Analyte sensor and sensing method for detecting creatinine

Abstract

Creatinine levels may be monitored as a measure of renal function. Typically, blood and / or urine testing is used for this purpose. An analyte sensor capable of monitoring creatinine in vivo may include a sensor tail including at least a first working electrode, a creatinine-responsive active region disposed on a surface of the first working electrode, a first membrane permeable to creatinine and covering the creatinine-responsive active region, and a second membrane permeable to creatinine and covering the creatinine-responsive active region. And an oxygen scavenger located aft of the sensor and proximate the creatinine responsive active region. The creatinine-responsive active region comprises a first electron transfer agent, a first polymer, and an enzyme system comprising a plurality of enzymes capable of acting synergistically to facilitate creatinine detection, particularly creatinine amide hydrolase, creatinine amidino hydrolase, and creatinine oxidase. Oxidase may be used as an oxygen scavenger, especially when detecting creatinine in fluids that also contain glucose, glucose oxidase may be used as an oxygen scavenger.

Description

[0001] This application is a divisional application of CN201980090556.5.

[0002] background

[0003] Measuring various analytes in an individual's body is sometimes essential to monitoring their health and well-being. Deviations from normal analyte levels often indicate an underlying physiological condition, such as a metabolic condition or disease, or exposure to a specific environmental condition. While a single analyte may be individually deregulated for a given physiological condition, there are times when multiple analytes are simultaneously deregulated, either due to the same physiological condition or due to comorbid (related) physiological conditions. When multiple analytes are simultaneously deregulated, the degree of deregulation may be different for each analyte. Therefore, it may be necessary to monitor each analyte to obtain a satisfactory assessment of an individual's health status.

[0004] Periodic ex vivo analyte monitoring using extracted body fluids may be sufficient to observe a given physiological condition for many individuals. However, ex vivo analyte monitoring may be inconvenient or painful for some people, particularly if body fluids need to be extracted or collected fairly frequently (e.g., several times a day). Continuous analyte monitoring using implanted in vivo analyte sensors may be a more ideal approach for individuals with severe analyte imbalances and / or rapidly fluctuating analyte levels, but it may also be beneficial to other individuals due to the convenience provided. Continuous analyte monitoring may allow an individual or physician to proactively address abnormal analyte levels before they have a chance to result in more serious health consequences (e.g., organ damage or failure). In many cases, subcutaneous, interstitial, or skin analyte sensors can provide sufficient measurement accuracy for this purpose while minimizing user discomfort.

[0005] Many analytes represent interesting targets for physiological analysis, provided that suitable detection chemistries can be identified. To this end, amperometric sensors configured for in vivo determination of glucose have been developed and improved in recent years to aid in monitoring the health status of diabetic individuals. Other analytes that are commonly deregulated in conjunction with glucose in diabetic individuals include, for example, lactate, oxygen, pH, A1c, ketones, etc. Sensors configured to detect analytes that are commonly deregulated in conjunction with glucose are known, but are currently rather crude.

[0006] In vivo analyte sensors are typically configured to analyze a single analyte to provide a specific analysis, typically using enzymes to provide high specificity for a given analyte. Due to this analytical specificity, existing in vivo analyte sensors configured to measure glucose are typically ineffective for analyzing other analytes that are often imbalanced in combination with glucose or caused by imbalanced glucose levels. At best, existing analyte monitoring methods require diabetic individuals to wear two different in vivo analyte sensors, one configured to measure glucose and the other configured to measure another analyte of interest. Analyte monitoring methods using multiple in vivo analyte sensors may be very inconvenient for users. In addition, when multiple in vivo analyte sensors are used for analyte monitoring, the cost burden of the equipment increases, and the statistical probability of at least one of the individual in vivo analyte sensors failing increases.

[0007] Individuals with diabetes are often particularly susceptible to comorbidities, which may be the result of poor management of their insulin levels or even of having well-managed diabetes for a long time. For example, diabetic neuropathy may be caused by high blood sugar levels and ultimately lead to kidney failure. Diabetic neuropathy is the leading cause of kidney failure in the United States, and a large number of diabetic individuals experience diabetic neuropathy within the first 10-20 years of illness. Diagnostic tests for assessing kidney function are currently based on the measurement of elevated creatinine levels in blood and / or urine samples. Although it is desirable to detect potential kidney failure as quickly as possible, current diagnostic test methods are typically performed over a long period of time (months to years) to verify whether creatinine levels remain elevated or are trending upward over time. If abnormal kidney function is not detected early, the infrequency of routine creatinine monitoring may increase the risk of kidney failure.

[0008] BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The following drawings are included to illustrate certain aspects of the present disclosure and should not be considered to be 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.

[0010] Figure 1 A diagram of an exemplary sensing system that may incorporate an analyte sensor of the present disclosure is shown.

[0011] Figure 2A and 2B An example of a cooperative enzyme system that can be used to detect creatinine according to the present disclosure is shown.

[0012] Figures 3A-3C Shown is a cross-sectional view of an exemplary analyte sensor having an active area suitable for detecting creatinine.

[0013] Figures 4A-4C A cross-sectional view of an exemplary analyte sensor having a single working electrode and an active area suitable for detecting creatinine and glucose is shown.

[0014] Figure 5 A cross-sectional view of an exemplary analyte sensor having two working electrodes and an active area suitable for detecting creatinine and glucose is shown.

[0015] Figures 6A-6D Shown is a perspective view of an exemplary analyte sensor featuring electrodes that are concentrically disposed relative to each other.

[0016] Fig. 7A and 7B A diagram of a creatinine-responsive active area having a membrane and an oxygen scavenger disposed thereon is shown.

[0017] Figure 8 Shown are exemplary graphs of three replicate current responses of a sensor comprising a creatinine-responsive active area covered with glucose oxidase when exposed to different creatinine concentrations.

[0018] Fig. 9 Shown are exemplary graphs of the current response of a single sensor comprising a creatinine-responsive active area covered with glucose oxidase when exposed to various creatinine concentrations.

[0019] Details

[0020] The present disclosure generally describes analyte sensors that use multiple enzymes to detect one or more analytes, and more specifically, describes analyte sensors that use multiple enzymes to detect at least creatinine and optionally other analytes and corresponding methods of use.

[0021] As discussed above, due to the frequent specificity of enzymes for particular substrates or substrate classes, analyte sensors employing enzymes are typically used to monitor a single analyte, such as glucose. Other analytes may also be monitored, provided that a suitable detection chemistry can be identified. Monitoring of multiple analytes may be complicated by the need to use a corresponding number of analyte sensors to detect each analyte separately. This approach may be problematic or undesirable, particularly when monitoring multiple analytes in vivo, due to issues such as the cost of multiple analyte sensors, user discomfort when wearing multiple analyte sensors, and the increased statistical likelihood of individual analyte sensor failures.

[0022] Glucose-responsive analyte sensors are a well-studied and still developing field to help diabetic individuals better manage their health. Despite the prevalence of comorbid conditions in diabetic individuals, sensor chemistries suitable for in vivo monitoring of other analytes that commonly bind dysregulated glucose have lagged significantly behind the more developed glucose sensing chemistry. For example, creatinine may be an analyte of particular interest for monitoring individuals susceptible to renal failure, especially those with diabetes who are at risk for diabetic neuropathy.

[0023] The present disclosure provides an analyte sensor responsive to creatinine. Specifically, the present disclosure provides an analyte sensor that can be worn on the body to continuously or nearly continuously monitor creatinine levels in vivo. Analyzing creatinine levels with the analyte sensor disclosed herein can provide a more accurate characterization of renal function for an individual or a medical care provider over an extended period of time than regular ex vivo laboratory measurements. By analyzing creatinine levels according to the present disclosure, earlier health care interventions can make it possible to limit potential renal damage and improve the overall health outcomes of an individual.

[0024] It is not feasible to use a single enzymatic reaction to electrochemically detect creatinine because there is no known enzyme that can transfer electrons directly from creatinine to the working electrode. The present disclosure alleviates this defect by providing a sensor chemical substance suitable for detecting creatinine with good response stability within a certain range of creatinine concentrations. Specifically, the present disclosure utilizes an enzyme system comprising multiple enzymes that can synergize to promote creatinine detection. As used herein, the term "synergy" refers to a coupled enzymatic reaction, wherein the product of the first enzymatic reaction becomes the substrate of the second enzymatic reaction, and the second enzymatic reaction is used as a basis for measuring the concentration of the substrate (analyte) reacted during the first enzymatic reaction. Although defined according to two coupled enzymatic reactions, it should be understood that more than two enzymatic reactions can be coupled in some cases. For example, the product of the first enzymatic reaction may become the substrate of the second enzymatic reaction, and the product of the second enzymatic reaction may become the substrate of the third enzymatic reaction, and the third enzymatic reaction is used as a basis for determining the concentration of the substrate (analyte) reacted during the first enzymatic reaction. As discussed further below, suitable enzyme systems for detecting creatinine according to the present disclosure employ three enzymes acting in concert, along with a fourth enzyme or other oxygen scavenger to facilitate oxygen scavenging. The fourth enzyme or other oxygen scavenger does not directly participate in the concerted enzymatic reaction, but rather prevents unwanted side reactions with oxygen.

[0025] When a single enzyme cannot facilitate detection, such as in the case of creatinine, it may be necessary to utilize two or more enzymes that act synergistically with each other to detect the analyte of interest. Situations where a single enzyme may be ineffective for facilitating analyte detection include, for example, situations where the enzyme is inhibited by one or more reaction products, cannot cycle between an oxidized state and a reduced state when placed in an analyte sensor, and / or where the required reaction pathways required to facilitate detection are unknown. In the case of creatinine, the enzymatic conversion of creatinine to creatine occurs hydrolytically and does not result in a change in oxidation state to provide current to the working electrode to facilitate detection of the analyte. Enzyme systems comprising multiple enzymes that act synergistically as disclosed herein can alleviate this difficulty.

[0026] The creatinine sensors disclosed herein may be useful for monitoring creatinine levels (and renal function) of any individual who may be at risk of renal damage or failure, but due to the prevalence of diabetic neuropathy, they may be particularly beneficial for diabetic individuals. Although monitoring creatinine levels alone may be beneficial, diabetic individuals may also monitor their glucose and creatinine levels to improve health outcomes, especially considering that diabetic individuals have routinely performed glucose monitoring. The present disclosure provides monitoring of glucose and creatinine using one or more in vivo analyte sensors responsive to each analyte, and in a particularly advantageous configuration, a single analyte sensor responsive to two analytes in vivo may be used. Advantageously and unexpectedly, an analyte sensor combining sensing functions for both glucose and creatinine on a single sensor tail can be manufactured by adopting the disclosure herein.

[0027] As referenced below Figure 2A and 2BFurther discussed, the creatinine response active region of the present disclosure can utilize oxygen scavengers to promote the detection of creatinine using the enzyme system shown therein. In some sensor configurations, oxidases can be used as oxygen scavengers. Glucose oxidase may be a particularly advantageous oxygen scavenger because glucose is widely present in body fluids that also contain creatinine, in which case glucose can be used as a reagent for removing oxygen (see reaction 1 below). The oxygen scavenger can be insulated from the creatinine response active region by a membrane so as not to generate a signal at the working electrode with the creatinine response active region (i.e., when removing oxygen by promoting an oxidation reaction). The oxygen scavenger can be located on the membrane to promote effective oxygen removal in the creatinine response active region. In addition to being arranged on the membrane, the oxygen scavenger can be located on a second position on the sensor tail away from the membrane, wherein the oxygen scavenger can play different roles (e.g., by promoting the detection of the glucose response active region) away from the position. Depending on the placement and position away from the oxygen scavenger, in addition to its oxygen removal function, the oxygen scavenger can be active or inactive for promoting the detection of another analyte (particularly glucose). When inactive for promoting glucose detection, the glucose oxidase can be insulated from the working electrode so that the oxidation reaction promoted by the enzyme (scavenging oxygen) does not result in the generation of current on the working electrode. When the glucose oxidase is active for promoting glucose detection and scavenging oxygen, the glucose oxidase can be present in a glucose-responsive active region that is located on a second working electrode or on a working electrode with a creatinine-responsive active region so that a separate signal can be obtained from each region. The strategy of simultaneously providing a creatinine-responsive active region and a glucose-responsive active region at the tail of a single sensor is further discussed below.

[0028] Even with the right detection chemistry at hand, combining two different types of active areas on a single analyte sensor is sometimes not a simple matter. Analyte sensors often employ a membrane covering the active area to act as a mass transfer limiting membrane and / or to improve biocompatibility. Using a mass transfer limiting membrane to limit access of the analyte to the active area helps to avoid sensor overload (saturation), thereby improving detection performance and accuracy. When a single analyte sensor is used to measure multiple analytes, different analytes may exhibit different permeability values ​​through a given mass transfer limiting membrane, which may result in very different sensitivities for each analyte. In some cases, combining a different mass transfer limiting membrane on each active area may be problematic. Surprisingly and advantageously, glucose and creatinine can be successfully analyzed using a mass transfer limiting membrane of the same composition at each location, thereby simplifying the manufacture of analyte sensors with detection capabilities for both analytes.

[0029] Before describing the analyte sensors of the present disclosure in greater detail, a brief overview of suitable in vivo analyte sensor configurations and sensor systems employing analyte sensors will first be provided so that embodiments of the present disclosure may be better understood. Figure 1 Shown is a diagram of an exemplary sensing system of an analyte sensor that can be combined with the present disclosure, particularly including a creatinine response active area and an optional glucose response active area. As shown, sensing system 100 includes sensor control device 102 and reader device 120, which are configured to communicate with each other through local communication paths or links, and local communication paths or links can be wired or wireless, unidirectional or bidirectional, encrypted or non-encrypted. According to some embodiments, reader device 120 can be configured to view the analyte concentration determined by sensor 104 or a processor associated therewith and an output medium for alarm or notification and allowing one or more user inputs. Reader device 120 can be a multi-function smart phone or a dedicated electronic reader instrument. Although only one reader device 120 is shown, multiple reader devices 120 can be present in some cases. Reader device 120 can also communicate with remote terminal 170 and / or trusted computer system 180 via communication path / link 141 and / or 142, respectively, and communication path / link 141 and / or 142 can also be wired or wireless, unidirectional or bidirectional, encrypted or non-encrypted. The reader device 120 may also or alternatively communicate with a network 150 (e.g., a mobile phone network, the Internet, or a cloud server) via a communication path / link 151. The network 150 may also be communicatively coupled to a remote terminal 170 via a communication path / link 152 and / or to a trusted computer system 180 via a communication path / link 153. Alternatively, the sensor 104 may communicate directly with the remote terminal 170 and / or the trusted computer system 180 without the presence of an intermediate reader device 120. For example, according to some embodiments, the sensor 104 may communicate with the remote terminal 170 and / or the trusted computer system 180 via a direct communication link to the network 150, as described in U.S. Patent Application Publication 2011 / 0213225 and incorporated herein by reference in its entirety. Any suitable electronic communication protocol may be used for each communication path or link, such as near field communication (NFC), radio frequency identification (RFID), or Low energy protocols, WiFi, etc. According to some embodiments, remote terminal 170 and / or trusted computer system 180 may be accessed by individuals other than the primary user who are interested in the user's analyte level. Reader device 120 may include a display 122 and an optional input component 121. According to some embodiments, display 122 may include a touch screen interface.

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

[0031] The sensor 104 is adapted to be at least partially inserted into a tissue of interest, such as within the dermis or subcutaneous layer of the skin. The sensor 104 may include a sensor tail of sufficient length to be inserted to a desired depth in a given tissue. The sensor tail may include at least one working electrode and a creatinine responsive active region disposed thereon. Optionally, a glucose responsive active region, further optionally in combination with a second working electrode, may be located in the sensor tail to facilitate detection of the analyte. A counter electrode may be present in combination with at least one working electrode. Reference Figures 3A-7B The specific electrode configuration on the sensor tail is described in more detail below.

[0032] At least one mass transfer limiting membrane can cover the creatinine response active region and the optional glucose response active region (when present) also described in further detail below. The glucose response active region (when present) can include a glucose response enzyme. The mass transfer limiting membrane can also cover an oxygen scavenger (e.g., glucose oxidase), in which case the oxygen scavenger can be inserted between the membrane layers separated.

[0033] As referenced below Figure 2A and 2B Described, creatinine response active region may comprise an enzyme system, and this enzyme system comprises multiple enzymes that can act synergistically to promote the detection of creatinine. According to various embodiments, creatinine response active region and glucose response active region (when present) may comprise a polymer, which is covalently bonded to the enzyme. Glucose oxidase located outside the glucose response active region may also be covalently bonded to the polymer in the analyte sensor disclosed herein. According to the present disclosure, creatinine and optional glucose can be monitored in any biological fluid of interest, such as: dermal fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, etc. In specific embodiments, the analyte sensor of the present disclosure may be applicable to measuring dermal fluid or interstitial fluid to determine the concentration of creatinine and / or glucose in vivo.

[0034] Refer to Figure 1, the sensor 104 can automatically forward the data to the reader device 120. For example, analyte concentration data (i.e., creatinine and / or glucose concentration) can be automatically and periodically communicated, such as when the data is obtained or the data is stored in a memory before transmission and communicated at a certain frequency (e.g., every minute, every five minutes or other predetermined time period) after a certain time period has passed. In other embodiments, the sensor 104 can communicate with the reader device 120 in a non-automatic manner rather than according to a set schedule. For example, when the sensor electronics enter the communication range of the reader device 120, RFID technology can be used to communicate data from the sensor 104. Until the communication with the reader device 120 is completed, the data can remain stored in the memory of the sensor 104. Therefore, the user does not have to stay close to the reader device 120 all the time, but can upload data at a convenient time. In other embodiments, a combination of automatic and non-automatic data transmission can be implemented. For example, data transmission can continue on an automatic basis until the reader device 120 is no longer within the communication range of the sensor 104.

[0035] The introducer may temporarily exist to facilitate the introduction of the sensor 104 into the tissue. In an exemplary embodiment, the introducer may include a needle or similar sharp. It should be appreciated that other types of introducers, such as a cannula or a blade, may exist in alternative embodiments. More specifically, a needle or other introducer may temporarily reside near the sensor 104 before the tissue is inserted, and then withdrawn afterwards. When present, a needle or other introducer may facilitate the insertion of the sensor 104 into the tissue by opening an entry path for the sensor 104 to follow. For example, according to one or more embodiments, a needle may facilitate the penetration of the epidermis as a path into the dermis to allow the sensor 104 to be implanted. After opening the entry path, the needle or other introducer may be withdrawn so that there is no sharp danger. In an exemplary embodiment, the cross-section of a suitable needle may be solid or hollow, beveled or non-beveled, and / or circular or non-circular. In a more specific embodiment, a suitable needle may be comparable to an acupuncture needle in terms of cross-sectional diameter and / or tip design, and an acupuncture needle may have a cross-sectional diameter of about 250 microns. However, it will be appreciated that a suitable needle may have a larger or smaller cross-sectional diameter if desired for a particular application.

[0036] In some embodiments, the tip of the needle (when present) may be angled above the end of the sensor 104 so that the needle first penetrates the tissue and opens an access path for the sensor 104. In other exemplary embodiments, the sensor 104 may reside within a lumen or groove of the needle, which similarly opens an access path for the sensor 104. In either case, the needle is subsequently withdrawn after facilitating sensor insertion.

[0037] Reference Figure 2A and2B Further details are provided for suitable enzyme systems that can be used to detect creatinine according to the disclosure herein. As shown, creatinine can undergo a reversible and hydrolysis reaction to form creatine in the presence of creatinine amidohydrolase (CNH). Creatine can then be catalytically hydrolyzed in the presence of creatine amidinohydrolase (CRH) to form sarcosine. None of these reactions will generate an electron flow (e.g., oxidation or reduction) to provide a basis for the electrochemical detection of creatinine.

[0038] like Figure 2A and 2B As further shown, sarcosine produced by creatine hydrolysis can be oxidized in the presence of an oxidized form of sarcosine oxidase (SOX-ox) to form glycine and formaldehyde, thereby generating a reduced form of sarcosine oxidase (SOX-red) in the process. Hydrogen peroxide ( Figure 2B The reduced form of sarcosine oxidase can then be reoxidized in the presence of an oxidized form of an electron transfer agent (e.g., Os(III)) to produce the corresponding reduced form of the electron transfer agent (e.g., Os(II)) and deliver electron flow to the working electrode.

[0039] Oxygen may interfere with the coordinated reaction sequence for detecting creatinine according to the present disclosure. Specifically, Figure 2B As shown, the reduced form of sarcosine oxidase can react with oxygen to reform the corresponding oxidized form of the enzyme, but without exchanging electrons with the electron transfer agent. Although the enzyme remains active when reacting with oxygen, no electrons flow to the working electrode. Without being bound by theory or mechanism, it is believed that the competitive reaction with oxygen is caused by kinetic effects. That is, it is believed that the oxidation of the reduced form of sarcosine oxidase by oxygen occurs faster than oxidation promoted by the electron transfer agent. Hydrogen peroxide is also formed in the presence of oxygen.

[0040] exist Figure 2A The desired reaction pathway for facilitating creatinine detection is shown in . Oxidation of the reduced form of sarcosine oxidase can be facilitated by including an oxygen scavenger in the vicinity of the enzyme system. As discussed above, a variety of oxygen scavengers and their disposal may be suitable, including oxidases such as glucose oxidase. Small molecule oxygen scavengers may also be suitable, but they may be completely consumed before the sensor life is otherwise completely exhausted. In contrast, the enzyme may undergo reversible oxidation and reduction, thereby providing a longer sensor life. By preventing the reduced form of sarcosine oxidase from being oxidized by oxygen, a slower electron exchange reaction with an electron transfer agent can occur, allowing current to be generated at the working electrode. The magnitude of the current generated is proportional to the amount of creatinine initially reacted.

[0041] In any embodiment of the present disclosure, for promoting Figure 2AThe oxygen scavenger for the desired reaction pathway in the present invention can be an oxidase. Any oxidase can be used to promote oxygen scavenging near the enzyme system, provided that a suitable substrate for the enzyme is also present in the fluid containing creatinine, thereby providing a reagent that reacts with oxygen in the presence of the oxidase. Oxidases that may be suitable for oxygen scavenging in the present disclosure include, but are not limited to, glucose oxidase, lactate oxidase, xanthine oxidase, and the like. Since glucose is readily available in various body fluids, glucose oxidase may be an oxidase that is particularly suitable for use in the present disclosure. Reaction 1 below shows an enzymatic reaction promoted by glucose oxidase to provide oxygen scavenging.

[0042] β-D-glucose + O2--→ D-glucono-1,5-lactone + H2O2

[0043] Reaction 1

[0044] The concentration of available lactate in the body is lower than that of glucose, but still sufficient to promote oxygen removal.

[0045] The oxidase (e.g., glucose oxidase) can be located at any position suitable for promoting oxygen removal in the analyte sensor disclosed herein. For example, the glucose oxidase can be located on the tail of the sensor so that the glucose oxidase is functional and / or non-functional for promoting glucose detection. When it is non-functional for promoting glucose detection, the glucose oxidase can be located on the tail of the sensor so that the electrons generated during glucose oxidation cannot reach the working electrode that receives the electrons generated when oxidizing creatine. The method of insulating the glucose oxidase from the working electrode is described in more detail below. When it is functional for promoting glucose detection, the glucose oxidase can be located in the glucose response active region on the working electrode, so that in addition to removing oxygen near the creatinine response active region, the working electrode receives the electrons generated during the glucose oxidation process. The working electrode having the glucose response active region thereon can be the same working electrode as the working electrode having the creatinine response active region or a different working electrode. Suitable methods for arranging glucose oxidase in the glucose response active region on a particular working electrode are also discussed below. Any combination of the aforementioned methods for arranging glucose oxidase on the tail of the sensor can be used in the analyte sensor disclosed herein.

[0046] Figure 2A An alternative detection strategy to that depicted in can omit the glucose oxidase, the membrane insulating the glucose oxidase from the working electrode, and the electron transfer agent. In such a detection method, creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase can work in concert as depicted in the figure, where oxygen promotes the formation of hydrogen peroxide and interconverts the oxidized and reduced forms of sarcosine oxidase. Hydrogen peroxide can be detected at the working electrode as a basis for determining creatinine in this type of sensor configuration.

[0047] The analyte sensors disclosed herein are characterized by at least one creatinine responsive active area on a working electrode, combined with at least one additional electrode, which can be a counter electrode, a reference electrode and / or a counter electrode / reference electrode. Analyte sensors characterized by a creatinine responsive active area and a glucose responsive active area can combine the creatinine responsive active area and the glucose responsive active area on separate working electrodes or on the same working electrode. Exemplary configurations of each possibility are discussed below.

[0048] Sensor configurations featuring creatinine-responsive active regions instead of glucose-responsive active regions can employ two-electrode or three-electrode detection phantoms, such as those described in reference Figures 3A-3C Sensor configurations featuring both creatinine-responsive active regions and glucose-responsive active regions on separate working electrodes or on the same working electrode are described below with reference to Figures 4A-6D Sensor configurations with multiple working electrodes may be particularly advantageous for combining creatinine-responsive and glucose-responsive active regions in the same sensor tail, as the signal contribution from each active region can be more easily determined.

[0049] When there is a single working electrode in the analyte sensor, the three-electrode sensor configuration may include a working electrode, a counter electrode and a reference electrode. The related two-electrode sensor configuration may include a working electrode and a second electrode, wherein the second electrode may be used as a counter electrode and a reference electrode (i.e., a counter electrode / reference electrode). Various electrodes may be at least partially stacked (layered) on each other and / or laterally spaced apart from each other on the sensor tail. Suitable sensor configurations may be substantially flat shapes or substantially cylindrical shapes, wherein the creatinine response active region and the optional glucose response active region are laterally spaced apart on the working electrode. In any sensor configuration disclosed herein, various electrodes may be insulated from each other by dielectric materials or similar insulators.

[0050] An analyte sensor featuring multiple working electrodes may similarly include at least one additional electrode. When one additional electrode is present, one additional electrode may be used as a counter electrode / reference electrode for each of the multiple working electrodes. When two additional electrodes are present, one of the additional electrodes may be used as a counter electrode for each of the multiple working electrodes, and the other of the additional electrodes may be used as a reference electrode for each of the multiple working electrodes.

[0051] Figure 3AA diagram of an exemplary dual electrode analyte sensor configuration is shown, which is compatible with the disclosure herein. As shown, the analyte sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter electrode / reference electrode 216. Alternatively, the working electrode 214 and the counter electrode / reference electrode 216 can be located on the same side of the substrate 212, with a dielectric material interposed therebetween (configuration not shown). A 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 points or a single point configured to detect creatinine, as further discussed herein.

[0052] Refer to Figure 3A According to some embodiments, the membrane 220 covers at least the creatinine response active area 218, and may optionally cover part or all of the working electrode 214 and / or the counter electrode / reference electrode 216, or the entire analyte sensor 200. One or both faces of the analyte sensor 200 may be covered with the membrane 220. The membrane 220 may include one or more polymer membrane materials having the ability to limit the flow of the analyte to the active area 218 (i.e., the membrane 220 is a mass transfer limiting membrane with a certain permeability to creatinine). The composition and thickness of the membrane 220 may vary to facilitate the desired creatinine flux to the creatinine response active area 218, thereby providing the desired signal strength and stability. The analyte sensor 200 may be operated by any of coulometric, amperometric, voltammetric or potentiometric electrochemical detection techniques to measure creatinine.

[0053] Figure 3B and 3C FIG. 2 shows an exemplary three-electrode analyte sensor configuration, which is also compatible with the disclosure herein. In addition to including additional electrodes 217 ( Figure 3B and 3C ), the three-electrode analyte sensor configuration can be similar to Figure 3A 200. Using the additional electrode 217, the counter electrode / reference electrode 216 can then be used as a counter electrode or a reference electrode, and the additional electrode 217 performs other electrode functions that are not otherwise considered. The working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed on the working electrode 214 or the electrode 216 with an insulating layer of dielectric material therebetween. For example, as Figure 3B As depicted in FIG. 1 , dielectric layers 219a, 219b, and 219c separate and insulate electrodes 214, 216, and 217 from one another. Alternatively, as Figure 3CAs shown, at least one of electrodes 214, 216, and 217 may be located on opposite sides of substrate 212. Therefore, in some embodiments, electrode 214 (working electrode) and electrode 216 (counter electrode) may be located on opposite sides of substrate 212, and electrode 217 (reference electrode) may be located on one of electrodes 214 or 216 and separated therefrom by a dielectric material. Reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, and the location of reference material layer 230 is not limited to Figure 3B and 3C As depicted in Figure 3A As shown in sensor 200 in FIG. 1 , the creatinine responsive active area 218 in analyte sensors 201 and 202 may include multiple points or a single point. In addition, analyte sensors 201 and 202 may be operated by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques to measure creatinine.

[0054] As with analyte sensor 200, membrane 220 may also cover creatinine responsive active area 218 and other sensor components in analyte sensors 201 and 202, thereby acting as a mass transfer limiting membrane. In some embodiments, additional electrode 217 may be covered with membrane 220. Figure 3B and 3C All electrodes 214, 216, and 217 have been depicted as being covered by membrane 220, but it should be appreciated that in some embodiments only working electrode 214 may be covered. In addition, the thickness of membrane 220 at each electrode 214, 216, and 217 may be the same or different. Figure 3A ), one or both faces of the analyte sensors 201 and 202 may be Figure 3B and 3C In the sensor configuration of , the membrane 220 may be covered, or the entire analyte sensor 201 and 202 may be covered. Figure 3B and 3C The three-electrode sensor configuration shown in should be understood to be non-limiting to the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of the present disclosure.

[0055] An analyte sensor having a creatinine-responsive active region and a glucose-responsive active region on a single working electrode or on multiple working electrodes will refer to Figures 4A-6D Described in further detail.

[0056] Figure 4AAn exemplary configuration of a sensor 203 having a single working electrode with a creatinine-responsive active region and a glucose-responsive active region disposed thereon is shown. In addition to the two active regions present on the working electrode 214, creatinine-responsive active region 218a and glucose-responsive active region 218b, which are laterally spaced apart from each other on the surface of the working electrode 214, Figure 4A Similar to Figure 3. Active areas 218a and 218b may contain multiple spots or a single spot configured for detecting each analyte. The composition of membrane 220 may vary or be identical in composition at active areas 218a and 218b.

[0057] Figure 4B and 4C Cross-sectional views of exemplary three-electrode sensor configurations are shown for sensors 204 and 205, respectively, each featuring a single working electrode having both a creatinine-responsive active region 218a and a glucose-responsive active region 218b disposed thereon. Figure 4B and 4C In other respects similar to Figure 3B and 3C And by reference Figure 3B and 3C can be better understood. Figure 4A As shown, the composition of the film 220 may vary or be compositionally identical at the active regions 218a and 218b.

[0058] An exemplary sensor configuration with multiple working electrodes, in particular two working electrodes, is shown in FIG. Figure 5-6D Further detailed description. Although the following description is mainly directed to a sensor configuration with two working electrodes, it should be understood that more than two working electrodes can be incorporated by extension of the disclosure herein. In addition to creatinine and glucose sensing, additional working electrodes can be used to give the analyte sensor additional sensing capabilities.

[0059] Figure 5A cross-sectional view of an exemplary analyte sensor configuration having two working electrodes, one reference electrode and one counter electrode is shown, which is compatible with the disclosure herein. As shown, the analyte sensor 300 includes working electrodes 304 and 306 disposed on opposite sides of a substrate 302. A creatinine-responsive active region 310a is disposed on the surface of the working electrode 304, and a glucose-responsive active region 310b is disposed on the surface of the working electrode 306. The counter electrode 320 is insulated from the working electrode 304 by a dielectric layer 322, while the reference electrode 321 is insulated from the working electrode 306 by a dielectric layer 323. Outer dielectric layers 330 and 332 are located on the reference electrode 321 and the counter electrode 320, respectively. According to various embodiments, a membrane 340 may cover at least the active regions 310a and 310b, and other components of the analyte sensor 300 or the entire analyte sensor 300 may also be optionally covered with a membrane 340. Again, if desired, membrane 340 may vary in composition at active regions 310a and 310b in order to provide suitable permeability values ​​to differentially modulate analyte flux at each location.

[0060] Having multiple working electrodes and Figure 5 Alternative sensor configurations that differ from the configuration shown may feature counter / reference electrodes rather than separate counter and reference electrodes 320, 321, and / or feature layer and / or film arrangements different from those explicitly depicted. For example, the positioning of the counter electrode 320 and the reference electrode 321 may be different from Figure 5 Furthermore, working electrodes 304 and 306 need not be located on opposite sides of substrate 302 in the manner shown in FIG.

[0061] While suitable sensor configurations may be characterized by electrodes that are substantially planar in nature, it should be understood that sensor configurations that are characterized by non-planar electrodes may be advantageous and particularly suitable for use with the disclosure herein. Specifically, as described below, substantially cylindrical electrodes arranged concentrically with each other can facilitate the deposition of mass transport limiting films. Figures 6A-6D A perspective view of an analyte sensor is shown featuring two working electrodes arranged concentrically relative to 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.

[0062] Fig. 6AA perspective view of an exemplary sensor configuration is shown, wherein a plurality of electrodes are substantially cylindrical and are concentrically arranged relative to each other around a central substrate. As shown, the analyte sensor 400 includes a central substrate 402, and all electrodes and dielectric layers are concentrically arranged relative to each other around the central substrate. Specifically, a working electrode 410 is disposed on a surface of the central substrate 402, and a dielectric layer 412 is disposed on a portion of the working electrode 410 away from the sensor tip 404. A working electrode 420 is disposed on the dielectric layer 412, and a dielectric layer 422 is disposed on a portion of the working electrode 420 away from the sensor tip 404. A counter electrode 430 is disposed on the dielectric layer 422, and a dielectric layer 432 is disposed on a portion of the counter electrode 430 away from the sensor tip 404. A reference electrode 440 is disposed on the dielectric layer 432, and a dielectric layer 442 is disposed on a portion of the reference electrode 440 away from the sensor tip 404. Thus, the exposed surfaces of working electrode 410 , working electrode 420 , counter electrode 430 , and reference electrode 440 are spaced apart from one another along longitudinal axis B of analyte sensor 400 .

[0063] Refer to Fig. 6A , creatinine responsive active region 414a and glucose responsive active region 414b are disposed on the exposed surfaces of working electrodes 410 and 420, respectively, thereby allowing contact with fluid for sensing of creatinine and / or glucose. Fig. 6A 414a and 414b are depicted as three discrete points, but it should be understood that there may be fewer or more than three points in alternative sensor configurations. In addition, the positioning of the creatinine responsive active region 414a and the glucose responsive active region 414b may be different from Fig. 6A The opposite of what is depicted in .

[0064] exist Fig. 6A 4 , the sensor 400 is partially coated with a membrane 450 over the working electrodes 410 and 420 and the active regions 414a and 414b disposed thereon. Figure 6B An alternative sensor configuration is shown in which sensor 401 is substantially entirely covered by membrane 450. Membrane 450 may be compositionally identical or compositionally different at active regions 414a and 414b.

[0065] It should also be understood that Fig. 6A and 6B The positioning of the various electrodes in the embodiment may be different from that explicitly depicted. For example, the positions of the counter electrode 430 and the reference electrode 440 may be different from those explicitly depicted. Fig. 6A and 6B Similarly, the positions of working electrodes 410 and 420 are not limited to Fig. 6A and 6B Those clearly described in. Figure 6C Shows Figure 6B , wherein sensor 405 includes counter electrode 430 and reference electrode 440 located closer to sensor tip 404 and working electrodes 410 and 420 located further away from sensor tip 404. A sensor configuration in which working electrodes 410 and 420 are located further away from sensor tip 404 may be advantageous in that it provides a greater surface area for deposition of active regions 414a and 414b ( Figure 6C Five discrete sensing points are exemplarily shown in FIG. 4 ) thereby facilitating increased signal strength in some cases.

[0066] Although Figures 6A-6C Sensor configurations have been depicted each supported on a central substrate 402, but it will be appreciated that alternative sensor configurations may instead have electrodes supported without the central substrate 402. In particular, the innermost concentric electrode may be used to support other electrodes and dielectric layers. Fig.6D Shows Figure 6C , wherein sensor 406 does not contain central substrate 402, and counter electrode 430 is the innermost concentric electrode for sequentially disposing reference electrode 440, working electrodes 410 and 420, and dielectric layers 432, 442, 412, and 422 thereon. In view of the disclosure herein, it is again recognized that other electrode and dielectric layer configurations may be employed in sensor configurations lacking central substrate 402. Thus, Fig.6D The sensor configurations depicted in should be considered exemplary in nature and non-limiting.

[0067] As mentioned above, an oxygen scavenger may be located near the creatinine responsive active region to promote oxidation of the reduced form of sarcosine oxidase with an electron transfer agent rather than with oxygen. Oxidase, particularly glucose oxidase, may be used for this purpose in the various sensor configurations disclosed herein. When only the creatinine responsive active region is present, a glucose oxidase that is non-functional for glucose detection may be located near the creatinine responsive active region. When both the creatinine responsive active region and the glucose responsive active region are present, the glucose oxidase in the glucose responsive active region may effectively promote oxygen scavenging, optionally in combination with a glucose oxidase that is non-functional for glucose detection.

[0068] exist Fig. 7A and 7B An exemplary configuration of glucose oxidase is shown to be non-functional for glucose detection in the creatinine responsive active region. Specifically, Fig. 7A and 7BGlucose oxidase 407 is shown disposed on membrane 409, which covers the creatinine responsive active area 403 located on the working electrode 500. Membrane 409 insulates glucose oxidase 407 from the working electrode 500 so that electrons generated when glucose is oxidized are not transferred to the working electrode 500 to limit oxygen exposure. Membrane 408 then covers glucose oxidase 407 to provide a mass transfer limiting function thereto. In various embodiments of the present disclosure, membranes 408 and 409 may be identical in composition. Although Fig. 7A Glucose oxidase 407 has been shown disposed directly on creatinine responsive active region 403, but it should be understood that creatinine responsive active region 403 and glucose oxidase 407 may be laterally spaced apart from one another, again assuming that glucose oxidase 407 is excluded from transferring electrons to working electrode 500 when oxidizing glucose. Further alternatively, in other sensor configurations, glucose oxidase 407 may additionally be located on the opposite face of the sensor. Figure 7B As shown, membrane 409 need not extend the same lateral distance over working electrode 500 as membrane 408 .

[0069] In the sensor configuration disclosed herein, the creatinine-responsive active area and the glucose-responsive active area (if present) may include one or more discrete points (e.g., one to about ten points, or even more discrete points) whose sizes may range from about 0.01 mm to about 0.1 mm. 2 To about 1mm 2 , however, larger or smaller individual spots within the active area are also contemplated herein. The total active area can be selected to provide the desired sensitivity for each analyte.

[0070] 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 have been discussed in more detail above. Considerations for deploying the sensor tail at a specific location within a given tissue are discussed above.

[0071] Therefore, the analyte sensor disclosed herein can include a sensor tail, which includes at least a first working electrode, an enzyme system comprising a first electron transfer agent, a first polymer, and a plurality of enzymes that can act synergistically to promote creatinine detection, a first membrane that can permeate creatinine and cover the creatinine response active area, and an oxygen scavenger located near the creatinine response active area at the sensor tail. The enzyme system includes creatinine amide hydrolase, creatine amidinohydrolase, and sarcosine oxidase. According to a specific embodiment of the present disclosure, the oxygen scavenger can be separated from the creatinine response active area by the first membrane. In some embodiments, an oxidase (e.g., glucose oxidase) can constitute at least a portion of the oxygen scavenger.

[0072] When arranged near the creatinine responsive active region, the oxidase (e.g., glucose oxidase) can be covalently bonded to a second polymer. Suitable polymers for covalently bonding to the glucose oxidase are not particularly limited and can be polyvinylpyridine in a particular embodiment of the present disclosure. The covalently bonded polymer can help to fix the glucose oxidase in a desired position relative to the creatinine responsive active region.

[0073] In any embodiment of the present disclosure, creatinine amidohydrolase, creatine amidinohydrolase and sarcosine oxidase can be covalently bonded to the first polymer in the creatinine responsive active region. Suitable polymers for covalently bonding these enzymes are not considered to be particularly limited and can be polyvinylpyridine in a specific embodiment of the present disclosure. The first polymer in the creatinine responsive active region and the second polymer covalently bonded to the glucose oxidase can be the same polymer.

[0074] Creatinine response active region and glucose response active region (if present) can comprise electron transfer agent in any exemplary sensor configuration disclosed herein.When creatinine response active region and glucose response active region are both present, electron transfer agent can be the same or different, depending on the specific sensor configuration adopted.Suitable electron transfer agent can promote the transmission of electrons to the working electrode after enzymatic oxidation or reduction reaction occurs, thereby generating a current indicating the presence of a specific analyte and proportional to the amount of analyte present.For example, when creatinine response active region and glucose response active region are arranged on the same working electrode, the electron transfer agent in each active region can be different (for example, chemically different, so that the electron transfer agent exhibits different redox potentials).When multiple working electrodes are present, the electron transfer agent in each active region can be the same or different, because each working electrode can be separately queried when obtaining a signal.Electron transfer agent can be covalently bonded to the polymer in any active region disclosed herein.

[0075] According to various embodiments of the present disclosure, suitable electron transfer agents may include electroreducible and electrooxidizable ions, complexes or molecules (e.g., quinones) having a redox potential higher or lower than the redox potential of a standard calomel electrode (SCE) by hundreds of millivolts. According to some embodiments, suitable electron transfer agents may include low potential osmium complexes, such as those described in U.S. Patents 6,134,461 and 6,605,200, which are incorporated herein by reference in their entirety. Other examples of suitable electron transfer agents include those described in U.S. Patents 6,736,957, 7,501,053 and 7,754,093, the disclosures of which are incorporated herein by reference in their entirety. Other suitable electron transfer agents may include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinyl ferrocene or hexacyanoferrate) or cobalt, including, for example, their metallocene compounds. Suitable ligands for metal complexes may also include, for example, bidentate or higher dentate ligands, such as bipyridine, biimidazole, phenanthroline or pyridyl (imidazole). Other suitable bidentate ligands may include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes or o-diaminoarenes.Any combination of monodentate, bidentate, tridentate, tetradentate or higher dentate ligands may be present in the metal complex to achieve a complete coordination sphere.

[0076] Active regions suitable for detecting creatinine and / or glucose can also include polymers, to which electron transfer agents are covalently bonded. Any electron transfer agent disclosed herein can include suitable functional groups to promote covalent bonding with polymers in the active region. Suitable examples of polymer-bound electron transfer agents can include those described in U.S. Patents 8,444,834, 8,268,143 and 6,605,201, the disclosures of which are incorporated herein by reference as a whole. Suitable polymers included in the active region can include, but are not limited to, polyvinylpyridine (e.g., poly-(4-vinylpyridine)), polyvinylimidazole (e.g., poly-(1-vinylimidazole)) or any copolymer thereof. Exemplary copolymers suitable for inclusion in the active region include copolymers containing monomeric units such as styrene, acrylamide, methacrylamide or acrylonitrile. The polymer in each active region can be the same or different.

[0077] In a particular embodiment of the present disclosure, the mass transfer limiting membrane covering the creatinine response active region can include at least one cross-linked polyvinyl pyridine homopolymer or copolymer, including polyvinyl pyridine polystyrene copolymer. The mass transfer limiting membrane with similar composition can also cover oxygen scavengers, such as glucose oxidase. The composition of the mass transfer limiting membrane can be the same or different, wherein the mass transfer limiting membrane covers each active region. Suitable techniques for depositing the mass transfer limiting membrane on one or more active regions can include, for example, spraying, painting, inkjet printing, template printing, roller coating, dip coating, etc., and any combination thereof.

[0078] The covalent bonding mode between the electron transfer agent and the polymer in each active region is not considered to be particularly limited. The covalent bonding of the electron transfer agent to the polymer can occur by polymerizing a monomer unit with a covalently bonded electron transfer agent, or the electron transfer agent can react with the polymer alone after the polymer has been synthesized. According to some embodiments, a bifunctional spacer can covalently bond the electron transfer agent to the polymer in the active region, using a first functional group that reacts with the polymer (e.g., a functional group that can quaternize a pyridine nitrogen atom or an imidazole nitrogen atom) and a second functional group that reacts with the electron transfer agent (e.g., a functional group that reacts with a ligand that coordinates a metal ion).

[0079] Similarly, one or more enzymes in the active region can be covalently bonded to the polymer.When the enzyme system comprising multiple enzymes is present in a given active region, in some embodiments all multiple enzymes can be covalently bonded to the polymer, and in other embodiments, only a portion of multiple enzymes can be covalently bonded to the polymer.For example, one or more enzymes comprising the enzyme system can be covalently bonded to the polymer and at least one enzyme can be non-covalently associated with the polymer, so that the non-covalently bonded enzyme is physically entrained in the polymer.According to a more specific embodiment, the covalent bonding of one or more enzymes to the polymer in a given active region can occur by introducing a cross-linking agent of a suitable cross-linking agent. Suitable cross-linking agents for reacting with the free amino groups in the enzyme (for example, with the free side chain amines in lysine) can include cross-linking agents, such as polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin or its derivative variants. Suitable cross-linking agents for reacting with the free carboxylic acid groups in the enzyme can include, for example, carbodiimide. The cross-linking of the enzyme and the polymer is usually intermolecular, but can be intramolecular in some embodiments. In certain embodiments, all enzymes herein may be covalently bonded to a polymer.

[0080] The electron transfer agent and / or one or more enzymes can also be associated with the polymer in the active area by means other than covalent bonds. In some embodiments, the electron transfer agent and / or one or more enzymes can be associated with the polymer ionically or coordinately. For example, a charged polymer can be ionically bound to an electron transfer agent or enzyme with an opposite charge. In other embodiments, the electron transfer agent and / or one or more enzymes can be physically entrained in the polymer without being bonded thereto. Physically entrained electron transfer agents and / or one or more enzymes can still appropriately interact with the fluid to promote analyte detection without leaching substantially from the active area.

[0081] In some embodiments of the present disclosure, the creatinine-responsive analyte sensor may further incorporate a glucose-responsive active region for sensing creatinine and glucose. Figures 4A-6D As discussed, when both the creatinine response active region and the glucose response active region are present, the creatinine response active region and the glucose response active region can be present on the same working electrode or on different working electrodes. The consideration of combining the glucose response active region at any position will be discussed in more detail below. In any sensor configuration including both the creatinine response active region and the glucose response active region herein, the glucose oxidase that is non-functional for glucose detection can be disposed on a membrane covering the creatinine response active region or at another position where electrons cannot be transferred to the working electrode associated with the creatinine response active region.

[0082] As described below, when a creatinine-responsive active region and a glucose-responsive active region are arranged on a single working electrode, one of the active regions can be configured so that it can be interrogated separately to facilitate detection of each analyte. Specifically, the creatinine-responsive active region and the glucose-responsive active region can contain different electron transfer agents to allow 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.

[0083] In the embodiment where the creatinine response active region and the glucose response active region are arranged on a single working electrode, the redox potential associated with the glucose response active region can be separated from the creatinine response active region by at least about 100mV, or at least about 150mV, or at least about 200mV. The upper limit of separation between the redox potentials is determined by the working electrochemical window in vivo. By making the redox potentials of the two active regions fully separated from each other in amplitude, the electrochemical reaction occurs in one of the two active regions (that is, in the glucose response active region or the creatinine response active region) and does not substantially cause an electrochemical reaction in another active region. Therefore, the signal from one of the glucose response active region or the creatinine response active region can be equal to or higher than its corresponding redox potential (lower redox potential) but lower than the redox potential (higher redox potential) of the other in the glucose response active region and the creatinine response active region and is independently generated. On the contrary, when equal to or higher than the redox potential (higher redox potential) of other active regions that were not previously inquired, the electrochemical reaction may occur in both the glucose response active region and the creatinine response active region. Therefore, the signal obtained at or above the higher redox potential may include signal contributions from both the glucose-responsive active region and the creatinine-responsive active region, and the observed signal is a composite signal. The signal contribution from one active region (the glucose-responsive active region or the creatinine-responsive active region) at or above its corresponding redox potential can then be determined by subtracting the signal obtained from only either the glucose-responsive active region or the creatinine-responsive active region from the composite signal.

[0084] In more specific embodiments, when the active regions are located on the same working electrode, the glucose-responsive active region and the creatinine-responsive active region may contain different electron transfer agents to provide redox potentials that are sufficiently separated in magnitude from each other. More specifically, the glucose-responsive active region may include a first electron transfer agent and the creatinine-responsive active region may include a second electron transfer agent, wherein the first electron transfer agent and the second electron transfer agent are different. According to various embodiments of the present disclosure, the metal center and / or ligand present in a given electron transfer agent may be varied to provide sufficient separation of the redox potentials in the two active regions.

[0085] Ideally, the glucose response active region and the creatinine response active region on a single working electrode can be configured to quickly obtain steady-state current when operating the analyte sensor at a given potential. The rapid acquisition of steady-state current can be promoted by selecting a kind of electron transfer agent for each active region, and the electron transfer agent can rapidly change its oxidation state when exposed to a potential equal to or higher than its redox potential. Making the active region as thin as possible can also promote the rapid acquisition of steady-state current. For example, the suitable thickness of the glucose response active region and the creatinine response active region can be in the range of about 0.1 micron to about 10 microns. In some or other embodiments, in one or more active regions, combining conductive materials such as carbon nanotubes, graphene or metal nanoparticles can promote the rapid achievement of steady-state current. The suitable amount of conductive particles can be about 0.1 weight % to about 50 weight % of the active region, or about 1 weight % to about 50 weight %, or about 0.1 weight % to about 10 weight %, or about 1 weight % to about 10 weight %. Stabilizers can also be used to promote response stability.

[0086] It should also be understood that the sensitivity (output current) of the analyte sensor to each analyte can be changed by changing the coverage (area or size) of the active areas, the area ratio of the active areas relative to each other, the characteristics, thickness and / or composition of the mass transport limiting membrane covering the active areas. These parameters can be easily changed by one of ordinary skill in the art once having the benefit of the disclosure herein.

[0087] Other embodiments of the analyte sensor disclosed herein can be characterized by creatinine response active area and glucose response active area on the surface of different working electrodes. Such analyte sensor can also include a second working electrode, a glucose response active area arranged on the surface of the second working electrode and a second membrane that can permeate glucose and cover the glucose response active area. The glucose response active area can include a second electron transfer agent, a third polymer and a glucose oxidase covalently bonded to the third polymer. The third polymer can be the same or different from the first polymer and / or the second polymer that are respectively related to the creatinine response active area or the glucose oxidase that is non-functional for detecting glucose. When the creatinine response active area and the glucose response active area are arranged on separate working electrodes, the electron transfer agent associated with each active area can be the same or different.

[0088] Therefore, certain analyte sensors capable of detecting both creatinine and glucose of the present disclosure may include: a sensor tail, which includes a first working electrode and a second working electrode; a creatinine response active region disposed on the surface of the first working electrode, the creatinine response active region comprising a first electron transfer agent, a first polymer, and an enzyme system comprising multiple enzymes that can act synergistically to promote the detection of creatinine, the enzyme system comprising: creatinine amide hydrolase, creatine amidinohydrolase, and sarcosine oxidase; a first membrane permeable to creatinine and covering the creatinine response active region; a glucose oxidase covalently bonded to the second polymer and located on the first membrane; a glucose response active region disposed on the surface of the second working electrode, the glucose response active region comprising a second electron transfer agent, a third polymer, and a glucose oxidase covalently bonded to the third polymer; and a second membrane permeable to glucose and covering the glucose response active region. This analyte sensor may also include a third membrane covering glucose oxidase, the third membrane being disposed on the first membrane. The first membrane, the second membrane, and the third membrane (if present) may be identical in composition in a particular embodiment.

[0089] The detection method for determining creatinine may include: exposing an analyte sensor to a fluid containing at least creatinine, wherein the analyte sensor includes a sensor tail, the sensor tail includes at least a first working electrode, a creatinine response active region disposed on its surface, a first membrane permeable to creatinine and covering the creatinine response active region, and an oxygen scavenger located near the creatinine response active region at the sensor tail. The creatinine response active region includes a first electron transfer agent, a first polymer, and an enzyme system including multiple enzymes that can act synergistically to promote creatinine detection. The enzyme system includes creatinine amide hydrolase, creatine amidinohydrolase, and sarcosine oxidase, all of which can be covalently bonded to the first polymer in a specific embodiment. The method may also include applying a potential to the first working electrode, obtaining a first signal equal to or higher than the redox potential of the creatinine response active region proportional to the creatinine concentration in the fluid, and associating the first signal with the creatinine concentration in the fluid. In a specific embodiment of the present disclosure, the fluid may be a biological fluid. In a more specific embodiment, glucose may be present in the fluid in combination with creatinine.

[0090] In some embodiments, the first signal can be associated with a corresponding creatinine concentration by consulting a lookup table or calibration curve. The creatinine lookup table can be populated by measuring multiple samples with known creatinine concentrations and recording the sensor response at each concentration. Similarly, a calibration curve for creatinine can be determined by plotting the analyte sensor response as a function of creatinine concentration and determining a suitable calibration function within the calibration range (e.g., by regression, particularly linear regression).

[0091] The processor may determine which sensor response value in the lookup table is closest to the value measured for the sample with unknown analyte concentration and then report the analyte concentration accordingly. In some or other embodiments, if the sensor response value for the sample with unknown analyte concentration is between the recorded values ​​in the lookup table, the processor may interpolate between the two lookup table values ​​to estimate the analyte concentration. The interpolation may assume that there is a linear concentration change between the two values ​​reported in the lookup table. Interpolation may be employed when the sensor response differs sufficiently from a given value in the lookup table, such as a change of about 10% or more.

[0092] Likewise, according to some or other various embodiments, the processor may input the sensor response value for a sample having an unknown analyte concentration into a corresponding calibration function. The processor may then report the analyte concentration accordingly.

[0093] The sensor tail may further include a second working electrode having a glucose responsive active region disposed thereon, and wherein the glucose responsive active region may include a second electron transfer agent, a third polymer, and a glucose oxidase covalently bonded to the third polymer. Therefore, the method may further include: applying a potential to the second working electrode, obtaining a second signal equal to or higher than the redox potential of the glucose responsive active region proportional to the glucose concentration in the fluid, and correlating the second signal with the glucose concentration in the fluid.

[0094] The detection method of analyzing creatinine and / or glucose using an analyte sensor having a creatinine response active region and a glucose response 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 a sensor tail, which includes at least one working electrode, particularly a single working electrode, and at least one creatinine response active region and a glucose response active region disposed on the working electrode surface and spaced apart from each other. In addition to the glucose oxidase in the glucose response active region, the film may cover the creatinine response active region, and the glucose oxidase covalently bonded to the polymer may be disposed on the film. The creatinine response active region includes an enzyme system, which includes two or more enzymes that can act synergistically to promote creatinine detection, a first polymer covalently bonded to the enzyme, and a first electron transfer agent covalently bonded to the first polymer. The glucose response active region includes glucose oxidase, a third polymer covalently bonded to the glucose oxidase, and a second electron transfer agent covalently bonded to the third polymer. As discussed in more detail herein, when the glucose-responsive active region and the creatinine-responsive active region are located on a single working electrode, the first electron transfer agent and the second electron transfer agent are different in composition 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 allow a signal to be generated from one of the active regions. The method further includes: obtaining a first signal that is equal to or higher than the lower redox potential but lower than 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 that is equal to or higher than the higher redox potential, such that the second signal is a composite signal that includes a signal contribution from the glucose-responsive active region and a signal contribution from the creatinine-responsive active region; and subtracting the first signal from the second signal to obtain a difference signal, the difference signal being proportional to the concentration of one of glucose and creatinine.

[0095] In more specific embodiments, the redox potential associated with the creatinine-responsive active region can be separated from the redox potential of the glucose-responsive active region by at least about 100 mV, or at least about 150 mV, or at least about 200 mV to provide sufficient separation for independently generating a signal from the first active region. The different redox potentials may be due to the addition of different electron transfer agents in the active region.

[0096] A detection method for measuring creatinine and / or glucose using an analyte sensor may include: exposing the analyte sensor to a fluid containing at least one of glucose and creatinine. The analyte sensor includes a sensor tail including at least a first working electrode and a second working electrode, 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, the enzyme system including at least two enzymes that can act synergistically to facilitate creatinine detection.

[0097] The method may also include applying a potential to the first working electrode and applying a potential to the second working electrode, obtaining a first signal equal to or higher than the redox potential of the creatinine response active region, wherein the first signal is proportional to the concentration of creatinine in the fluid, obtaining a second signal equal to or higher than the redox potential of the glucose response active region, wherein the second signal is proportional to the concentration of glucose in the fluid, and correlating the first signal with the concentration of creatinine in the fluid and correlating the second signal with the concentration of glucose in the fluid.

[0098] According to more specific embodiments, the first signal and the second signal can be measured at different times. Therefore, 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 through 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 area can then be associated with the concentration of creatinine and glucose using a lookup table or calibration function in a similar manner to that discussed above.

[0099] Embodiments disclosed herein include:

[0100] A. An analyte sensor responsive to creatinine. The analyte sensor comprises: a sensor tail comprising at least a first working electrode; a creatinine responsive active region disposed on a surface of the first working electrode, the creatinine responsive active region comprising a first electron transfer agent, a first polymer, and an enzyme system comprising a plurality of enzymes capable of acting synergistically to facilitate detection of creatinine, the enzyme system comprising: creatinine amide hydrolase, creatine amidinohydrolase, and sarcosine oxidase; a first membrane permeable to creatinine and covering the creatinine responsive active region; and an oxygen scavenger located in the sensor tail proximal to the creatinine responsive active region.

[0101] B. An analyte sensor responsive to creatinine and glucose. The analyte sensor comprises: 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 creatinine responsive active region comprising a first electron transfer agent, a first polymer, and an enzyme system comprising a plurality of enzymes capable of synergistically acting to facilitate the detection of creatinine, the enzyme system comprising: creatinine amide hydrolase, creatine amidinohydrolase, and sarcosine oxidase; a first membrane permeable to creatinine and covering the creatinine responsive active region; a glucose oxidase covalently bonded to the second polymer and located on the first membrane; 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 a glucose oxidase covalently bonded to the third polymer; and a second membrane permeable to glucose and covering the glucose responsive active region.

[0102] C. A method for determining creatinine using an analyte sensor. The method includes: exposing the analyte sensor to a fluid containing at least creatinine; wherein the analyte sensor includes a sensor tail, the sensor tail including at least a first working electrode, a creatinine response active region disposed on the surface of the first working electrode, a first membrane permeable to creatinine and covering the creatinine response active region, and an oxygen scavenger located near the creatinine response active region at the sensor tail; wherein the creatinine response active region includes a first electron transfer agent, a first polymer, and an enzyme system including multiple enzymes that can act synergistically to promote the detection of creatinine, the enzyme system including: creatinine amide hydrolase, creatine amidinohydrolase, and sarcosine oxidase; applying a potential to the first working electrode; obtaining a first signal equal to or higher than the redox potential of the creatinine response active region, the first signal being proportional to the creatinine concentration in the fluid; and correlating the first signal with the concentration of creatinine in the fluid. Each of embodiments AD can have one or more of the following additional elements in any combination:

[0103] Element 1: wherein the oxygen scavenger is separated from the creatinine responsive active region by a first membrane.

[0104] Element 2: wherein the oxygen scavenger comprises an oxidase.

[0105] Element 3: wherein the oxygen scavenger comprises glucose oxidase.

[0106] Element 4: wherein the glucose oxidase is covalently bonded to the second polymer.

[0107] Element 5: wherein the oxygen scavenger is disposed on the first membrane, optionally wherein the oxygen scavenger comprises an oxidase, optionally wherein the oxygen scavenger comprises glucose oxidase, optionally wherein the glucose oxidase is covalently bonded to the second polymer.

[0108] Element 6: wherein creatinine amide hydrolase, creatine amidinohydrolase and sarcosine oxidase are each covalently bonded to the first polymer.

[0109] Element 7: wherein 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 permeable to glucose and covering the glucose responsive active region.

[0110] Element 8: wherein the analyte sensor further comprises glucose oxidase covalently bonded to the second polymer and disposed on the first membrane.

[0111] Element 9: wherein the first film and the second film are identical in composition.

[0112] Element 10: wherein the glucose oxidase located on the first membrane is covered by a third membrane which is also permeable to creatinine.

[0113] Element 11: wherein the first film, the second film and the third film are identical in composition.

[0114] Element 12: wherein the oxygen scavenger is disposed on the first membrane.

[0115] Element 13: wherein the oxygen scavenger comprises glucose oxidase covalently bonded to the second polymer.

[0116] Element 14: wherein the tail of the sensor also includes a second working electrode, the second working electrode having a glucose responsive active area disposed on the surface of the second working electrode, the glucose responsive active area comprising a second electron transfer agent, a third polymer and a glucose oxidase covalently bonded to the third polymer, the method further comprising: applying a potential to the second working electrode; obtaining a second signal having a redox potential equal to or higher than that of the glucose responsive active area, the second signal being proportional to the glucose concentration in the fluid; and correlating the second signal with the glucose concentration in the fluid.

[0117] Element 15: wherein the first signal and the second signal are obtained at different times.

[0118] Element 16: wherein the first signal and the second signal are obtained simultaneously through the first channel and the second channel.

[0119] As non-limiting examples, exemplary combinations suitable for 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; Exemplary combinations for B include 10 and 11; and 8 and 9. 9 and 10; 6 and 10; Exemplary combinations for C include 1 and 3; and 6 and 11. 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.

[0120] In order to facilitate a better understanding of the disclosure herein, the following examples of various representative embodiments are given. The following examples should in no way be read as limiting or defining the scope of the present invention. Example

[0121] A poly(vinylpyridine)-bound transition metal complex having the structure shown in Formula 1 was prepared. More details on the transition metal complex and its electron transfer are provided in co-owned U.S. Patent 6,605,200, which is incorporated herein by reference. The subscripts for each monomer represent exemplary atomic ratios and do not represent any particular order of the monomers.

[0122]

[0123] The buffered spotting formulation specified in Table 1 below (10 mM MES buffer) was deposited on the carbon working electrode to form a creatinine responsive active area. Deposition was performed with 15 nL of the spotting formulation to form a 0.12 mm 2The creatinine response active area of ​​a single point of the area. After deposition, the creatinine response active area was cured overnight at 25°C. Thereafter, the film was dip-coated to the creatinine response active area using a coating solution prepared by 4mL of 35mg / mL polyvinylpyridine-copolystyrene, 0.1mL 100mg / mLPEGDGE400 and 3.3μL PDMS in 80:20 ethanol / 10mM HEPES buffer (pH=8). No curing was performed at this stage.

[0124] Table 1

[0125] Components Concentration (mg / mL) CNH 20 CRH 40 SOX 5 The polymer of formula 1 8.5 PEGDGE400 6.5

[0126] After coating the membrane on the creatinine-responsive active area, a buffered spotting formulation containing glucose oxidase as specified in Table 2 was deposited on the membrane. Specifically, 15 nL of the spotting formulation was deposited at 0.05 mm 2 An area of ​​1000 μm was deposited on the membrane and then cured overnight at 25° C. Thereafter, dip coating was performed using the same coating solution as above to form a membrane on the deposited glucose oxidase.

[0127] Table 2

[0128]

[0129] A control electrode was prepared as above, except that the glucose oxidase deposition on the creatinine responsive active area was omitted.

[0130] Creatinine analysis was performed by immersing the electrode in the same solution containing different amounts of creatinine (20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 130 μM and 200 μM) and measuring the current response. A control electrode lacking glucose oxidase on the creatinine-responsive active area was also tested under the same conditions. Figure 8 An exemplary graph of three replicates of the current response of an analyte sensor comprising a creatinine-responsive active area covered with glucose oxidase when exposed to different creatinine concentrations at 33°C is shown. As shown, the current response increases within a few minutes after exposure to a new creatinine concentration and stabilizes thereafter. In contrast, two control sensors lacking glucose oxidase coverage on the creatinine-responsive active area did not respond to any concentration of creatinine. Fig. 9 The results are shown when exposed to different creatinine concentrations (20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 130 μM and 200 μM). As shown, the sensor response is essentially linear over the concentration range tested. The control sensor lacking glucose oxidase coverage again showed essentially no response to creatinine, likely due to interference of oxygen with the enzyme system.

[0131] Unless otherwise indicated, all numbers expressing quantities and the like in this specification and the associated claims should be understood as being modified in all instances by the term "about". Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that may vary depending on the desired properties sought to be obtained by embodiments of the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0132] One or more exemplary embodiments in combination with various features are presented herein. For the sake of clarity, all features of physical implementation are not described or shown in this application. It is understood that in the development of the physical implementation comprising the embodiments of the present invention, many decisions specific to implementation must be made to achieve the goals of the developer, such as complying with system-related, business-related, government-related and other restrictions, which vary from time to time due to implementation. Although the labor of the developer may be time-consuming, such labor will be routine work for those skilled in the art and will benefit from the present disclosure.

[0133] Although various systems, tools, and methods are described herein as "comprising" various components or steps, the systems, tools, and methods may also "consist essentially of" or "consist of" the various components and steps.

[0134] As used herein, the phrase "at least one of" preceding a series of items and with the terms "and" or "or" to separate any items, modifies the list as a whole, rather than modifying each member (i.e., each item) of the list. The phrase "at least one of" allows for a meaning that includes at least one of any of the items, and / or at least one of any combination of the items, and / or at least one of each. For 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.

[0135] Therefore, the disclosed systems, tools and methods are very suitable for achieving the mentioned purposes and advantages and those inherent therein. The specific embodiments disclosed above are exemplary only, because the teachings of the present disclosure can be modified and practiced in different but equivalent ways, which is obvious to those skilled in the art who benefit from the teachings herein. In addition, except for those described in the appended claims, it is not intended to limit the details of the construction or design shown here. It is therefore obvious that the specific exemplary embodiments disclosed above can be changed, combined or modified, and all these changes are considered to be within the scope of the present disclosure. In the absence of any element not specifically disclosed herein and / or any optional element disclosed herein, the systems, tools and methods disclosed exemplarily herein can be appropriately implemented. Although systems, tools and methods are described in terms of "comprising", "containing" or "including" various components or steps, systems, tools and methods can also be "essentially composed of various components and steps" or "composed of". All numbers and ranges disclosed above may vary. Whenever a numerical range with a lower limit and an upper limit is disclosed, any numerical value and any included range falling within the range are specifically disclosed. Specifically, each range of values ​​disclosed herein (in the form of "about a to about b," or equivalently, "about a to b," or equivalently, "about ab") should be understood to list each number and range within the broader numerical range. In addition, unless otherwise clearly and unambiguously defined by the patentee, the terms in the claims have their plain, ordinary meaning. In addition, the indefinite articles "a" or "an" used in the claims are defined herein to mean the element or elements it introduces. If the usage of a word or term in this specification conflicts with one or more patents or other documents that may be incorporated herein by reference, the definition consistent with this specification should be adopted.

Claims

1. An analyte sensor comprising: a sensor tail comprising at least a first working electrode; A creatinine responsive active region is disposed on the surface of the first working electrode, the creatinine responsive active region comprising a first electron transfer agent, a first polymer, and an enzyme system comprising a plurality of enzymes that can act synergistically to facilitate the detection of creatinine, the enzyme system comprising: 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 is located at the tail of the sensor and near the creatinine-responsive active region. 2 . The analyte sensor of claim 1 , wherein the oxygen scavenger is separated from the creatinine-responsive active region by the first membrane. The analyte sensor of claim 1 , wherein the oxygen scavenger comprises an oxidase. The analyte sensor of claim 3 , wherein the oxygen scavenger comprises glucose oxidase. The analyte sensor of claim 4 , wherein the glucose oxidase is covalently bonded to a second polymer. The analyte sensor of claim 1 , wherein the oxygen scavenger is disposed on the first membrane. The analyte sensor of claim 6 , wherein the oxygen scavenger comprises an oxidase. The analyte sensor of claim 7 , wherein the oxygen scavenger comprises glucose oxidase.

9. The analyte sensor of claim 8, wherein the glucose oxidase is covalently bonded to a second polymer.

10. The analyte sensor of claim 1, wherein the creatinine amide hydrolase, the creatine amidinohydrolase, and the sarcosine oxidase are each covalently bonded to the first polymer.

11. The analyte sensor of claim 1 , further comprising: 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 is permeable to glucose and covers the glucose responsive active area.

12. The analyte sensor of claim 11, further comprising: Glucose oxidase is covalently bonded to a second polymer and disposed on the first membrane.

13. The analyte sensor of claim 11, wherein the first membrane and the second membrane are identical in composition.

14. An analyte sensor comprising: a sensor tail comprising a first working electrode and a second working electrode; A creatinine responsive active region is disposed on the surface of the first working electrode, the creatinine responsive active region comprising a first electron transfer agent, a first polymer, and an enzyme system comprising a plurality of enzymes that can act synergistically to facilitate creatinine detection, the enzyme system comprising: 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 the second polymer and located on the first membrane; 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 is permeable to glucose and covers the glucose responsive active area.

15. The analyte sensor of claim 14, wherein the glucose oxidase disposed in the first membrane is covered by a third membrane, the third membrane also being permeable to creatinine.

16. The analyte sensor of claim 15, wherein the first membrane, the second membrane, and the third membrane are identical in composition.

17. The analyte sensor of claim 14, wherein the first membrane and the second membrane are identical in composition.

18. The analyte sensor of claim 14, wherein the creatinine amide hydrolase, the creatine amidinohydrolase, and the sarcosine oxidase are each covalently bonded to the first polymer.

19. A method comprising: exposing the analyte sensor to a fluid comprising at least creatinine; wherein the analyte sensor comprises a sensor tail, the sensor tail comprising at least a first working electrode, a creatinine-responsive active region disposed on a surface of the first working electrode, a first membrane permeable to creatinine and covering the creatinine-responsive active region, and an oxygen scavenger located in the sensor tail and proximate to the creatinine-responsive active region; The creatinine-responsive active region comprises a first electron transfer agent, a first polymer, and an enzyme system comprising a plurality of enzymes that can act synergistically to facilitate the detection of creatinine, the enzyme system comprising: Creatinine amidohydrolase, Creatine amidinohydrolase, and Sarcosine oxidase; applying a potential to the first working electrode; Obtaining a first signal that is equal to or higher than the redox potential of the creatinine-responsive active region, the first signal being proportional to the creatinine concentration in the fluid; and The first signal is correlated to a concentration of creatinine in the fluid.

20. The method of claim 19, wherein the creatinine amidohydrolase, the creatine amidinohydrolase, and the sarcosine oxidase are each covalently bonded to the first polymer.

21. The method of claim 19, wherein the oxygen scavenger is disposed on the first film.

22. The method of claim 21, wherein the oxygen scavenger comprises glucose oxidase covalently bonded to a second polymer.

23. The method of claim 20, wherein the sensor tail further comprises a second working electrode having a glucose responsive active region disposed on a surface of the second working electrode, the glucose responsive active region comprising a second electron transfer agent, a third polymer, and a glucose oxidase covalently bonded to the third polymer, the method further comprising: applying a potential to the second working electrode; obtaining a second signal that is equal to or higher than the redox potential of the glucose-responsive active region, the second signal being proportional to the glucose concentration in the fluid; and The second signal is correlated to a glucose concentration in the fluid. The method of claim 23 , wherein the first signal and the second signal are obtained at different times.

25. The method of claim 23, wherein the first signal and the second signal are obtained simultaneously through a first channel and a second channel.

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