contains an electron transfer agent comprising NAD... +The analyte is composed of: NADH or any combination thereof; an NAD-dependent dehydrogenase; and an enzyme system of flavin dehydrogenases; and an analyte-permeable membrane coating at least a first active region; wherein the transfer of electrons from the first active region to the first working electrode is rate-limited with respect to the flavin dehydrogenase, thereby the first active region responding 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, the first signal being proportional to the concentration of the inhibitor in the fluid; and correlating the first signal with the concentration of the inhibitor in the fluid. Optionally, the analyte-permeable membrane coating the first active region may be omitted, provided that NAD or NADH can be sufficiently retained in the first active region (e.g., by physical entrainment or covalent bonding to the polymer constituting the first active region). Any flavin dehydrogenase inhibitor can be tested with the analyte sensors disclosed herein, including those specified above. Any suitable manner discussed above may cause the transfer of electrons to the first working electrode to be rate-limited with respect to the flavin dehydrogenase.

[0098] In specific instances, the NAD-dependent dehydrogenase can be an NAD-dependent glucose dehydrogenase with glucose as the substrate. Because glucose is readily ubiquitous in biological fluids, this substrate / dehydrogenase combination can be particularly advantageous in providing an electron supply to facilitate the detection of flavoprotein inhibitors.

[0099] In some implementations, the first signal can be correlated with the corresponding concentration of the flavoprotein inhibitor by consulting a lookup table or calibration curve. A lookup table for a specific inhibitor can be constructed by testing 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 the inhibitor concentration and determining a suitable calibration function within a calibration range (e.g., through regression, particularly linear regression).

[0100] The processor can determine which sensor response value in the lookup table is closest to the value measured for a sample with an unknown analyte concentration, and then report the analyte concentration accordingly. In some or other embodiments, if the sensor response value for a sample with an unknown analyte concentration falls between the values ​​recorded in the lookup table, the processor can interpolate between the two lookup table values ​​to estimate the analyte concentration. Interpolation can assume a linear concentration change between the two values ​​reported in the lookup table. Interpolation can be used when the difference between the sensor response and a given value in the lookup table is sufficiently large, for example, about 10% or greater.

[0101] Similarly, according to some or other various implementations, the processor can input the sensor response value for a sample with an unknown analyte concentration into a corresponding calibration function. The processor can then report the analyte concentration accordingly.

[0102] The sensor tail may also include a second working electrode having an active region thereon that responds differently to analytes than the inhibitors, such as a glucose-responsive active region. Thus, the method may further include obtaining a second signal at or above the redox potential of the glucose-responsive active region, the signal being proportional to the concentration of glucose in the fluid, and correlating the second signal with the concentration of glucose in the fluid. Other analytes can be analyzed similarly by using appropriate active regions and applied potentials.

[0103] According to a more specific embodiment, the first and second signals can be measured at different times. Thus, in this embodiment, the potential can be applied alternately to the first and second working electrodes. In other specific embodiments, the first and second signals can be measured simultaneously via a first channel and a second channel, in which case the potential can be applied to both working electrodes simultaneously. In both cases, the signals associated with each active region can then be correlated with the concentrations of a flavoxin inhibitor and another analyte, such as glucose or a similar analyte, using a lookup table or calibration function in a manner similar to that discussed above. Detailed Implementation

[0104] The implementation methods disclosed herein include:

[0105] A. An analyte sensor responsive to a flavin transactivator inhibitor. The analyte sensor comprises: 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 comprising an electron transfer agent and an enzyme system comprising nicotinamide adenine dinucleotide (NAD), reduced NAD, or any combination thereof, an NAD-dependent dehydrogenase, and a flavin transactivator; wherein the transfer of electrons from the first active region to the first working electrode is rate-limited with respect to the flavin transactivator, thereby the first active region is responsive to a flavin transactivator inhibitor.

[0106] B. A method for testing a flavin-delivering enzyme inhibitor. The method comprises: exposing an analyte sensor to a fluid containing a substrate of a nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase and a flavin-delivering enzyme inhibitor; wherein the analyte sensor includes at least a sensor tail comprising a first working electrode, and a first active region disposed on the surface of the first working electrode, the first active region comprising an electron transfer agent and an enzyme system comprising 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-limited with respect to the flavin-delivering enzyme, such that the first active region responds 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, the first signal being proportional to the concentration of the inhibitor in the fluid; and correlating the first signal with the concentration of the inhibitor in the fluid.

[0107] Implementation A may have one or more of the following additional elements in any combination:

[0108] Element 1: Among them, the NAD-dependent dehydrogenase is the NAD-dependent glucose dehydrogenase.

[0109] Element 2: wherein the first active region contains a yellow transducer in an amount that is electron rate-limited with respect to the transfer to the first working electrode, the yellow transducer being modified to become electron rate-limited with respect to the transfer to the first working electrode, or any combination thereof.

[0110] Element 3: The flavin inhibitor comprises at least one compound selected from warfarin, dicumarol, N-methylmaleimide, diphenyliodonium, 5,6-dimethylxanthonone-4-acetic acid, flavonoid-8-acetic acid, dimethylbenzylalkylammonium chloride, 7,8-dihydroxyflavone, flavonoidin, and any combination thereof.

[0111] Element 4: The analyte sensor further comprises an analyte-permeable membrane covering at least a first active region; wherein the analyte-permeable membrane is inhibitor-permeable.

[0112] Element 5: The analyte sensor further includes a second active region that responds to analytes that are different from the inhibitor.

[0113] Element 6: The second active region is a glucose-responsive active region containing a glucose-responsive enzyme arranged on the tail of the sensor.

[0114] Element 6A: The glucose-permeable analyte is coated with a second active region by a permeable membrane.

[0115] Element 7: The analyte sensor further includes a second working electrode, a second active region disposed on the surface of the second working electrode, and an analyte-permeable membrane covering the second active region.

[0116] Element 8: The tail of the sensor is configured for insertion into the tissue.

[0117] Element 9: wherein at least an electron transfer agent, a yellow transdermal enzyme, and an NAD-dependent dehydrogenase are covalently bound to the polymer constituting the first active region.

[0118] Element 10: The first active region also contains albumin.

[0119] As a non-limiting example, exemplary combinations of A that are possible 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, 6 A 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; 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.

[0120] Implementation B may have one or more of the following additional elements in any combination:

[0121] Element 11: The NAD-dependent dehydrogenase is an NAD-dependent glucose dehydrogenase and the substrate is glucose.

[0122] Element 12: wherein the first active region contains a yellow transducer in an amount that is electron rate-limited with respect to the transfer to the first working electrode, the yellow transducer being modified to become electron rate-limited with respect to the transfer to the first working electrode, or any combination thereof.

[0123] Element 13: wherein the inhibitor comprises at least one compound selected from warfarin, dicumarol, N-methylmaleimide, diphenyliodonium, 5,6-dimethylxanthonone-4-acetic acid, flavonoid-8-acetic acid, dimethylbenzylalkylammonium chloride, 7,8-dihydroxyflavone, flavonoidin, and any combination thereof.

[0124] Element 14: wherein the analyte-permeable membrane covers at least the first active region, and the analyte-permeable membrane is permeable to the inhibitor.

[0125] Element 15: The sensor tail also includes a second active region that responds to analytes that are different from the inhibitor.

[0126] Element 16: wherein the second active region is a glucose-responsive active region containing a glucose-responsive enzyme arranged on the tail of the sensor, and the method further includes: 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 fluid; and correlating the second signal with the concentration of glucose in the fluid.

[0127] Element 16A: The substrate is glucose.

[0128] Element 16B: Wherein the glucose-permeable analyte permeable membrane covers the second active region.

[0129] Element 17: wherein the second active region is arranged on the surface of the second working electrode, and 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.

[0130] Element 18: The analyte is permeable to the membrane and encapsulates the second active region.

[0131] Element 19: The first and second signals are obtained at different times.

[0132] Element 20: Wherein the first signal and the second signal are obtained simultaneously via the first channel and the second channel.

[0133] Element 21: wherein at least an electron transfer agent, a yellow transdermal enzyme, and an NAD-dependent dehydrogenase are covalently bound to the polymer constituting the first active region.

[0134] Element 22: The first active region also contains albumin.

[0135] Element 23: wherein the fluid is a biological fluid and the analyte sensor is exposed to the biological fluid in vivo.

[0136] As a non-limiting example, exemplary combinations of B that are feasible include, but are not limited to: 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 and 16B; 11, 15 and 17; 11, 15, 17 and 18; 11, 15, 17, 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, 1 5, 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 and 22; 13 and 23; 14 and 15; 14-16; 14, 15, 1 6 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.

[0137] To facilitate a better understanding of the embodiments described herein, the following examples of various representative embodiments are provided. These examples should not be construed in any way as limiting or defining the scope of the invention.

[0138] Example

[0139] Preparation of poly(vinylpyridine)-bonded transition metal complexes having the structure shown in Formula 1. Further details regarding this transition metal complex and electron transfer therewith are provided in commonly owned US Patent 6,605,200, which is incorporated herein by reference. The subscripts of the monomers represent exemplary atomic ratios and do not indicate any particular monomer order.

[0140]

[0141] Formula 1

[0142] Example 1: Inhibition of flavoxerase by dicoumarin. For this example, the spotting formulations shown in Table 1 below were coated onto separate carbon working electrodes. 35 nL of each formulation was deposited onto the separate carbon working electrodes using a microsyringe to achieve a deposition diameter of approximately 0.2 mm. 2 Individual points of area. After deposition, the working electrode is cured overnight at 25°C.

[0143] Table 1

[0144]

[0145] The electrode was exposed to fresh phosphate-buffered saline (PBS) solution, and then varying amounts of NADH and dicumarol were titrated into the buffer solution. No glucose or GDH was added to "drive" the sensor and complete the enzyme system specified above. Figure 3B Titrate NADH up to 30 μM. Then titrate coumarin up to 100 μM in a buffer solution containing 30 μM NADH. After the coumarin concentration has been titrated to 100 μM, finally titrate the NADH concentration to 40 μM. Figure 10 The figure shows the results of titrating NADH and dicoumarin (DCM) in PBS solution exposed to the analyte sensors (sensors 1-4) of Example 1. As shown, the NADH concentration increases with all sensor responses, but with increasing dicoumarin concentration, only sensors 2 and 4 (each containing low concentrations of electron transfer agent) exhibit a sudden and sensitive signal decrease. As a result of the higher concentration of the flavoxelase, sensor 2 yields a higher signal. Further optimization work focuses on optimizing the sensor response at low flavoxelase loadings, with the goal of making the sensor response flavoxelase-limited.

[0146] Subsequently, several electrodes were fabricated using 10 mM MES buffer, as described above, containing 0.2 mg / mL flavotransferase, 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 dicumarol were then titrated into PBS solution exposed to the analyte sensor. In this case, NADH was titrated up to 160 μM, and dicumarol up to 80 μM. Figure 11A and 11B The figure shows the results of titrating NADH and dicoumarin (DCM) in PBS solution exposed to an electrode with varying amounts of electron transfer agent in its active region. Figure 11A Displaying the original current response, while Figure 11BIt displays a standardized current response. Figure 12 The graph shows the sensor response as a function of NADH concentration for an electrode with varying amounts of electron transfer agent in its active region; while... Figure 13 The graph shows the normalized sensor response as a function of dicumarol concentration for electrodes with varying amounts of electron transfer agent in their active regions. As shown, a concentration of 0.4 mg / mL of electron transfer agent provides the optimal combination of strong inhibition and good sensitivity for detecting flavolacase inhibitors.

[0147] Example 2: Detection of dicumarol using an analyte sensor with an active region containing glucose dehydrogenase and rate-limited electron transfer to the working electrode. For this example, the spotting formulations shown in Table 2 below were coated onto separate carbon working electrodes. 35 nL of each formulation was deposited onto the separate carbon working electrodes using a microsyringe to a depth of approximately 0.2 mm. 2 Individual points of area. After deposition, the working electrode is cured overnight at 25°C.

[0148] Table 2

[0149]

[0150] Glucose and dicoumarin were then titrated into the PBS solution immersed in the sensor. First, 500 μM of NAD was added to the buffer solution, followed by three additions of glucose up to 50 μM. Then, dicoumarin (DCM) was titrated up to 160 μM. Figure 14A and 14B The figure shows the results of titrating glucose and dicoumarin (DCM) in PBS solution exposed to an electrode with varying amounts of NAD-dependent glucose dehydrogenase in its active region. Figure 14A Displaying the original current response, while Figure 14B The normalized current response is shown. As shown, the signal response increases with increasing amounts of glucose dehydrogenase during glucose addition and during DCM addition. Overall, the DCM response decreases with increasing amounts of DCM added.

[0151] Unless otherwise specified, all figures expressing quantities, etc., in this specification and its claims shall be understood to be modified by the term "about" in all cases. Accordingly, unless indicated to the contrary, the numerical parameters described in the specification and appended claims are approximate values ​​and may vary depending on the desired characteristics sought to be obtained by embodiments of the invention. At least and not as a limitation to the application of the doctrine of equivalents to the scope of the claims, each numerical parameter shall be interpreted at least according to the number of significant figures reported and with the application of ordinary rounding methods.

[0152] This document presents one or more exemplary implementations incorporating various features. For clarity, this application does not describe or show all the features of the physical implementation. It should be understood that in the development of physical implementations incorporated into this invention, many implementation-specific decisions must be made to achieve the developer's objectives, such as compliance with system relevance, business relevance, governmental relevance, and other restrictions, which vary with implementation and over time. While the developer's efforts may be time-consuming, such efforts will still be routine for those skilled in the art and have the benefits of this disclosure.

[0153] Although various systems, tools, and methods are described herein as "comprising" various components or steps, they can also be "substantially composed of" or "consisting of" "the various components and steps".

[0154] As used herein, the phrase "at least one" following a series of items separated by the terms "and" or "or" modifies the entire list, not the individual members (i.e., items) of the list. The phrase "at least one" allows for the meaning of at least one of any of the items, and / or at least one combination of the items, and / or at least one item. As an example, the phrases "at least one of A, B, and C" or "at least one of A, B, or C" each refer to only A, only B, or only C; any combination of A, B, and C; and / or at least one of A, B, and C.

[0155] Therefore, the disclosed systems, tools, and methods are well-suited to achieving the mentioned objectives and advantages, as well as those inherent therein. The specific embodiments disclosed above are merely exemplary, as the teachings of this disclosure can be modified and implemented in different but equivalent ways, as will be apparent to those skilled in the art who are aware of the benefits taught herein. Furthermore, it is not intended to limit the details of the constructions or designs shown herein beyond the description in the appended claims. Therefore, it is apparent that the particular exemplary embodiments disclosed above can be changed, combined, or modified, and all such changes are considered to fall within the scope of this disclosure. The systems, tools, and methods disclosed herein can be suitably implemented where no element specifically disclosed herein and / or any optional element disclosed herein is present. While systems, tools, and methods are described as "comprising," "containing," or "including" various components or steps, they can also be "substantially composed of" or "consisting of" "the various components and steps." All figures and ranges disclosed above can vary by quantity. In the case of the lower and upper limits of the disclosed numerical ranges, any figures falling within that range and any included ranges are specified. In particular, each range of values ​​disclosed herein (in the form of "from about a to about b" or equivalently "from about a to b" or equivalently "from about ab") should be understood to describe each number and range covered within that wider range of values. Furthermore, the terms in the claims have their clear and ordinary meaning unless otherwise expressly and clearly defined by the patentee. Additionally, the indefinite articles "an" or "a" used in the claims are defined herein as meaning that the element they introduce is one or more. If any wording or terminology used in this specification conflicts with one or more patents or other documents that may be incorporated herein by reference, the definitions consistent with this specification shall prevail.

Claims

1. Analyte sensor, including: The sensor tail section includes at least the first working electrode; and A first active region is disposed on the surface of a first working electrode, the first active region comprising an electron transfer agent and an enzyme system, the enzyme system comprising: Nicotinamide adenine dinucleotide (NAD), reduced NAD, or any combination thereof, NAD-dependent dehydrogenases, and Yellow enzyme; The transfer of electrons from the first active region to the first working electrode is rate-limited by the yellow transfectant, thus the first active region is responsive to yellow transfectant inhibitors.

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

3. The analyte sensor of claim 1, wherein the first active region contains a yellow transducer in an amount that is electron rate-limited with respect to transfer to the first working electrode, the yellow transducer being modified to become electron rate-limited with respect to transfer to the first working electrode, or any combination thereof.

4. The analyte sensor of claim 1, wherein the flavoxin inhibitor comprises at least one compound selected from warfarin, dicumarol, N-methylmaleimide, diphenyliodonium, 5,6-dimethylxanthonone-4-acetic acid, flavonoid-8-acetic acid, dimethylbenzylalkylammonium chloride, 7,8-dihydroxyflavone, flavonoidin, and any combination thereof.

5. The analyte sensor of claim 1, further comprising: An analyte-permeable membrane covering at least the first active region; The analyte-permeable membrane is permeable to the inhibitor.

6. The analyte sensor of claim 1, further comprising: The second active region of the analyte is different from that of the inhibitor.

7. The analyte sensor of claim 6, wherein the second active region is a glucose-responsive active region comprising a glucose-responsive enzyme disposed at the tail of the sensor.

8. The analyte sensor of claim 6, further comprising: The second working electrode has a second active region arranged on its surface. and An analyte-permeable membrane that encapsulates the second active region.

9. The analyte sensor of claim 1, wherein the sensor tail is configured for insertion into tissue.

10. The analyte sensor of claim 1, wherein at least the electron transfer agent, the flavoprotein enzyme, and the NAD-dependent dehydrogenase are covalently bound to the polymer constituting the first active region.

11. The analyte sensor of claim 1, wherein the first active region further comprises albumin.

12. A method comprising: The analyte sensor is exposed to a fluid containing a substrate of nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase and a flavoprotein inhibitor; The analyte sensor includes at least a sensor tail containing a first working electrode, and a first active region disposed on the surface of the first working electrode, the first active region containing an electron transfer agent and an enzyme system, the enzyme system containing NAD, reduced NAD or any combination thereof; and an NAD-dependent dehydrogenase. The transfer of electrons from the first active region to the first working electrode is rate-limited by the flavoprotein enzyme, thus the first active region is responsive to the inhibitor; Apply a potential to the first working electrode; A first signal is obtained at or above the redox potential of the first active region, and the first signal is proportional to the concentration of the inhibitor in the fluid. and The first signal is correlated with the concentration of the inhibitor in the fluid.

13. The method of claim 12, wherein the NAD-dependent dehydrogenase is an NAD-dependent glucose dehydrogenase and the substrate is glucose.

14. The method of claim 12, wherein the first active region contains a yellow transfectant in an amount that is electron rate-limiting with respect to the transfer to the first working electrode, the yellow transfectant being modified to become electron rate-limiting with respect to the transfer to the first working electrode, or any combination thereof.

15. The method of claim 12, wherein the inhibitor comprises at least one compound selected from warfarin, dicumarol, N-methylmaleimide, diphenyliodonium, 5,6-dimethylxanthonone-4-acetic acid, flavonoid-8-acetic acid, dimethylbenzylalkylammonium chloride, 7,8-dihydroxyflavone, flavonoidin, and any combination thereof.

16. The method of claim 12, wherein the analyte-permeable membrane covers at least a first active region, said analyte-permeable membrane being inhibitor-permeable.

17. The method of claim 12, wherein the sensor tail further comprises a second active region that responds to an analyte different from the inhibitor.

18. The method of claim 17, wherein the second active region is a glucose-responsive active region comprising a glucose-responsive enzyme disposed on the tail of the sensor, the method further comprising: A second signal is obtained at or above the redox potential of the glucose-responsive active region, and the second signal is proportional to the concentration of glucose in the fluid; and The second signal is correlated with the concentration of glucose in the fluid.

19. The method of claim 17, wherein the second active region is disposed on the surface of the second working electrode, and 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.

20. The method of claim 19, wherein the analyte-permeable membrane coats the second active region.

21. The method of claim 19, wherein the first signal and the second signal are obtained at different times.

22. The method of claim 19, wherein the first signal and the second signal are obtained simultaneously via the first channel and the second channel.

23. The method of claim 12, wherein at least the electron transfer agent, the flavoprotein enzyme, and the NAD-dependent dehydrogenase are covalently bound to the polymer constituting the first active region.

24. The method of claim 12, wherein the first active region further comprises albumin.

25. The method of claim 12, wherein the fluid is a biological fluid and the analyte sensor is exposed to the biological fluid in vivo.

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