Analyte Sensor and Detection Method for Detecting Creatinine

Abstract

To provide an analyte sensor capable of monitoring creatinine inside an organism.SOLUTION: An analyte sensor comprises: a sensor tail including at least a first working electrode, a creatinine responsiveness active area placed on a front surface of the first working electrode, and a first membrane for allowing permeation of creatinine so as to cover the creatinine responsiveness active area; and an oxygen scavenger placed on the sensor tail and also in the neighborhood of the creatinine responsiveness active area. The creatinine responsiveness active area includes a first electron transfer agent, a first polymer, and a plurality of enzymes to work in cooperation to facilitate detection of creatinine, especially, an enzyme system containing creatinine amide hydrolase, creatine amidinohydrolase, and sarcosine oxidase. An oxidase enzyme functions as the oxygen scavenger, especially as glucose oxidase when detecting creatinine in a fluid containing glucose.SELECTED DRAWING: Figure 2A

Description

Background Art

[0001] The detection of various analytes within an individual can sometimes be essential for monitoring health and well-being. Deviations from normal analyte levels often indicate underlying physiological conditions such as metabolic states or diseases, or exposure to specific environmental conditions. While a single analyte may become dysregulated alone with respect to a particular physiological state, multiple analytes may become dysregulated simultaneously due to the same physiological state or as a result of co-existing (related) physiological states. When multiple analytes become dysregulated simultaneously, the degree of dysregulation may vary for each analyte. Therefore, it is necessary to monitor each analyte in order to accurately assess an individual's health status.

[0002] Regular in vitro analyte monitoring using collected body fluids may be sufficient to observe a given physiological state in many individuals. However, in vitro analyte monitoring can be inconvenient or painful for some individuals, especially when it is necessary to perform body fluid collection or sampling fairly frequently (e.g., several times a day). Continuous analyte monitoring using implanted in vivo analyte sensors is a more desirable approach for individuals with severe analyte dysregulation and / or rapidly fluctuating analyte levels, but may also be beneficial to other individuals due to the convenience provided. Continuous analyte monitoring allows an individual or physician to proactively address abnormal analyte levels before reaching a point where more serious health effects such as organ damage or impairment may occur. Subcutaneous, interstitial, or dermal analyte sensors can often provide sufficient measurement accuracy for this purpose while minimizing user discomfort.

[0003] If suitable detection chemistries can be identified, many analytes become interesting targets for physiological analysis. For this purpose, amperometric sensors configured to assay glucose in vivo have been developed and improved in recent years to assist in monitoring the health of diabetic patients. Other analytes that are commonly dysregulated along with glucose in diabetic patients include, for example, lactate, oxygen, pH, A1c, ketones, and the like. Sensors configured to detect analytes that are commonly dysregulated along with glucose are known, but currently have fairly low accuracy.

[0004] In vivo analyte sensors are typically configured to analyze a single analyte to provide a specific analysis and often use enzymes to provide high specificity for a given analyte. Because of such analytical specificity, most current in vivo analyte sensors configured to assay glucose are ineffective at assaying other analytes that are often dysregulated along with glucose or that are due to dysregulated glucose levels. At best, current analyte monitoring approaches require diabetic patients to wear two different in vivo analyte sensors, one configured to analyze glucose and the other configured to analyze another analyte of interest. Analyte monitoring approaches that use multiple in vivo analyte sensors can be very inconvenient for the user. Further, when multiple in vivo analyte sensors are used for analyte monitoring, the cost burden on the device increases and the statistical likelihood that at least one of the individual in vivo analyte sensors will fail also increases.

[0005] Diabetic patients are often particularly vulnerable to co - morbidities, which can be due to mismanagement of insulin levels or can result even when diabetes has been appropriately managed over a long period. As an example, diabetic neuropathy can be caused by hyperglycaemia and can ultimately lead to kidney failure. Diabetic neuropathy is a major cause of kidney failure in the United States and is experienced by a significant number of diabetic patients within the first 10 - 20 years of the disease. Diagnostic tests for assessing kidney function currently rely on measuring elevated creatinine levels in blood and / or urine samples. It is desirable to detect potential kidney failure as early as possible, but the current diagnostic test approach is typically carried out over a long period (from months to years) to confirm whether creatinine levels are continuously increasing or trending upwards over time. If the frequency of conventional creatinine monitoring is low, the risk of kidney failure occurring increases if abnormalities in kidney function are not detected early enough.

Brief Description of the Drawings

[0006] The accompanying drawings are included to illustrate particular aspects of the present disclosure and should not be regarded as exclusive embodiments. The disclosed subject matter is capable of considerable modification, alteration, combination, and equivalents in form and function without departing from the scope of the present disclosure.

Figure 1

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 3C

Figure 4A

Figure 4B

Figure 4C

Figure 5

Figure 6A

Figure 6B

Figure 6C

Figure 6D

Figure 7A

Figure 7B

Figure 8

Figure 9

DETAILED DESCRIPTION OF THE INVENTION

[0007] DETAILED DESCRIPTION The present disclosure generally describes analyte sensors that use multiple enzymes to detect one or more analytes, more specifically, analyte sensors that use multiple enzymes to detect at least creatinine and optionally other analytes, and corresponding methods for using them.

[0008] As described above, due to the well-known specificity of enzymes for specific substrates or classes of substrates, analyte sensors that use enzymes are typically used to monitor a single analyte such as glucose. If an appropriate detection chemistry can be identified, other analytes can also be monitored. Monitoring multiple analytes can be complex because it requires the use of a corresponding number of analyte sensors to detect each analyte individually. This approach can be problematic or undesirable, especially when monitoring multiple analytes in vivo, for example, due to issues such as the cost of multiple analyte sensors, the discomfort of the user when wearing multiple analyte sensors, and the increased statistical probability of failure of individual analyte sensors.

[0009] Glucose-responsive analyte sensors are a well-studied and still evolving field for assisting diabetic patients in better managing their health. Despite the prevalence of comorbidities in diabetic patients, sensor chemistries suitable for in vivo monitoring of other analytes that often become dysregulated along with glucose have lagged significantly behind the more developed glucose detection chemistries. For example, creatinine can be an analyte of particular interest for monitoring individuals at risk of renal insufficiency, especially diabetic patients at risk of diabetic neuropathy.

[0010] The present disclosure provides an analyte sensor that responds to creatinine. Specifically, the present disclosure provides an analyte sensor that can be worn on the body for continuously or substantially continuously monitoring creatinine levels in vivo. Analysis of creatinine levels using the analyte sensors disclosed herein can provide a more accurate representation of renal function over a longer period of time to an individual or a healthcare provider than is possible with regular in vitro laboratory measurements. By analyzing creatinine levels in accordance with the present disclosure, it may be possible to enable early medical intervention to limit potential kidney damage and improve the overall health outcome of an individual.

[0011] Since there is no known enzyme that can directly transfer electrons to the working electrode from creatinine, electrochemical detection of creatinine using a single enzyme reaction is not feasible. The present disclosure remedies this deficiency by providing a sensor chemistry suitable for detecting creatinine with good response stability over a wide creatinine concentration range. In particular, the present disclosure utilizes an enzyme system that includes a plurality of enzymes that can act cooperatively to facilitate the detection of creatinine. As used herein, the term "cooperatively" refers to a combined enzyme reaction in which the product of a first enzyme reaction serves as the substrate for a second enzyme reaction, and the second enzyme reaction functions as the basis for measuring the concentration of the substrate (analyte) that reacted during the first enzyme reaction. Although defined with respect to two combined enzyme reactions, it should be understood that in some cases, three or more enzyme reactions can be similarly combined. For example, the product of a first enzyme reaction can serve as the substrate for a second enzyme reaction, the product of the second enzyme reaction can serve as the substrate for a third enzyme reaction, and the third enzyme reaction functions as the basis for measuring the concentration of the substrate (analyte) that reacted during the first enzyme reaction. As further described below, a suitable enzyme system for detecting creatinine according to the present disclosure uses three enzymes that act cooperatively, along with a fourth enzyme or other oxygen scavenger to promote oxygen clearance. The fourth enzyme or other oxygen scavenger is not directly involved in the cooperative enzyme reaction, but instead prevents the occurrence of unwanted side reactions with oxygen.

[0012] When, as in the case of creatinine, a single enzyme cannot facilitate detection, it may be desirable to utilize two or more enzymes that act in concert with each other to detect the analyte of interest. Situations where a single enzyme may not be effective in promoting detection of an analyte include, for example, being inhibited by one or more reaction products, or when placed within an analyte sensor, being unable to cycle between an oxidized state and a reduced state, and / or when no enzyme is known to facilitate the desired reaction pathway necessary for detection to facilitate detection. In the case of creatinine, the enzymatic conversion of creatinine to creatine occurs hydrolytically and does not result in a change in the oxidized state to provide a current to the working electrode to facilitate detection of this analyte. An enzyme system comprising a plurality of enzymes acting in concert in accordance with the disclosure herein may mitigate this difficulty.

[0013] The creatinine sensor disclosed herein may be advantageous for monitoring creatinine levels (and kidney function) in individuals potentially at risk of kidney damage or renal failure, but may be particularly beneficial for individuals with diabetes due to the prevalence of diabetic neuropathy. While it may also be beneficial to monitor creatinine levels alone, especially considering that glucose monitoring is already routinely performed by diabetic patients on a daily basis, it is also possible for diabetic patients to monitor both glucose and creatinine levels to improve health outcomes. The present disclosure provides for the monitoring of both glucose and creatinine using one or more in vivo analyte sensors that respond to each analyte, and in particularly advantageous configurations, a single analyte sensor that responds to both analytes in vivo can be used. Advantageously and surprisingly, an analyte sensor incorporating the detection functions of both glucose and creatinine on a single sensor tail can be manufactured by using the disclosure herein.

[0014] As further described below with reference to FIGS. 2A and 2B, the creatinine-responsive active region of the present disclosure can utilize an oxygen scavenger to facilitate the detection of creatinine using the enzyme system shown therein. The oxidase enzyme can function as an oxygen scavenger in certain sensor configurations. Since glucose is widely present in body fluids including creatinine, glucose oxidase may be a particularly advantageous oxygen scavenger, in which case glucose can function as a reagent for removing oxygen (see Reaction 1 below). The oxygen scavenger can be electrically isolated from the creatinine-responsive active region by a membrane so as not to generate a signal at the working electrode having the creatinine-responsive active region (i.e., when removing oxygen by promoting an oxidation reaction). To facilitate effective oxygen removal within the creatinine-responsive active region, the oxygen scavenger can be disposed on the membrane. In addition to being disposed on the membrane, the oxygen scavenger may be disposed at a second location on the sensor tail separated from the membrane, where the separated oxygen scavenger can function differently at the separated location (e.g., by facilitating the detection of glucose in the glucose-responsive active region). Depending on how and where the separated oxygen scavenger is disposed, the oxygen scavenger can be activated or inactivated to facilitate the detection of another analyte, particularly glucose, in addition to its oxygen removal function. When it is inactive in facilitating the detection of glucose, glucose oxidase can be electrically isolated from the working electrode, and as a result, the oxidation reaction (oxygen removal) promoted by this enzyme does not result in current generation at the working electrode. When glucose oxidase is active for both facilitating glucose detection and oxygen removal, glucose oxidase can be disposed on a second working electrode or present in a glucose-responsive active region disposed on the working electrode having the creatinine-responsive active region, from which separate signals can be obtained. Strategies for disposing both the creatinine-responsive active region and the glucose-responsive active region on a single sensor tail are further described below.

[0015] Even when the appropriate detection chemistry is understood, it may not be straightforward to incorporate two different types of active regions into a single analyte sensor. Analyte sensors often use a membrane that covers the active region to function as a mass transport limiting membrane and / or to improve biocompatibility. Limiting access of the analyte to the active region using a mass transport limiting membrane can avoid sensor overload (saturation) and thereby improve detection performance and accuracy. When assaying multiple analytes using a single analyte sensor, different permeability values may be exhibited by the various analytes passing through a given mass transport limiting membrane, and the sensitivity of each analyte may vary widely. Incorporating different mass transport limiting membranes into each active region can, in some cases, be problematic. Surprisingly and advantageously, glucose and creatinine can be analyzed without issue using a mass transport limiting membrane that is compositionally the same at each location, thereby simplifying the manufacture of an analyte sensor having the ability to detect both analytes.

[0016] Before describing the analyte sensor of the present disclosure in more detail, a brief overview of a suitable in vivo analyte sensor configuration and a sensor system using the analyte sensor is provided to better understand the embodiments of the present disclosure. FIG. 1 shows a diagram of an exemplary detection system that can incorporate the analyte sensor of the present disclosure, specifically an analyte sensor including a creatinine-responsive active region and optionally a glucose-responsive active region. As shown, the detection system 100 includes a sensor control device 102 and a reader device 120 configured to communicate with each other via a local communication path or link that is wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. The reader device 120 can, according to some embodiments, constitute an output medium for displaying analyte concentration and warnings or notifications determined by the sensor 104 or an associated processor, and can also enable one or more user inputs. The reader device 120 can be a multi-purpose smartphone or a dedicated electronic reader device. Only one reader device 120 is shown, but in certain cases, multiple reader devices 120 may exist. The reader device 120 can also communicate with a remote terminal 170 and / or a reliable computer system 180 via communication paths / links 141 and / or 142, respectively, which are also wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. In addition or alternatively, the reader device 120 can communicate with a network 150 (e.g., a cellular phone network, the Internet, or a cloud server) via a communication path / link 151. The network 150 can be further communicatively connected to the remote terminal 170 via a communication path / link 152 and / or to the reliable computer system 180 via a communication path / link 153. Alternatively, the sensor 104 can communicate directly with the remote terminal 170 and / or the reliable computer system 180 without the presence of an intervening reader device 120.For example, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is hereby incorporated by reference in its entirety, according to some embodiments, sensor 104 can communicate with remote terminal 170 and / or trusted computer system 180 via a direct communication link to network 150. Any suitable electronic communication protocol such as Near Field Communication (NFC), Radio Frequency Identification (RFID), BLUETOOTH® or BLUETOOTH® Low Energy protocol, WiFi® can be used for each of the communication paths or links. Remote terminal 170 and / or trusted computer system 180 can be accessible, according to some embodiments, by individuals other than the primary user who are interested in the user's analyte level. Reader device 120 can include a display 122 and optional input component 121. Display 122 can include a touch screen interface, according to some embodiments.

[0017] Sensor control device 102 includes a sensor housing 103 that can accommodate a circuit and a power source for operating sensor 104. Optionally, the power source and / or active circuit may be omitted. A processor (not shown) may be communicatively connected to sensor 104, and the processor is physically disposed within sensor housing 103 or reader device 120. Sensor 104 projects from the lower side of sensor housing 103 and extends through adhesive layer 105. Adhesive layer 105 is adapted, according to some embodiments, to adhere sensor housing 103 to a tissue surface such as the skin.

[0018] Sensor 104 is adapted to be at least partially inserted into the tissue of interest, such as within the dermis or subcutaneous layer of the skin. Sensor 104 may include a sensor tail of sufficient length to insert to a desired depth within a given tissue. The sensor tail may include at least one working electrode and a creatinine-responsive active region disposed thereon. Optionally, a glucose-responsive active region may be disposed on the sensor tail, optionally in combination with a second working electrode, to facilitate detection of this analyte. A counter electrode may be present in combination with the at least one working electrode. Specific electrode configurations on the sensor tail are described in more detail below with reference to FIGS. 3A-7B.

[0019] As will be described in more detail below, when a creatinine-responsive active region and an optional glucose-responsive active region are present, at least one mass transport limiting membrane can cover them. The glucose-responsive active region, if present, may include a glucose-responsive enzyme. The mass transport limiting membrane can also cover an oxygen scavenger (e.g., glucose oxidase), in which case the oxygen scavenger can be inserted between separate membrane layers.

[0020] The creatinine-responsive active region may include an enzyme system including a plurality of enzymes that can act in concert to facilitate detection of creatinine, as described below with reference to FIGS. 2A and 2B. According to various embodiments, the creatinine-responsive active region, and, if present, the glucose-responsive active region, may include an enzyme-conjugated polymer. Glucose oxidase disposed outside the glucose-responsive active region may also be covalently bound to the polymer within the analyte sensor disclosed herein. According to the present disclosure, creatinine and optionally glucose can be monitored in any biological fluid of the subject, such as dermal fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid. In certain embodiments, the analyte sensor of the present disclosure can be adapted to assay dermal fluid or interstitial fluid to determine the concentration of creatinine and / or glucose in vivo.

[0021] Referring further to FIG. 1, sensor 104 can automatically transfer data to reader device 120. For example, analyte concentration data (i.e., creatinine and / or glucose concentration) can be stored in memory when the data is acquired and communicated automatically and periodically at a specific frequency or after a specific period of time (e.g., every minute, every 5 minutes, or other predetermined period) until it is transmitted. In other embodiments, sensor 104 can communicate with reader device 120 in a non-automatic manner rather than according to a set schedule. For example, data can be communicated from sensor 104 using RFID technology when the sensor electronics enter the communication range of reader device 120. The data may remain stored in the memory of sensor 104 until it is communicated to reader device 120. Thus, the user does not need to always be in close proximity to reader device 120 and can instead upload data at a convenient time. In still other embodiments, a combination of automatic and non-automatic data transfer can be implemented. For example, data transfer can continue automatically until reader device 120 moves out of the communication range of sensor 104.

[0022] To facilitate the introduction of sensor 104 into the tissue, an introducer may be temporarily present. In an exemplary embodiment, the introducer may include a needle or similar sharp object. In alternative embodiments, it should be recognized that other types of introducers, such as sheaths or blades, may exist. More specifically, the needle or other introducer may be temporarily present in the vicinity of sensor 104 prior to tissue insertion and then withdrawn thereafter. While present, the needle or other introducer may facilitate the insertion of sensor 104 into the tissue by opening an access path for sensor 104 to follow. For example, according to one or more embodiments, the needle may facilitate penetration of the epidermis as an access path to the dermis, enabling the implantation of sensor 104. After opening the access path, the needle or other introducer can be withdrawn to avoid presenting a sharp hazard. In an exemplary embodiment, a suitable needle may be solid or hollow, angled or non-angled, and / or have a circular or non-circular cross-section. In a more particular embodiment, a suitable needle may be comparable to an acupuncture needle that may have a cross-sectional diameter of about 250 microns in cross-sectional diameter and / or tip design. However, it should be recognized that a suitable needle may have a larger or smaller cross-sectional diameter if required for a particular application.

[0023] In some embodiments, the tip of the needle (while present) may be angled on the end of sensor 104 such that the needle first penetrates the tissue and opens an access path for sensor 104. In other exemplary embodiments, sensor 104 may be present within the lumen or groove of the needle, and the needle, likewise, opens an access path for sensor 104. In either case, once the needle has facilitated the insertion of the sensor, it is then withdrawn.

[0024] A suitable enzyme system that can be used to detect creatinine in accordance with the disclosure of this specification will be described in more detail with reference to FIGS. 2A and 2B. As shown, creatinine can react reversibly and hydrolytically in the presence of creatinine amidohydrolase (CNH) to form creatine. Next, creatine can undergo catalytic hydrolysis in the presence of creatine amidinohydrolase (CRH) to form sarcosine. Neither of these reactions generates an electron flow (e.g., oxidation or reduction) to provide a basis for the electrochemical detection of creatinine.

[0025] As further shown in FIGS. 2A and 2B, the sarcosine produced by the hydrolysis of creatine undergoes oxidation in the presence of oxidized sarcosine oxidase (SOX-ox) to form glycine and formaldehyde, thereby generating reduced sarcosine oxidase (SOX-red) during the process. Hydrogen peroxide can also be generated in the presence of oxygen (FIG. 2B). Next, the reduced sarcosine oxidase undergoes reoxidation in the presence of an oxidized electron transfer agent (e.g., Os(III)), thereby generating the corresponding reduced electron transfer agent (e.g., Os(II)) and delivering an electron flow to the working electrode.

[0026] Oxygen can interfere with the coordinated series of reactions used to detect creatinine according to this disclosure. Specifically, as shown in FIG. 2B, reduced sarcosine oxidase can react with oxygen to reform the corresponding oxidized form of this enzyme, but does not exchange electrons with the electron transfer agent. Although all the enzymes remain active even when the reaction with oxygen occurs, no electrons flow to the working electrode. Without being bound by theory or mechanism, the competitive reaction with oxygen is thought to be due to a kinetic effect. That is, the oxidation of reduced sarcosine oxidase by oxygen is thought to occur faster than the oxidation promoted by the electron transfer agent. Hydrogen peroxide is also formed in the presence of oxygen.

[0027] A desirable reaction pathway for facilitating creatinine detection is shown in FIG. 2A. Oxidation of reduced sarcosine oxidase may be promoted by including an oxygen scavenger near the enzyme system. As noted above, various oxygen scavengers and their arrangements, including oxidase enzymes such as glucose oxidase, may be appropriate. Small molecule oxygen scavengers may also be suitable, but may be completely consumed before the sensor's lifetime is fully exhausted. In contrast, enzymes can undergo reversible oxidation and reduction, thereby lengthening the sensor's lifetime. By preventing the oxidation of reduced sarcosine oxidase by oxygen, the electron exchange reaction with the electron transfer agent is slowed, thereby enabling the generation of current at the working electrode. The magnitude of the generated current is proportional to the amount of creatinine that reacted first.

[0028] The oxygen scavenger used to facilitate the desirable reaction pathway in FIG. 2A can be an oxidase enzyme in any embodiment of the present disclosure. If an appropriate substrate for the enzyme is also present in the creatinine-containing fluid, thereby providing a reagent for reacting with oxygen in the presence of the oxidase enzyme, any oxidase enzyme can be used to promote oxygen removal near the enzyme system. Oxidase enzymes that may be suitable for oxygen removal in the present disclosure include, but are not limited to, glucose oxidase, lactate oxidase, xanthine oxidase, and the like. Glucose oxidase can be a particularly suitable oxidase enzyme for use in the present disclosure because glucose is readily available in various body fluids. The following Reaction 1 shows an enzymatic reaction that is promoted by glucose oxidase to remove oxygen.

[0029] β-D-glucose + O2 --→ D-glucono-1,5-lactone + H2O2 Reaction 1 The concentration of lactate available in vivo is lower than that of glucose, but is still sufficient to promote oxygen removal.

[0030] An oxidase enzyme, such as glucose oxidase, can be located anywhere suitable for facilitating oxygen removal in the analyte sensors disclosed herein. For example, glucose oxidase can be located on the sensor tail such that it is functional and / or non-functional for facilitating glucose detection. If it is non-functional for facilitating glucose detection, glucose oxidase can be located on the sensor tail to prevent electrons generated during glucose oxidation from reaching the working electrode, which receives electrons generated during sarcosine oxidation. Approaches for electrically isolating glucose oxidase from the working electrode are addressed in more detail below. If it is functional for facilitating glucose detection, glucose oxidase can be located in a glucose-responsive active region on the working electrode, such that in addition to scavenging oxygen near the creatinine-responsive active region, electrons generated during glucose oxidation are received by the working electrode. The working electrode having a glucose-responsive active region thereon can be the same working electrode as the one having the creatinine-responsive active region or a different working electrode. Suitable approaches for placing glucose oxidase within the glucose-responsive active region on a particular working electrode are also discussed below. Any combination of the aforementioned approaches for disposing glucose oxidase on the sensor tail can be used in the analyte sensors disclosed herein.

[0031] An alternative detection strategy to that shown in Figure 2A may omit the glucose oxidase, the membrane separating it from the working electrode, and the electron transfer agent. In such a detection approach, creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase act in concert as shown, with oxygen promoting the formation of hydrogen peroxide and interconverting the oxidized and reduced forms of sarcosine oxidase. Hydrogen peroxide is detected at the working electrode and can serve as the basis for assaying creatinine with this type of sensor configuration.

[0032] The analyte sensors disclosed herein are characterized by a combination of at least a creatinine-responsive active region on a working electrode and at least one additional electrode that can be a counter electrode, a reference electrode, and / or a counter / reference electrode. Analyte sensors characterized by both a creatinine-responsive active region and a glucose-responsive active region can incorporate the creatinine-responsive active region and the glucose-responsive active region on separate working electrodes or on the same working electrode. Each possible exemplary configuration is described below.

[0033] Sensor configurations that are characterized by a creatinine-responsive active region but not by a glucose-responsive active region can use a two-electrode or three-electrode detection motif, as further described herein with reference to FIGS. 3A - 3C. Sensor configurations that are characterized by both a creatinine-responsive active region and a glucose-responsive active region on separate working electrodes or on the same working electrode are then described separately with reference to FIGS. 4A - 6D. Sensor configurations having multiple working electrodes can be particularly advantageous for incorporating both a creatinine-responsive active region and a glucose-responsive active region within the same sensor tail, because the signal contributions from each active region can be more readily determined.

[0034] When a single working electrode is present in the analyte sensor, the three-electrode sensor configuration can include a working electrode, a counter electrode, and a reference electrode. The related two-electrode sensor configuration can include a working electrode and a second electrode that can function as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). The various electrodes can be at least partially stacked on top of each other (layered) and / or laterally spaced from each other on the sensor tail. Suitable sensor configurations can be substantially flat or substantially cylindrical in shape, and the creatinine-responsive active region and any optional glucose-responsive active region are laterally spaced on the working electrode. In any of the sensor configurations disclosed herein, the various electrodes can be electrically insulated from each other by a dielectric material or a similar insulator.

[0035] An analyte sensor characterized by a plurality of working electrodes may similarly include at least one additional electrode. If one additional electrode is present, that one additional electrode may function as a counter / reference electrode for each of the plurality of working electrodes. If two additional electrodes are present, one of the additional electrodes may function as a counter electrode for each of the plurality of working electrodes, and the other of the additional electrodes may function as a reference electrode for each of the plurality of working electrodes.

[0036] FIG. 3A shows a diagram of an exemplary two-electrode analyte sensor configuration suitable for use in the disclosure herein. As shown, the analyte sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter / reference electrode 216. Alternatively, the working electrode 214 and the counter / reference electrode 216 may be disposed on the same side of the substrate 212 with a dielectric material inserted therebetween (configuration not shown). The creatinine-responsive active region 218 is disposed as at least one layer on at least a portion of the working electrode 214. The creatinine-responsive active region 218 may include a plurality of spots or a single spot configured for the detection of creatinine, as further described herein.

[0037] Continuing to refer to FIG. 3A, in some embodiments, the membrane 220 can cover at least the creatinine-responsive active region 218 and, optionally, some 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 the membrane 220. The membrane 220 can include one or more polymeric membrane materials having the ability to restrict analyte flux to the active region 218 (i.e., the membrane 220 is a mass transfer limiting membrane having some permeability to creatinine). The composition and thickness of the membrane 220 can be varied to facilitate a desired creatinine flux to the creatinine-responsive active region 218, thereby providing a desired signal strength and stability. The analyte sensor 200 can be operable to assay creatinine by any of the electrochemical detection techniques of coulometry, amperometry, voltammetry, or potentiometry.

[0038] Figures 3B and 3C show diagrams of exemplary three - electrode analyte sensor configurations that are also compatible with the uses disclosed herein. The three - electrode analyte sensor configuration can be made the same as that shown for analyte sensor 200 in FIG. 3A, except that additional electrode 217 is included in analyte sensors 201 and 202 (FIGS. 3B and 3C). With the additional electrode 217, counter / reference electrode 216 can function either as a counter electrode or a reference electrode, and the additional electrode 217 serves the other electrode function. Working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed either on working electrode 210 or on electrode 216, sandwiching a dielectric separation layer therebetween. For example, as shown in FIG. 2B, dielectric layers 219a, 219b, and 219c separate electrodes 214, 216, and 217 from each other and provide electrical insulation. Alternatively, at least one of electrodes 214, 216, and 217 can be disposed on the opposite face of substrate 212, as shown in FIG. 3C. Thus, in some embodiments, electrode 214 (working electrode) and electrode 216 (counter electrode) can be disposed on the opposite face of substrate 212, and electrode 217 (reference electrode) can be disposed on one of electrodes 214 or 216 and be separated therefrom by a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, and the position of reference material layer 230 is not limited to that shown in FIGS. 2B and 2C. Similar to sensor 200 shown in FIG. 3A, the creatinine - responsive active regions 218 of analyte sensors 201 and 202 can include multiple spots or a single spot. Further, analyte sensors 201 and 202 can be operable to assay creatinine by any of the electrochemical detection techniques of coulometry, amperometry, voltammetry, or potentiometry.

[0039] Similar to the analyte sensor 200, the membrane 220 can also cover the creatinine-responsive active regions 218 and other sensor components in the analyte sensors 201 and 202, thereby functioning as a mass transfer limiting membrane. In some embodiments, the additional electrode 217 can be covered with the membrane 220. In FIGS. 3B and 2C, all of the electrodes 214, 216, and 217 are depicted as being covered with the membrane 220, but it should be recognized that in some embodiments, only the working electrode 214 may be covered. Further, the thickness of the membrane 220 at each of the electrodes 214, 216, and 217 may be the same or different. As in the case of the two-electrode analyte sensor configuration (FIG. 3A), one or both sides of the analyte sensors 201 and 202 may be covered with the membrane 220 in the sensor configurations of FIGS. 3B and 2C, or the entire analyte sensors 201 and 202 may be covered. Thus, the three-electrode sensor configurations shown in FIGS. 3B and 3C should be understood as non-limiting examples of the embodiments disclosed herein with alternative electrode and / or layer configurations within the scope of the present disclosure.

[0040] An analyte sensor having both a creatinine-responsive active region and a glucose-responsive active region on a single working electrode or on multiple working electrodes will be described in further detail with reference to FIGS. 4A-6D.

[0041] FIG. 4A shows an exemplary configuration of a sensor 203 having a single working electrode on which both a creatinine-responsive active region and a glucose-responsive active region are disposed. FIG. 4A is similar to FIG. 3A except for the presence of two active regions on the working electrode 214: the creatinine-responsive active region 218a and the glucose-responsive active region 218b. These active regions are laterally spaced from each other on the surface of the working electrode 214. The active regions 218a and 218b can include multiple spots or a single spot configured for the detection of each analyte. The composition of the membrane 220 may vary or be compositionally the same in the active regions 218a and 218b.

[0042] FIG. 4B and 4C each show a cross-sectional view of an exemplary three-electrode sensor configuration of sensors 204 and 205, respectively, featuring a single working electrode with both a creatinine-responsive active region 218a and a glucose-responsive active region 218b disposed on the surface. FIGS. 4B and 4C are otherwise similar to FIGS. 3B and 3C and can be better understood by referring to them. Similar to FIG. 4A, the composition of the membrane 220 may vary in the active regions 218a and 218b or may be compositionally the same.

[0043] An exemplary sensor configuration having a plurality of working electrodes, specifically two working electrodes, will be described in further detail with reference to FIGS. 5 - 6D. The following description is primarily directed to a sensor configuration having two working electrodes, but it should be understood that by extending the disclosure herein, three or more working electrodes may also be incorporated. The use of additional working electrodes can provide not only the detection of creatinine and glucose to the analyte sensor but also additional sensing capabilities.

[0044] Figure 5 shows a cross-sectional view of an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode that is suitable for use as disclosed herein. As shown, analyte sensor 300 includes working electrodes 304 and 306 disposed on opposing surfaces of substrate 302. Creatinine-responsive active region 310a is disposed on the surface of working electrode 304, and glucose-responsive active region 310b is disposed on the surface of working electrode 306. Counter electrode 320 is electrically insulated from working electrode 304 by dielectric layer 322, and reference electrode 321 is electrically insulated from working electrode 306 by dielectric layer 323. Outer dielectric layers 330 and 332 are disposed on reference electrode 321 and counter electrode 320, respectively. Membrane 340 can, according to various embodiments, cover at least active regions 310a and 310b, and optionally, other components of analyte sensor 300 or the entire analyte sensor 300 may also be covered by membrane 340. Also in this case, membrane 340 can vary compositionally at active regions 310a and 310b, if necessary, to provide appropriate permeability values for separately regulating analyte flux at each location.

[0045] Alternative sensor configurations having multiple working electrodes and different from the configuration shown in FIG. 5 may feature a counter / reference electrode and / or an arrangement of layers and / or membranes different from those explicitly shown, instead of separate counter and reference electrodes 320, 321. For example, the arrangement of counter electrode 320 and reference electrode 321 can be reversed from that shown in FIG. 5. Further, working electrodes 304 and 306 do not necessarily have to be present on opposing surfaces of substrate 302 in the manner shown in FIG. 4.

[0046] A suitable sensor configuration may feature electrodes that are substantially planar in nature, but it should be understood that sensor configurations featuring non-planar electrodes are also advantageous for use in the present disclosure and may be particularly suitable. In particular, substantially cylindrical electrodes arranged concentrically with each other can facilitate the deposition of a mass transfer limiting membrane, as described below herein. FIGS. 6A-6D show perspective views of an analyte sensor featuring two working electrodes arranged concentrically with each other. It should be understood that sensor configurations having a concentric electrode arrangement but lacking a second working electrode are also possible in the present disclosure.

[0047] FIG. 6A shows a perspective view of an exemplary sensor configuration in which a plurality of electrodes are substantially cylindrical and arranged concentrically with each other around a central substrate. As shown, the analyte sensor 400 includes a central substrate 402 around which all the electrodes and dielectric layers are arranged concentrically with each other. In particular, the working electrode 410 is disposed on the surface of the central substrate 402, and the dielectric layer 412 is disposed on a portion of the working electrode 410 distal to the sensor tip 404. The working electrode 420 is disposed on the dielectric layer 412, and the dielectric layer 422 is disposed on a portion of the working electrode 420 distal to the sensor tip 404. The counter electrode 430 is disposed on the dielectric layer 422, and the dielectric layer 432 is disposed on a portion of the counter electrode 430 distal to the sensor tip 404. The reference electrode 440 is disposed on the dielectric layer 432, and the 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, the working electrode 420, the counter electrode 430, and the reference electrode 440 are spaced apart from each other along the longitudinal axis B of the analyte sensor 400.

[0048] Continuing to refer to FIG. 6A, the creatinine-responsive active region 414a and the glucose-responsive active region 414b are respectively disposed on the exposed surfaces of the working electrodes 410 and 420, thereby enabling contact with a fluid to detect creatinine and / or glucose. The active regions 414a and 414b are depicted as three separate spots in FIG. 6A, but it should be understood that in alternative sensor configurations, there may be fewer or more than three spots. Further, the arrangement of the creatinine-responsive active region 414a and the glucose-responsive active region 414b may be reversed from that shown in FIG. 6A.

[0049] In FIG. 6A, the sensor 400 is partially covered with a membrane 450 over the working electrodes 410 and 420 and the active regions 414a and 414b disposed thereon. FIG. 6B shows an alternative sensor configuration in which substantially the entire sensor 401 is covered with a membrane 450. The membrane 450 may be the same or compositionally different over the active regions 414a and 414b.

[0050] It should be further understood that the various electrode arrangements of FIGS. 6A and 6B may differ from those explicitly shown. For example, the positions of the counter electrode 430 and the reference electrode 440 can be reversed from the configurations shown in FIGS. 6A and 6B. Similarly, the positions of the working electrodes 410 and 420 are not limited to those explicitly shown in FIGS. 6A and 6B. FIG. 6C shows an alternative sensor configuration relative to that shown in FIG. 6B, where the sensor 405 includes a counter electrode 430 and a reference electrode 440 disposed more proximally to the sensor tip 404, and working electrodes 410 and 420 disposed more distally to the sensor tip 404. A sensor configuration in which the working electrodes 410 and 420 are disposed more distally relative to the sensor tip 404 can be advantageous by providing a larger surface area for the deposition of the active regions 414a and 414b (five separate detection spots exemplarily shown in FIG. 6C). Thereby, in some cases, it facilitates an increase in signal strength.

[0051] Figures 6A - 6C show sensor configurations each supported on a central substrate 402, although it should be understood that alternative sensor configurations may instead be supported on electrodes and lack the central substrate 402. In particular, the innermost concentric electrode can be utilized to support the other electrodes and dielectric layers. Figure 6D shows an alternative sensor configuration to that shown in Figure 6C, where sensor 406 does not include the central substrate 402, counter electrode 430 is the innermost concentric electrode, and is used to sequentially dispose the reference electrode 440, working electrodes 410 and 420, and dielectric layers 432, 442, 412, and 422 thereon. Considering the disclosure herein, it should again be understood that other electrode and dielectric layer configurations can be used in sensor configurations lacking the central substrate 402 and can be used in different positional configurations. Thus, the sensor configuration shown in Figure 6D should be considered exemplary and non - limiting in nature.

[0052] As described above, an oxygen scavenger can be disposed near the creatinine - responsive active region to promote the oxidation of reduced sarcosine oxidase using an electron transfer agent instead of oxygen. Oxidase enzymes, particularly glucose oxidase, can be used for this purpose in the various sensor configurations disclosed herein. If only the creatinine - responsive active region is present, glucose oxidase that does not function for glucose detection can be located near the creatinine - responsive active region. If both a creatinine - responsive active region and a glucose - responsive active region are present, the glucose oxidase in the glucose - responsive active region can optionally be combined with glucose oxidase that does not function for glucose detection to effectively promote oxygen removal.

[0053] Exemplary arrangements of glucose oxidase that do not function for glucose detection with respect to the creatinine-responsive active region are shown in FIGS. 7A and 7B. In particular, FIGS. 7A and 7B show diagrams of the arrangement of glucose oxidase 407 on a membrane 409 covering the creatinine-responsive active region 403 disposed on the working electrode 500. The membrane 409 electrically isolates the glucose oxidase 407 from the working electrode 500, such that electrons generated when glucose is oxidized to limit oxygen exposure are not transmitted to the working electrode 500. Subsequently, the membrane 408 covers the glucose oxidase 407 and provides a mass transfer limiting function thereto. The membranes 408 and 409 may be compositionally the same in various embodiments of the present disclosure. FIG. 7A shows glucose oxidase 407 disposed directly on the creatinine-responsive active region 403, but it should be understood that the creatinine-responsive active region 403 and the glucose oxidase 407 may be laterally spaced from each other as long as the glucose oxidase 407 inhibits the transfer of electrons to the working electrode 500 when oxidizing glucose. Additionally alternatively, the glucose oxidase 407 may be further disposed on the opposite side surface of the sensor in yet other sensor configurations. As shown in FIG. 7B, the membrane 409 does not necessarily extend the same lateral distance as the membrane 408 does on the working electrode 500.

[0054] In the sensor configurations disclosed herein, the creatinine-responsive active region and, if present, the glucose-responsive active region may include one or more individual spots (e.g., from 1 to about 10 spots, or even more individual spots). These may range in size from about 0.01 mm 2 to about 1 mm 2 although larger or smaller individual active region spots are also contemplated herein. The total active region can be selected to provide the desired sensitivity to each analyte.

[0055] In some or other embodiments, the analyte sensor of the present disclosure may include a sensor tail configured for insertion into tissue. Suitable tissues are not considered to be particularly limited and are addressed in more detail above. Considerations in placing the sensor tail at a particular location within a given tissue are addressed above.

[0056] Accordingly, the analyte sensor disclosed herein includes at least a first working electrode, a first electron transfer agent, a first polymer, and an enzyme system including a plurality of enzymes that can act in cooperation to facilitate the detection of creatinine, disposed on the surface of the first working electrode, a creatinine-responsive active region including a sensor tail, a first membrane that allows creatinine to permeate and covers the creatinine-responsive active region, and an oxygen scavenger disposed near the creatinine-responsive activity on the sensor tail. The enzyme system includes creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase. The oxygen scavenger may be separated from the creatinine-responsive active region by the first membrane according to a particular embodiment of the present disclosure. Oxidase enzymes such as glucose oxidase may constitute at least a part of the oxygen scavenger in some embodiments.

[0057] When an oxidase enzyme such as glucose oxidase is disposed near the creatinine-responsive active region, it may covalently bind to a second polymer. Suitable polymers for covalently binding glucose oxidase are not particularly limited and may be polyvinylpyridine in a particular embodiment of the present disclosure. The covalently bound polymer can help immobilize glucose oxidase at a desired position relative to the creatinine-responsive active region.

[0058] Creatininamide hydrolase, creatine amidinohydrolase, and sarcosine oxidase can be covalently attached to the first polymer of the creatinine-responsive active region in any embodiment of the present disclosure. Polymers suitable for covalently attaching these enzymes are not considered to be particularly limited and, in certain embodiments of the present disclosure, can be polyvinylpyridine. The first polymer of the creatinine-responsive active region and the second polymer covalently attached to glucose oxidase can be the same polymer.

[0059] In any of the exemplary sensor configurations disclosed herein, the creatinine-responsive active region and, if present, the glucose-responsive active region can each contain an electron transfer agent. When both a creatinine-responsive active region and a glucose-responsive active region are present, the electron transfer agent can be the same or different depending on the particular sensor configuration used. Suitable electron transfer agents facilitate the transfer of electrons to the working electrode after an enzymatic oxidation or reduction reaction has occurred, thereby indicating the presence of a particular analyte and generating a current proportional to the amount of analyte present. For example, when the creatinine-responsive active region and the glucose-responsive active region are disposed on the same working electrode, the electron transfer agents within each active region can be different (e.g., chemically different such that the electron transfer agents exhibit different redox potentials). When multiple working electrodes are present, the electron transfer agents within each active region can be the same or different because signals can be acquired by individually responding to each working electrode when obtaining signals. The electron transfer agent can be covalently attached to the polymer in any of the active regions disclosed herein.

[0060] According to various embodiments of the present disclosure, suitable electron transfer agents can include electroreductive and electrooxidative ions, complexes, or molecules (e.g., quinones) having a redox potential several hundred millivolts above or below the redox potential of a standard calomel electrode (SCE). According to some embodiments, suitable electron transfer agents can include low-potential osmium complexes such as those described in U.S. Pat. Nos. 6,134,461 and 6,605,200, which are incorporated herein by reference in their entirety. Additional examples of suitable electron transfer agents include those described in U.S. Pat. Nos. 6,736,957, 7,501,053, and 7,754,093, the entire disclosures of each of which are incorporated herein by reference. Other suitable electron transfer agents can include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt (e.g., including its metallocene compounds). Ligands suitable for metal complexes can include, for example, bidentate or higher denticity ligands such as bipyridine, biimidazole, phenanthroline, or pyridyl(imidazole). Other suitable bidentate ligands can include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. To achieve a complete coordination sphere, any combination of monodentate, bidentate, tridentate, tetradentate, or higher denticity ligands can be present in the metal complex.

[0061] An active region suitable for detecting creatinine and / or glucose may also include a polymer to which an electron transfer agent is covalently bonded. Any of the electron transfer agents disclosed herein may include suitable functionality to facilitate covalent bonding to the polymer within the active region. Suitable examples of polymer-bound electron transfer agents may include those described in U.S. Patent Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are hereby incorporated by reference in their entirety. Polymers suitable for inclusion in the active region may include, but are not limited to, polyvinyl pyridine (e.g., poly(4-vinyl pyridine)), polyvinyl imidazole (e.g., poly(1-vinyl imidazole)), or any copolymer thereof. Exemplary copolymers that may be suitable for inclusion in the active region may include those containing monomer units such as, for example, styrene, acrylamide, methacrylamide, or acrylonitrile. The polymers within each active region may be the same or different.

[0062] In certain embodiments of the present disclosure, the mass transfer limiting membrane covering the creatinine-responsive active region may include a crosslinked polyvinyl pyridine homopolymer or copolymer comprising at least a polyvinyl pyridine-co-styrene polymer. A mass transfer limiting membrane having a similar composition may also cover an oxygen scavenger such as glucose oxidase. The composition of the mass transfer limiting membrane may be the same or different at the locations where the mass transfer limiting membrane covers each active region. Suitable techniques for depositing the mass transfer limiting membrane over the active region may include, for example, spray coating, painting, inkjet printing, stenciling, roller coating, dip coating, etc., and any combination thereof.

[0063] The method of covalent bonding between the electron transfer agent and the polymer in each active region is not considered to be particularly limited. Covalent bonding of the electron transfer agent to the polymer can occur by polymerizing monomer units having the covalently bonded electron transfer agent, or, if the polymer has already been synthesized, the electron transfer agent can be reacted separately with the polymer. According to some embodiments, a bifunctional spacer can covalently bond an electron transfer agent to a polymer within the active region, where the first functional group is reactive with the polymer (e.g., a functional group capable of quaternizing a pyridine nitrogen atom or an imidazole nitrogen atom), and the second functional group is reactive with the electron transfer agent (e.g., a functional group that reacts with a ligand that coordinates a metal ion).

[0064] Similarly, one or more enzymes within the active region can be covalently bonded to the polymer. If an enzyme system containing multiple enzymes is present in a given active region, in some embodiments, all of the multiple enzymes can be covalently bonded to the polymer, and in other embodiments, only some of the multiple enzymes can be covalently bonded to the polymer. For example, one or more enzymes containing an enzyme system can be covalently bonded to the polymer, and at least one enzyme can be non-covalently bonded to the polymer such that the non-covalently bonded enzyme is physically entrained within the polymer. According to more specific embodiments, covalent bonding of an enzyme to a polymer in a given active region can occur via a crosslinking agent introduced with a suitable crosslinking agent. Suitable crosslinking agents for reaction with free amino groups in the enzyme (e.g., with free side chain amines in lysine) can include crosslinking agents such as polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanogen chloride, N-hydroxysuccinimide, imido esters, epichlorohydrin, or derivatized variants thereof. Suitable crosslinking agents for reaction with free carboxylic acid groups in the enzyme can include, for example, carbodiimides. Crosslinking of the enzyme to the polymer is generally intermolecular, but in some embodiments can be intramolecular. In certain embodiments, all enzymes herein can be covalently bonded to the polymer.

[0065] The electron transfer agent and / or enzyme can also be bound to the polymer within the active region by means other than covalent bonding. In some embodiments, the electron transfer agent and / or enzyme can be ionically or coordinatively bound to the polymer. For example, a charged polymer can form an ionic bond with an oppositely charged electron transfer agent or enzyme. In yet other embodiments, the electron transfer agent and / or enzyme can be physically entrained within the polymer without being bound to the polymer. Physically entrained electron transfer agents and / or enzymes can still interact appropriately with the fluid to facilitate the detection of the analyte without substantially leaching from the active region.

[0066] In some embodiments of the present disclosure, the creatinine-responsive analyte sensor can further incorporate a glucose-responsive active region for detecting both creatinine and glucose. When both a creatinine-responsive active region and a glucose-responsive active region are present, the creatinine-responsive active region and the glucose-responsive active region can be present on the same working electrode or different working electrodes as described above with reference to FIGS. 4A-6D. Considerations for incorporating the glucose-responsive active region anywhere are described in more detail below. In any of the sensor configurations herein that include both a creatinine-responsive active region and a glucose-responsive active region, glucose oxidase that does not function for glucose detection can be placed on a membrane covering the creatinine-responsive active region or in another location that cannot transfer electrons to the working electrode associated with the creatinine-responsive active region.

[0067] When the creatinine-responsive active region and the glucose-responsive active region are disposed on a single working electrode, one of the active regions can be configured to respond separately to facilitate the detection of each analyte, as described below. In particular, the creatinine-responsive active region and the glucose-responsive active region can include different electron transfer agents to enable one of the active regions to generate a signal independently of the other. Either the creatinine-responsive active region or the glucose-responsive active region can be configured to generate a signal independently of the other active region.

[0068] In an embodiment where a creatinine-responsive active region and a glucose-responsive active region are disposed on a single working electrode, the redox potential associated with the glucose-responsive active region can be separated from the redox potential of the creatinine-responsive active region by at least about 100 mV, or at least about 150 mV, or at least about 200 mV. The upper limit of the separation between the redox potentials is determined in vivo by the working electrochemical window. By sufficiently separating the magnitudes of the redox potentials of the two active regions from each other, an electrochemical reaction can occur within one of the two active regions (i.e., the glucose-responsive active region or the creatinine-responsive active region) without substantially inducing an electrochemical reaction within the other active region. Thus, a signal from one of the glucose-responsive active region or the creatinine-responsive active region can be generated independently above its corresponding redox potential (lower redox potential) but below the redox potential of the other of the glucose-responsive active region and the creatinine-responsive active region (higher redox potential). In contrast, above the redox potential (higher redox potential) of the other active region that has not been previously interrogated, an electrochemical reaction can occur in both the glucose-responsive active region and the creatinine-responsive active region. Thus, a signal obtained above the higher redox potential can include signal contributions from both the glucose-responsive active region and the creatinine-responsive active region, and the observed signal is a composite signal. Next, the signal contribution above its redox potential from one active region (either the glucose-responsive active region or the creatinine-responsive active region) can be determined by subtracting the signal above that redox potential obtained only from the glucose-responsive active region or the creatinine-responsive active region from the composite signal.

[0069] In more specific embodiments, the glucose-responsive active region and the creatinine-responsive active region may include different electron transfer agents in order to sufficiently separate the magnitudes of the redox potentials from each other when the active regions are located on the same working electrode. More specifically, the glucose-responsive active region may include a first electron transfer agent, the creatinine-responsive active region may include a second electron transfer agent, and the first electron transfer agent and the second electron transfer agent are different. According to various embodiments of the present disclosure, the redox potentials of the two active regions can be sufficiently separated by changing the metal center and / or ligand present in a given electron transfer agent.

[0070] Ideally, the glucose-responsive active region and the creatinine-responsive active region disposed on a single working electrode may be configured to rapidly achieve a steady-state current when operating the analyte sensor at a given potential. The rapid achievement of the steady-state current can be facilitated by selecting an electron transfer agent that rapidly changes its oxidation state when exposed to a potential above its redox potential for each active region. Making the active regions as thin as possible can also facilitate the rapid achievement of the steady-state current. For example, suitable thicknesses for the glucose-responsive active region and the creatinine-responsive active region can range from about 0.1 micron to about 10 microns. In some or other embodiments, for example, combining one or more conductive materials such as carbon nanotubes, graphene, or metal nanoparticles within the one or more active regions can facilitate the rapid achievement of the steady-state current. Suitable amounts of the conductive particles can range from about 0.1 wt% to about 50 wt%, or about 1 wt% to about 50 wt%, or about 0.1 wt% to about 10 wt%, or about 1 wt% to about 10 wt% of the active region. Stabilizers can also be used to promote the stability of the response.

[0071] It should also be understood that the sensitivity (output current) of the analyte sensor to each analyte can be altered by changing the coverage (area or size) of the active region, the area ratio of the active regions to each other, and the nature, thickness, and / or composition of the mass transfer limiting membrane covering the active region. Changes to these parameters can be readily implemented by those skilled in the art provided the benefits of the disclosure herein.

[0072] Other embodiments of the analyte sensor disclosed herein may feature creatinine-responsive and glucose-responsive active regions on the surfaces of different working electrodes. Such an analyte sensor may further include a second working electrode, a glucose-responsive active region disposed on the surface of the second working electrode, and a second membrane that is permeable to glucose and covers the glucose-responsive active region. The glucose-responsive active region may include a second electron transfer agent, a third polymer, and glucose oxidase covalently bound to the third polymer. The third polymer may be the same as or different from the first and / or second polymers respectively associated with the creatinine-responsive active region or a glucose oxidase that does not function for glucose detection. If the creatinine-responsive and glucose-responsive active regions are disposed on separate working electrodes, the electron transfer agents associated with each active region may be the same or different.

[0073] Accordingly, a particular analyte sensor of the present disclosure capable of detecting both creatinine and glucose includes: a sensor tail including a first working electrode and a second working electrode; a creatinine-responsive active region disposed on the surface of the first working electrode, the enzyme system including a first electron transfer agent, a first polymer, and a plurality of enzymes capable of acting in cooperation to facilitate the detection of creatinine, the enzyme system including creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase; a first membrane permeable to creatinine and covering the creatinine-responsive active region; glucose oxidase covalently bound to a second polymer and disposed on the first membrane; a glucose-responsive active region disposed on the surface of the second working electrode, the glucose-responsive active region including a second electron transfer agent, a third polymer, and glucose oxidase covalently bound to the third polymer; and a second membrane permeable to glucose and covering the glucose-responsive active region. Such an analyte sensor may further include a third membrane covering the glucose oxidase disposed on the first membrane. The first membrane, the second membrane, and, if present, the third membrane may be compositionally the same in certain embodiments.

[0074] The detection method for assaying creatinine comprises exposing an analyte sensor to a fluid containing at least creatinine, the analyte sensor comprising at least a first working electrode, a sensor tail comprising a creatinine-responsive active region disposed on the surface of the first working electrode, a first membrane that is permeable to creatinine and covers the creatinine-responsive active region, and an oxygen scavenger disposed on the sensor tail and near the creatinine-responsive active region. The creatinine-responsive active region comprises an enzyme system comprising a first electron transfer agent, a first polymer, and a plurality of enzymes that can act in concert to facilitate the detection of creatinine. The enzyme system comprises creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase, all of which can be covalently attached to the first polymer in certain embodiments. The method can further comprise applying a potential to the first working electrode, obtaining a first signal that is proportional to the concentration of creatinine in the fluid at an oxidation-reduction potential above that of the creatinine-responsive active region, and correlating the first signal to the concentration of creatinine in the fluid. In certain embodiments of the present disclosure, the fluid can be a biological fluid. In further certain embodiments, glucose can be present in the fluid together with creatinine.

[0075] In some embodiments, the first signal can be correlated to the corresponding creatinine concentration by reference to a look-up table or calibration curve. The look-up table for creatinine can be created by analyzing a plurality of samples having known creatinine concentrations and recording the sensor response at each concentration. Similarly, the calibration curve for creatinine can be determined by plotting the analyte sensor response as a function of creatinine concentration and determining an appropriate calibration function (e.g., by regression, particularly linear regression) over a calibration range.

[0076] The processor can determine which sensor response value in the look-up table is closest to that measured for a sample having an unknown analyte concentration and report the analyte concentration accordingly. In some or other embodiments, if the sensor response value of a sample having an unknown analyte concentration is between the values recorded in the look-up table, the processor can interpolate between two look-up table values to estimate the analyte concentration. The interpolation can be based on a linear concentration variation between the two values reported in the look-up table. Interpolation can be used when the response of the sensor is sufficiently different from a given value in the look-up table (such as a variation of about 10% or more).

[0077] Similarly, according to some or other various embodiments, the processor can input the sensor response value of a sample having an unknown analyte concentration into a corresponding calibration function. The processor can then report the analyte concentration accordingly.

[0078] The sensor tail may further include a second working electrode with a glucose-responsive active region disposed on the surface, and the glucose-responsive active region may include a second electron transfer agent, a third polymer, and glucose oxidase covalently bonded to the third polymer. Thus, the method may further include: applying a potential to the second working electrode, obtaining a second signal proportional to the concentration of glucose in the fluid above the redox potential of the glucose-responsive active region, and correlating the second signal to the concentration of glucose in the fluid.

[0079] A detection method for assaying creatinine and / or glucose using an analyte sensor characterized by a creatinine-responsive active region and a glucose-responsive active region on a single working electrode may include exposing the analyte sensor to a fluid containing at least one of creatinine and glucose. The analyte sensor may include at least a working electrode, particularly a single working electrode, and a sensor tail disposed on the surface of the working electrode and including at least a creatinine-responsive active region and a glucose-responsive active region spaced apart from each other. The creatinine-responsive active region may be covered by a membrane, and glucose oxidase covalently bound to a polymer may be disposed on the membrane in addition to the glucose oxidase in the glucose-responsive active region. The creatinine-responsive active region includes an enzyme system including two or more enzymes that can act cooperatively to facilitate the detection of creatinine, a first polymer covalently bound to the enzyme, and a first electron transfer agent covalently bound to the first polymer. The glucose-responsive active region includes glucose oxidase, a third polymer covalently bound to the glucose oxidase, and a second electron transfer agent covalently bound to the third polymer. When the glucose-responsive active region and the creatinine-responsive active region are located on a single working electrode, as described in more detail herein, the first and second electron transfer agents are compositionally different from each other. Each active region has a redox potential, and the redox potential of the creatinine-responsive active region is sufficiently separated from the redox potential of the glucose-responsive active region to enable the generation of a signal from one of the active regions. The method further includes obtaining a first signal at a potential above the lower redox potential but below the higher redox potential such that the first signal is proportional to the concentration of one of glucose or creatinine in the fluid; obtaining a second signal at a potential above the higher redox potential such that the second signal is a composite signal including a signal contribution from the glucose-responsive active region and a signal contribution from the creatinine-responsive active region; and obtaining a differential signal proportional to the concentration of one of glucose and creatinine by subtracting the first signal from the second signal.

[0080] In a more specific embodiment, to provide sufficient separation for the independent generation of signals from the first active region, the redox potential associated with the creatinine-responsive active region may be at least about 100 mV, or at least about 150 mV, or at least about 200 mV away from the redox potential of the glucose-responsive active region. By incorporating different electron transfer agents into the active regions, different redox potentials can be produced.

[0081] A detection method for assaying creatinine and / or glucose using an analyte sensor characterized by a creatinine-responsive active region and a glucose-responsive active region on separate working electrodes may include exposing the analyte sensor to a fluid containing at least one of glucose and creatinine. The analyte sensor includes at least a first working electrode and a second working electrode, a sensor tail including a creatinine-responsive active region disposed on the surface of the first working electrode, a glucose-responsive active region disposed on the surface of the second working electrode, and a first membrane covering the creatinine-responsive active region and a second membrane covering the glucose-responsive active region. The glucose-responsive active region includes a glucose-responsive enzyme such as glucose oxidase, and the creatinine-responsive active region includes an enzyme system including at least two enzymes that can act cooperatively to facilitate the detection of creatinine.

[0082] This method may further include applying a potential to the first working electrode and applying a potential to the second working electrode, obtaining a first signal proportional to the concentration of creatinine in the fluid above the redox potential of the creatinine-responsive active region, obtaining a second signal proportional to the concentration of glucose in the fluid above the redox potential of the glucose-responsive active region, and correlating the first signal to the concentration of creatinine in the fluid and the second signal to the glucose concentration in the fluid.

[0083] According to more specific embodiments, the first signal and the second signal can be measured at different times. Thus, in such embodiments, the potential can be applied alternately to the first working electrode and the second working electrode. In other specific embodiments, the first signal and the second signal can be measured simultaneously via the first channel and the second channel, in which case the potential can be applied to both electrodes simultaneously. In either case, the signal associated with each active region can be correlated to the concentrations of creatinine and glucose in a similar manner as described above using a look-up table or a calibration function.

[0084] The embodiments disclosed herein include the following. A. A creatinine-responsive analyte sensor. The analyte sensor includes: a sensor tail including at least a first working electrode; a creatinine-responsive active region disposed on the surface of the first working electrode, the enzyme system including a first electron transfer agent, a first polymer, and a plurality of enzymes that can act cooperatively to facilitate the detection of creatinine, the enzyme system including an enzyme system including creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase; a first membrane that is permeable to creatinine and covers the creatinine-responsive active region; and an oxygen scavenger disposed on the sensor tail and near the creatinine-responsive active region.

[0085] B. Creatinine and Glucose-Responsive Analyte Sensor. The analyte sensor includes: a sensor tail including a first working electrode and a second working electrode; a creatinine-responsive active region disposed on the surface of the first working electrode, the enzyme system including a first electron transfer agent, a first polymer, and a plurality of enzymes that can cooperate to facilitate the detection of creatinine, the enzyme system including creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase; a first membrane that is permeable to creatinine and covers the creatinine-responsive active region; glucose oxidase covalently bonded to a second polymer and disposed on the first membrane; a glucose-responsive active region disposed on the surface of the second working electrode, the glucose-responsive active region including a second electron transfer agent, a third polymer, and glucose oxidase covalently bonded to the third polymer; and a second membrane that is permeable to glucose and covers the glucose-responsive active region.

[0086] C. Method for Assaying Creatinine Using an Analyte Sensor. The method includes: exposing the analyte sensor to a fluid containing at least creatinine; the analyte sensor including a sensor tail including at least a first working electrode, a creatinine-responsive active region disposed on the surface of the first working electrode, a first membrane that is permeable to creatinine and covers the creatinine-responsive active region, and an oxygen scavenger disposed on the sensor tail and near the creatinine-responsive active region; the creatinine-responsive active region being an enzyme system including a first electron transfer agent, a first polymer, and a plurality of enzymes that can cooperate to facilitate the detection of creatinine, the enzyme system including creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase; applying a potential to the first working electrode; obtaining a first signal that is proportional to the concentration of creatinine in the fluid at an oxidation-reduction potential above that of the creatinine-responsive active region; and correlating the first signal to the concentration of creatinine in the fluid. Each of Embodiments A - D may optionally have one or more of the following additional elements in any combination: Element 1: The oxygen scavenger is separated from the creatinine-responsive active region by a first membrane.

[0087] Element 2: The oxygen scavenger contains an oxidase enzyme. Element 3: The oxygen scavenger contains glucose oxidase. Element 4: The glucose oxidase is covalently bonded to a second polymer.

[0088] Element 5: The oxygen scavenger is disposed on the first membrane, optionally the oxygen scavenger contains an oxidase enzyme, optionally the oxygen scavenger contains glucose oxidase, and optionally the glucose oxidase is covalently bonded to a second polymer.

[0089] Element 6: Creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase are each covalently bonded to a first polymer. Element 7: The analyte sensor further comprises a second working electrode; a glucose-responsive active region disposed on the surface of the second working electrode, the glucose-responsive active region comprising a second electron transfer agent, a third polymer, and glucose oxidase covalently bonded to the third polymer; and a second membrane that is permeable to glucose and covers the glucose-responsive active region.

[0090] Element 8: The analyte sensor further comprises glucose oxidase covalently bonded to a second polymer and disposed on the first membrane. Element 9: The first membrane and the second membrane are compositionally the same.

[0091] Element 10: The glucose oxidase disposed on the first membrane is covered by a third membrane that is permeable to creatinine. Element 11: The first membrane, the second membrane, and the third membrane are compositionally the same.

[0092] Element 12: The oxygen scavenger is disposed on the first membrane. Element 13: The oxygen scavenger includes glucose oxidase covalently bonded to a second polymer.

[0093] Element 14: The sensor tail further includes a second working electrode having a glucose-responsive active region disposed on the surface of the second working electrode. The glucose-responsive active region includes a second electron transfer agent, a third polymer, and glucose oxidase covalently bonded to the third polymer. The method further includes: applying a potential to the second working electrode; obtaining a second signal that is proportional to the concentration of glucose in the fluid at an oxidation-reduction potential equal to or higher than that of the glucose-responsive active region; and correlating the second signal with the concentration of glucose in the fluid.

[0094] Element 15: The first signal and the second signal are obtained at different times. Element 16: The first signal and the second signal are obtained simultaneously via a first channel and a second channel.

[0095] As non-limiting examples, exemplary combinations applicable to A include: 1 and 2; 1 and 3; 1, 3 and 4; 1 and 5; 1, 5 and 6; 1 and 7; 1, 3 and 7; 1, 3, 4 and 7; 1, 5 and 7; 1 and 5-7; 1 and 8; 1, 7 and 8; 1, 3, 4 and 7; 1, 5 and 7; 1 and 5-7; 1, 6 and 7; 1, 3, 4, 6 and 7; 1, 3, 4 and 6-8; 1, 3, 4, 6, 7 and 9; 1 and 5-10; 1 and 5-11; 1 and 5-12; 2 and 3; 2-4; 2 and 6; 2 and 7; 3 and 6; 3, 6 and 7; 3 and 7; 3 and 8; 3, 7 and 8; 3, 8 and 9; 3, 7 and 8; 3 and 7-9; 3 and 8-10; 3 and 8-11; 3 and 8-12; 3, 4 and 7; 3, 4, 8 and 9; 3, 4 and 7-10; 3, 4 and 7-11; 4 and 5; 5 and 6; 5-7; 5 and 7; 7 and 9; 7, 9 and 10; 7 and 9-11; 7 and 8; and 8 and 9. Exemplary combinations applicable to B include 10 and 11; 9 and 10; 6 and 10; and 6 and 11. Exemplary combinations applicable to C include 1 and 3; 1 and 6; 1, 3 and 6; 1 and 9; 1, 3 and 9; 1, 3 and 10; 1, 3, 10 and 11; 6 and 12; 6 and 13; 6 and 14; 6 and 15; 6 and 16; 12 and 13; 12 and 14; 12 and 15; 12 and 16; 13 and 14; 13 and 15; 13 and 16; 14 and 15; and 14 and 16.

[0096] To facilitate a better understanding of the disclosure herein, the following examples of various representative embodiments are provided. The following examples should not be construed as limiting or defining the scope of the invention.

[0097] Examples A poly(vinylpyridine) - bound transition metal complex having the structure shown in Formula 1 was prepared. Further details regarding this transition metal complex and the electron transfer thereby are provided in commonly owned U.S. Patent No. 6,605,200, which is incorporated by reference above. The subscript of each monomer represents an exemplary atomic ratio and does not indicate the order of the particular monomers.

[0098]

Chemical formula

[0099] The buffer spotting formulation (10 mM MES buffer) specified in Table 1 below was deposited on the carbon working electrode to form a creatinine-responsive active region. The deposition was carried out using 15 nL of the spotting formulation to form a single spot having an area of 0.12 mm 2 . After deposition, the creatinine-responsive active region was cured overnight at 25 °C. Then, a coating solution prepared by mixing 4 mL of 35 mg / mL polyvinylpyridine-co-styrene, 0.1 mL of 100 mg / mL PEGDGE400, and 3.3 μL of PDMS in 80:20 ethanol / 10 mM HEPES buffer (pH = 8) was used to dip-coat the membrane over the creatinine-responsive active region. No curing was performed at this stage.

[0100]

Table 1

[0101] After coating the membrane over the creatinine-responsive active region, a buffer spotting formulation containing glucose oxidase as specified in Table 2 was deposited on the membrane. Specifically, 15 nL of the spotting formulation was deposited on the membrane with an area of 0.05 mm 2 , and then curing was carried out overnight at 25 °C. Then, dip-coating was performed using the same coating solution as above to form a membrane over the deposited glucose oxidase.

[0102]

Table 2

[0103] A control electrode was prepared as described above, except that the deposition of glucose oxidase on the creatinine-responsive active region was omitted. Creatinine analysis was performed by immersing the electrode in 5 mM glucose solutions containing various amounts of creatinine (20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 130 μM, and 200 μM) and measuring the current response. Control electrodes without glucose oxidase in the creatinine-responsive active region were also tested under the same conditions. Figure 8 shows exemplary plots of the current responses of three replicates of an analyte sensor containing a creatinine-responsive active region covered with glucose oxidase when exposed to various creatinine concentrations at 33 °C. As shown, the current response increased within a few minutes after exposure to the new creatinine concentration and then stabilized. In contrast, two control sensors whose creatinine-responsive active regions were not covered with glucose oxidase did not respond to creatinine at any concentration. Figure 9 shows an exemplary plot of the current response of a single analyte sensor containing a creatinine-responsive active region covered with glucose oxidase when exposed to various creatinine concentrations (20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 130 μM, and 200 μM). As shown, the response of the sensor was essentially linear over the concentration range tested. Again, the control sensors not covered with glucose oxidase essentially did not show a response to creatinine, presumably due to oxygen interference with the enzyme system.

[0104] Unless otherwise specified, all numbers expressing quantities, etc. in this specification and the related patent claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended patent claims are approximations that may vary depending upon the desired properties sought to be obtained by embodiments of the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the reported significant digits and by applying ordinary rounding techniques.

[0105] One or more exemplary embodiments incorporating various features are presented herein. For clarity, not all features of physical implementations are described or shown in this application. In developing a physical implementation incorporating embodiments of the present invention, it will be understood that numerous implementation-specific decisions must be made to achieve the developer's goals, such as compliance with system-related, business-related, government-related, and other constraints. These vary depending on the implementation and, in some cases, the circumstances. Although the developer's efforts may be time-consuming, such efforts are routine for those of ordinary skill in the art and will be beneficial from the present disclosure.

[0106] Although various systems, tools, and methods are described herein in terms of "including" various components or steps, the systems, tools, and methods can also be "consisting essentially of" or "consisting of" various components and steps.

[0107] As used herein, the phrase "at least one" preceding a series of items and accompanied by the term "and" or "or" separating any of the items modifies the entire list rather than each member of the list (i.e., each item). The phrase "at least one of" permits the meaning of including at least one of any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. By way of example, the phrases "at least one of A, B, and C" or "at least one of A, B, or C" each refer to only A, only B, or only C; any combination of A, B, and C; and / or at least one of each of A, B, and C.

[0108] Accordingly, the disclosed systems, tools, and methods are well adapted to attain the recited objects and advantages, as well as those inherent therein. Since the teachings of the present disclosure can be modified and implemented in different equivalent manners apparent to those skilled in the art having the benefit of the teachings herein, the specific embodiments disclosed above are merely illustrative. Further, it is not intended to be limited to the details of the structure or design shown herein, except as described in the appended claims. Thus, it is apparent that the specific exemplary embodiments disclosed above can be varied, combined, or modified, and all such variations are considered to be within the scope of the present disclosure. The systems, tools, and methods illustratively disclosed herein can be suitably implemented without the elements specifically disclosed herein and / or without any optional elements disclosed herein. The systems, tools, and methods are described from the perspective of "comprising" various components or steps, but the systems, tools, and methods can also "consist essentially of" or "consist of" various components and steps. All numbers and ranges disclosed above may vary somewhat. Whenever a numerical range having a lower and upper limit is disclosed, any number and any included range within that range are specifically disclosed. In particular, all range values disclosed herein (in the form of "about a to about b", or equivalently "about a to b", or equivalently "about a~b") should be understood as describing all numbers and ranges included within the broader range of values. Also, unless expressly and unambiguously defined by the patentee, the terms of the claims have their ordinary and original meaning. In the event of any conflict in the usage of words or terms between this specification and one or more patent documents or other documents that may be incorporated herein by reference, the definitions in this specification shall prevail.

Claims

1. An analyte sensor for detecting creatinine in vivo, comprising: a first working electrode; a creatinine-responsive active region for detecting creatinine, disposed on the surface of the first working electrode, creatinine amidohydrolase, creatinine amidinohydrolase, and sarcosine oxidase the creatinine-responsive active region comprising an enzyme system containing and a first electron transfer agent; and an oxygen scavenger disposed near the creatinine-responsive active region, wherein the analyte sensor is configured to be at least partially inserted into a user's skin such that a distal portion of the analyte sensor contacts interstitial fluid, thereby detecting creatinine in vivo.

2. The analyte sensor according to claim 1, wherein the oxygen scavenger comprises an oxidase enzyme.

3. The analyte sensor according to claim 1 or 2, wherein the oxygen scavenger comprises glucose oxidase.

4. The analyte sensor according to any one of claims 1 to 3, wherein the creatinine-responsive active region comprises a first polymer, and creatinine amidohydrolase, creatinine amidinohydrolase, and sarcosine oxidase are each covalently bonded to the first polymer.

5. The analyte sensor according to any one of claims 1 to 4, further comprising a first mass transfer limiting membrane disposed on the creatinine-responsive active region, the first mass transfer limiting membrane restricting the creatinine flux to the creatinine-responsive active region and electrically isolating the oxygen scavenger from the creatinine-responsive active region.

6. The analyte sensor according to claim 5, wherein the oxygen scavenger is separated from the creatinine-responsive active region by the first mass transfer limiting membrane.

7. The analyte sensor according to claim 5, wherein the oxygen scavenger is disposed on the first mass transfer limiting membrane.

8. The analyte sensor according to any one of claims 5 to 7, wherein a second mass transfer limiting membrane is disposed on the oxygen scavenger and the first mass transfer limiting membrane.

9. a second working electrode, and a glucose-responsive active region for detecting glucose, disposed on the surface of the second working electrode Further comprising, wherein the glucose-responsive active region contains glucose oxidase, the analyte sensor according to any one of claims 5 to 7.

10. A second mass transfer limiting membrane disposed on the glucose-responsive active region, further comprising the second mass transfer limiting membrane for limiting the glucose flux to the glucose-responsive active region, the analyte sensor according to claim 9.

11. The analyte sensor according to claim 10, wherein the first mass transfer limiting membrane and the second mass transfer limiting membrane have the same composition.

12. The analyte sensor according to claim 10 or 11, wherein the oxygen scavenger is covered by a third mass transfer limiting membrane for limiting the creatinine flux to the creatinine-responsive active region.

13. The analyte sensor according to claim 12, wherein the first mass transfer limiting membrane, the second mass transfer limiting membrane, and the third mass transfer limiting membrane are compositionally the same.

14. A method for assaying creatinine in vivo, exposing the analyte sensor according to any one of claims 1 to 13 to a fluid containing at least creatinine, applying a potential to the first working electrode, acquiring a first signal proportional to the concentration of creatinine in the fluid at an oxidation-reduction potential equal to or higher than that of the creatinine-responsive active region, and determining the concentration of creatinine in the fluid based on the first signal A method comprising.

15. A method for assaying glucose and creatinine in vivo, exposing the analyte sensor according to any one of claims 9 to 13 to a fluid containing glucose and creatinine, applying a potential to the first working electrode and the second working electrode, acquiring a first signal proportional to the concentration of creatinine in the fluid at an oxidation-reduction potential equal to or higher than that of the creatinine-responsive active region, acquiring a second signal proportional to the concentration of glucose in the fluid at an oxidation-reduction potential equal to or higher than that of the glucose-responsive active region, determining the concentration of creatinine in the fluid based on the first signal, and determining the concentration of glucose in the fluid based on the second signal A method comprising.

16. The method according to claim 15, wherein the first signal and the second signal are acquired at different times.

17. The method according to claim 15, wherein the first signal and the second signal are simultaneously acquired via a first channel and a second channel.

Images