A test substance sensor characterized by low potential detection and a detection method

Abstract

To solve a problem where an analyte flux across a membrane may vary considerably as a function of temperature and / or the length of time the analyte sensor has been implanted in a tissue.SOLUTION: An analyte sensor that responds at a low working electrode potential may include an active area on the surface of the working electrode, the active area including a polymer, a redox mediator covalently bound to the polymer, and at least one enzyme that responds to the analyte and is covalently bound to the polymer. Certain redox mediators that react at low potentials may have a structure of (I), where G is a linking group that covalently attaches the redox mediator to the polymer. A mass transfer limiting membrane permeable to the analyte may cover the active area. In some sensor configurations, the mass transfer limiting membrane may comprise a membrane polymer crosslinked by a branched crosslinking agent comprising three or more crosslinking groups, such as polyethylene glycol tetraglycidyl ether.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to a test substance sensor and a detection method.

Background Art

[0002] The detection of various test substances within an individual can sometimes be important for monitoring health status. Deviations from normal test substance levels can indicate many physiological conditions. Glucose levels, for example, are particularly important for detection and monitoring in individuals with diabetes. By monitoring glucose levels with sufficient regularity, individuals with diabetes can take corrective measures before serious physiological discomfort occurs (e.g., by injecting insulin to lower glucose levels or by eating to raise glucose levels). For other physiological conditions, it may be desirable to monitor other test substances. In some cases, it may also be desirable to monitor multiple test substances, particularly for concurrent conditions that cause abnormal regulation of two or more test substances in combination with each other.

[0003] If suitable detection chemistries can be identified, many test substances become interesting targets for physiological analysis. For this purpose, amperometric sensors configured to continuously assay glucose in vivo have been developed and improved in recent years and are useful for monitoring the health of individuals with diabetes. Other test substances that are commonly dysregulated simultaneously with glucose in individuals with diabetes include, for example, lactate, oxygen, pH, A1c, and ketones. It may also be desirable to monitor these together with other test substances that are independent of glucose dysregulation. Test substance sensors configured to detect test substances other than glucose in vivo are known, but are not currently sufficiently improved. Low sensitivity to small amounts of test substances can be particularly problematic.

[0004] Personal analyte monitoring can be performed periodically or continuously over a period of time. Periodic analyte monitoring can be performed by collecting samples of body fluids, such as blood or urine, at set time intervals and analyzing them in vitro. Periodic in vitro analyte monitoring is sufficient to determine the physiological state of many individuals. However, in vitro analyte monitoring can be inconvenient or painful in some cases. Furthermore, there is no way to fill in missing data if the analyte measurement is not obtained at the appropriate time. Continuous analyte monitoring may be performed using one or more sensors that are at least partially implanted within an individual's tissue, such as in the skin, subcutaneous, or intravenous, so that the analysis can be performed in vivo. Depending on an individual's specific health needs and / or previously measured analyte levels, an implantable sensor may collect analyte data on demand, at a determined schedule, or continuously. Analyte monitoring using an in vivo implantable sensor is a more desirable approach for individuals with severe analyte dysregulation and / or rapidly fluctuating analyte levels. However, it can also be beneficial to other individuals as well. Since implantable analyte sensors often remain within an individual's tissue for extended periods of time, it may be highly desirable to manufacture such analyte sensors from stable materials that exhibit high biocompatibility.

[0005] To improve biocompatibility, the analyte sensor can include a membrane disposed over the entire embedded portion of the sensor, particularly a membrane covering at least the active region of the sensor. In addition to promoting biocompatibility, the membrane may be permeable or semi-permeable to the analyte of interest and restrict the total analyte flux to the active region of the sensor. Such a mass transfer limiting membrane can assist in avoiding overloading (saturation) of the sensing component within the active region, thereby improving sensor performance and accuracy. For example, in the case of a sensor performing enzyme-based detection, restricting the mass transfer of the analyte to the active region can make the kinetics of the detection process analyte-limited rather than enzyme-limited, thereby enabling easy correlation of the sensor output to the amount of analyte present.

[0006] One problem associated with equipping the analyte sensor with a membrane is that the analyte flux across the membrane can vary significantly depending on temperature and / or the length of time the analyte sensor is embedded in tissue. Some membrane materials are less affected by temperature-dependent analyte flux than others. If necessary, a calibration factor or calibration equation can be employed to evaluate the variability of the analyte flux with temperature, but this can significantly complicate the use of the sensor. Composition changes in the membrane can occur over time, particularly due to extraction losses and / or membrane degradation or metabolism of various membrane components, and can cause the permeability value of the analyte to vary depending on the degree of composition change. It can be difficult to quantitatively address composition changes that will cause the membrane permeability value to vary, and it can be difficult to determine when the membrane permeability will stabilize sufficiently to enable accurate measurement of the analyte concentration. Often, when a new analyte sensor is implanted in the body, an equilibration period of several hours or more may be required for the analyte flux across the membrane to stabilize.

Brief Description of the Drawings

[0007] The following drawings are included to illustrate certain aspects of the present disclosure and should not be regarded as exclusive embodiments. The disclosed subject matter is capable of numerous modifications, variations, combinations, and equivalents in form and function without departing from the scope of the present disclosure.

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Mode for Carrying Out the Invention

[0018] The present disclosure generally describes an analyte sensor suitable for in vivo use, and more specifically, the analyte sensor comprises components including low potential operating ability and / or characteristics of a stabilized membrane material. Depending on the sensor configuration, the analyte sensor of the present disclosure can be configured to detect one analyte or multiple analytes simultaneously or almost simultaneously. A series of dip coating operations can be performed to introduce different membrane compositions at specific multiple positions on the analyte sensor.

[0019] The components of various analyte sensors can cause certain impairments during the monitoring of some analytes or combinations of analytes. The redox mediator used to facilitate the transfer of electrons to the working electrode may require operating the analyte sensor at a relatively high potential, and the operation of the analyte sensor at a high potential may lead to electrochemical side reactions that complicate the detection of small amounts of analytes. Changes in the composition of the mass transfer limiting membrane within the analyte sensor can lead to undesirable changes in the permeability of the analyte during the period when the analyte sensor is implanted in the body, particularly during a long-term sensor wearing period. Further, when analyzing multiple analytes using a single analyte sensor, different analyte permeation characteristics may require the use of different mass transfer limiting membranes at various positions.

[0020] To address the foregoing needs, the present disclosure provides a redox mediator for promoting electron transfer at an operating electrode potential lower than that of previously used materials. Use of such a "low potential" redox mediator can reduce the occurrence of electrochemical side reactions by enabling detection of the analyte at a potential lower than would otherwise be possible. By reducing the occurrence of electrochemical side reactions and associated signal noise, detection of small amounts of analytes such as ketones can be performed more readily than would otherwise be possible at a higher operating electrode potential. Such low potential redox mediators can be advantageous when used in combination with detection of multiple analytes, as will be further discussed below. A redox mediator capable of promoting detection of an analyte at a low operating electrode potential is further described below.

[0021] Various crosslinked polyvinylimidazole and polyvinylpyridine film polymers can be used in analyte sensors to improve biocompatibility and provide properties that limit mass transfer. Functionalization on the membrane material can be selected to modify the permeability of the analyte and to limit analyte permeability changes due to temperature. Such membrane polymers can be crosslinked to a linear glycidyl ether having two crosslinking groups, such as polyethylene glycol diglycidyl ether (PEGDGE), but an equilibration period may be required after sensor implantation to enable stabilization of the analyte flux. Without being bound by theory or mechanism, it is believed that the composition of the membrane changes during the equilibration period in which a small amount of extractable material is released from the membrane, thereby potentially changing the permeability of the analyte. Surprisingly, the present disclosure shows that a branched crosslinking agent containing three or more crosslinking groups, more specifically a branched polyethylene glycol glycidyl ether such as polyethylene glycol tetraglycidyl ether, can reduce the amount of extractable material released from the membrane after sensor implantation. A reduction in the production of extractable material from the membrane can be achieved even if the crosslink density and the amount (mass) of the crosslinking agent are substantially the same as those provided by a linear glycidyl ether having two crosslinking groups. That is, the amount of a given crosslinking group from a branched crosslinking agent such as polyethylene glycol tetraglycidyl ether can result in a reduction in extractable material for a given membrane material as compared to the amount of extractable material resulting from a substantially similar amount of crosslinking groups obtained from a linear glycidyl ether crosslinking agent such as polyethylene glycol diglycidyl ether. Advantageously, as disclosed herein, the reduction in extractable material can be derived from a membrane material crosslinked with a branched crosslinking agent, thereby providing an improved toxicity profile. Further, the reduction in compositional changes provided by the membrane materials disclosed herein may provide a shorter sensor equilibration time and potentially extend the wear life of the sensor after the sensor is implanted.

[0022] Before further elaborating on the analyte sensors of the present disclosure and their components, an overview of a suitable in-vivo analyte sensor configuration and a sensor system using such analyte sensors is first provided so that embodiments of the present disclosure can be better understood. FIG. 1 shows a diagram of an exemplary detection system that can incorporate the analyte sensors of the present disclosure. As shown in the figure, the detection system 100 includes a sensor control device 102 and a reading device 120 configured to communicate with each other via a local communication path or link, either wired or wireless, in one direction or two directions, and either encrypted or unencrypted. According to some embodiments, the reading device 120 may constitute an output device for viewing the concentration of the analyte and warnings or notifications determined by the sensor 104 or an associated processor, and may similarly enable input from one or more users. The reading device 120 can be a multi-purpose smartphone or a dedicated electronic reading device. Although only one reading device 120 is shown, multiple reading devices 120 may exist in certain cases. The reading device 120 can also communicate with a remote terminal 170 and / or a reliable computer system 180 via communication paths / links 141 and / or 142, respectively, either wired or wireless, in one direction or two directions, and either encrypted or unencrypted. In addition or alternatively, the reading device 120 can communicate with a network 150 (e.g., a mobile telephone network, the Internet, or a cloud server) via a communication path / link 151. The network 150 can be further communicatively connected to the remote terminal 170 via a communication path / link 152 and / or to the reliable computer system 180 via a communication path / link 153. Alternatively, the sensor 104 can communicate directly with the remote terminal 170 and / or the reliable computer system 180 without the presence of an intervening reading device 120.For example, according to some embodiments, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is hereby incorporated by reference in its entirety, sensor 104 can communicate with remote terminal 170 and / or reliable computer system 180 via a direct communication link to network 150. Any suitable electronic communication protocol can be used for each of the communication paths or links, such as near field communication (NFC), radio frequency identification (RFID), BLUETOOTH® or BLUETOOTH® low energy protocol, or WiFi. According to some embodiments, remote terminal 170 and / or reliable computer system 180 can be accessible by individuals other than the first user who is interested in the user's analyte level. Reader device 120 can include display portion 122 and any input element 121. According to some embodiments, display portion 122 can include a touch screen interface.

[0023] Sensor control device 102 includes sensor housing 103 and can house the electrical circuits and power source for operating sensor 104. Optionally, the power source and / or active electrical circuits can be removed. A processor (not shown) may be communicatively connected to sensor 104 and the processor is physically incorporated within sensor housing 103 or reader device 120. Sensor 104 protrudes from the lower side of sensor housing 103 and extends through adhesive layer 105 and is suitable for adhering sensor housing 103 to a tissue surface such as skin according to some embodiments.

[0024] Sensor 104 is adapted to be at least partially inserted into a target tissue such as the dermis or subcutaneous layer of the skin. Sensor 104 may include a sensor tail that is long enough to be inserted to a desired depth in a given tissue. The sensor tail may include at least one working electrode. In a particular configuration, it is further discussed that the sensor tail may include a ketone-responsive active region and, in certain cases, a low-potential redox mediator. A counter electrode may be present in combination with at least one working electrode. The specific electrode configuration on the sensor tail is described in further detail below.

[0025] One or more mass transfer limiting membranes, particularly mass transfer limiting membranes crosslinked by a branched glycidyl ether such as polyethylene glycol tetra glycidyl ether, may cover the active region, as will be described in further detail below. The active region may be configured to detect a particular analyte. For example, a glucose-responsive active region may include a glucose-responsive enzyme, a lactate-responsive active region may include a lactate-responsive enzyme, and a ketone-responsive active region may include an enzyme system including at least two enzymes that can work together to facilitate the detection of ketones. A suitable enzyme system for detecting ketones is described in further detail below with reference to FIGS. 6A through 6C. According to various embodiments, each active region may include a polymer that covalently binds to at least some of the enzymes.

[0026] In any embodiment of the present disclosure, one or more analytes may be monitored in a target body fluid such as skin fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, or amniotic fluid. In a particular embodiment, the analyte sensor of the present disclosure may be adapted for an assay of dermal or interstitial fluid to determine the concentration of one or more analytes in vivo.

[0027] Referring further to FIG. 1, sensor 104 can automatically transfer data to the reader device 120. For example, the analyte concentration data (i.e., glucose concentration and / or ketone concentration) can be automatically and periodically communicated using the data stored in memory until the data is transmitted, such as at a particular frequency at which the data is obtained or after a particular period of time has elapsed (e.g., every minute, every 5 minutes, or other predetermined time period). In other embodiments, sensor 104 can communicate with reader device 120 in a non-automatic manner and not according to a set schedule. For example, the data can be communicable from sensor 104 using RFID technology when the sensor electronics are brought within the communication range of reader device 120. The data can remain stored in the memory of sensor 104 until it is communicated to reader device 120. Thus, the user does not need to constantly remain in the vicinity of reader device 120 and instead can upload the data at a convenient time. In yet other embodiments, a combination of automatic and non-automatic data transfer can be implemented. For example, data transfer can continue automatically until reader device 120 is no longer present within the communication range of sensor 104.

[0028] The introducer may be present temporarily to facilitate the introduction of the sensor 104 into the tissue. In an exemplary embodiment, the introducer may comprise a needle or a similar sharp portion. It should be recognized that other types of introducers, such as sheaths or blades, may exist in alternative embodiments. More specifically, the needle or other introducer may be present near the sensor 104 temporarily before insertion into the tissue and may then be 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 the penetration of the epithelium as an access path to the dermis to enable the implantation of the sensor 104. The needle or other introducer may be withdrawn after opening the access path so as not to present a danger due to the sharp tip. In an exemplary embodiment, a suitable needle may be solid or hollow, have an acute or non-acute angle, and / or be circular or non-circular in cross-section. In a more particular embodiment, a suitable needle may be comparable to a needle for acupuncture in terms of the cross-sectional diameter and / or the tip design and may have a cross-sectional diameter of about 250 microns (250 μm). However, it should be recognized that a suitable needle may have a larger or smaller cross-sectional diameter if required for a particular application.

[0029] In some embodiments, the tip of the needle (while present) may be angled on the end of the sensor 104 such that the needle first penetrates the tissue and opens an access path for the sensor 104. In other exemplary embodiments, the sensor 104 may be present in the lumen or groove of the needle such that the needle similarly opens an access path for the sensor 104. In either case, the needle is then withdrawn after facilitating the insertion of the sensor.

[0030] A sensor configuration including a single active region configured to detect a corresponding single analyte may employ a two - electrode or three - electrode detection motif, as further described herein with reference to FIGS. 2A through 2C. A sensor configuration including two different active regions for the detection of different analytes, either on separate working electrodes or on the same working electrode, will be described separately later with reference to FIGS. 3A through 5C. A sensor configuration having multiple working electrodes can be particularly advantageous for incorporating two different active regions within the same sensor tail because the signal contributed by each active region can be more easily determined. Further, the deposition of different film compositions on each active region can be readily performed by a series of dip - coating operations when the active regions are present on a second working electrode.

[0031] When a single working electrode is present in the analyte sensor, the three - electrode sensor configuration can include a working electrode, a counter electrode, and a reference electrode. The related two - electrode sensor configuration may include a working electrode and a second electrode, where the second electrode can function as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). The various electrodes may be at least partially stacked on top of each other (layered), and / or laterally spaced apart from each other on the sensor tail. A suitable sensor configuration may be in a substantially flat form or in a substantially cylindrical form. In any of the sensor configurations disclosed herein, the various electrodes can be electrically insulated from each other by a dielectric material or a similar insulator.

[0032] An analyte sensor having multiple working electrodes may similarly include at least one additional electrode. When one additional electrode is present, the one additional electrode can function as a counter / reference electrode for each of the multiple working electrodes. When 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.

[0033] Figure 2A shows a schematic diagram of an exemplary two - electrode analyte sensor configuration, which is suitable for use in the disclosure of this specification. As shown in the figure, 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 can be disposed on the same side of the substrate 212 together with an intervening dielectric material (the configuration is not shown). The active region 218 is disposed as at least one layer on at least a portion of the working electrode 214. The active region 218 can include a plurality of spots or a single spot configured to detect an analyte at a low working electrode potential, as further discussed herein.

[0034] Referring further to Figure 2A, the membrane 220, according to some embodiments, can cover at least the active region 218, optionally covering some or all of the working electrode 214 and / or the counter / reference electrode 216, or the entire analyte sensor 200. One or both sides of the analyte sensor 200 can be covered with the membrane 220. The membrane 220 can include one or more polymeric membrane materials capable of restricting the analyte flux to the active region 218 (i.e., the membrane 220 is a mass transfer - limiting membrane that is permeable to the analyte of interest). According to the disclosure of this specification, the membrane 220 can be cross - linked using a branching cross - linker in a particular sensor configuration. The composition and thickness of the membrane 220 can be varied to promote the desired analyte flux to the active region 218, thereby providing the desired signal strength and stability. The analyte sensor 200 is operable to assay an analyte by any electrochemical detection technique of coulometry, amperometry, voltammetry, or potentiometry.

[0035] Figures 2B and 2C show schematic diagrams of exemplary three - electrode analyte sensors, and such sensor configurations are also suitable for use in the disclosure herein. The three - electrode analyte sensor configuration may be similar to the configuration shown as analyte sensor 200 in Figure 2A, except that it includes additional electrodes 217 in analyte sensors 201 and 202 (Figures 2B and 2C). When additional electrode 217 is present, counter / reference electrode 216 may then function as either the counter electrode or the reference electrode, and additional electrode 217 serves the other electrode function that is not configured. Working electrode 214 continues to perform its original function. Additional electrode 217 may be disposed on either working electrode 214 or electrode 216 with a separation layer of dielectric material sandwiched therebetween. For example, as shown in Figure 2B, dielectric layers 219a, 219b, and 219c separate electrodes 214, 216, and 217 from each other and provide electrical insulation. Alternatively, at least one of electrodes 214, 216, and 217 may be disposed on both sides of substrate 212, as shown in Figure 2C. Thus, in some embodiments, electrode 214 (working electrode) and electrode 216 (counter electrode) may be disposed on both sides of substrate 212, and electrode 217 (reference electrode) is disposed on one of electrode 214 or electrode 216 and spaced apart from each other with a dielectric material sandwiched therebetween. Reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, and the arrangement of reference material layer 230 is not limited to the arrangements shown in Figures 2B and 2C. Similar to sensor 200 shown in Figure 2A, the active regions 218 in analyte sensors 201 and 202 may include multiple spots or a single spot. Further, analyte sensors 201 and 202 may be operated to assay an analyte by any coulometric, amperometric, voltammetric, or potentiometric electrochemical detection technique.

[0036] In analyte sensors such as analyte sensor 200, analyte sensors 201 and 202, membrane 220 may also cover active region 218, as in other sensor configurations, thereby serving as a mass transport limiting membrane. Additional electrodes 217 may be covered by membrane 220 in some embodiments. FIGS. 3B and 2C show all electrodes 214, 216, and 217 covered by membrane 220, although it should be appreciated that in some embodiments only working electrode 214 may be covered. Further, the thickness of membrane 220 at each of electrodes 214, 216, and 217 may be the same or different, and / or the composition of the membrane may vary locally. As in the two-electrode analyte sensor configuration (FIG. 2A), one or both sides of analyte sensors 201 and 202 may be covered by membrane 220 in the sensor configurations of FIGS. 2B and 2C, or analyte sensors 201 and 202 may be entirely covered. It should further be understood that the three-electrode sensor configurations shown in FIGS. 2B and 2C are not limited to the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of this disclosure.

[0037] FIG. 3A shows an exemplary configuration of sensor 203 having a single working electrode with two different active regions disposed thereon. FIG. 3A is similar to FIG. 2A except that there are two active regions on working electrode 214. First active region 218a and second active region 218b respond to different analytes and are laterally spaced apart from each other 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 be different or compositionally the same in active regions 218a and 218b. First active region 218a and second active region 218b may be configured to detect corresponding analytes at different working electrode potentials, as further discussed below.

[0038] FIG. 3B and FIG. 3C each show a cross-sectional view of an exemplary three-electrode sensor configuration for sensors 204 and 205, and feature a single working electrode having a first active region 218a and a second active region 218b disposed on the single working electrode. FIGS. 3B and 3C are otherwise similar to FIGS. 2B and 2C, which may be better understood by reference. Similar to FIG. 3A, the composition of the membrane 220 may vary or be compositionally the same in the active regions 218a and 218b.

[0039] Exemplary sensor configurations having multiple working electrodes, particularly two working electrodes, are described in further detail with reference to FIGS. 4 through 5C. The following description is primarily directed to sensor configurations having two working electrodes, although it should be recognized that configurations having more than two working electrodes may be incorporated by extension of the disclosure herein. Additional working electrodes may be used to provide additional sensing functionality to the analyte sensor for analytes other than the first analyte and the second analyte.

[0040] FIG. 4 shows a cross-sectional view of an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode, and is suitable for use in the disclosure of this specification. As shown in the figure, the analyte sensor 300 includes working electrodes 304 and 306 disposed on both sides of a substrate 302. The first active region 310a is disposed on the surface of the working electrode 304, and the second active region 310b is disposed on the surface of the working electrode 306. The counter electrode 320 is electrically insulated from the working electrode 304 by a dielectric layer 322, and the reference electrode 321 is electrically insulated from the working electrode 306 by a dielectric layer 323. The outer dielectric layers 330 and 332 are disposed on the reference electrode 321 and the counter electrode 320, respectively. The film 340 may, according to various embodiments, cover at least the active regions 310a and 310b, and similarly, other components of the analyte sensor 300 or the entire analyte sensor 300 may optionally be covered by the film 340. Further, if necessary, the film 340 may be compositionally different between the active regions 310a and 310b to provide a permeability value suitable for differentially regulating the analyte flux at each location. For example, the film 340 may be homogeneous at the location covering the active region 310a and heterogeneous at the location covering the active region 310b.

[0041] An alternative sensor configuration having a plurality of working electrodes and different from the configuration shown in FIG. 4 may comprise one counter / reference electrode instead of separate counter and reference electrodes 320, 321, and / or may be characterized in that the explicitly shown arrangement of layers and / or membranes is different. For example, the arrangement of counter electrode 320 and reference electrode 321 may be reversed from the arrangement shown in FIG. 4. Further, working electrodes 304 and 306 do not necessarily have to be present on both sides of substrate 302 in the arrangement shown in FIG. 4. Instead, working electrodes 304 and 306 may be present on the same surface of substrate 302 and may be spaced apart from each other. In particular, working electrode 304 may be arranged in the direction of the more distal end (tip) of the analyte sensor or in the direction of the more proximal end, compared to working electrode 306. If there is a sufficiently large spacing between working electrodes 304 and 306, membrane 340 may be deposited on working electrodes 304 and 306 by a series of dip coating operations, and the composition of membrane 340 may vary locally. In particular, the first dip coating operation may deposit a first membrane polymer on both of working electrodes 304 and 306, and the second dip coating operation may deposit a second membrane polymer having a different composition only on working electrode 304, thereby defining two layers on working electrode 304 and leaving a uniform membrane on working electrode 306. Thus, the layer under the two-layer membrane and the uniform membrane may contain the same membrane polymer. Instead, to define the two-layer membrane and the uniform membrane, the first dip coating operation may deposit a first membrane polymer on working electrode 304, and the second dip coating operation may deposit a second membrane polymer having a different composition on both of working electrodes 304 and 306. In this case, the upper layer of the two-layer membrane and the uniform membrane may contain the same membrane polymer.

[0042] The distance between the working electrodes 304 and 306 is large enough to provide sufficient margin for at least electrical insulation between the two electrodes, and more typically, it is large enough to enable a series of dip coating operations (e.g., by allowing the analyte sensor to be immersed at different depths to preferentially cover one of the working electrodes in at least one dipping step). In other words, the distance can provide a margin of error for lowering the analyte sensor to a specific depth within a particular dip coating agent in order to facilitate film formation on one of the working electrodes preferentially over the other.

[0043] A suitable sensor configuration may feature electrodes of a substantially flat nature, including a flat sensor configuration with spaced-apart working electrodes. However, it should be understood that sensor configurations featuring non-flat electrodes can be advantageous for use in the disclosure herein and may be particularly suitable. In particular, cylindrical electrodes arranged concentrically with each other and spaced apart along the length of the sensor tail can facilitate the formation of mass transfer limiting films of different compositions, as will be further described below. In particular, concentric working electrodes spaced apart along the length of the sensor tail can facilitate film formation by a series of dip coating operations, similar to the method described above for a substantially flat sensor configuration. FIGS. 5A through 5C show perspective views of an analyte sensor comprising two working electrodes arranged concentrically with each other. It should also be understood that sensor configurations having a concentric electrode arrangement but no second working electrode are possible in the present disclosure.

[0044] FIG. 5A shows a perspective view of an exemplary sensor configuration in which a plurality of electrodes are substantially cylindrical and arranged concentrically with respect to a central substrate. As shown in the figure, the analyte sensor 400 includes a central substrate 402 on which all the electrodes and dielectric layers are arranged concentrically with each other. In particular, the working electrode 410 is disposed on the surface of the central substrate 402, and the dielectric layer 412 is disposed distally with respect to the sensor tip 404 over a portion of the working electrode 410. The working electrode 420 is disposed on the dielectric layer 412, and the dielectric layer 422 is disposed distally with respect to the sensor tip 404 over a portion of the working electrode 420. The counter electrode 430 is disposed on the dielectric layer 422, and the dielectric layer 432 is disposed distally with respect to the sensor tip 404 over a portion of the counter electrode 430. The reference electrode 440 is disposed on the dielectric layer 432, and the dielectric layer 442 is disposed distally with respect to the sensor tip 404 over a portion of the reference electrode 440. Thus, the exposed surfaces of the working electrode 410, the working electrode 420, the counter electrode 430, and the reference electrode 440 are spaced apart from each other along the longitudinal axis B of the analyte sensor 400. This electrode arrangement may enable the film 450 to be deposited by a series of dip coating operations, and as discussed below, may enable a bilayer film portion to be disposed on the working electrode 410 and a uniform film portion to be disposed on the working electrode 420. The bilayer film portion and the uniform film portion may be adjacent to each other.

[0045] Referring further to FIG. 5A, a first active region 414a and a second active region 414b that respond to different analytes are disposed on the exposed surfaces of the working electrodes 410 and 420, respectively, thereby enabling contact with the fluid to be made for sensing. The active regions 414a and 414b are shown as three separate spots in FIG. 5A, but it should be understood that they may include a continuous layer of active regions and that alternative sensor configurations may exist with fewer or more than three spots.

[0046] In FIG. 5A, sensor 400 is partially coated by membrane 450 over working electrodes 410 and 420 and active regions 414a and 414b disposed thereon. FIG. 5B shows an alternative sensor configuration in which substantially the entire sensor 401 is covered by membrane 450. Membrane 450 may be compositionally the same or different in active regions 414a and 414b. For example, membrane 450 may include a bilayer membrane portion covering active region 414a and may be a uniform membrane portion covering active region 414b. Membrane 450 may be deposited by a series of dip coating operations to deposit a bilayer membrane portion over working electrode 410 and active region 414a and a uniform membrane portion over working electrode 420 and active region 414b.

[0047] It should further be understood that the various electrode arrangements in Figures 5A and 5B may differ from those explicitly shown. For example, the arrangement of the counter electrode 430 and the reference electrode 440 may be inverted from the configuration shown in Figures 5A and 5B. Similarly, the arrangement of the working electrodes 410 and 420 is not limited to the arrangement explicitly shown in Figures 5A and 5B. Figure 5C shows an alternative sensor configuration to that shown in Figure 5B, where the sensor 405 includes the counter electrode 430 and the reference electrode 440 disposed more proximally relative to the sensor tip 404, and the working electrodes 410 and 420 disposed more distally relative to the sensor tip 404. A sensor configuration in which the working electrodes 410 and 420 are disposed more distally relative to the sensor tip 404 may be advantageous by providing a larger surface area for deposition of the active areas 414a and 414b (five separate sensing spots are illustratively shown in Figure 5C), thereby facilitating enhanced signal strength in some cases. Furthermore, by having working electrodes 410 and 420 concentric with one another and spaced apart along the sensor tail, a bilayer film portion may be deposited on the working electrode located near the sensor tip 404 and a uniform film portion may be deposited on the working electrode distal to the sensor tip 404 by a series of dip coating operations. Similarly, the central substrate 402 may be eliminated in any of the concentric sensor configurations disclosed herein, with the farthest electrode instead supporting subsequent deposition layers.

[0048] It should also be understood that the analyte sensor capable of operating at low potential according to the following disclosure may include a mass transport limiting membrane having a bilayer membrane portion and a uniform membrane portion. A series of dip coating operations may be advantageous for deposition of such a membrane. However, the present disclosure also contemplates an analyte sensor further equipped with a mass transport limiting membrane including a bilayer membrane portion and a uniform membrane portion, although detection is not necessarily performed at low potential.

[0049] As a result, according to the present disclosure, an analyte sensor operable at a low potential includes a sensor tail portion including at least a first working electrode, and a first active region disposed on the surface of the first working electrode and responsive to a first analyte at a low potential, the first active region including a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme responsive to the first analyte covalently bonded to the first polymer, and a mass transfer limiting membrane permeable to the first analyte covering at least the first active region. As used herein, the term "low potential" refers to a potential higher than the redox potential of the first redox mediator, and when measured relative to an Ag / AgCl reference electrode, represents a potential of less than about +200 mV, including less than about +100 mV, less than about -50 mV, less than about -80 mV, or less than about -100 mV. Exemplary redox potentials of the first redox mediator that can facilitate operation at a potential such as the working electrode potential can be less than about -200 mV, such as from about -400 mV to about -200 mV, or from about -350 mV to about -250 mV, or from about -300 mV to about -250 mV when measured relative to an Ag / AgCl reference electrode.

[0050] A suitable example of a first redox mediator that can facilitate operation at a low potential may have a structure represented by Formula 1, [Chemical Formula] wherein M is osmium, ruthenium, vanadium, cobalt, or iron, and L 1 to L 6 are independent heteroaromatic ligands coordinately bonded to M, and two or more of L 1 to L 6 may optionally be bonded together to form a bidentate, tridentate, or higher dentate ligand, and at least one of L 1 to L 6 includes a linking group for bonding the first redox mediator to the first polymer, and L 1 to L 6At least one of them is functionalized by an electron-donating group. The electron-donating group is separated from and different from the linking group.

[0051] Particularly suitable bidentate ligands characterized by a heteroaromatic ligand contained in a low-potential redox mediator include optionally substituted 2,2'-biimidazole ligands, 2-(2-pyridyl)imidazole ligands, and 2,2'-bipyridine ligands.

[0052] Examples of 2,2'-biimidazole ligands are represented by Formula 2,

Chemical Formula

[0053] Examples of 2-(2-pyridyl)imidazole ligands may have a structure represented by Formula 3,

Chemical Formula

[0054] Examples of 2,2'-bipyridine ligands may have a structure represented by formula 4,

Chemical formula

[0055] Particularly suitable examples of redox mediators capable of promoting electron transfer at low potentials may have a structure represented by formula 5, L 1 and L 2 , L 3 and L 4 , and L 5 and L 6 are all bonded to form a bidentate ligand, and one of the bidentate ligands has a linking group G that covalently bonds the redox mediator to the polymer, L 1 -L 2 , L 3 -L 4 , and L 5 -L6 At least one of them has an electron-donating group. The electron-donating group is separated from and different from the linking group. The electron-donating group may be on the same bidentate ligand containing the linking group G, or on different bidentate ligands. L 1 -L 2 、L 3 -L 4 、and L 5 -L 6 may include, for example, a bidentate ligand represented by one or more of Formula 2 to Formula 4. [Chemical formula]

[0056] In some embodiments, the redox mediator may be charged with a positive charge (e.g., a charge in the range of +1 to +5). Alternatively, when the ligand or the main chain is derivatized by a sufficient number of negatively charged functional groups such as, for example, carboxylic acid groups, phosphate groups, or sulfonic acid groups, the redox mediator is charged with a negative charge (e.g., a charge in the range of -1 to -5). One or more counterions are used to balance the charge. Examples of suitable counterions are anions such as halide ions (e.g., fluoride ions, chloride ions, bromide ions, or iodide ions), sulfate ions, phosphate ions, hexafluorophosphate ions, and tetrafluoroborate ions, and cations, particularly monovalent cations such as lithium ions, sodium ions, potassium ions, tetraalkylammonium ions, and ammonium ions.

[0057] In a more specific example, the redox mediator in the first active region may have a structure represented by Formula 6, [Chemical formula] In the formula, G is a linking group that covalently attaches a redox mediator to the polymer in the active region, and D is an electron-donating group. Specific examples of suitable electron-donating groups include, for example, a hydroxyl group, an alkoxy group (e.g., a methoxy group or an ethoxy group), an amino group, or an alkyl group or a dialkylamino group (e.g., a methylamino group, an ethylamino group, a dimethylamino group, or a diethylamino group). In a more specific example, the electron-donating group can be located at the 4-position of the pyridine ring, as shown in Formula 7.

Chemical formula

Chemical formula

[0058] In at least one embodiment, the linking group G can include a reactive group for promoting covalent bonding to the polymer. This reactive group reacts with a complementary reactive group located on the polymer or within the polymer precursor to promote covalent bonding thereto. In some embodiments, an amide group can be present within the linking group G.

[0059] The backbone of any suitable polymer may be present in the active region to facilitate detection of analytes at low potentials by covalent attachment of a redox mediator and an enzyme thereto. Examples of suitable polymers within the active region include poly(4-vinylpyridine) and poly(N-vinylimidazole) or copolymers thereof. For example, quaternized pyridine groups and quaternized imidazole groups serve as attachment sites for the redox mediator or enzyme thereto. Other suitable polymers that may be present in the active region include, but are not limited to, polymers such as those described in U.S. Patent No. 6,605,200, all of which are hereby incorporated by reference herein, including poly(acrylic acid), styrene / maleic anhydride copolymer, methyl vinyl ether / maleic anhydride copolymer (GANTREZ polymer), poly(vinylbenzyl chloride), poly(allylamine), polylysine, poly(4-vinylpyridine) quaternized with carboxypentyl groups, and poly(sodium 4-styrenesulfonate).

[0060] In the first active region, it is believed that enzymes covalently attached to a polymer capable of promoting detection at low potentials are not particularly limited. Suitable enzymes may include enzymes capable of detecting glucose, lactate, ketones, creatinine, etc. In some cases, at least one enzyme covalently attached to the polymer in the first active region may include a plurality of enzymes that respond as a group to an analyte at low potentials. The enzyme system may be particularly desirable for detecting ketones and creatinine.

[0061] In a more specific embodiment, the first active region may include an enzyme system capable of detecting ketones. As mentioned previously, ketones are typically present in low biological amounts and can benefit from detection at low potentials according to the disclosure herein. Now, referring to FIGS. 6A through 6C, a specific enzyme system that can be used to detect ketones will be described in more detail. In the shown enzyme reaction, β-hydroxybutyric acid serves as a surrogate for ketones formed in vivo. As shown in FIG. 6A, a pair of cooperative enzymes that can be used to detect ketones according to the disclosure herein are β-hydroxybutyrate dehydrogenase (HBDH) and diaphorase, which can be deposited within a ketone-responsive active region on the surface of at least one working electrode, as further described herein. When the ketone-responsive active region contains this pair of cooperative enzymes, β-hydroxybutyrate dehydrogenase converts β-hydroxybutyric acid and oxidized nicotinamide adenine dinucleotide (NAD + ) to acetoacetate and reduced nicotinamide adenine dinucleotide (NADH), respectively. The enzyme cofactors NAD + and NADH serve to facilitate the cooperative enzyme reaction disclosed herein. NADH may then undergo oxidation via diaphorase, and the transfer of electrons during this process provides the basis for ketone detection at the working electrode. Thus, there is a 1:1 molar correspondence between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyric acid converted, thereby providing the basis for ketone detection and quantification based on measurement of the current flow of the working electrode. The transfer of electrons that results in NADH oxidation at the working electrode can be effected by a redox mediator capable of promoting operation at low potentials. Albumin may be present as a stabilizer along with this pair of cooperative enzymes. According to a particular embodiment, β-hydroxybutyrate dehydrogenase and diaphorase can be covalently bound to a polymer within the ketone-responsive active region of the analyte sensor. NAD + may or may not be covalently bound to the polymer, and if NAD + is not covalently bound, it can physically remain within the ketone-responsive active region. The membrane covering the ketone-responsive active region may contain NAD +Helps to retain and allows sufficient internal diffusion of ketones to enable detection of ketones.

[0062] Other suitable chemistries for enzymatically detecting ketones are shown in FIGS. 6B and 6C. In both cases, there is a further 1:1 molar correspondence between the amount of electrons transferred to the working electrode and the amount of conversion of β-hydroxybutyric acid, thereby providing a basis for ketone detection.

[0063] As shown in FIG. 6B, β-hydroxybutyrate dehydrogenase (HBDH) can further convert β-hydroxybutyric acid and NAD + to acetoacetate and NADH, respectively. Instead of the transfer of electrons to the working electrode completed by diaphorase (shown in FIG. 6A) and a suitable redox mediator, the reduced form of NADH oxidase (NADHOx(Red)) undergoes a reaction to form the corresponding oxidized form (NADHOx(Ox)). NADHOx(Red) can then be reformed by a reaction using molecular oxygen to produce superoxide, which can then be converted to hydrogen peroxide via superoxide dismutase (SOD). The hydrogen peroxide may then be oxidized at the working electrode to provide a signal that can correlate with the amount of ketone initially present. According to various embodiments, SOD can be covalently bound to the polymer in the ketone-responsive active region. Like the enzyme system shown in FIG. 6A, β-hydroxybutyrate dehydrogenase and NADH oxidase may be covalently bound to the polymer in the ketone-responsive active region, NAD may or may not be covalently bound to the polymer in the ketone-responsive active region. NAD + If not covalently bound, it may be physically retained within the ketone-responsive active region using a membrane polymer that promotes retention of NAD + within the ketone-responsive active region.

[0064] As shown in FIG. 6C, another enzyme detection chemistry for ketones is β-hydroxybutyric acid and NAD +β-Hydroxybutyrate dehydrogenase (HBDH) may be utilized to convert each into acetoacetate and NADH, respectively. In this case, the electron transfer cycle is completed by the oxidation of 1,10-phenanthroline-5,6-dione at the working electrode to reform NAD. 1,10-Phenanthroline-5,6-dione may or may not be covalently bonded to the polymer within the ketone-responsive active region. As in the enzyme system shown in FIG. 6A, β-hydroxybutyrate dehydrogenase may be covalently bonded to the polymer in the ketone-responsive active region, and NAD may or may not be covalently bonded to the polymer in the ketone-responsive active region. The inclusion of albumin in the active region can provide a surprising improvement in reaction stability. A suitable membrane polymer can promote the retention of NAD + within it.

[0065] The analyte sensor of the present disclosure can further be configured to analyze a second or subsequent analyte in addition to the analyte detectable at a low potential in the first active region. To facilitate the detection of the second analyte, the analyte sensor of the present disclosure includes a second working electrode and a second active region disposed on the surface of the second working electrode and responsive to a second analyte different from the first analyte, the second active region including a second polymer, a second redox mediator different from the first redox mediator covalently bonded to the second polymer, and at least one enzyme responsive to the second analyte covalently bonded to the second polymer. A second portion of the mass transfer limiting membrane may cover the second active region. The at least one enzyme responsive to the second analyte may include an enzyme system including a plurality of enzymes that respond to the second analyte as a group. In the second active region, the second redox mediator does not necessarily need to be capable of promoting electron transfer at a low potential, but may be capable of promoting electron transfer at a low potential.

[0066] Suitable redox mediators included in the second active region may include osmium complexes and other transition metal complexes, including but not limited to those described in U.S. Patent No. 6,134,461 and U.S. Patent No. 6,605,200, all of which are hereby incorporated by reference. Additional examples of suitable redox mediators include those described in U.S. Patent No. 6,736,957, U.S. Patent No. 7,501,053, and U.S. Patent No. 7,754,093, all of which are also hereby incorporated by reference in their respective disclosures. Other suitable redox mediators included in the second active region may include metal compounds or metal complexes containing ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt, e.g., its metallocene compounds. Suitable ligands for the metal complexes may include, for example, ligands having a coordination number of two or more, such as bipyridine, biimidazole, phenanthroline, or pyridyl(imidazole). Other suitable bidentate ligands may include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or more highly dentate ligands may be present in the metal complex to achieve a complete coordination sphere.

[0067] The active region for promoting the detection of an analyte according to the disclosure herein may include a polymer to which the redox mediator is covalently bound. Suitable examples of polymer-bound redox mediators include those described in U.S. Patent No. 8,444,834, U.S. Patent No. 8,268,143, and U.S. Patent No. 6,605,200, the disclosures of which may include all that are hereby incorporated by reference. Suitable polymers included in the active region may include, but are not limited to, polyvinylpyridine (e.g., poly(4-vinylpyridine)), polyvinylimidazole (e.g., poly(1-vinylimidazole)), or any copolymer thereof. Exemplary copolymers that may be included in the active region and may be suitable include those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile. The polymers within each active region may be the same or different.

[0068] In certain examples, the second active region can be configured to detect glucose in combination with an analyte detectable at a low potential in the first active region. Thus, in certain embodiments of the present disclosure, the second enzyme can be glucose oxidase. Further, in a more specific example, the first analyte may be a ketone detectable by an enzyme system as described herein, and the second analyte may be glucose detectable by glucose oxidase.

[0069] The detection of each analyte can include applying a potential separately to each working electrode such that separate signals are obtained from each analyte. The signals obtained from each analyte can then be associated with the concentration of the analyte by use of a calibration curve or function, or by using a look-up table. The correlation between the analyte signal and the analyte concentration can, in certain examples, be derived by use of a processor.

[0070] In other analyte sensor configurations, the first and second active regions can be disposed on a single working electrode. The first signal may be obtained at a low potential from the first active region, and the second signal, including the signal contributions from both active regions, may be obtained at a higher potential. Next, subtracting the first signal from the second signal can enable determination of the signal contribution generated from the second analyte. Next, similar to the method described for a sensor configuration having multiple working electrodes, the signal contribution from each analyte can be associated with the analyte concentration.

[0071] As a result, the present disclosure provides a substance sensor including a sensor tail portion including at least a first working electrode, a first active region disposed on the surface of the first working electrode and responsive to a first analyte at a low potential, the first active region including a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme responsive to the first analyte covalently bonded to the first polymer, the redox mediator having a structure represented by any one of Formulas 1 to 8 described above, G being a linking group that covalently bonds the first redox mediator to the first polymer, the first active region, and a mass transfer limiting membrane that is permeable to the first analyte and covers at least the first active region; applying a low potential to the first working electrode; obtaining a first signal that is proportional to the concentration of the first analyte in a fluid in contact with the first active region and is equal to or higher than the redox potential of the first active region; and correlating the concentration of the first analyte in the fluid with the first signal.

[0072] When measured relative to an Ag / AgCl reference electrode, the low potential may include less than about +100 mV, less than about -50 mV, less than about -80 mV, or less than about -100 mV, and may be less than about +200 mV. The low potential may also be higher than the redox potential of the first redox mediator. Exemplary redox potentials of the first redox mediator that may facilitate operation at a low working electrode potential may be less than about -200 mV, such as from about -400 mV to about -200 mV, or from about -350 mV to about -250 mV, or from about -300 mV to about -250 mV when measured relative to an Ag / AgCl reference electrode.

[0073] The analyte sensor disclosed in this specification further includes a mass transfer restriction membrane that is permeable to the analyte covering at least the first active region. When there are a plurality of active regions, the mass transfer restriction membrane may have different compositions on different active regions and may cover each active region such that, for example, a bilayer film portion can be created on the working electrode near the tip of the sensor, which can be achieved by a series of dip coating operations. The mass transfer restriction membrane may include, for example, a membrane polymer such as a homopolymer or copolymer of polyvinylpyridine or polyvinylimidazole, and may be further cross-linked with a suitable cross-linking agent. The membrane polymer may include a copolymer of vinylpyridine and styrene in certain embodiments. In a more specific example, the membrane polymer covering one or more active regions may be cross-linked by a branched cross-linking agent containing three or more cross-linking groups such as polyethylene glycol tetraglycidyl ether, and the amount of extractable substances obtained from the mass transfer restriction membrane as described above may be surprisingly reduced. More specifically, the mass transfer restriction membrane may include polyvinylpyridine or a copolymer of vinylpyridine and styrene cross-linked by a branched glycidyl ether cross-linking agent containing three cross-linking groups such as polyethylene glycol tetraglycidyl ether. In particular, the epoxy groups of polyethylene glycol tetraglycidyl ether may react with the pyridine nitrogen atom or imidazole nitrogen atom, and the ring-opening of the epoxide ring may promote the covalent bonding of the cross-linking groups. A hydroxyalkyl group that cross-links the cross-linking agent body to the heterocycle of the membrane polymer may be provided.

[0074] Suitable copolymers of vinylpyridine and styrene can have styrene in the range of about 0.01% to about 50% molar%, or about 0.05% to about 45% molar%, or about 0.1% to about 40% molar%, or about 0.5% to about 35% molar%, or about 1% to about 30% molar%, or about 2% to about 25% molar%, or about 5% to about 20% molar%. Substituted styrenes can be used in the same manner and in similar amounts. Suitable copolymers of vinylpyridine and styrene can have a molecular weight of 5 kDa or more, or about 10 kDa or more, or about 15 kDa or more, or about 20 kDa or more, or about 25 kDa or more, or about 30 kDa or more, or about 40 kDa or more, or about 50 kDa or more, or about 75 kDa or more, or about 90 kDa or more, or about 100 kDa or more. By way of non-limiting example, suitable copolymers of vinylpyridine and styrene can have a molecular weight in the range of about 5 kDa to about 150 kDa, or about 10 kDa to about 125 kDa, or about 15 kDa to about 100 kDa, or about 20 kDa to about 80 kDa, or about 25 kDa to about 75 kDa, or about 30 kDa to about 60 kDa.

[0075] As a result, at least some of the analyte sensors described herein can include a sensor tail portion that includes at least a first working electrode, a first active region disposed on the surface of the first working electrode, and a mass transfer limiting membrane that is permeable to at least the first analyte that covers at least the first active region. The first active region includes a first polymer and at least one enzyme covalently bonded to the first polymer that responds to the first analyte. The mass transfer limiting membrane includes, for example, a membrane polymer crosslinked by a branched glycidyl ether crosslinking agent that includes three or more crosslinking groups such as polyethylene glycol tetra glycidyl ether.

[0076] Crosslinking can occur intermolecularly in more specific embodiments. The polyethylene glycol tetraglycidyl ether used to promote intermolecular crosslinking between two or more film polymer backbones can exhibit a wide range of appropriate molecular weights. Up to four polymer backbones can be crosslinked by a single molecule of the polyethylene glycol tetraglycidyl ether crosslinking agent. In certain examples, the molecular weight of the polyethylene glycol tetraglycidyl ether can range from about 1000 g / mol to about 5000 g / mol. The number of repeating units of ethylene glycol in each arm of the polyethylene glycol tetraglycidyl ether may be the same or different and may typically vary over the range for a given sample that gives an average molecular weight. The structure of the polyethylene glycol tetraglycidyl ether prior to crosslinking is represented by the following formula 9, [Chemical formula] wherein n1, n2, n3 and n4 are all integers of 0 or more, usually 1 or more, and n1, n2, n3 and n4 may be the same or different. The sum of n1, n2, n3 and n4 can be selected such that the molecular weight of the polyethylene glycol tetraglycidyl ether falls within the above range. In other words, to produce a polyethylene glycol tetraglycidyl ether having a molecular weight within the above range, the sum of n1, n2, n3 and n4 may range from about 14 to about 110, or from about 15 to about 104, including any sub-range between these values, and n1, n2, n3 and n4 may each be any integer of 0 or more, or an integer of 1 or more.

[0077] The crosslink density indicates the number of side chains of the membrane polymer that have a crosslinking agent attached to the side chains of the membrane polymer. For example, a membrane polymer crosslinked by a branched glycidyl ether such as polyethylene glycol tetraglycidyl ether or a similar polyethylene oxide crosslinking agent having three or more crosslinking groups can have a crosslink density that varies over a wide range. In certain examples, a portion of the side chains that can have a crosslinking agent added to the side chains can be about 0.1% or more of the available heterocycles in the membrane polymer, or about 0.2% or more of the available heterocycles in the membrane polymer, or about 0.3% or more of the available heterocycles in the membrane polymer, or about 0.4% or more of the available heterocycles in the membrane polymer, or about 0.5% or more of the available heterocycles in the membrane polymer, or about 0.6% or more of the available heterocycles in the membrane polymer, or about 0.7% or more of the available heterocycles in the membrane polymer, or about 0.8% or more of the available heterocycles in the membrane polymer, or about 0.9% or more of the available heterocycles in the membrane polymer, or about 1.0% or more of the available heterocycles in the membrane polymer, or about 1.2% or more of the available heterocycles in the membrane polymer, or about 1.4% or more of the available heterocycles in the membrane polymer, or about 1.6% or more of the available heterocycles in the membrane polymer, or about 1.8% or more of the available heterocycles in the membrane polymer, or about 2.0% or more of the available heterocycles in the membrane polymer, or about 2.2% or more of the available heterocycles in the membrane polymer, or about 2.4% or more of the available heterocycles in the membrane polymer, or about 2.6% or more of the available heterocycles in the membrane polymer, or about 2.8% or more of the available heterocycles in the membrane polymer, or about 3.0% or more of the available heterocycles in the membrane polymer, or about 3.5% or more of the available heterocycles in the membrane polymer, or about 4.0% or more of the available heterocycles in the membrane polymer, or about 4.5% or more of the available heterocycles in the membrane polymer, or about 5.It may be 0% or more, or may be about 5.5% or more of the available heterocycles in the membrane polymer, or may be about 6.0% or more of the available heterocycles in the membrane polymer, or may be about 6.5% or more of the available heterocycles in the membrane polymer, or may be about 7.0% or more of the available heterocycles in the membrane polymer, or may be about 7.5% or more of the available heterocycles in the membrane polymer, or may be about 8.0% or more of the available heterocycles in the membrane polymer, or may be about 8.5% or more of the available heterocycles in the membrane polymer, or may be about 9.0% or more of the available heterocycles in the membrane polymer, or may be about 9.5% or more of the available heterocycles in the membrane polymer, or may be about 10% or more of the available heterocycles in the heterocyclic polymer. In more specific embodiments, the crosslinking agent can be added between about 1% and about 20% of the available heterocycles in the membrane polymer, or between about 2% and about 10% of the available heterocycles in the membrane polymer, or between about 3% and about 8% of the available heterocycles in the membrane polymer, or between about 4% and about 9% of the available heterocycles in the membrane polymer, or between about 5% and about 12% of the available heterocycles in the membrane polymer.

[0078] Suitable membrane polymers may further include one or more polyether arms (side chains) that bind to the nitrogen atoms of the monomer units of pyridine or imidazole. Some of the membrane polymers disclosed herein may further include one or more polyether arms. The polyether arms are distinguished from crosslinking groups formed from polyethylene glycol tetraglycidyl ether or similar crosslinking agents, and the polyether arms do not extend between different polymer chains or terminate intramolecularly within a single polymer chain. Thus, the polyether arms are separate and different from the crosslinking groups formed from crosslinking agents. The polyether arms may include polyethylene oxide blocks and polypropylene oxide blocks, and in particular, polyether arms having polypropylene oxide blocks are inserted between two polyethylene oxide blocks. The bond between the polyether arm and the nitrogen atom of the heterocycle may occur by any reactive functional group capable of forming a bond with the nitrogen atom of the heterocycle in the membrane polymer. The bond between the polyether arm and the nitrogen atom of the heterocycle may occur by an alkyl group, a hydroxyl-functionalized alkyl group, or a carbonyl. In other specific cases, the polyether arm may also include an amine group away from the nitrogen atom of the heterocycle or may be amine-free.

[0079] The polyether arm of the membrane polymer may include at least one polyethylene oxide block and at least one polypropylene oxide block, thereby providing at least a diblock arrangement of monomer units of polyethylene oxide and polypropylene oxide bonded to the nitrogen atom of the heterocyclic ring by a spacer. Either the polyethylene oxide block or the polypropylene oxide block may be bonded to the spacer. In other more specific embodiments, the polyether arm may include, in order, a spacer, a first polyethylene oxide block, a polypropylene oxide block, and a second polyethylene oxide block (i.e., an A-B-A repeating pattern) or, in order, a spacer, a first polypropylene oxide block, a polyethylene oxide block, and a second polypropylene oxide block (i.e., a B-A-B repeating pattern). The amine group may mediate between the polyethylene oxide block and the polypropylene oxide block in the amine-containing polyether arm. Thus, the polyether arm in the membrane polymer disclosed herein may have a structure generally defined by Formulas 10 to 13 below, [Chemical formula] In the formula, PE represents a polyethylene oxide block, PP represents a polypropylene oxide block, A is an amino group, and J is a spacer group. The spacer group J can be bonded to the heterocyclic ring of the membrane polymer. Suitable spacer groups J include, but are not limited to, alkyl, hydroxy-functionalized alkyl, carbonyl, carboxylic acid ester, and carboxamide. The variable q, r, s, and t are positive integers that define the number of monomer units in each block and the number of block repetitions. Under the conditions of a diblock arrangement, the variable t can be 0, and the variable s can be 1. According to some embodiments, the variable q is an integer in the range between about 2 and about 50 or between about 6 and about 20, the variable r is an integer in the range between about 2 and about 60 or between about 10 and about 40, and the variable t is an integer in the range between about 2 and about 50 or between about 10 and about 30. According to some or other various embodiments, the variable s is an integer in the range between 1 and about 20 or between 1 and about 10. In some embodiments, the variable s is 1.

[0080] According to more specific embodiments of the present disclosure, the amine-free polyether arm having a triblock arrangement of polyethylene oxide, polypropylene oxide, and polyethylene oxide (similar to Formula 10) arms may have a structure defined by Formula 14,

Chemical formula

[0081] In other specific embodiments of the present disclosure, a polyether arm having a triblock arrangement of polyethylene oxide, polypropylene oxide, and polyethylene oxide and having an amine group that mediates between the polyethylene oxide block and the polypropylene oxide block (similar to Formula 12) may have a structure defined by Formula 15,

Chemical formula

[0082] The polyether arms disclosed herein can be attached to the nitrogen atom of a heterocycle as a reactive functional group in a polyether arm precursor. Suitable reactive functional groups can include, for example, halogen or epoxide. As shown in Formulas 14 and 15 above (where n = 1 in Formulas 14 and 15), for example, epoxide leads to the formation of a hydroxyalkyl spacer group that attaches the polyether arm to the nitrogen atom of the heterocycle of the membrane polymer. In contrast, a halogen-functionalized polyether arm precursor may lead to an alkyl spacer (where n = 0 in Formulas 14 and 15), and suitable alkyl groups may be linear or branched and may contain from 2 to about 20 carbon atoms.

[0083] In some embodiments, arms containing sulfonic acid can be added as side chains in at least a portion of the membrane polymers disclosed herein. Arms containing sulfonic acid can be present in any suitable ratio in combination with polyether arms and / or crosslinking agents. Any of the membrane polymers disclosed herein can contain a greater amount of polyether arms or crosslinking groups than arms containing sulfonic acid. Arms containing sulfonic acid are attached to the membrane polymer by an alkyl group. According to various embodiments, the alkyl group can contain from 1 to about 6 carbon atoms, or from 2 to about 4 carbon atoms. Suitable reagents for introducing arms containing sulfonic acid into the membrane polymers disclosed herein can include halosulfonic acid compounds such as chloromethanesulfonic acid, or bromoethanesulfonic acid, or cyclic sulfonic acids (sultones).

[0084] Polydimethylsiloxane (PDMS) can be incorporated into some of the mass transfer restriction membranes disclosed herein.

[0085] When a first active region and a second active region configured to assay different analytes are disposed on separate working electrodes, the mass transfer limiting membrane can have different permeability values for the first analyte and the second analyte. The thickness of the membrane and / or the size of the active region of each working electrode can be varied so that the sensitivity is equal for each analyte, but this approach can significantly complicate the manufacture of the analyte sensor. As a solution, the mass transfer limiting membrane covering at least one of the active regions can include a mixed membrane of a first membrane polymer and a second membrane polymer or a bilayer membrane of the first membrane polymer and the second membrane polymer. The uniform membrane may cover the active region not covered by the mixed membrane or the bilayer membrane, and the uniform membrane includes only one of the first membrane polymer or the second membrane polymer. Advantageously, the structure of the analyte sensor disclosed herein readily permits a continuous membrane having a uniform membrane portion disposed on the first active region of the analyte sensor and a multi-component membrane portion disposed on the second active region, thereby equalizing the permeability values for each analyte and simultaneously improving the sensitivity and detection accuracy. The formation of the continuous membrane can be performed by a series of dip coating operations in certain embodiments.

[0086] The embodiments disclosed herein include the following.

[0087] A. An analyte sensor capable of detecting an analyte at a low potential. An analyte sensor comprising a sensor tail including at least a first working electrode, and a first active region disposed on the surface of the first working electrode and responsive to a first analyte at a low potential, wherein the first active region includes a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme responsive to the first analyte covalently bonded to the first polymer, and the first redox mediator is

Chemical formula

[0088] B. A method for detecting an analyte using an analyte sensor capable of low-potential detection. A sensor tail including at least a first working electrode, and a first active region disposed on the surface of the first working electrode and responding to the first analyte at a low potential, wherein the first active region includes a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme that responds to the first analyte covalently bonded to the first polymer, and the first redox mediator is

Chemical formula

[0089] B1. A method for detecting an analyte using an analyte sensor capable of low-potential detection. A sensor tail including at least a first working electrode, and a first active region disposed on the surface of the first working electrode and responding to the first analyte at a low potential, wherein the first active region includes a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme that responds to the first analyte covalently bonded to the first polymer, and the first redox mediator is

Chemical formula

[0090] C. An analyte sensor comprising a mass transfer limiting membrane crosslinked with a branched glycidyl ether crosslinking agent. An analyte sensor comprising a sensor tail portion including at least a first working electrode, and a first active region disposed on the surface of the first working electrode, the first active region including a first polymer and at least one enzyme covalently bonded to the first polymer and responsive to a first analyte, and a mass transfer limiting membrane that is permeable to at least the first analyte covering the first active region, the mass transfer limiting membrane including a membrane polymer crosslinked with a branched crosslinking agent including three or more crosslinking groups.

[0091] A method for detecting a test substance using a test substance sensor including a mass transfer limiting membrane crosslinked by a branched glycidyl ether crosslinking agent. A sensor tail including at least a first working electrode, a first active region disposed on the surface of the first working electrode, the first active region including a first polymer and at least one enzyme covalently bonded to the first polymer and responsive to a first test substance, and a mass transfer limiting membrane permeable to the first test substance and covering at least the first active region, the mass transfer limiting membrane including a membrane polymer crosslinked by a branched crosslinking agent including three or more crosslinking groups, providing a test substance sensor including the same; applying a potential to the first working electrode; obtaining a first signal proportional to the concentration of the first test substance in a fluid in contact with the first active region and equal to or higher than the redox potential of the first active region; and correlating the concentration of the first test substance in the fluid with the first signal.

[0092] Embodiments A to D may have one or more of the following elements in any combination.

[0093] Element 1: The at least one enzyme includes an enzyme system including a plurality of enzymes that respond as a group to the first test substance.

[0094] Element 2: The first test substance includes one or more ketones.

[0095] Element 3: The mass transfer limiting membrane includes a membrane polymer crosslinked by a branched crosslinking agent including three or more crosslinking groups.

[0096] Element 4: The membrane polymer includes polyvinylpyridine or polyvinylimidazole.

[0097] Element 5: The membrane polymer includes a copolymer of vinylpyridine and styrene.

[0098] Element 6: The branched crosslinking agent includes polyethylene glycol tetraglycidyl ether.

[0099] Element 7: The analyte sensor further includes a second working electrode, a second active region disposed on the surface of the second working electrode and responsive to a second analyte different from the first analyte, the second active region including a second polymer, a second redox mediator covalently bonded to the second polymer and different from the first redox mediator, and at least one enzyme responsive to the second analyte covalently bonded to the second polymer, and a second portion of a mass transfer limiting membrane covering the second active region.

[0100] Element 8: The at least one enzyme responsive to the second analyte includes an enzyme system including a plurality of enzymes that respond as a group to the second analyte.

[0101] Element 9: The second analyte includes glucose.

[0102] Element 10: The low potential is higher than the redox potential of the first redox mediator and less than about -80 mV with respect to the Ag / AgCl reference electrode.

[0103] Element 10A: The redox potential of the first redox mediator is in the range of about -200 mV to about -400 mV with respect to the Ag / AgCl reference electrode.

[0104] Element 11: The polyethylene glycol tetra glycidyl ether has a molecular weight in the range of about 1000 g / mol to about 5000 g / mol.

[0105] By way of non-limiting example, typical combinations applicable to A and B include, but are not limited to, 1 and 2; 1 to 3; 1, 3 and 6; 1 and 4; 1 and 7; 1 and 9; 1 and 10 or 10A; 2 and 3; 2, 3 and 6; 2 and 7; 2 and 9; 2 and 10 or 10A; 3 and 4; 3 and 6; 3, 4 and 6; 3, 5 and 6; 3 and 7; 3 and 9; 3 and 10 or 10A; 4 and 7; 5 and 7; 4 or 5, and 9; and 4 or 5, and 10 or 10A. Further by way of non-limiting example, typical combinations applicable to C and D include, but are not limited to, 6 and 11; 4 and 6; 5 and 6; 6 and 7; 6 to 8; 4, 6 and 11; 5, 6 and 11; 6, 7 and 11; and 6 to 8 and 11.

[0106] Further embodiments disclosed herein include the following.

[0107] A’: A method for forming a mass transfer limiting film by dip coating. Providing an analyte sensor comprising a sensor tail including a first working electrode and a second working electrode spaced apart from each other along at least the length of the sensor tail, and a first active region disposed on the surface of the first working electrode and a second active region disposed on the surface of the second working electrode, the first active region and the second active region being responsive to different analytes; and depositing a mass transfer limiting film on the first active region and the second active region by a series of dip coating operations, the mass transfer limiting film including a bilayer film portion covering the first active region and a uniform film portion covering the second active region.

[0108] B’: A test substance sensor having a dip-coated mass transfer limiting membrane. A test substance sensor comprising a sensor tail portion including a first working electrode and a second working electrode spaced apart from each other along at least the length of the sensor tail, a first active region disposed on the surface of the first working electrode, and a second active region disposed on the surface of the second working electrode, wherein the first active region and the second active region respond to different test substances, and a dip-coated mass transfer limiting membrane disposed on the first active region and the second active region, the dip-coated mass transfer limiting membrane including a dip-coated bilayer membrane portion covering the first active region and a dip-coated uniform membrane portion covering the second active region.

[0109] Embodiments A’ and B’ may have one or more of the following elements in any combination.

[0110] Element 1’: The bilayer membrane portion and the uniform membrane portion are adjacent to each other.

[0111] Element 2’: The first working electrode and the first active region are located closer to the tip of the test substance sensor than the second working electrode and the second active region.

[0112] Element 3’: The upper layers of the bilayer membrane portion and the uniform membrane portion contain the same membrane polymer.

[0113] Element 4’: The first dip-coating operation deposits a first membrane polymer on the first active region, the second dip-coating operation deposits a second membrane polymer on both the first active region and the second active region, defining the bilayer membrane portion on the first active region and the uniform membrane portion on the second active region, and the first membrane polymer and the second membrane polymer are different from each other.

[0114] Element 5’: The lower layers of the bilayer membrane portion and the uniform membrane portion contain the same membrane polymer.

[0115] Element 6’: In the first dip coating operation, a first film polymer is deposited over both the first active region and the second active region. In the second dip coating operation, a second film polymer is deposited over the first active region to define the bilayer film portion over the first active region. The first film polymer and the second film polymer are different from each other.

[0116] Element 7’: The first active region responds to glucose, lactic acid, ketone, or creatinine.

[0117] Element 8’: The first active region responds to ketone.

[0118] Element 9’: The second active region responds to glucose.

[0119] Element 10’: At least a portion of the mass transfer limiting membrane comprises a homopolymer or copolymer of crosslinked polyvinyl pyridine.

[0120] Element 11’: At least a portion of the mass transfer limiting membrane comprises a membrane polymer crosslinked by a branched crosslinking agent containing three or more crosslinking groups.

[0121] Element 12’: The branched crosslinking agent includes polyethylene glycol tetraglycidyl ether.

[0122] Element 13’: The dip-coated bilayer film portion and the dip-coated uniform film portion are adjacent to each other.

[0123] Element 14’: The first working electrode and the first active region are located closer to the tip of the analyte sensor than the second working electrode and the second active region.

[0124] Element 15’: The upper layer of the dip-coated bilayer film portion and the dip-coated uniform film portion comprises the same membrane polymer.

[0125] Element 16': The lower layers of the dip-coated two-layer film portion and the dip-coated uniform film portion contain the same film polymer.

[0126] Element 17': The first active region responds to ketones.

[0127] Element 18': The second active region responds to glucose.

[0128] As non-limiting examples, typical combinations applicable to A' include, but are not limited to, 1' and 2'; 1' to 3'; 1' to 4'; 1', 2' and 5'; 1', 2', 5' and 6'; 1' and 7'; 1' and 8'; 1', 8' and 9'; 1' and 10'; 2' and 3'; 2' to 4'; 2' to 5'; 2', 5' and 6'; 2' and 7'; 2' and 8'; 2', 3', 4' and 8'; 2', 3', 4', 8' and 9'; 2', 5', 6' and 8'; 2', 5', 6', 8' and 9'; 3' and 4'; 3', 4' and 7'; 3', 4' and 8'; 3', 4', 8' and 9'; 3' and 7'; 3' and 8'; 3', 8' and 9'; 5' and 6'; 5', 6' and 8'; 5', 6', 8' and 9'; and 8' and 9'. Typical combinations applicable to B' include, but are not limited to, 13' and 14'; 13', 14' and 15'; 13' 14' and 16'; 13', 14' and 17'; 13', 14', 17' and 18'; 14' and 15'; 14' and 16'; 14' and 17'; 14' and 18'; 15' and 17'; 15', 17' and 18'; 16' and 17'; 16', 17' and 18'; and 17' and 18'.

[0129] To facilitate the understanding of the disclosure herein, examples of various representative embodiments are given below. The scope of the invention should not be limited or defined by reading the following embodiments.

[0130] Examples Example 1: Detection of ketones at low potentials using a test substance sensor having both diaphorase and β-hydroxybutyrate dehydrogenase working together. For this example, the enzyme system of Figure 6A uses the formulations of the active regions described in Tables 1 and 2 below, and either an unbound formula 8 transition metal complex (Table 1) or a formula 8 transition metal complex bound to polyvinylpyridine-co-styrene (Table 2) is used as a redox mediator to facilitate the detection of ketones (HBHD = β-hydroxybutyrate dehydrogenase; HSA = human serum albumin; PEGDGE400 = polyethylene glycol diglycidyl ether). The formulations of the detection active regions shown in Tables 1 and 2 were coated onto a carbon-coated working electrode by depositing one spot having an area of approximately 0.2 mm 2 and cured at 25°C for 24 hours. After curing, a polyvinylpyridine film having the formulation described in Table 3 was applied onto the active region by a total of 4 dip coatings. After the application of the film, the sensor was cured at 25°C for 24 hours and then at 56°C for 48 hours.

Table 1

Table 2

Table 3

[0131] Figure 7 shows cyclic voltammograms for the transition metal complex of formula 8 or the polymer-bound form of the transition metal complex of formula 8. The cyclic voltammograms were deoxygenated by bubbling with nitrogen, maintained at a temperature of 33°C, and obtained in a 100 mM PBS solution at pH 7.4. The investigation was carried out from -0.5 V to 0.1 V at a scan rate of 5 mV / s using a carbon counter electrode and an Ag / AgCl reference electrode. From the cyclic voltammograms, the E of the transition metal complex of formula 8 1 / 2was measured to be -0.29 V vs. Ag / AgCl, and the E of the polymer-bound type 8 transition metal complex 1 / 2 was measured to be -0.24 V vs. Ag / AgCl. A redox mediator with insufficient N,N-dimethylamino substitution showed a much smaller negative E 1 / 2 value of -0.08 V vs. Ag / AgCl (no data).

[0132] Figure 8 shows a plot of current vs. time for a ketone sensor containing a polymer-bound type 8 transition metal complex at various working electrode potentials. The sensor was held at one of four potentials in the range of +40 mV to -200 mV in 100 mM PBS at pH 7.4, and various amounts of β-hydroxybutyric acid were titrated at 33 °C until a final ketone concentration of 8 mM was reached. As shown in the figure, the response of the sensor stabilized rapidly after addition of ketone at each potential. All four potentials investigated showed a linear response to ketone concentration (shown in Figure 9).

[0133] Example 2: Extractables from a membrane crosslinked with polyethylene glycol tetraglycidyl ether. Polymer membrane samples crosslinked with polyethylene glycol tetraglycidyl ether (molecular weight ~2500) were prepared. The base membrane polymer was a copolymer of vinylpyridine and styrene containing amine-free polyether arms that bind to at least a portion of the pyridine moieties in the base membrane polymer. Comparative polymer membrane samples were prepared using the same membrane polymer crosslinked with polyethylene glycol diglycidyl ether (molecular weight ~1000). The comparative polymer membrane samples were crosslinked with polyethylene glycol diglycidyl ether in an amount similar to the crosslinking agent of polyethylene glycol tetraglycidyl ether used in the other samples, thereby providing a similar crosslink density.

[0134] The membrane polymer was cast as a film on the bottom of a sample vial using 0.5 mL of a solution containing the membrane polymer. The solvent was evaporated and then the membrane was cured at 25 °C for 48 hours and at 56 °C for 56 hours. Water or 95% ethanol (3 mL) was added to the cast polymer film and extraction was carried out at room temperature for 72 hours (water) or 144 hours (ethanol). The vials were agitated on a rocker during the extraction period. The water extract was analyzed without dilution by UV-Vis spectrophotometry (Figure 10), and the ethanol extract was diluted 1:9 prior to UV-Vis analysis (Figure 11). In both cases, the samples crosslinked with polyethylene glycol tetraglycidyl ether, as indicated by the intensity of the UV-Vis absorbance, showed a lower amount of extractable material.

[0135] Example 3: Equilibrium time for sensors with membranes crosslinked with polyethylene glycol tetraglycidyl ether. The glucose-responsive analyte sensor was coated with a membrane polymer crosslinked with polyethylene glycol tetraglycidyl ether (as shown in Example 2). A comparative sensor was prepared using a comparative membrane polymer crosslinked with polyethylene glycol diglycidyl ether. Each sensor coated with the membrane was placed in a 30 mM glucose solution in 1000 mM PBS (pH = 7.4) at 37 °C. Figure 12 shows a comparative plot of sensor output versus time over 10 hours for an analyte sensor coated with a membrane polymer crosslinked with polyethylene glycol tetraglycidyl ether compared to an analyte sensor coated with a membrane polymer crosslinked with polyethylene glycol diglycidyl ether. As shown in the figure, both membranes provided a stable sensor output after an equilibrium of approximately 1 hour. Figure 13 shows an expanded plot of the 1-hour sensor equilibrium and it was found that the membrane crosslinked with polyethylene glycol tetraglycidyl ether provided a more rapidly stabilized response than the membrane crosslinked with polyethylene glycol diglycidyl ether.

[0136] Unless otherwise indicated, all numbers expressing quantities such as those in this specification and the claims associated therewith are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and 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 be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0137] One or more exemplary embodiments incorporating various features are presented herein. Not all features of a physical implementation are necessarily described or shown in this application for clarity. In developing a physical implementation incorporating embodiments of the present invention, it will be understood that numerous implementation-specific decisions must be made to achieve the developer's goals, such as compliance with system, business, government, and other constraints, which vary by implementation and by circumstance. The developer's efforts may be time-consuming, but such efforts would still be routine undertakings for those of ordinary skill in the art and would be within the benefits of this disclosure.

[0138] While various systems, tools, and methods are described herein in terms of "comprising" various components or steps, the systems, tools, and methods may also be "consisting essentially of" or "consisting of" various components or steps.

[0139] As used herein, the phrase "at least one of" with the terms "and" or "or" that precede a series of items and separate some of the items, modifies the entire list rather than each member of the list (i.e., each item). The phrase "at least one of" allows a meaning that includes at least one of any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. By way of example, the phrases "at least one of A, B, and C" or "at least one of A, B, or C" each denote only A, only B, or only C; any combination of A, B, and C; and / or at least one of each of A, B, and C.

[0140] Accordingly, the disclosed systems, tools, and methods are well suited to achieve the stated objectives and advantages, as well as those inherent therein. The teachings of this disclosure will be apparent to those skilled in the art having the benefit of the teachings herein, and may be modified and practiced in different but equivalent manners, and the specific embodiments disclosed above are merely examples. Further, unless otherwise specified in the following claims, the details of the configurations or designs set forth herein are not intended to be limiting. Thus, the specific, particular embodiments disclosed above may be varied, combined, or modified, and it is clear that all such variations are considered to be within the scope of this disclosure. The systems, tools, and methods exemplified herein may be appropriately implemented in the absence of any element specifically disclosed herein and / or in the absence of any element disclosed herein. The systems, tools, and methods are described in terms of comprising, containing, or including various components or steps, but the systems, tools, and methods may also consist essentially of or consist of various components or steps. All of the numbers and ranges disclosed above may vary somewhat. Whenever a numerical range having a lower limit and an upper limit is disclosed, any number and any included range falling within that range are specifically disclosed. In particular, all ranges of values disclosed herein (in the form of "about a to about b", or equivalently "approximately a to b", or equivalently "about a - b") are understood to set forth all numbers and ranges included within the broader range of values. Also, in the claims, terms have their plain and ordinary meaning unless explicitly and clearly defined by the patentee. Further, as used in the claims, the indefinite articles "a" or "an" are hereby defined to mean more than one of the element they introduce. Where there are some inconsistencies in the usage of words or terms in this specification and one or more patent documents or other documents incorporated herein by reference, the definitions consistent with this specification should be adopted.

Claims

1. A test substance sensor, comprising: i) a Ag / AgCl reference electrode; ii) a first working electrode; and iii) a second working electrode; and iv) a first active area disposed on the surface of the first working electrode and responsive to ketones at a low potential, the first active area comprising a first polymer, a first redox mediator covalently bound to the first polymer, and an enzyme system comprising a plurality of enzymes covalently bound to the first polymer and cooperatively responsive to ketones, the low potential being greater than a redox potential of the first redox mediator and less than −80 mV versus the Ag / AgCl reference electrode, the redox potential of the first redox mediator being in the range of −200 mV to −400 mV versus the Ag / AgCl reference electrode, the first redox mediator having the structure: 【Chemistry 1】 wherein G is a linking group that covalently bonds the first redox mediator to the first polymer; v) a second active area disposed on a surface of the second working electrode and responsive to a second analyte, the second active area comprising a second polymer, a second redox mediator covalently bound to the second polymer and different from the first redox mediator, and at least one enzyme responsive to the second analyte covalently bound to the second polymer; vi) a mass transport limiting membrane covering at least the first active area and permeable to ketones; The analyte sensor is configured to be partially insertable into tissue such that a distal portion of the analyte sensor contacts interstitial fluid for detecting ketones in vivo.

2. The test substance sensor described in claim 1, wherein the enzyme system includes β-hydroxybutyrate dehydrogenase (HBDH) and diaphorase.

3. A test substance sensor as described in claim 1 or 2, wherein the second test substance is glucose.

4. The test substance sensor described in claim 3, wherein at least one enzyme that responds to glucose is glucose oxidase.

5. The test substance sensor according to any one of claims 1 to 4, wherein the mass transport limiting membrane is a two-layer membrane consisting of a lower layer and an upper layer.

6. The test substance sensor of claim 5, wherein the lower layer and the upper layer of the bilayer membrane cover a first active area.

7. A test substance sensor as described in claim 5 or 6, wherein the upper layer of the bilayer membrane covers the second active area.

8. A test substance sensor described in any one of claims 5 to 7, wherein the lower layer includes a first membrane polymer.

9. The test substance sensor of claim 8, wherein the first membrane polymer includes polyvinylpyridine.

10. The test substance sensor according to claim 8 or 9, wherein the lower layer includes a first crosslinking agent.

11. The test substance sensor of claim 10, wherein the first crosslinking agent contains two or more crosslinkable groups.

12. A test substance sensor as described in claim 10 or 11, wherein the first crosslinking agent is polyethylene glycol diglycidyl ether.

13. A test substance sensor as described in claim 10 or 11, wherein the first crosslinking agent is polyethylene glycol tetraglycidyl ether.

14. A test substance sensor as described in any one of claims 5 to 13, wherein the upper layer includes a second membrane polymer.

15. The analyte sensor of claim 14, wherein the second membrane polymer comprises polyvinylpyridine-co-styrene.

16. A test substance sensor as described in claim 14 or 15, wherein the upper layer contains a second crosslinking agent.

17. The test substance sensor of claim 16, wherein the second crosslinking agent contains two or more crosslinkable groups.

18. A test substance sensor as described in claim 16 or 17, wherein the second crosslinking agent is polyethylene glycol diglycidyl ether.

19. A test substance sensor as described in claim 16 or 17, wherein the second crosslinking agent is polyethylene glycol tetraglycidyl ether.

20. A method for controlling a test substance sensor according to any one of claims 1 to 19, which is already introduced into tissue, comprising: applying the low potential to the first working electrode; obtaining a first signal at or above the redox potential of the first active area, the first signal being proportional to a ketone concentration in a fluid contacting the first active area; Correlating said first signal to a ketone concentration in said fluid.

16. A method for controlling an analyte sensor comprising:

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