Analyte sensor for detecting creatinine and detection method

Abstract

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

Description

[Background technology]

[0001] Detection of various analytes in an individual can sometimes be essential for monitoring the state of health and well-being. Deviations from normal analyte levels often indicate an underlying physiological state, such as a metabolic state or disease, or exposure to a particular environmental condition. While a single analyte may be dysregulated in isolation for a particular physiological state, multiple analytes may be dysregulated simultaneously due to the same physiological condition or as a result of coexisting (related) physiological conditions. When multiple analytes are dysregulated simultaneously, the degree of dysregulation may vary from analyte to analyte. Therefore, monitoring each analyte is necessary to properly assess the health status of an individual.

[0002] Periodic in vitro analyte monitoring using sampled bodily fluids may be sufficient to monitor a given physiological condition for many individuals. However, in vitro analyte monitoring can be inconvenient or painful for some individuals, especially if the sample or collection of bodily fluids requires frequent sampling (e.g., several times per day). Continuous analyte monitoring using implanted in vivo analyte sensors is a more desirable approach for individuals with severe analyte dysregulation and / or rapidly fluctuating analyte levels, but may also be beneficial to other individuals due to the convenience it offers. Continuous analyte monitoring allows individuals or physicians to proactively address abnormal analyte levels before they have the opportunity to lead to more serious health consequences, such as organ damage or failure. Subcutaneous, interstitial, or cutaneous analyte sensors can often provide sufficient measurement accuracy for this purpose while minimizing user discomfort.

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

[0004] In-vivo analyte sensors are typically configured to analyze a single analyte to provide a specific analysis, often using enzymes to provide high specificity for a given analyte. Because of such analytical specificity, most current in-vivo analyte sensors configured to assay glucose are ineffective at assaying other analytes that are often dysregulated along with glucose or that result from dysregulated glucose levels. At best, current analyte monitoring approaches require diabetics to wear two different in-vivo analyte sensors, one configured to analyze glucose and the other configured to analyze another analyte of interest. Analyte monitoring approaches using multiple in-vivo analyte sensors can be very inconvenient for users. Furthermore, when multiple in-vivo analyte sensors are used for analyte monitoring, the equipment costs increase and the likelihood of at least one of the individual in-vivo analyte sensors failing increases statistically.

[0005] Diabetic patients are often particularly susceptible to comorbidities, which can result from mismanagement of insulin levels or even from adequately managing diabetes over a long period of time. As an example, diabetic neuropathy can result from high blood glucose levels and ultimately lead to kidney failure. Diabetic neuropathy is the leading cause of kidney failure in the United States, experienced by a significant number of diabetic patients within the first 10 to 20 years of the disease. Diagnostic tests for assessing kidney function currently rely on measuring elevated creatinine levels in blood and / or urine samples. While detecting potential kidney failure as early as possible is desirable, current diagnostic testing approaches typically require long-term testing (months to years) to confirm that creatinine levels are persistently elevated or trend upward over time. The infrequent use of traditional creatinine monitoring may increase the risk of developing kidney failure if abnormalities in kidney function are not detected early enough. [Brief explanation of the drawings]

[0006] The accompanying drawings are included to illustrate certain aspects of the present disclosure and should not be considered as exclusive embodiments. The disclosed subject matter is capable of considerable modification, alteration, combination, and equivalents in form and function without departing from the scope of the present disclosure. [Figure 1] 1 shows a diagram of an exemplary sensing system that can incorporate an analyte sensor of the present disclosure. [Figure 2A] 1 shows an example of a cooperative enzyme system that can be used to detect creatinine according to the present disclosure. [Figure 2B] Same as above. [Figure 3A] 1 shows a cross-sectional view of an exemplary analyte sensor having an active area suitable for detecting creatinine. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 4A]FIG. 1 shows a cross-sectional view of an exemplary analyte sensor having a single working electrode and an active area suitable for detecting creatinine and glucose. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 5] 1 shows a cross-sectional view of an exemplary analyte sensor having two working electrodes and an active area suitable for detecting creatinine and glucose. [Figure 6A] 1 shows a perspective view of an exemplary analyte sensor featuring electrodes arranged concentrically with respect to one another. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 7A] 1 shows a diagram of a membrane and a creatinine-responsive active region with an oxygen scavenger disposed thereon. [Figure 7B] Same as above. [Figure 8] 1 shows an exemplary plot of current response for three replicates of a sensor containing a creatinine-responsive active region coated with glucose oxidase when exposed to various creatinine concentrations. [Figure 9] 1 shows an exemplary plot of the current response of a single sensor containing a creatinine-responsive active region coated with glucose oxidase when exposed to various creatinine concentrations. DETAILED DESCRIPTION OF THE INVENTION

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

[0008] As mentioned above, due to the common specificity of enzymes for particular substrates or classes of substrates, enzyme-based analyte sensors are typically used to monitor a single analyte, such as glucose. Other analytes can also be monitored if appropriate detection chemistries can be identified. Monitoring multiple analytes can be complicated by the need to use a corresponding number of analyte sensors to detect each analyte individually. This approach can be problematic or undesirable, especially when monitoring multiple analytes in vivo, due to issues such as the cost of multiple analyte sensors, user discomfort when wearing multiple analyte sensors, and an increased statistical probability of individual analyte sensor failure.

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

[0010] The present disclosure provides analyte sensors that are responsive to creatinine. Specifically, the present disclosure provides analyte sensors that can be worn on the body for continuous or near-continuous in vivo monitoring of creatinine levels. Analysis of creatinine levels using the analyte sensors disclosed herein may provide an individual or healthcare provider with a more accurate indication of kidney function over time than is possible with periodic ex vivo laboratory measurements. Analyzing creatinine levels in accordance with the present disclosure may enable early medical intervention to limit potential kidney damage and improve an individual's overall health outcomes.

[0011] Due to the lack of known enzymes capable of directly transferring electrons from creatinine to a working electrode, electrochemical detection of creatinine using a single enzyme reaction is not feasible. The present disclosure addresses this deficiency by providing a sensor chemistry suitable for detecting creatinine with good response stability over a wide creatinine concentration range. In particular, the present disclosure utilizes an enzyme system including multiple enzymes that can act in concert to facilitate creatinine detection. As used herein, the term "in concert" refers to coupled enzyme reactions in which the product of a first enzyme reaction serves as a substrate for a second enzyme reaction, and the second enzyme reaction serves as the basis for measuring the concentration of the substrate (analyte) reacted during the first enzyme reaction. While defined in terms of two coupled enzyme reactions, it should be understood that in some cases, three or more enzyme reactions may be similarly coupled. For example, the product of a first enzyme reaction may serve as a substrate for a second enzyme reaction, and the product of the second enzyme reaction may serve as a substrate for a third enzyme reaction, which serves as the basis for measuring the concentration of the substrate (analyte) reacted during the first enzyme reaction. As discussed further below, a suitable enzyme system for detecting creatinine according to the present disclosure uses three enzymes acting in concert with a fourth enzyme or other oxygen scavenger to facilitate oxygen clearance that does not directly participate in the concerted enzymatic reaction but instead prevents unwanted side reactions with oxygen from occurring.

[0012] When a single enzyme cannot facilitate detection, such as in the case of creatinine, it may be desirable to utilize two or more enzymes acting in concert with each other to detect the analyte of interest. Situations in which a single enzyme may be ineffective in facilitating detection of the analyte include, for example, situations in which the enzyme is inhibited by one or more reaction products or is unable to cycle between oxidized and reduced states when placed in the analyte sensor, and / or situations in which no enzyme is known to promote the desired reaction pathway required for detection to facilitate detection. In the case of creatinine, the enzymatic conversion of creatinine to creatine occurs hydrolytically and does not result in a change in oxidation state to provide a current at the working electrode to facilitate detection of this analyte. An enzyme system comprising multiple enzymes acting in concert according to the disclosure herein may alleviate this difficulty.

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

[0014] As further described below with reference to Figures 2A and 2B, the creatinine-responsive active region of the present disclosure can utilize an oxygen scavenger to facilitate the detection of creatinine using the enzyme system shown therein. Oxidase enzymes can function as oxygen scavengers in certain sensor configurations. Glucose oxidase may be a particularly advantageous oxygen scavenger because glucose is widely present in bodily fluids that also contain creatinine, where glucose can function as a reagent to remove oxygen (see Reaction 1 below). The oxygen scavenger can be electrically isolated from the creatinine-responsive active region by a membrane so that it does not generate a signal at the working electrode with the creatinine-responsive active region (i.e., when removing oxygen by promoting an oxidation reaction). To facilitate effective oxygen removal within the creatinine-responsive active region, the oxygen scavenger can be disposed on the membrane. In addition to being disposed on the membrane, an oxygen scavenger may be disposed at a second location on the sensor tail remote from the membrane, where the remote oxygen scavenger may function differently at the remote location (e.g., by facilitating glucose detection in the glucose-responsive active region). Depending on how and where the remote oxygen scavenger is disposed, the oxygen scavenger may be active or inactive to facilitate the detection of another analyte, particularly glucose, in addition to its oxygen scavenging function. If inactive to facilitate glucose detection, glucose oxidase may be electrically isolated from the working electrode, such that the oxidation reaction (oxygen scavenging) promoted by this enzyme does not result in current generation at the working electrode. If glucose oxidase is active for both facilitating glucose detection and oxygen scavenging, glucose oxidase may be present in a glucose-responsive active region disposed on a second working electrode or on a working electrode with a creatinine-responsive active region, resulting in separate signals from each. Strategies for placing both creatinine- and glucose-responsive active regions on a single sensor tail are discussed further below.

[0015] Even with the appropriate detection chemistry, incorporating two different types of active regions into a single analyte sensor can be challenging. Analyte sensors often use a membrane covering the active region to act as a mass transport limiting membrane and / or to improve biocompatibility. Using a mass transport limiting membrane to restrict analyte access to the active region can prevent sensor overload (saturation), thereby improving detection performance and accuracy. When a single analyte sensor is used to assay multiple analytes, different permeability values ​​may be exhibited by various analytes passing through a given mass transport limiting membrane, and the sensitivity of each analyte may vary significantly. Incorporating different mass transport limiting membranes into each active region can be problematic in some cases. Surprisingly and advantageously, glucose and creatinine can be successfully analyzed using compositionally identical mass transport limiting membranes at each location, thereby simplifying the fabrication of analyte sensors capable of detecting both analytes.

[0016] Before describing the analyte sensors of the present disclosure in more detail, a brief overview of suitable in-vivo analyte sensor configurations and sensor systems using analyte sensors is provided to enable a better understanding of embodiments of the present disclosure. FIG. 1 illustrates a diagram of an exemplary sensing system that can incorporate the analyte sensors of the present disclosure, specifically analyte sensors including creatinine-responsive active regions and, optionally, glucose-responsive active regions. As shown, sensing system 100 includes a sensor control device 102 and a reader device 120 configured to communicate with each other via a local communication path or link that may be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. Reader device 120, according to some embodiments, may provide an output medium for displaying analyte concentrations and alerts or notifications determined by sensor 104 or its associated processor, as well as allowing for one or more user inputs. Reader device 120 may be a general-purpose smartphone or a dedicated electronic reader instrument. While only one reader device 120 is shown, multiple reader devices 120 may be present in certain cases. Reader device 120 may also communicate with remote terminal 170 and / or trusted computer system 180 via communication paths / links 141 and / or 142, respectively, which may also be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. Additionally or alternatively, reader device 120 may communicate with network 150 (e.g., a cellular network, the Internet, or a cloud server) via communication path / link 151. Network 150 may be further communicatively connected to remote terminal 170 via communication path / link 152 and / or to trusted computer system 180 via communication path / link 153. Alternatively, sensor 104 may communicate directly with remote terminal 170 and / or trusted computer system 180 without the intervening reader device 120.For example, sensor 104 may communicate with remote terminal 170 and / or trusted computer system 180 via a direct communication link to network 150, according to some embodiments, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is incorporated herein by reference in its entirety. Any suitable electronic communication protocol, such as near field communication (NFC), radio frequency identification (RFID), BLUETOOTH® or BLUETOOTH® low energy protocol, WiFi®, or the like, may be used for each communication path or link. Remote terminal 170 and / or trusted computer system 180, according to some embodiments, are accessible by individuals other than the primary user who are interested in the user's analyte levels. Reader device 120 may include a display 122 and an optional input component 121. Display 122, according to some embodiments, may include a touchscreen interface.

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

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

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

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

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

[0022] An introducer may be temporarily present 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 in alternative embodiments, other types of introducers, such as a sheath or blade, may be present. More specifically, the needle or other introducer may be temporarily present near the sensor 104 prior to tissue insertion and then withdrawn. While present, the needle or other introducer may facilitate the insertion of the sensor 104 into the tissue by opening an access path for the sensor 104 to follow. For example, according to one or more embodiments, the needle may facilitate penetration of the epidermis as an access path to the dermis, allowing implantation of the sensor 104 to occur. After opening the access path, the needle or other introducer may be withdrawn to avoid presenting a sharps hazard. In an exemplary embodiment, a suitable needle may be solid or hollow, beveled or non-beveled, and / or circular or non-circular in cross section. In more particular embodiments, suitable needles may be comparable in cross-sectional diameter and / or tip design to acupuncture needles, which may have a cross-sectional diameter of about 250 microns. However, it should be recognized that suitable needles may have larger or smaller cross-sectional diameters as needed for a particular application.

[0023] In some embodiments, the tip of the needle (while present) may be angled on the end of the sensor 104 so that the needle penetrates the tissue first, opening an access pathway for the sensor 104. In other exemplary embodiments, the sensor 104 may reside within a lumen or channel of the needle, which similarly opens an access pathway for the sensor 104. In either case, the needle facilitates insertion of the sensor and is then withdrawn.

[0024] Suitable enzyme systems that can be used to detect creatinine in accordance with the present disclosure are described in further detail with reference to Figures 2A and 2B. As shown, creatinine can react reversibly and hydrolytically to form creatine in the presence of creatinine amidohydrolase (CNH). Creatine can then undergo catalytic hydrolysis in the presence of creatine amidinohydrolase (CRH) to form sarcosine. Neither of these reactions generates electron flow (e.g., oxidation or reduction) to provide the basis for electrochemical detection of creatinine.

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

[0026] Oxygen can interfere with the concerted reaction sequence used to detect creatinine according to the present disclosure. Specifically, as shown in FIG. 2B, reduced sarcosine oxidase can react with oxygen to reform the corresponding oxidized form of the enzyme, but without exchanging electrons with the electron transfer agent. When the reaction with oxygen occurs, all enzymes remain active, but no electrons flow to the working electrode. Without being bound by theory or mechanism, the competing reaction with oxygen is believed to be due to kinetic effects. That is, the oxidation of reduced sarcosine oxidase by oxygen is believed to occur faster than the oxidation promoted by the electron transfer agent. Hydrogen peroxide is also formed in the presence of oxygen.

[0027] A desirable reaction pathway for facilitating creatinine detection is shown in Figure 2A. The oxidation of reduced sarcosine oxidase may be facilitated by including an oxygen scavenger near the enzyme system. As noted above, various oxygen scavengers and their placement may be suitable, including oxidase enzymes such as glucose oxidase. Small molecule oxygen scavengers may also be suitable, but may be completely consumed before the sensor's lifetime is fully exhausted. In contrast, the enzyme can undergo reversible oxidation and reduction, thereby extending the sensor's lifetime. Preventing the oxidation of reduced sarcosine oxidase by oxygen allows for a slower electron exchange reaction with the electron transfer agent, thereby allowing current generation at the working electrode. The magnitude of the current generated is proportional to the amount of creatinine initially reacted.

[0028] The oxygen scavenger used to promote the desired reaction pathway in Figure 2A may be an oxidase enzyme in any embodiment of the present disclosure. Any oxidase enzyme may be used to promote oxygen removal near the enzyme system, so long as the appropriate substrate for the enzyme is also present in the creatinine-containing fluid, thereby providing a reagent for reaction with oxygen in the presence of the oxidase enzyme. Oxidase enzymes that may be suitable for oxygen removal in the present disclosure include, but are not limited to, glucose oxidase, lactate oxidase, xanthine oxidase, and the like. Due to the ready availability of glucose in various bodily fluids, glucose oxidase may be a particularly suitable oxidase enzyme for use in the present disclosure. Reaction 1 below illustrates the enzymatic reaction promoted by glucose oxidase to remove oxygen.

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

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

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

[0032] The analyte sensors disclosed herein feature at least a creatinine-responsive active area on a working electrode in combination with at least one additional electrode, which may be a counter electrode, a reference electrode, and / or a counter / reference electrode. Analyte sensors featuring both a creatinine-responsive active area and a glucose-responsive active area can incorporate 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 are discussed below.

[0033] Sensor configurations featuring a creatinine-responsive active region, but not a glucose-responsive active region, may use two-electrode or three-electrode detection motifs, as further described herein with reference to Figures 3A-3C. Sensor configurations featuring both creatinine-responsive and glucose-responsive active regions on separate working electrodes or on the same working electrode are subsequently described separately with reference to Figures 4A-6D. Sensor configurations with multiple working electrodes can be particularly advantageous for incorporating both creatinine-responsive and glucose-responsive active regions within the same sensor tail, because the signal contribution from each active region can be more easily determined.

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

[0035] Analyte sensors featuring multiple working electrodes may also include at least one additional electrode. If one additional electrode is present, the one additional electrode may function as a counter / reference electrode for each of the multiple working electrodes. If two additional electrodes are present, one of the additional electrodes may function as a counter electrode for each of the multiple working electrodes, and the other of the additional electrodes may function as a reference electrode for each of the multiple working electrodes.

[0036] 3A shows a diagram of an exemplary two-electrode analyte sensor configuration suitable for use in the present disclosure. As shown, the analyte sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter / reference electrode 216. Alternatively, the working electrode 214 and the counter / reference electrode 216 may be disposed 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 multiple spots or a single spot configured for the detection of creatinine, as described further herein.

[0037] 3A , according to some embodiments, membrane 220 covers at least creatinine-responsive active region 218 and can optionally cover part or all of working electrode 214 and / or counter / reference electrode 216, or the entire analyte sensor 200. One or both sides of analyte sensor 200 may be covered with membrane 220. Membrane 220 may comprise one or more polymeric membrane materials capable of restricting analyte flux to active region 218 (i.e., membrane 220 is a mass-transport limiting membrane with some permeability to creatinine). The composition and thickness of membrane 220 may be varied to facilitate a desired creatinine flux to creatinine-responsive active region 218, thereby providing a desired signal strength and stability. Analyte sensor 200 may be operable to assay creatinine by any of the following electrochemical detection techniques: coulometry, amperometry, voltammetry, or potentiometry.

[0038] 3B and 3C show diagrams of exemplary three-electrode analyte sensor configurations suitable for use herein. The three-electrode analyte sensor configuration can be similar to that shown for analyte sensor 200 in FIG. 3A, except for the inclusion of an additional electrode 217 in analyte sensors 201 and 202 (FIGS. 3B and 3C). With the additional electrode 217, counter / reference electrode 216 can function as either a counter electrode or a reference electrode, while the additional electrode 217 performs the other electrode function. Working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed on either working electrode 210 or electrode 216, with a dielectric separating layer therebetween. For example, as shown in FIG. 2B, dielectric layers 219a, 219b, and 219c separate electrodes 214, 216, and 217 from one another and provide electrical insulation. Alternatively, at least one of electrodes 214, 216, and 217 may be disposed on opposite sides of substrate 212, as shown in FIG. 3C. Thus, in some embodiments, electrode 214 (working electrode) and electrode 216 (counter electrode) may be disposed on opposite sides of substrate 212, and electrode 217 (reference electrode) may be disposed on one of electrodes 214 or 216 and separated therefrom by a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, and the location of reference material layer 230 is not limited to that shown in FIGS. 2B and 2C. Similar to sensor 200 shown in FIG. 3A, creatinine-responsive active region 218 of analyte sensors 201 and 202 may include multiple spots or a single spot. Additionally, analyte sensors 201 and 202 may be operable to assay creatinine by any of the following electrochemical detection techniques: coulometry, amperometry, voltammetry, or potentiometry.

[0039] Similar to analyte sensor 200, membrane 220 can also cover creatinine-responsive active region 218, as well as other sensor components, in analyte sensors 201 and 202, thereby functioning as a mass transport limiting membrane. In some embodiments, additional electrode 217 can be covered with membrane 220. While FIGS. 3B and 2C depict electrodes 214, 216, and 217 as all being covered with membrane 220, it should be appreciated that in some embodiments, only working electrode 214 may be covered. Furthermore, the thickness of membrane 220 on each of electrodes 214, 216, and 217 can be the same or different. As in the two-electrode analyte sensor configuration (FIG. 3A), one or both sides of analyte sensors 201 and 202 can be covered with membrane 220 in the sensor configurations of FIGS. 3B and 2C, or the entire analyte sensors 201 and 202 can be covered. Therefore, the three-electrode sensor configuration shown in Figures 3B and 3C should be understood as a non-limiting example of the embodiments disclosed herein, with alternative electrode and / or layer configurations falling within the scope of the present disclosure.

[0040] Analyte sensors having both creatinine- and glucose-responsive active regions on a single working electrode or on multiple working electrodes are described in further detail with reference to Figures 4A-6D.

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

[0042] Figures 4B and 4C show cross-sectional views of exemplary three-electrode sensor configurations for sensors 204 and 205, respectively, each featuring a single working electrode with both creatinine-responsive active region 218a and glucose-responsive active region 218b disposed thereon. Figures 4B and 4C are otherwise similar to Figures 3B and 3C and may be better understood by reference thereto. As with Figure 4A, the composition of membrane 220 may vary in active regions 218a and 218b or may be compositionally the same.

[0043] Exemplary sensor configurations having multiple working electrodes, specifically two working electrodes, are described in further detail with reference to Figures 5-6D. While the following description is primarily directed to sensor configurations having two working electrodes, it should be understood that the disclosure herein can be extended to incorporate three or more working electrodes. Additional working electrodes can be used to provide the analyte sensor with additional sensing capabilities beyond just creatinine and glucose sensing.

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

[0045] 5 may feature counter / reference electrodes instead of separate counter and reference electrodes 320, 321, and / or different layer and / or film arrangements than those explicitly shown. For example, the arrangement of counter electrode 320 and reference electrode 321 may be reversed from that shown in FIG. 5. Furthermore, working electrodes 304 and 306 do not necessarily have to be on opposite sides of substrate 302 in the manner shown in FIG. 4.

[0046] While suitable sensor configurations may feature electrodes that are substantially planar in nature, it should be understood that sensor configurations featuring non-planar electrodes may also be advantageous and particularly suitable for use in the present disclosure. In particular, substantially cylindrical electrodes arranged concentrically with respect to one another may facilitate deposition of a mass transport limiting film, as described herein below. Figures 6A-6D show perspective views of analyte sensors featuring two working electrodes arranged concentrically with respect to one another. It should be understood that sensor configurations having a concentric electrode arrangement but lacking a second working electrode are also possible in the present disclosure.

[0047] 6A shows a perspective view of an exemplary sensor configuration in which multiple electrodes are substantially cylindrical and concentrically arranged around a central substrate. As shown, the analyte sensor 400 includes a central substrate 402 around which all of the electrodes and dielectric layers are concentrically arranged. In particular, a working electrode 410 is disposed on the surface of the central substrate 402, and a dielectric layer 412 is disposed on a portion of the working electrode 410 distal to the sensor tip 404. A working electrode 420 is disposed on the dielectric layer 412, and a dielectric layer 422 is disposed on a portion of the working electrode 420 distal to the sensor tip 404. A counter electrode 430 is disposed on the dielectric layer 422, and a dielectric layer 432 is disposed on a portion of the counter electrode 430 distal to the sensor tip 404. A reference electrode 440 is disposed on the dielectric layer 432, and a dielectric layer 442 is disposed on a portion of the reference electrode 440 distal to the sensor tip 404. As such, 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 .

[0048] Continuing with reference 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 enabling fluid contact for creatinine and / or glucose sensing. While active regions 414a and 414b are depicted as three separate spots in FIG. 6A, it should be understood that fewer or more than three spots may be present in alternative sensor configurations. Additionally, the arrangement of creatinine-responsive active region 414a and glucose-responsive active region 414b may be reversed from that shown in FIG. 6A.

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

[0050] It should be further understood that the arrangement of the various electrodes in FIGS. 6A and 6B may differ from that explicitly shown. For example, the positions of the counter electrode 430 and the reference electrode 440 may be reversed from the configuration shown in FIGS. 6A and 6B. Similarly, the positions of the working electrodes 410 and 420 are not limited to those explicitly shown in FIGS. 6A and 6B. FIG. 6C illustrates an alternative sensor configuration to that shown in FIG. 6B, in which the sensor 405 includes the counter electrode 430 and the reference electrode 440 positioned more proximally to the sensor tip 404 and the working electrodes 410 and 420 positioned more distally to the sensor tip 404. A sensor configuration in which the working electrodes 410 and 420 are positioned more distally to the sensor tip 404 may be advantageous by providing a larger surface area for deposition of the active regions 414a and 414b (the five distinct sensing spots illustratively shown in FIG. 6C), potentially facilitating increased signal strength.

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

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

[0053] An exemplary placement of glucose oxidase, which does not function in glucose detection, relative to a creatinine-responsive active region is shown in Figures 7A and 7B. In particular, Figures 7A and 7B show diagrams illustrating the placement of glucose oxidase 407 on a membrane 409 covering a creatinine-responsive active region 403 disposed on a working electrode 500. The membrane 409 electrically isolates the glucose oxidase 407 from the working electrode 500 so that electrons generated upon oxidizing glucose are not transferred to the working electrode 500 to limit oxygen exposure. A membrane 408 then covers the glucose oxidase 407 and provides it with a mass transport limiting function. The membranes 408 and 409 can be compositionally identical in various embodiments of the present disclosure. 7A shows glucose oxidase 407 disposed directly on creatinine-responsive active region 403, it should be understood that creatinine-responsive active region 403 and glucose oxidase 407 can be laterally spaced apart from one another so long as glucose oxidase 407 inhibits the transfer of electrons to working electrode 500 when oxidizing glucose. Alternatively, glucose oxidase 407 can also be disposed on the opposite side of the sensor in yet other sensor configurations. As shown in FIG. 7B, membrane 409 does not necessarily extend the same lateral distance over working electrode 500 as membrane 408 does.

[0054] In the sensor configurations disclosed herein, the creatinine-responsive active region and, if present, the glucose-responsive active region may comprise one or more discrete spots (e.g., from 1 to about 10 spots, or even more discrete spots), which may be about 0.01 mm in size. 2 ~about 1mm 2 The total active area can range from 0.01 to 0.1, although larger or smaller individual active area spots are also contemplated herein. The total active area can be selected to provide the desired sensitivity for each analyte.

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

[0056] Thus, the analyte sensor disclosed herein may include at least a first working electrode, a sensor tail disposed on the first working electrode and including a creatinine-responsive active region containing an enzyme system including a first electron transfer agent, a first polymer, and multiple enzymes capable of acting in concert to facilitate creatinine detection, a first membrane permeable to creatinine and covering the creatinine-responsive active region, and an oxygen scavenger disposed near the creatinine-responsive active region on the sensor tail. The enzyme system includes creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase. According to certain embodiments of the present disclosure, the oxygen scavenger may be separated from the creatinine-responsive active region by the first membrane. In some embodiments, an oxidase enzyme, such as glucose oxidase, may constitute at least a portion of the oxygen scavenger.

[0057] An oxidase enzyme, such as glucose oxidase, can be covalently attached to a second polymer when positioned near the creatinine-responsive active region. Suitable polymers for covalently attaching glucose oxidase are not particularly limited and, in certain embodiments of the present disclosure, can be polyvinylpyridine. The covalently attached polymer can help immobilize the glucose oxidase in a desired location relative to the creatinine-responsive active region.

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

[0059] In any of the exemplary sensor configurations disclosed herein, the creatinine-responsive active region and, if present, the glucose-responsive active region may each include an electron transfer agent. When both a creatinine-responsive active region and a glucose-responsive active region are present, the electron transfer agents may be the same or different, depending on the particular sensor configuration used. A suitable electron transfer agent may facilitate the transfer of electrons to the working electrode after an enzymatic oxidation or reduction reaction occurs, thereby indicating the presence of a particular analyte and generating a current proportional to the amount of analyte present. For example, when a creatinine-responsive active region and a glucose-responsive active region are disposed on the same working electrode, the electron transfer agents in each active region may be different (e.g., chemically distinct so that the electron transfer agents exhibit different redox potentials). When multiple working electrodes are present, the electron transfer agents in each active region may be the same or different, allowing each working electrode to respond individually when acquiring a signal. The electron transfer agent may be covalently attached to the polymer in any active region disclosed herein.

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

[0061] Active regions suitable for detecting creatinine and / or glucose can also include a polymer to which an electron transfer agent is covalently attached. Any of the electron transfer agents disclosed herein can include appropriate functionality to facilitate covalent attachment to the polymer in the active region. Suitable examples of polymer-bound electron transfer agents can include those described in U.S. Patent Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are incorporated herein by reference in their entireties. Polymers suitable for inclusion 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 copolymers thereof. Exemplary copolymers that may be suitable for inclusion in the active region include those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile. The polymers in each active region can be the same or different.

[0062] In certain embodiments of the present disclosure, the mass transport limiting membrane covering the creatinine-responsive active area can comprise at least a cross-linked polyvinylpyridine homopolymer or copolymer, including polyvinylpyridine-co-styrene polymer. A mass transport limiting membrane with a similar composition can also cover an oxygen scavenger, such as glucose oxidase. The composition of the mass transport limiting membrane can be the same or different where it covers each active area. Suitable techniques for depositing the mass transport limiting membrane on the active area include, for example, spray coating, painting, inkjet printing, stenciling, roller coating, dip coating, etc., and any combination thereof.

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

[0064] Similarly, one or more enzymes within an active region can be covalently bound to a polymer. When an enzyme system comprising multiple enzymes is present in a given active region, in some embodiments, all of the enzymes may be covalently bound to the polymer, while in other embodiments, only a portion of the enzymes may be covalently bound to the polymer. For example, one or more enzymes comprising the enzyme system may be covalently bound to the polymer, and at least one enzyme may be non-covalently bound to the polymer, such that the non-covalently bound enzyme is physically entrained within the polymer. According to more specific embodiments, covalent binding of an enzyme to a polymer in a given active region can occur via a cross-linking agent introduced with an appropriate cross-linking agent. Suitable cross-linking agents for reaction with free amino groups in an enzyme (e.g., with the free side-chain amine in lysine) can include, for example, polyethylene glycol diglycidyl ether (PEG-DGE) or other polyepoxides, cyanogen chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatized variants thereof. Suitable cross-linking agents for reaction with free carboxylic acid groups in an enzyme can include, for example, carbodiimides. Cross-linking of the enzyme to the polymer is generally intermolecular, but in some embodiments can be intramolecular. In certain embodiments, all enzymes herein can be covalently bound to the polymer.

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

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

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

[0068] In embodiments in which the creatinine-responsive active region and the glucose-responsive active region are disposed on a single working electrode, the redox potential associated with the glucose-responsive active region may be separated from the redox potential of the creatinine-responsive active region by at least about 100 mV, or at least about 150 mV, or at least about 200 mV. The upper limit of the separation between the redox potentials is determined in vivo by the working electrochemical window. By separating the redox potentials of the two active regions sufficiently, an electrochemical reaction can occur within one of the two active regions (i.e., the glucose-responsive active region or the creatinine-responsive active region) without substantially inducing an electrochemical reaction within the other active region. Thus, a signal from one of the glucose-responsive active region or the creatinine-responsive active region can be independently generated at or above its corresponding redox potential (lower redox potential) but below the redox potential (higher redox potential) of the other of the glucose-responsive active region and the creatinine-responsive active region. In contrast, above the redox potential (higher redox potential) of the other, previously uninterrogated, active region, electrochemical reactions can occur in both the glucose-responsive active region and the creatinine-responsive active region. Thus, the signal obtained above the higher redox potential can include signal contributions from both the glucose-responsive active region and the creatinine-responsive active region, and the observed signal is a composite signal. The signal contribution above that redox potential from one active region (either the glucose-responsive active region or the creatinine-responsive active region) can then be determined by subtracting from the composite signal the signal above that redox potential obtained from only the glucose-responsive active region or the creatinine-responsive active region.

[0069] In more specific embodiments, the glucose-responsive active region and the creatinine-responsive active region can contain different electron transfer agents to ensure that the redox potentials of the two active regions are sufficiently separated when the active regions are located on the same working electrode. More specifically, the glucose-responsive active region can contain a first electron transfer agent, and the creatinine-responsive active region can contain a second electron transfer agent, where the first and second electron transfer agents are different. According to various embodiments of the present disclosure, the metal center and / or ligands present in a given electron transfer agent can be varied to ensure that the redox potentials of the two active regions are sufficiently separated.

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

[0071] It should also be understood that the sensitivity (output current) of the analyte sensor to each analyte can be altered by varying the coverage (area or size) of the active regions, the area ratio of the active regions to each other, the nature, thickness, and / or composition of the mass transport limiting membrane covering the active regions, etc. Variations in these parameters can be readily implemented by one of ordinary skill in the art given the benefit of this disclosure.

[0072] Other embodiments of the analyte sensors disclosed herein may feature a creatinine-responsive active region and a glucose-responsive active region on the surface of different working electrodes. Such analyte sensors may further include a second working electrode, a glucose-responsive active region disposed on the surface of the second working electrode, and a second membrane permeable to glucose covering the glucose-responsive active region. The glucose-responsive active region may include a second electron transfer agent, a third polymer, and glucose oxidase covalently bound to the third polymer. The third polymer may be the same as or different from the first and / or second polymers associated with the creatinine-responsive active region or the glucose oxidase, respectively, which do not function in glucose detection. When the creatinine-responsive active region and the glucose-responsive active region are disposed on separate working electrodes, the electron transfer agents associated with each active region may be the same or different.

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

[0074] A detection method for assaying creatinine includes exposing an analyte sensor to a fluid containing at least creatinine, the analyte sensor including at least a first working electrode, a sensor tail including a creatinine-responsive active region disposed on the surface of the first working electrode, a first membrane permeable to creatinine and covering the creatinine-responsive active region, and an oxygen scavenger disposed on the sensor tail and near the creatinine-responsive active region. The creatinine-responsive active region includes a first electron transfer agent, a first polymer, and an enzyme system including multiple enzymes capable of acting in concert to facilitate creatinine detection. The enzyme system includes creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase, all of which may be covalently attached to the first polymer in certain embodiments. The method may further include applying a potential to the first working electrode, obtaining a first signal at or above the redox potential of the creatinine-responsive active region, the first signal being proportional to the concentration of creatinine in the fluid, and correlating the first signal to the concentration of creatinine in the fluid. In certain embodiments of the present disclosure, the fluid may be a biological fluid. In more particular embodiments, glucose may be present in the fluid along with creatinine.

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

[0076] The processor can determine which sensor response value in the lookup table is closest to that measured for the sample with the unknown analyte concentration and report the analyte concentration accordingly. In some or other embodiments, if the sensor response value of the sample with the unknown analyte concentration falls between the values ​​recorded in the lookup table, the processor can interpolate between the two lookup table values ​​to estimate the analyte concentration. The interpolation can assume a linear concentration variation between the two values ​​reported in the lookup table. Interpolation can be used when the sensor response differs sufficiently from a given value in the lookup table (e.g., by about 10% or more).

[0077] Similarly, according to some or various other embodiments, the processor can input the sensor response values ​​of samples having unknown analyte concentrations into a corresponding calibration function, and the processor can then report the analyte concentration accordingly.

[0078] The sensor tail may further include a second working electrode having a glucose-responsive active region disposed thereon, the glucose-responsive active region including a second electron transfer agent, a third polymer, and glucose oxidase covalently bound to the third polymer. Thus, the method may further include: applying a potential to the second working electrode, obtaining a second signal at or above the redox potential of the glucose-responsive active region that is proportional to the concentration of glucose in the fluid, and correlating the second signal to the concentration of glucose in the fluid.

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

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

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

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

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

[0084] Embodiments disclosed herein include the following: A. Creatinine-Responsive Analyte Sensor. The analyte sensor includes: a sensor tail including at least a first working electrode; a creatinine-responsive active region disposed on the surface of the first working electrode, the creatinine-responsive active region including a first electron transfer agent, a first polymer, and an enzyme system including a plurality of enzymes capable of acting in concert to facilitate the detection of creatinine, the enzyme system including creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase; a first membrane permeable to creatinine and covering the creatinine-responsive active region; and an oxygen scavenger disposed on the sensor tail and near the creatinine-responsive active region.

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

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

[0087] Element 2: The oxygen scavenger comprises an oxidase enzyme. Element 3: Oxygen scavengers include glucose oxidase. Element 4: Glucose oxidase is covalently attached to a second polymer.

[0088] Element 5: An oxygen scavenger is disposed on the first membrane, optionally the oxygen scavenger comprises an oxidase enzyme, optionally the oxygen scavenger comprises glucose oxidase, optionally the glucose oxidase is covalently bound to a second polymer.

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

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

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

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

[0093] Element 14: The sensor tail further includes a second working electrode having a glucose-responsive active region disposed on a surface of the second working electrode, the glucose-responsive active region including a second electron transfer agent, a third polymer, and glucose oxidase covalently bound to the third polymer, and the method further includes: applying a potential to the second working electrode; obtaining a second signal at or above the redox potential of the glucose-responsive active region, the second signal being proportional to the concentration of glucose in the fluid; and correlating the second signal to the concentration of glucose in the fluid.

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

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

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

[0097] Example A poly(vinylpyridine)-bound transition metal complex was prepared having the structure shown in Formula 1. Further details regarding this transition metal complex and electron transfer therethrough are provided in commonly owned U.S. Patent No. 6,605,200, incorporated by reference above. The subscripts for each monomer represent exemplary atomic ratios and do not indicate a particular monomer order.

[0098] [ka]

[0099] The buffered spotting formulation (10 mM MES buffer) identified in Table 1 below was deposited onto the carbon working electrode to form the creatinine-responsive active area. The deposition was performed to form a creatinine-responsive active area of ​​0.12 mm. 2 The spotting mixture was used to form a single spot with an area of ​​15 nL. After deposition, the creatinine-responsive active area was cured overnight at 25 °C. The membrane was then dip-coated onto the creatinine-responsive active area using a coating solution consisting of 4 mL of 35 mg / mL polyvinylpyridine-co-styrene, 0.1 mL of 100 mg / mL PEG-DGE400, and 3.3 μL of PDMS in 80:20 ethanol / 10 mM HEPES buffer (pH = 8). No curing was performed at this stage.

[0100] [Table 1]

[0101] After coating the membrane on the creatinine-responsive active area, a buffered spotting formulation containing glucose oxidase as specified in Table 2 was deposited onto the membrane. Specifically, 15 nL of the spotting formulation was applied to a 0.05 mm 2 The coating solution was then dip-coated using the same coating solution as above to form a film on the deposited glucose oxidase.

[0102] [Table 2]

[0103] A control electrode was prepared as above, except that the glucose oxidase deposition onto the creatinine-responsive active area was omitted. Creatinine analysis was performed by immersing the electrodes in 5 mM glucose solutions containing various amounts of creatinine (20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 130 μM, and 200 μM) and measuring the current response. A control electrode without glucose oxidase in the creatinine-responsive active area was also tested under the same conditions. Figure 8 shows an example plot of the current response of three replicate analyte sensors containing a creatinine-responsive active area coated with glucose oxidase upon exposure to various creatinine concentrations at 33 °C. As shown, the current response increased for several minutes after exposure to a new creatinine concentration and then stabilized. In contrast, two control sensors without glucose oxidase-coated creatinine-responsive active areas did not respond to creatinine at any concentration. Figure 9 shows an exemplary plot of the current response of a single-analyte sensor containing a creatinine-responsive active region coated with glucose oxidase when exposed to various creatinine concentrations (20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 130 μM, and 200 μM). As shown, the sensor response was essentially linear over the concentration range tested. Again, a control sensor not coated with glucose oxidase showed essentially no response to creatinine, likely due to oxygen interference with the enzyme system.

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

[0105] One or more exemplary embodiments incorporating various features are presented herein. For clarity, not all features of a physical implementation are described or shown herein. It is understood that in developing a physical embodiment incorporating embodiments of the present invention, numerous implementation-specific decisions must be made to achieve the developer's goals, such as compliance with system-related, business-related, government-related, and other constraints. These will vary from implementation to implementation and from case to case. While the developer's efforts may be time-consuming, such efforts would nevertheless be routine for one of ordinary skill in the art and would have the benefit of this disclosure.

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

[0107] As used herein, the phrase "at least one of" followed by the word "and" or "or" preceding a list of items and separating any of the items modifies the list as a whole, rather than each member (i.e., each item) of the list. The phrase "at least one of" allows for the meaning of including at least one of any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. By way of example, the phrase "at least one of A, B, and C" or "at least one of A, B, or C" refers, respectively, to A only, B only, or C only; any combination of A, B, and C; and / or at least one of each of A, B, and C.

[0108] Thus, the disclosed systems, tools, and methods are well adapted to achieve the objects and advantages mentioned, as well as those inherent therein. The specific embodiments disclosed above are illustrative only, as the teachings of the present disclosure may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Moreover, no limitations are intended to the details of construction or design shown herein, except as described in the appended claims. Accordingly, it will be apparent that the specific exemplary embodiments disclosed above may be altered, combined, or modified, and all such variations are deemed to be within the scope of the present disclosure. The systems, tools, and methods illustratively disclosed herein may suitably be practiced in the absence of elements not specifically disclosed herein and / or optional elements disclosed herein. While systems, tools, and methods are described in terms of "including" various components or steps, the systems, tools, and methods may also "consist essentially of" or "consist of" the various components and steps. All of the numbers and ranges disclosed above may be subject to some variation. Whenever a numerical range with a lower and upper limit is disclosed, any number within that range and any included range is specifically disclosed. Specifically, all range values ​​disclosed herein (in the form "about a to about b," or, equivalently, "about a to b," or, equivalently, "about a to b") should be understood to describe all numbers and ranges within that broader range. Additionally, unless expressly and unambiguously defined by the patent owner, claim terms have their ordinary meanings. If there is a discrepancy in the usage of a word or term between this specification and one or more patents or other documents incorporated herein by reference, the definition in this specification shall control.

Claims

1. 1. An analyte sensor comprising: a sensor tail including at least a first working electrode; a creatinine-responsive active region disposed on a surface of a first working electrode, the enzyme system comprising a first electron transfer agent, a first polymer, and a plurality of enzymes capable of acting in concert to facilitate the detection of creatinine; creatinine amidohydrolase, creatine amidinohydrolase, and Sarcosine oxidase a creatinine-responsive active region comprising an enzyme system comprising: a first membrane permeable to creatinine and covering a creatinine-responsive active area; and An analyte sensor comprising an oxygen scavenger disposed on the sensor tail and near a creatinine-responsive active region.

2. 10. The analyte sensor of claim 1, wherein the oxygen scavenger is separated from the creatinine-responsive active region by a first membrane.

3. 10. The analyte sensor of claim 1, wherein the oxygen scavenger comprises an oxidase enzyme.

4. The analyte sensor of claim 3 , wherein the oxygen scavenger comprises glucose oxidase.

5. 5. The analyte sensor of claim 4, wherein the glucose oxidase is covalently bound to the second polymer.

6. 10. The analyte sensor of claim 1, wherein the oxygen scavenger is disposed on the first membrane.

7. The analyte sensor of claim 6 , wherein the oxygen scavenger comprises an oxidase enzyme.

8. The analyte sensor of claim 7 , wherein the oxygen scavenger comprises glucose oxidase.

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

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

11. 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 bound to the third polymer; and 10. The analyte sensor of claim 1, further comprising a second membrane that is permeable to glucose and that covers the glucose-responsive active region.

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

13. 12. The analyte sensor of claim 11, wherein the first membrane and the second membrane are compositionally the same.

14. 1. An analyte sensor comprising: a sensor tail including a first working electrode and a second working electrode; a creatinine-responsive active region disposed on a surface of a first working electrode, the enzyme system comprising a first electron transfer agent, a first polymer, and a plurality of enzymes capable of acting in concert to facilitate the detection of creatinine; creatinine amidohydrolase, creatine amidinohydrolase, and Sarcosine oxidase a creatinine-responsive active region comprising an enzyme system comprising: a first membrane permeable to creatinine and covering a creatinine-responsive active area; glucose oxidase covalently bonded to a second polymer and disposed on the first membrane; a glucose-responsive active region disposed on the surface of the second working electrode, the glucose-responsive active region comprising a second electron transfer agent, a third polymer, and glucose oxidase covalently bound to the third polymer; and An analyte sensor comprising a second membrane that is permeable to glucose and that covers the glucose-responsive active area.

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

16. 16. The analyte sensor of claim 15, wherein the first membrane, the second membrane, and the third membrane are compositionally the same.

17. 15. The analyte sensor of claim 14, wherein the first membrane and the second membrane are compositionally the same.

18. 15. The analyte sensor of claim 14, wherein the creatinine amidohydrolase, creatine amidinohydrolase, and sarcosine oxidase are each covalently bound to the first polymer.

19. 1. A method comprising: exposing the analyte sensor to a fluid containing at least creatinine; wherein the analyte sensor comprises a sensor tail including at least a first working electrode, a creatinine-responsive active area disposed on a surface of the first working electrode, a first membrane permeable to creatinine and covering the creatinine-responsive active area, and an oxygen scavenger disposed on the sensor tail and near the creatinine-responsive active area; The creatinine-responsive active region is an enzyme system comprising a first electron transfer agent, a first polymer, and a plurality of enzymes capable of acting in concert to facilitate the detection of creatinine, creatinine amidohydrolase, creatine amidinohydrolase, and Sarcosine oxidase containing an enzyme system comprising: applying a potential to the first working electrode; obtaining a first signal proportional to the concentration of creatinine in the fluid at or above the redox potential of the creatinine-responsive active region; and correlating the first signal to a concentration of creatinine in the fluid.

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

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

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

23. The sensor tail further includes a second working electrode having a glucose-responsive active region disposed on a surface of the second working electrode, the glucose-responsive active region including a second electron transfer agent, a third polymer, and glucose oxidase covalently bound to the third polymer, and the method further includes: applying a potential to the second working electrode; obtaining a second signal proportional to the concentration of glucose in the fluid at or above the redox potential of the glucose-responsive active region; and 21. The method of claim 20, comprising correlating the second signal to the concentration of glucose in the fluid.

24. 24. The method of claim 23, wherein the first signal and the second signal are acquired at different times.

25. 24. The method of claim 23, wherein the first signal and the second signal are acquired simultaneously via the first channel and the second channel.

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