Analyte sensors and sensing methods characterized by low-potential detection

Abstract

To provide analyte sensors suitable for in vivo use.SOLUTION: Analyte sensors responsive at low working electrode potentials may comprise an active area upon a surface of a working electrode, where the active area comprises a polymer, a redox mediator covalently bonded to the polymer, and at least one analyte-responsive enzyme covalently bonded to the polymer. A specific redox mediator responsive at low potential may have a structure of (I), where G is a linking group covalently bonding the redox mediator to the polymer. A mass transport limiting membrane permeable to the analyte may overcoat the active area. In some sensor configurations, the mass transport limiting membrane may comprise a membrane polymer crosslinked with a branched crosslinker comprising three or more crosslinkable groups, such as polyethylene glycol tetraglycidyl ether.SELECTED DRAWING: None

Description

[Technical Field]

[0001] TECHNICAL FIELD The present disclosure relates to analyte sensors and sensing methods. [Background technology]

[0002] Detection of various analytes in an individual may be important for monitoring health conditions. Deviations from normal analyte levels may indicate many physiological conditions. Glucose levels are particularly important for detection and monitoring in, for example, diabetic individuals. By monitoring glucose levels with sufficient regularity, diabetic individuals may take corrective action before serious physiological problems occur (e.g., by injecting insulin to lower glucose levels or by eating to raise glucose levels). It may be desirable to monitor other analytes for other physiological conditions. In some cases, monitoring multiple analytes may be desirable, especially for co-occurring conditions that, in combination with each other, simultaneously result in dysregulation of two or more analytes.

[0003] Many analytes are interesting targets for physiological analysis if appropriate detection chemistries can be identified. To this end, amperometric sensors configured to continuously assay glucose in vivo have been developed and improved in recent years to aid in health monitoring in diabetic individuals. Other analytes that are commonly co-regulated with glucose in diabetic individuals include, for example, lactate, oxygen, pH, A1c, and ketones. It may also be desirable to monitor these and other analytes independently of glucose dysregulation. Analyte sensors configured to detect analytes other than glucose in vivo are known but have not yet been sufficiently improved. Low sensitivity to low amounts of analyte can be particularly problematic.

[0004] Analyte monitoring of an individual can be performed periodically or continuously over a period of time. Periodic analyte monitoring can be performed by collecting samples of bodily fluids, such as blood or urine, at set intervals and analyzing them ex vivo. Periodic ex vivo analyte monitoring is sufficient to determine the physiological status of many individuals. However, ex vivo analyte monitoring can be inconvenient or painful in some cases. Furthermore, if analyte measurements are not obtained at the appropriate time, there is no way to make up the missing data. Continuous analyte monitoring may be performed using one or more sensors that remain at least partially implanted within the individual's tissue, such as cutaneously, subcutaneously, or intravenously, so that analysis can be performed in vivo. Depending on the individual's particular health needs and / or previously measured analyte levels, implanted sensors may collect analyte data on demand, on a set schedule, or continuously. Analyte monitoring using in vivo implanted sensors is a more desirable approach for individuals with severe analyte dysregulation and / or rapidly fluctuating analyte levels, although it may be beneficial for other individuals as well. Because implantable analyte sensors often remain within an individual's tissues for extended periods of time, it may be highly desirable to fabricate such analyte sensors from stable materials that exhibit a high degree of biocompatibility.

[0005] To improve biocompatibility, the analyte sensor can include a membrane disposed over the entire implantable portion of the sensor, particularly covering at least the active area of the sensor. In addition to promoting biocompatibility, the membrane may be permeable or semi-permeable to the analyte of interest and limit the total analyte flux to the active area of the sensor. Such a mass-transport-limiting membrane can help avoid overloading (saturation) of the sensing component within the active area, thereby improving sensor performance and accuracy. For example, in the case of a sensor performing enzyme-based detection, limiting the mass transport of the analyte to the active area can make the chemical kinetics of the sensing process analyte-limited rather than enzyme-limited, thereby allowing the sensor output to be easily correlated to the amount of analyte present.

[0006] One problem associated with incorporating a membrane on an analyte sensor is that the analyte flux across the membrane can vary significantly with temperature and / or the length of time the analyte sensor is implanted in tissue. Some membrane materials are less susceptible to temperature-dependent analyte flux than others. If necessary, calibration coefficients or equations can be employed to account for the variability of analyte flux with temperature, but doing so can significantly complicate the use of the sensor. Changes in membrane composition can occur over time, particularly due to extraction loss and / or membrane degradation or metabolism of various membrane components, which can cause different analyte permeability values depending on the extent of the compositional changes. It can be difficult to quantitatively address compositional changes that result in different membrane permeability values, and it can be difficult to determine when membrane permeability has stabilized sufficiently to allow accurate measurement of analyte concentration. Often, when a new analyte sensor is implanted in vivo, an equilibration period of several hours or more is required for the analyte flux across the membrane to stabilize. [Brief explanation of the drawings]

[0007] The following drawings are included to illustrate certain aspects of the present disclosure and should not be viewed 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.

[0008] [Figure 1] 1 shows a schematic diagram of an exemplary sensing system incorporating an analyte sensor of the present disclosure.

[0009] [Figure 2A] 1 shows a cross-sectional view of an analyte sensor including a single active area. [Figure 2B] 1 shows a cross-sectional view of an analyte sensor including a single active area. [Figure 2C] 1 shows a cross-sectional view of an analyte sensor including a single active area.

[0010] [Figure 3A] 1 shows a cross-sectional view of an analyte sensor including two active areas. [Figure 3B] 1 shows a cross-sectional view of an analyte sensor including two active areas. [Figure 3C] 1 shows a cross-sectional view of an analyte sensor including two active areas. [Figure 4] 1 shows a cross-sectional view of an analyte sensor including two active areas.

[0011] [Figure 5A] FIG. 1 shows a perspective view of an analyte sensor containing two active areas on separate working electrodes. [Figure 5B] FIG. 1 shows a perspective view of an analyte sensor containing two active areas on separate working electrodes. [Figure 5C] FIG. 1 shows a perspective view of an analyte sensor containing two active areas on separate working electrodes.

[0012] [Figure 6] A, B, and C show schematic diagrams of enzyme systems that can be used to detect ketones in an analyte sensor.

[0013] [Figure 7] 1 shows cyclic voltammograms of unbound and polymer-bound low potential redox mediators.

[0014] [Figure 8] 1 shows plots of current versus time at various working electrode potentials for a ketone sensor incorporating a low-potential redox mediator.

[0015] [Figure 9] 1 shows plots of current versus ketone concentration at various working electrode potentials for a ketone sensor incorporating a low-potential redox mediator.

[0016] [Figure 10] 1 shows UV-VIS absorbance data for extracts obtained from membrane polymers crosslinked with polyethylene glycol tetraglycidyl ether compared to membrane polymers crosslinked with polyethylene glycol diglycidyl ether. [Figure 11] 1 shows UV-VIS absorbance data for extracts obtained from membrane polymers crosslinked with polyethylene glycol tetraglycidyl ether compared to membrane polymers crosslinked with polyethylene glycol diglycidyl ether.

[0017] [Figure 12] 1 shows a plot of sensor output versus time for an analyte sensor coated with a membrane polymer cross-linked with polyethylene glycol tetraglycidyl ether compared to an analyte sensor coated with a membrane polymer cross-linked with polyethylene glycol diglycidyl. [Figure 13] 1 shows a plot of sensor output versus time for an analyte sensor coated with a membrane polymer cross-linked with polyethylene glycol tetraglycidyl ether compared to an analyte sensor coated with a membrane polymer cross-linked with polyethylene glycol diglycidyl. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present disclosure generally describes analyte sensors suitable for in vivo use, and more specifically, analyte sensors comprising components including low-potential operating capabilities and / or stabilized membrane material features. Depending on the sensor configuration, analyte sensors of the present disclosure can be configured to detect a single analyte or multiple analytes simultaneously or near-simultaneously. A series of dip-coating operations can be performed to introduce different membrane compositions at specific locations on the analyte sensor.

[0019] Various analyte sensor components can present particular challenges when monitoring several analytes or combinations of analytes. Redox mediators used to facilitate electron transfer to the working electrode can require the analyte sensor to operate at relatively high potentials, which can lead to electrochemical side reactions that can complicate the detection of small amounts of analyte. Compositional changes in the mass transport limiting membrane within the analyte sensor can lead to undesirable changes in analyte permeability during periods when the analyte sensor is implanted in vivo, especially during long-term sensor wear. Furthermore, when analyzing multiple analytes using a single analyte sensor, different analyte permeability characteristics may require the use of different mass transport limiting membranes at various locations.

[0020] To address the aforementioned needs, the present disclosure provides redox mediators for facilitating electron transfer at lower working electrode potentials than those previously used. The use of such "low potential" redox mediators may reduce the occurrence of electrochemical side reactions by allowing analyte detection to occur at lower potentials than would otherwise be possible. By reducing the occurrence of electrochemical side reactions and associated signal noise, low-abundance analytes, such as ketones, may be detected more easily than would otherwise be possible at higher working electrode potentials. Such low-potential redox mediators may be advantageous when used in conjunction with the detection of multiple analytes, as discussed further below. Redox mediators capable of facilitating analyte detection at low working electrode potentials are further described below.

[0021] Various cross-linked polyvinylimidazole and polyvinylpyridine membrane polymers can be used in analyte sensors to improve biocompatibility and provide mass transport-limiting properties. Functional groups on the membrane material can be selected to modify analyte permeability and limit temperature-dependent changes in analyte permeability. Such membrane polymers can be cross-linked to linear glycidyl ethers with two cross-linking groups, such as polyethylene glycol diglycidyl ether (PEG-DGE), but an equilibration period may be required after sensor implantation to stabilize analyte flux. Without being bound by theory or mechanism, it is believed that the membrane composition may change during the equilibration period, during which small amounts of extractable substances are released from the membrane, thereby altering analyte permeability. Surprisingly, the present disclosure demonstrates that branched cross-linkers containing three or more cross-linking groups, such as branched polyethylene glycol glycidyl ether, and more specifically, polyethylene glycol tetraglycidyl ether, can reduce the amount of extractable substances released from the membrane after sensor implantation. Reduced production of extractables from membranes can be achieved even when the crosslink density and amount (mass) of crosslinker are substantially the same as those provided by linear glycidyl ethers with two crosslinking groups. That is, a given amount of crosslinking groups from a branched crosslinker, such as polyethylene glycol tetraglycidyl ether, can result in reduced extractables for a given membrane material compared to the amount of extractables provided by a substantially similar amount of crosslinking groups from a linear glycidyl ether crosslinker, such as polyethylene glycol diglycidyl ether. Advantageously, as disclosed herein, reduced extractables can result from membrane materials crosslinked with branched crosslinkers, thereby providing an improved toxicity profile. Furthermore, the reduced compositional changes provided by the membrane materials disclosed herein can result in shorter sensor equilibration times after sensor implantation, potentially extending the wear life of the sensor.

[0022] Before describing the analyte sensors and their components of the present disclosure in further detail, an overview of suitable in-vivo analyte sensor configurations and sensor systems using the analyte sensors will first be provided so that embodiments of the present disclosure can be better understood. FIG. 1 shows a diagram of an exemplary sensing system that may incorporate the analyte sensors of the present disclosure. As shown, sensing system 100 includes a sensor controller 102 and a reader 120 configured to communicate with each other via a wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted local communication path or link. According to some embodiments, reader 120 may constitute an output device for displaying analyte concentration and alerts or notifications determined by sensor 104 or its associated processor, as well as allowing for one or more user inputs. Reader 120 may be a general-purpose smartphone or a dedicated electronic reading device. While only one reader 120 is shown, multiple readers 120 may be present in certain cases. Reader 120 may also communicate with remote terminal 170 and / or trusted computer system 180 via wired or wireless, unidirectional or bidirectional, encrypted or unencrypted communication path(s) / link(s) 141 and / or 142, respectively. Additionally or alternatively, reader 120 may communicate with network 150 (e.g., a mobile telephone network, the Internet, or a cloud server) via communication path / link 151. Network 150 may further be communicatively coupled to remote terminal 170 via communication path / link 152 and / or to trusted computer system 180 via communication path / link 153. Alternatively, sensor 104 may communicate directly with remote terminal 170 and / or trusted computer system 180 without the presence of intervening reader 120.For example, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is incorporated herein by reference in its entirety, according to some embodiments, sensor 104 may communicate with remote terminal 170 and / or trusted computer system 180 via a direct communication link to network 150. Any suitable electronic communication protocol may be used for each communication path or link, 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 trusted computer system 180 may be accessible by individuals other than the original user who are interested in the user's analyte levels. Reader 120 may include a display 122 and an optional input element 121. According to some embodiments, display 122 may include a touchscreen interface.

[0023] The sensor control device 102 includes a sensor housing 103 and may house electrical circuitry and a power source for operating the sensor 104. Optionally, the power source and / or active electrical circuitry may be omitted. A processor (not shown) may be communicatively coupled to the sensor 104, the processor being physically embedded in the sensor housing 103 or the reader 120. The sensor 104 protrudes from the underside of the sensor housing 103 and extends through an adhesive layer 105, which, according to some embodiments, is suitable for adhering the sensor housing 103 to a tissue surface, such as skin.

[0024] The sensor 104 is adapted to be at least partially inserted into a target tissue, such as the dermis or subcutaneous layer of the skin. The sensor 104 may include a sensor tail of sufficient length to insert to a desired depth in a given tissue. The sensor tail may include at least one working electrode. In certain configurations, the sensor tail may include a ketone-responsive active area and, in certain cases, a low-potential redox mediator, as further discussed. A counter electrode may be present in combination with the at least one working electrode. Specific electrode configurations on the sensor tail are described in further detail below.

[0025] One or more mass transport limiting membranes, particularly those crosslinked with branched glycidyl ethers such as polyethylene glycol tetraglycidyl ether, may cover the active areas, as described in further detail below. The active areas may be configured to detect specific analytes. For example, a glucose-responsive active area may include a glucose-responsive enzyme, a lactate-responsive active area may include a lactate-responsive enzyme, and a ketone-responsive active area may include an enzyme system including at least two enzymes that can work together to facilitate the detection of ketones. Suitable enzyme systems for detecting ketones are further described below with reference to Figures 6A-6C. According to various embodiments, each active area may include a polymer covalently bonded to at least some of the enzymes.

[0026] In any embodiment of the present disclosure, one or more analytes may be monitored in a bodily fluid of interest, such as dermal fluid, interstitial fluid, plasma, blood, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, or amniotic fluid, etc. In certain embodiments, the analyte sensors of the present disclosure may be adapted for assaying dermal or interstitial fluid to determine the concentration of one or more analytes in vivo.

[0027] Continuing with reference to FIG. 1 , the sensor 104 may automatically transfer data to the reader 120. For example, analyte concentration data (i.e., glucose and / or ketone concentrations) may be automatically and periodically communicated using data stored in memory until the data is transmitted (e.g., every minute, every five minutes, or other predetermined time period), such as at a specific frequency when data is obtained or after a specific period of time has elapsed. In other embodiments, the sensor 104 may communicate with the reader 120 in a non-automatic manner and not according to a set schedule. For example, data may be communicated from the sensor 104 using RFID technology when the sensor electronics are brought within communication range of the reader 120. The data may remain stored in the sensor 104's memory until communicated to the reader 120. Thus, the user need not be constantly near the reader 120 but can instead upload the data at a convenient time. In still other embodiments, a combination of automatic and non-automatic data transfer may be performed. For example, data transfer may continue automatically until the reader 120 is no longer within communication range of the sensor 104.

[0028] The introducer may be temporarily present to facilitate the introduction of the sensor 104 into the tissue. In an exemplary embodiment, the introducer may comprise a needle or similar sharp portion. It should be appreciated that other types of introducers, such as a sheath or blade, may be present in alternative embodiments. More specifically, the needle or other introducer may be temporarily present near the sensor 104 before insertion into the tissue and then withdrawn. While present, the needle or other introducer may facilitate the insertion of the sensor 104 into the tissue by opening an access path for the sensor 104 to follow. For example, according to one or more embodiments, the needle may facilitate penetration of the epithelium as an access path to the dermis to enable implantation of the sensor 104. After opening the access path, the needle or other introducer may be withdrawn so as not to present a risk due to the sharp tip. In an exemplary embodiment, a suitable needle may be solid or hollow, sharp or non-sharp, and / or circular or non-circular in cross-section. In more particular embodiments, suitable needles may be comparable in cross-sectional diameter and / or tip design to acupuncture needles and may have a cross-sectional diameter of approximately two hundred fifty microns (250 μm). However, it should be recognized that suitable needles may have larger or smaller cross-sectional diameters as needed 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 so that the needle first penetrates the tissue and opens an access path for the sensor 104. In other exemplary embodiments, the sensor 104 may reside in a lumen or groove of the needle so that the needle similarly opens an access path for the sensor 104. In either case, the needle is then withdrawn after facilitating insertion of the sensor.

[0030] Sensor configurations including a single active area configured to detect a corresponding single analyte may employ two-electrode or three-electrode detection motifs, as further described herein with reference to Figures 2A through 2C. Sensor configurations including two different active areas for detection of separate analytes, either on separate working electrodes or on the same working electrode, are described separately below with reference to Figures 3A through 5C. Sensor configurations with multiple working electrodes may be particularly advantageous over incorporating two different active areas within the same sensor tail, since the signal contributed by each active area may be more easily determined. Furthermore, deposition of different film compositions on each active area may be easily achieved by a series of dip-coating operations when the active area is present on the second working electrode.

[0031] When a single working electrode is present in the analyte sensor, a three-electrode sensor configuration may include a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration may include a working electrode and a second electrode, where the second electrode may 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 (layered) on one another and / or spaced apart laterally on the sensor tail. Suitable sensor configurations may be substantially planar or substantially cylindrical. In any of the sensor configurations disclosed herein, the various electrodes may be electrically insulated from one another by a dielectric material or similar insulator.

[0032] An analyte sensor with multiple working electrodes may also include at least one additional electrode. If one additional electrode is present, the one additional electrode may function as a counter / reference electrode for each of the multiple working electrodes. If two additional electrodes are present, one of the additional electrodes may function as a counter electrode for each of the multiple working electrodes, and the other additional electrode may function as a reference electrode for each of the multiple working electrodes.

[0033] 2A shows a schematic diagram of an exemplary two-electrode analyte sensor configuration suitable for use in the present disclosure. As shown, analyte sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter / reference electrode 216. Alternatively, working electrode 214 and counter / reference electrode 216 may be disposed on the same side of substrate 212 with an intervening dielectric material (configuration not shown). Active area 218 is disposed as at least one layer on at least a portion of working electrode 214. Active area 218 may include multiple spots or a single spot configured to detect analyte at low working electrode potentials, as discussed further herein.

[0034] 2A , according to some embodiments, membrane 220 covers at least active area 218 and may optionally cover some or all of working electrode 214 and / or counter / reference electrode 216, or the entire analyte sensor 200. One or both sides of analyte sensor 200 may be covered with membrane 220. Membrane 220 may include one or more polymeric membrane materials capable of limiting analyte flux to active area 218 (i.e., membrane 220 is a mass transport limiting membrane permeable to the analyte of interest). According to the disclosure herein, membrane 220 may be crosslinked using a branched crosslinker in certain sensor configurations. The composition and thickness of membrane 220 may be modified to promote desired analyte flux to active area 218, thereby providing desired signal strength and stability. Analyte sensor 200 is operable to assay an analyte by any coulometric, amperometric, voltammetric, or potentiometric electrochemical detection technique.

[0035] 2B and 2C show schematic diagrams of exemplary three-electrode analyte sensor configurations suitable for use in the present disclosure. The three-electrode analyte sensor configuration may be similar to the configuration shown as analyte sensor 200 in FIG. 2A, except for the inclusion of additional electrode 217 in analyte sensors 201 and 202 (FIGS. 2B and 2C). When additional electrode 217 is present, counter / reference electrode 216 may then function as either a counter electrode or a reference electrode, with additional electrode 217 performing the function of the other electrode that is not configured. Working electrode 214 continues to perform its original function. Additional electrode 217 may be positioned on either working electrode 214 or electrode 216, with a separating layer of dielectric material between them. For example, as shown in FIG. 2B, dielectric layers 219a, 219b, and 219c separate electrodes 214, 216, and 217 from one another and provide electrical insulation. Alternatively, at least one of electrodes 214, 216, and 217 may be disposed on both sides of substrate 212, as shown in FIG. 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 electrodes 214 or 216 and spaced apart by a dielectric material. A 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 arrangement shown in FIGS. 2B and 2C. Similar to sensor 200 shown in FIG. 2A, active area 218 in analyte sensors 201 and 202 may include multiple spots or a single spot. Additionally, analyte sensors 201 and 202 can be operated to assay analytes by any coulometric, amperometric, voltammetric, or potentiometric electrochemical detection technique.

[0036] Like analyte sensor 200, in analyte sensors 201 and 202, membrane 220 may also cover active area 218, thereby serving as a mass transport limiting membrane, as in other sensor configurations. Additional electrode 217 may be covered by membrane 220 in some embodiments. While FIGS. 3B and 2C show all electrodes 214, 216, and 217 covered by membrane 220, it should be appreciated that in some embodiments, only working electrode 214 may be covered. Furthermore, 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 the entire analyte sensors 201 and 202 may be covered. Furthermore, it should be understood that the three-electrode sensor configuration shown in Figures 2B and 2C is not limited to the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of the present disclosure.

[0037] FIG. 3A shows an exemplary configuration of a sensor 203 having a single working electrode with two distinct active areas disposed thereon. FIG. 3A is similar to FIG. 2A , except that there are two active areas on the working electrode 214: a first active area 218a and a second active area 218b, which respond to different analytes and are spaced laterally apart from each other on the surface of the working electrode 214. The active areas 218a and 218b may include multiple spots or a single spot configured for detection of each analyte. The composition of the membrane 220 may be different in the active areas 218a and 218b, or may be compositionally the same. The first active area 218a and the second active area 218b may be configured to detect corresponding analytes at different working electrode potentials, as discussed further below.

[0038] 3B and 3C show cross-sectional views of exemplary three-electrode sensor configurations for sensors 204 and 205, respectively, featuring a single working electrode with first and second active areas 218a and 218b disposed thereon. Figures 3B and 3C are otherwise similar to Figures 2B and 2C, which may be better understood by reference thereto. Similar to Figure 3A, the composition of membrane 220 may be different in active areas 218a and 218b, or may be compositionally the same.

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

[0040] FIG. 4 illustrates a cross-sectional view of an exemplary analyte sensor configuration suitable for use in the present disclosure, having two working electrodes, a reference electrode, and a counter electrode. As shown, analyte sensor 300 includes working electrodes 304 and 306 disposed on opposite sides of substrate 302. A first active area 310a is disposed on the surface of working electrode 304, and a second active area 310b is disposed on the surface of working electrode 306. Counter electrode 320 is electrically insulated from working electrode 304 by dielectric layer 322, and reference electrode 321 is electrically insulated from working electrode 306 by dielectric layer 323. Outer dielectric layers 330 and 332 are disposed on reference electrode 321 and counter electrode 320, respectively. According to various embodiments, membrane 340 may cover at least active areas 310a and 310b, and optionally other components of analyte sensor 300 or the entire analyte sensor 300 may also be covered by membrane 340. Furthermore, if desired, membrane 340 may be compositionally different between active areas 310a and 310b to provide permeability values suitable for differentially adjusting analyte flux at each location. For example, membrane 340 may be homogeneous over active area 310a and heterogeneous over active area 310b.

[0041] Alternative sensor configurations having multiple working electrodes that differ from the configuration shown in FIG. 4 may include a single counter / reference electrode instead of separate counter and reference electrodes 320, 321, and / or may feature different layer and / or film arrangements than those explicitly shown. For example, the arrangement of counter electrode 320 and reference electrode 321 may be reversed from that shown in FIG. 4. Furthermore, working electrodes 304 and 306 need not necessarily be present on opposite 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 spaced apart from each other. In particular, working electrode 304 may be positioned toward either the more distal end (tip) or the more proximal end of the analyte sensor relative to working electrode 306. If a sufficiently large gap exists between the working electrodes 304 and 306, the film 340 may be deposited on the working electrodes 304 and 306 by a series of dip-coating operations, and the composition of the film 340 may vary locally. In particular, a first dip-coating operation may deposit a first film polymer on both the working electrodes 304 and 306, and a second dip-coating operation may deposit a second film polymer having a different composition only on the working electrode 304, thereby defining a bilayer on the working electrode 304 and leaving a uniform film on the working electrode 306. Thus, the lower layer of the bilayer film and the uniform film may comprise the same film polymer. Alternatively, to define a bilayer film and a uniform film, a first dip-coating operation may deposit a first film polymer on the working electrode 304, and a second dip-coating operation may deposit a second film polymer having a different composition on both the working electrodes 304 and 306. In this case, the upper layer of the bilayer film and the uniform film may comprise the same film polymer.

[0042] The size of the gap between the working electrodes 304 and 306 is large enough to at least provide electrical isolation between the two electrodes, and more typically is large enough to facilitate a series of dip-coating operations (e.g., by allowing the analyte sensor to be immersed to different depths to preferentially cover one of the working electrodes in at least one immersion step). In other words, the size of the gap may provide a margin of error for lowering the analyte sensor to a particular depth in a particular dip-coating agent to facilitate deposition on one of the working electrodes in preference to the other.

[0043] While suitable sensor configurations may feature electrodes of a substantially planar nature, including planar sensor configurations with spaced apart working electrodes, it should be understood that sensor configurations featuring non-planar electrodes may be advantageous and particularly suitable for use in the present disclosure. In particular, cylindrical electrodes spaced apart along the length of the sensor tail may facilitate deposition of mass transport limiting films of different compositions, as further described below. In particular, concentric working electrodes spaced apart along the length of the sensor tail may facilitate deposition by a series of dip-coating operations, similar to the method described above for substantially planar sensor configurations. Figures 5A-5C show perspective views of an analyte sensor with two working electrodes spaced apart concentrically. It should also be understood that sensor configurations without a second working electrode but with a concentric electrode arrangement are possible in the present disclosure.

[0044] 5A shows a perspective view of an exemplary sensor configuration in which multiple electrodes are substantially cylindrical and concentrically arranged relative to a central substrate. As shown, the analyte sensor 400 includes a central substrate 402 on which all electrodes and dielectric layers are concentrically arranged relative to one another. In particular, a working electrode 410 is disposed on the surface of the central substrate 402, and a dielectric layer 412 is disposed on a portion of the working electrode 410 distal to the sensor tip 404. A working electrode 420 is disposed on the dielectric layer 412, and a dielectric layer 422 is disposed on a portion of the working electrode 420 distal to the sensor tip 404. A counter electrode 430 is disposed on the dielectric layer 422, and a dielectric layer 432 is disposed on a portion of the counter electrode 430 distal to the sensor tip 404. A reference electrode 440 is disposed on the dielectric layer 432, and a dielectric layer 442 is disposed on a portion of the reference electrode 440 distal to the sensor tip 404. As such, the exposed surfaces of working electrode 410, working electrode 420, counter electrode 430, and reference electrode 440 are spaced apart from one another along the longitudinal axis B of analyte sensor 400. This electrode arrangement may allow for membrane 450 to be deposited by a series of dip-coating operations, which may allow for a bilayer membrane portion to be placed on working electrode 410 and a uniform membrane portion to be placed on working electrode 420, as discussed below. The bilayer membrane portion and the uniform membrane portion may be adjacent to one another.

[0045] 5A, first and second active areas 414a and 414b responsive to different analytes are disposed on the exposed surfaces of working electrodes 410 and 420, respectively, to allow contact with the fluid for sensing. While active areas 414a and 414b are shown as three separate spots in FIG. 5A, it should be understood that fewer or more than three spots may be present in alternative sensor configurations, including a continuous layer of active areas.

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

[0047] It should be further understood that the various electrode arrangements in FIGS. 5A and 5B may differ from those explicitly shown. For example, the arrangement of the counter electrode 430 and reference electrode 440 may be reversed from the configuration shown in FIGS. 5A and 5B. Similarly, the arrangement of the working electrodes 410 and 420 is not limited to the arrangement explicitly shown in FIGS. 5A and 5B. FIG. 5C illustrates an alternative sensor configuration to that shown in FIG. 5B, in which the sensor 405 includes the counter electrode 430 and reference electrode 440 positioned more proximally relative to the sensor tip 404 and the working electrodes 410 and 420 positioned more distally relative to the sensor tip 404. A sensor configuration in which the working electrodes 410 and 420 are positioned 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 FIG. 5C), thereby facilitating enhanced signal strength in some cases. Furthermore, by having the 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 by a series of dip-coating operations, and a uniform film portion may be deposited on the working electrode distal from the sensor tip 404. 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 an analyte sensor operable 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 forming 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 does not necessarily have to be performed at low potential.

[0049] Thus, according to the present disclosure, an analyte sensor operable at low potentials may include a sensor tail including at least a first working electrode, a first active area disposed on the first working electrode and responsive to a first analyte at low potentials, the first active area including a first polymer, a first redox mediator covalently bound to the first polymer, and at least one enzyme responsive to the first analyte covalently bound to the first polymer, and a mass transport limiting membrane permeable to the first analyte covering at least the first active area. As used herein, the term "low potential" refers to a potential higher than the redox potential of the first redox mediator and 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, as measured relative to an Ag / AgCl reference electrode. Exemplary redox potentials of the first redox mediator that may facilitate operation at potentials 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 potential.

[0050] Suitable examples of first redox mediators capable of facilitating operation at low potentials may have a structure represented by Formula 1: [ka] In the formula, M is osmium, ruthenium, vanadium, cobalt, or iron, and L 1 From L 6 is an independent heteroaromatic ligand coordinatively bonded to M, and L 1 From L 6 two or more of may optionally combine to form bidentate, tridentate or higher dentate ligands, and L 1 From L 6 at least one of L includes a linking group that links the first redox mediator to the first polymer; 1 From L 6At least one of the groups is functionalized with an electron donating group, the electron donating group being separate and different from the linking group.

[0051] Particularly suitable bidentate ligands featuring heteroaromatic ligands included in low potential redox mediators include optionally substituted 2,2'-biimidazole ligands, 2-(2-pyridyl)imidazole ligands, and 2,2'-bipyridine ligands.

[0052] An example of a 2,2'-biimidazole ligand is shown by formula 2: [ka] In the formula, R 1 and R 2 are independently selected from optionally substituted alkyl, alkenyl, or aryl groups. 1 and R 2 is unsaturated C1 to C 12 In some embodiments, R 1 and R 2 where both are methyl. Q is an optional substituent attached to one or more carbon atoms of the imidazole ring (n=0, 1, or 2), and any Q substituent may, in some embodiments, be an electron-donating group or a linking group. Suitable electron-donating groups that may comprise one or more Q include, for example, alkyl, alkoxy, hydroxyl, amino, alkylamino, or dialkylamino. When Q is absent, that carbon atom bears a hydrogen atom.

[0053] An example of a 2-(2-pyridyl)imidazole ligand may have a structure represented by formula 3: [ka] In the formula, R 1 ' is unsaturated C1 to C 12or an optionally substituted alkyl group such as a C1 to C4 alkyl group, particularly methyl, an alkenyl group, or an aryl group. Q is an optional substituent attached to one or more carbon atoms of the imidazole or pyridine ring (imidazole n=0, 1, or 2, pyridine p=0, 1, 2, 3, or 4), and any Q substituent may, in some embodiments, be an electron-donating group or a linking group. Suitable electron-donating groups that may comprise one or more Qs include, for example, alkyl, alkoxy, hydroxyl, amino, alkylamino, or dialkylamino. When Q is absent, that carbon atom bears a hydrogen atom.

[0054] An example of a 2,2'-bipyridine ligand may have a structure represented by formula 4: [ka] wherein Q is an optional substituent attached to one or more carbon atoms of the pyridine ring (p=0, 1, 2, 3, or 4), and any Q substituent may, in some embodiments, be an electron-donating group or a linking group. Suitable electron-donating groups comprising one or more Q include, for example, alkyl, alkoxy, hydroxyl, amino, alkylamino, or dialkylamino. When Q is absent, that carbon atom bears a hydrogen atom.

[0055] Particularly suitable examples of redox mediators capable of facilitating electron transfer at low potentials may have a structure represented by Formula 5, where L 1 and L 2 , L 3 and L 4 , and L 5 and L 6 are linked to form a bidentate ligand, one of which has a linking group G that covalently bonds the redox mediator to the polymer, and L 1 -L 2 , L 3 -L 4 , and L 5 -L 6At least one of L has an electron donating group. The electron donating group is separate and different from the linking group G. The electron donating group may be on the same bidentate ligand containing the linking group G or on a different bidentate ligand. 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 Formulas 2 through 4. [ka]

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

[0057] In a more particular example, the redox mediator in the first active zone may have a structure represented by Formula 6: [ka] wherein G is a linking group that covalently bonds the redox mediator to the polymer in the active area, and D is an electron-donating group. Specific examples of suitable electron-donating groups include, for example, hydroxyl groups, alkoxy groups (e.g., methoxy or ethoxy groups), amino groups, or alkyl or dialkylamino groups (e.g., methylamino, ethylamino, dimethylamino, or diethylamino groups). 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. [ka] In any embodiment herein, the redox mediator capable of facilitating electron transfer at low potentials can have a structure represented by Formula 8: [ka]

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

[0059] Any suitable polymer backbone can be present in the active area to facilitate detection of analytes at low potentials by covalently binding redox mediators and enzymes thereto. Examples of suitable polymers in the active area include poly(4-vinylpyridine) and poly(N-vinylimidazole) or copolymers thereof, where, for example, quaternized pyridine and quaternized imidazole groups serve as attachment sites for the redox mediator or enzyme. Other suitable polymers that can be present in the active area include, but are not limited to, polymers such as 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), as described in U.S. Pat. No. 6,605,200, the entire contents of which are incorporated herein by reference.

[0060] The enzyme covalently attached to the polymer in the first active area that can facilitate detection at low potential is not believed to be particularly limited. Suitable enzymes may include enzymes capable of detecting glucose, lactate, ketones, creatinine, etc. In some cases, the at least one enzyme covalently attached to the polymer in the first active area may include multiple enzymes that collectively respond to the analyte at low potential. Enzyme systems may be particularly desirable for detecting ketones and creatinine.

[0061] In more specific embodiments, the first active area may include an enzyme system capable of detecting ketones. As previously mentioned, ketones are typically present in low biological amounts and may benefit from low-potential detection in accordance with the present disclosure. Referring now to FIGS. 6A through 6C, a particular enzyme system that may be used to detect ketones is described in further detail. In the enzymatic reaction shown, β-hydroxybutyrate serves as a surrogate for ketones formed in vivo. As shown in FIG. 6A, a pair of cooperating enzymes that may be used to detect ketones in accordance with the present disclosure are β-hydroxybutyrate dehydrogenase (HBDH) and diaphorase, which may be deposited within a ketone-responsive active area on the surface of at least one working electrode, as further described herein. When the ketone-responsive active area includes this pair of cooperating enzymes, β-hydroxybutyrate dehydrogenase converts β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD) to β-hydroxybutyrate. + ) to acetoacetate and reduced nicotinamide adenine dinucleotide (NADH), respectively. The enzyme cofactor NAD + and NADH serve to facilitate the concerted enzymatic reaction disclosed herein. NADH may then undergo oxidation via diaphorase, and the electron transfer during this process provides the basis for ketone detection at the working electrode. Thus, there is a 1:1 molar correspondence between the amount of electron transfer to the working electrode and the amount of β-hydroxybutyrate conversion, thereby providing the basis for ketone detection and quantification based on measuring the current flow at the working electrode. The electron transfer leading to NADH oxidation at the working electrode may be carried out by a redox mediator capable of facilitating operation at low potentials. Albumin may be present as a stabilizer along with this concerted enzyme pair. According to certain embodiments, β-hydroxybutyrate dehydrogenase and diaphorase may be covalently attached to a polymer within the ketone-responsive active area of the analyte sensor. NAD + may or may not be covalently attached to a polymer, and NAD + If NAD is not covalently bound, it may physically remain within the ketone-responsive active area. The membrane covering the ketone-responsive active area prevents NAD from being released into the ketone-responsive active area. +This helps to keep the ketones in place and allows sufficient diffusion of the ketones within to allow for their detection.

[0062] Other suitable chemistries for enzymatically detecting ketones are shown in Figures 6B and 6C. In both cases, there is also a 1:1 molar correspondence between the amount of electron transfer to the working electrode and the amount of β-hydroxybutyrate converted, thereby providing the basis for ketone detection.

[0063] As shown in Figure 6B, β-hydroxybutyrate dehydrogenase (HBDH) further decomposes β-hydroxybutyrate and NAD + acetoacetate and NADH, respectively. Instead of electron transfer to the working electrode being 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 reaction with molecular oxygen to generate superoxide, which can then be converted to hydrogen peroxide via superoxide dismutase (SOD). The hydrogen peroxide can then be oxidized at the working electrode, providing a signal that can be correlated to the amount of ketone initially present. SOD, according to various embodiments, can be covalently attached to a polymer in the ketone-responsive active area. As in the enzyme system shown in FIG. 6A, β-hydroxybutyrate dehydrogenase and NADH oxidase can be covalently attached to a polymer in the ketone-responsive active area, and NAD may or may not be covalently attached to a polymer in the ketone-responsive active area. NAD + When NAD is not covalently bound, it is not present in the ketone-responsive active area. + The ketone-responsive active area may be physically secured within the ketone-responsive active area using a membrane polymer that promotes retention of the ketone-responsive active area.

[0064] As shown in Figure 6C, another enzymatic detection chemistry for ketones is β-hydroxybutyrate and NAD. +β-hydroxybutyrate dehydrogenase (HBDH) may be utilized to convert β-hydroxybutyrate dehydrogenase (HBDH) to acetoacetate and NADH, respectively. The electron transfer cycle in this case is completed by 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 bound to a polymer in the ketone-responsive active area. As in the enzyme system shown in Figure 6A, β-hydroxybutyrate dehydrogenase may or may not be covalently bound to a polymer in the ketone-responsive active area, and NAD may or may not be covalently bound to a polymer in the ketone-responsive active area. The inclusion of albumin in the active area can provide a surprising improvement in reaction stability. A suitable membrane polymer can be used to convert NAD into acetoacetate and NADH in the ketone-responsive active area. + This may promote retention of

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

[0066] Suitable redox mediators for inclusion in the second active zone may include, but are not limited to, osmium complexes and other transition metal complexes as described in U.S. Patent Nos. 6,134,461 and 6,605,200, the entire disclosures of which are incorporated herein by reference. Additional examples of suitable redox mediators include those described in U.S. Patent Nos. 6,736,957, 7,501,053, and 7,754,093, the disclosures of which are also incorporated herein by reference in their entirety. Other suitable redox mediators for inclusion in the second active zone may include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt, including, for example, metallocene compounds thereof. Suitable ligands for the metal complexes may include, for example, bidentate or higher dentate ligands, such as, for example, bipyridine, biimidazole, phenanthroline, or pyridyl (imidazole). Other suitable bidentate ligands may include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher dentate ligands may be present in the metal complex to achieve a complete coordination sphere.

[0067] The active area for facilitating the detection of an analyte according to the present disclosure may comprise a polymer to which a redox mediator is covalently attached. Suitable examples of polymer-bound redox mediators are described in U.S. Patent Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which may include those incorporated herein by reference in their entirety. Suitable polymers for inclusion in the active area 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 suitable for inclusion in the active area include those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile. The polymers in each active area may be the same or different.

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

[0069] Detection of each analyte may involve applying a separate potential to each working electrode such that a separate signal is obtained from each analyte. The signal obtained from each analyte may then be correlated to the concentration of the analyte through the use of a calibration curve or function or by using a look-up table. The correlation between analyte signal and analyte concentration may, in certain instances, be derived through the use of a processor.

[0070] In other analyte sensor configurations, the first and second active areas can be disposed on a single working electrode. A first signal can be obtained from the first active area at a low potential, and a second signal including signal contributions from both active areas can be obtained at a higher potential. Subtracting the first signal from the second signal can then allow for determination of the signal contribution arising from the second analyte. Similar to the methods described for sensor configurations with multiple working electrodes, the signal contribution from each analyte can then be correlated with the analyte concentration.

[0071] Accordingly, the present disclosure provides a method for providing an analyte sensor comprising: a sensor tail including at least a first working electrode; a first active area disposed on a surface of the first working electrode and responsive to the first analyte at a low potential, the first active area comprising a first polymer, a first redox mediator covalently bound to the first polymer, and at least one enzyme responsive to the first analyte covalently bound to the first polymer, the redox mediator having a structure represented by any one of Formulas 1 through 8 set forth above, and G being a linking group covalently linking the first redox mediator to the first polymer; and a mass transport limiting membrane permeable to the first analyte covering at least the first active area; applying a low potential to the first working electrode; obtaining a first signal proportional to the concentration of the first analyte in a fluid in contact with the first active area and equal to or greater than the redox potential of the first active area; and correlating the first signal with the concentration of the first analyte in the fluid.

[0072] The low potential may be 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, as measured relative to an Ag / AgCl reference electrode. The low potential may also be higher than the redox potential of the first redox mediator. Exemplary redox potentials of first redox mediators that can facilitate operation at low working electrode potentials 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, as measured relative to an Ag / AgCl reference electrode.

[0073] The analyte sensor disclosed herein further includes a mass transport limiting membrane permeable to the analyte covering at least a first active area. When multiple active areas are present, the mass transport limiting membrane may have different compositions on different active areas and cover each active area, such that, for example, creating a bilayer membrane portion on the working electrode near the sensor tip can be achieved by a series of dip-coating operations. The mass transport limiting membrane may include a membrane polymer, such as a homopolymer or copolymer of polyvinylpyridine or polyvinylimidazole, and may be further crosslinked with a suitable crosslinking agent. In certain embodiments, the membrane polymer may include a copolymer of vinylpyridine and styrene. In more specific examples, the membrane polymer covering one or more active areas may be crosslinked with a branched crosslinking agent containing three or more crosslinking groups, such as polyethylene glycol tetraglycidyl ether, to surprisingly reduce the amount of extractables obtained from such a mass transport limiting membrane. More specifically, the mass transport limiting membrane may comprise a copolymer of vinylpyridine and styrene crosslinked with a branched glycidyl ether crosslinker containing three crosslinking groups, such as polyethylene glycol tetraglycidyl ether. In particular, the epoxy group of polyethylene glycol tetraglycidyl ether may react with a pyridine nitrogen atom or an imidazole nitrogen atom, facilitating covalent bonding of the crosslinking group by opening the epoxide ring. Hydroxyalkyl groups may be provided that bridge the crosslinker body to the heterocycle of the membrane polymer.

[0074] Suitable copolymers of vinylpyridine and styrene may contain styrene in the range of about 0.01% to about 50% by mole, or about 0.05% to about 45% by mole, or about 0.1% to about 40% by mole, or about 0.5% to about 35% by mole, or about 1% to about 30% by mole, or about 2% to about 25% by mole, or about 5% to about 20% by mole. Substituted styrenes may be used in the same manner and in similar amounts. Suitable copolymers of vinylpyridine and styrene may have a molecular weight of 5 kDa or greater, or about 10 kDa or greater, or about 15 kDa or greater, or about 20 kDa or greater, or about 25 kDa or greater, or about 30 kDa or greater, or about 40 kDa or greater, or about 50 kDa or greater, or about 75 kDa or greater, or about 90 kDa or greater, or about 100 kDa or greater. In non-limiting examples, suitable copolymers of vinylpyridine and styrene can have a molecular weight ranging from about 5 kDa to about 150 kDa, or from about 10 kDa to about 125 kDa, or from about 15 kDa to about 100 kDa, or from about 20 kDa to about 80 kDa, or from about 25 kDa to about 75 kDa, or from 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 including at least a first working electrode, a first active area disposed on the surface of the first working electrode, and a mass transport limiting membrane permeable to the first analyte covering at least the first active area. The first active area includes a first polymer and at least one enzyme covalently bonded to the first polymer and responsive to the first analyte. The mass transport limiting membrane includes a membrane polymer crosslinked by a branched glycidyl ether crosslinker containing three or more crosslinking groups, such as polyethylene glycol tetraglycidyl ether.

[0076] In more specific embodiments, crosslinking can occur between molecules. The polyethylene glycol tetraglycidyl ether used to promote intermolecular crosslinking between two or more membrane polymer backbones can exhibit a wide range of suitable molecular weights. Up to four polymer backbones can be crosslinked by a single molecule of polyethylene glycol tetraglycidyl ether crosslinker. In a specific example, the molecular weight of the polyethylene glycol tetraglycidyl ether can range from about 1000 g / mol to about 5000 g / mol. The number of ethylene glycol repeat units in each arm of the polyethylene glycol tetraglycidyl ether can be the same or different and can be varied over the range of a given sample, usually giving an average molecular weight. The structure of polyethylene glycol tetraglycidyl ether before crosslinking is represented by the following formula 9: [ka] wherein n1, n2, n3, and n4 are all integers equal to or greater than 0, typically equal to or greater than 1, and may be the same or different. The sum of n1, n2, n3, and n4 may be selected so that the molecular weight of the polyethylene glycol tetraglycidyl ether falls within the aforementioned range. In other words, to produce a polyethylene glycol tetraglycidyl ether having a molecular weight within the aforementioned 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 subrange therebetween, and n1, n2, n3, and n4 may independently be any integer equal to or greater than 0, or may be an integer equal to or greater than 1.

[0077] Crosslink density refers to the number of membrane polymer side chains that have a crosslinker attached to them. For example, membrane polymers crosslinked with branched glycidyl ethers such as polyethylene glycol tetraglycidyl ether or similar polyethylene oxide crosslinkers with three or more crosslinking groups can have crosslink densities that vary over a wide range. In certain examples, the portion of the side chains that may have a crosslinker attached thereto may represent about 0.1% or more of the available heterocycles in the membrane polymer, or may represent about 0.2% or more of the available heterocycles in the membrane polymer, or may represent about 0.3% or more of the available heterocycles in the membrane polymer, or may represent about 0.4% or more of the available heterocycles in the membrane polymer, or may represent about 0.5% or more of the available heterocycles in the membrane polymer, or may represent about 0.6% or more of the available heterocycles in the membrane polymer, or may represent about 0.7% or more of the available heterocycles in the membrane polymer, or may represent about 0.8% or more of the available heterocycles in the membrane polymer, or may represent about 0.9% or more of the available heterocycles in the membrane polymer, or may represent about 1.0% or more of the available heterocycles in the membrane polymer, or may represent about 1.2% or more of the available heterocycles in the membrane polymer. The heterocycles may be about 1.4% or more of the available heterocycles in the membrane polymer, or may be about 1.6% or more of the available heterocycles in the membrane polymer, or may be about 1.8% or more of the available heterocycles in the membrane polymer, or may be about 2.0% or more of the available heterocycles in the membrane polymer, or may be about 2.2% or more of the available heterocycles in the membrane polymer, or may be about 2.4% or more of the available heterocycles in the membrane polymer, or may be about 2.6% or more of the available heterocycles in the membrane polymer, or may be about 2.8% or more of the available heterocycles in the membrane polymer, or may be about 3.0% or more of the available heterocycles in the membrane polymer, or may be about 3.5% or more of the available heterocycles in the membrane polymer, or may be about 4.0% or more of the available heterocycles in the membrane polymer, or may be about 4.5% or more of the available heterocycles in the membrane polymer, or may be about 5.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 membrane polymer. In more specific embodiments, the crosslinker may be added to 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 comprise one or more polyether arms (side chains) attached to the nitrogen atoms of pyridine or imidazole monomer units. Some membrane polymers disclosed herein may further comprise one or more polyether arms. Polyether arms are distinct from crosslinking groups formed from polyethylene glycol tetraglycidyl ether or similar crosslinking agents; polyether arms do not extend between separate polymer chains or terminate intramolecularly within a single polymer chain. Thus, polyether arms are separate and distinct from crosslinking groups formed from crosslinking agents. Polyether arms may comprise polyethylene oxide blocks and polypropylene oxide blocks, and in particular, polyether arms having polypropylene oxide blocks are inserted between two polyethylene oxide blocks. Bonding of the polyether arm to the nitrogen atom of the heterocycle can occur via any reactive functional group capable of forming a bond with the nitrogen atom of the heterocycle in the membrane polymer. Bonding of the polyether arm to the nitrogen atom of the heterocycle can occur via an alkyl group, a hydroxyl-functionalized alkyl group, or a carbonyl. In certain other cases, the polyether arms may also contain amine groups spaced apart from the nitrogen atoms of the heterocycle, or may be amine-free.

[0079] The polyether arms of the membrane polymer may comprise at least one polyethylene oxide block and at least one polypropylene oxide block, thereby providing at least a diblock sequence of polyethylene oxide and polypropylene oxide monomer units linked to the nitrogen atom of the heterocycle 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 arms may comprise, in order, a spacer, a first polyethylene oxide block, a polypropylene oxide block, and a second polyethylene oxide block (i.e., an ABA 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 BAB repeating pattern). An amine group may be intermediate between the polyethylene oxide block and the polypropylene oxide block in the amine-containing polyether arm. Thus, the polyether arms in the membrane polymers disclosed herein may have a structure generally defined by the following Formulas 10 to 13: [ka] In the formula, PE represents a polyethylene oxide block, PP represents a polypropylene oxide block, A represents an amino group, and J represents a spacer group. The spacer group J can be attached to a heterocycle of the membrane polymer. Suitable spacer groups J can include, but are not limited to, alkyl, hydroxy-functionalized alkyl, carbonyl, carboxylic acid ester, carboxamide, and the like. The variables q, r, s, and t are positive integers that define the number of monomer units in each block and the number of times the block repeats. In the case 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 ranging from about 2 to about 50 or from about 6 to about 20, the variable r is an integer ranging from about 2 to about 60 or from about 10 to about 40, and the variable t is an integer ranging from about 2 to about 50 or from about 10 to about 30. According to some or various other embodiments, the variable s is an integer ranging 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, amine-free polyether arms 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: [ka] wherein R is an alkyl group, particularly a methyl group; the variable w is 0 or 1; the variable x is an integer ranging from about 4 to about 24 or from about 6 to about 20; the variable y is an integer ranging from about 8 to about 60 or from about 10 to about 40; and the variable z is an integer ranging from about 6 to about 36 or from about 10 to about 30. In more specific embodiments, the variable x may range from about 8 to about 16 or from about 9 to about 12; the variable y may range from about 10 to about 32, or from about 16 to about 30, or from about 12 to about 20; and the variable z may range from about 10 to about 20 or from about 14 to about 18. In some embodiments, the variable x may be smaller than the variable z, such that the second polyethylene oxide block is longer (larger) than the first polyethylene oxide block. When the variable w is 0, the amine-free polyether arms are attached directly to the membrane polymer by a two-carbon alkyl group, although longer alkyl groups are also contemplated by the present disclosure.

[0081] In other specific embodiments of the present disclosure, a polyether arm (similar to Formula 12) having a triblock arrangement of polyethylene oxide, polypropylene oxide, and polyethylene oxide and having an amine group interposed between the polyethylene oxide block and the polypropylene oxide block may have a structure defined by Formula 15: [ka] where w, x, y, z, and R are defined above in Formula 14. When the variable w is 0, the polyether arms are attached directly to the membrane polymer by a two-carbon alkyl group, although longer alkyl groups are also contemplated by the present disclosure.

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

[0083] In some embodiments, sulfonic acid-containing arms can be attached as side chains to at least a portion of the membrane polymers disclosed herein. The sulfonic acid-containing arms can be present in any suitable ratio in combination with polyether arms and / or crosslinkers. Any membrane polymer disclosed herein can contain more polyether arms or crosslinking groups than sulfonic acid-containing arms. The sulfonic acid-containing arms are attached to the membrane polymer by alkyl groups. According to various embodiments, the alkyl groups can contain between 1 and about 6 carbon atoms, or between 2 and about 4 carbon atoms. Suitable reagents for introducing sulfonic acid-containing arms 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) may be incorporated in some of the mass transport limiting membranes disclosed herein.

[0085] When first and second active areas configured to assay different analytes are placed on separate working electrodes, the mass transport limiting membrane may have different permeability values for the first and second analytes. While the membrane thickness and / or active area size of each working electrode can be varied to achieve equal sensitivity for each analyte, this approach can significantly complicate the fabrication of the analyte sensor. As a solution, the mass transport limiting membrane covering at least one of the active areas may comprise a blend of a first membrane polymer and a second membrane polymer, or a bilayer of a first membrane polymer and a second membrane polymer. A homogeneous membrane may cover the active areas not covered by the blend or bilayer, and the homogeneous membrane may comprise only one of the first membrane polymer or the second membrane polymer. Advantageously, the analyte sensor structures disclosed herein readily accommodate continuous membranes having a uniform membrane portion disposed on a first active area of the analyte sensor and a multi-component membrane portion disposed on a second active area, thereby providing uniform permeability values for each analyte while simultaneously improving sensitivity and detection accuracy. Deposition of the continuous membrane may, in certain embodiments, be accomplished by a series of dip-coating operations.

[0086] Embodiments disclosed herein include the following.

[0087] A. An analyte sensor capable of low-potential detection of an analyte, comprising: a sensor tail including at least a first working electrode; and a first active area disposed on a surface of the first working electrode and responsive to a first analyte at low potential, the first active area including a first polymer, a first redox mediator covalently bound to the first polymer, and at least one enzyme responsive to the first analyte covalently bound to the first polymer, the first redox mediator being: [ka] wherein G is a linking group covalently bonding the first redox mediator to the first polymer; and a mass transport limiting membrane permeable to the first analyte covering at least the first active area.

[0088] B. A method for detecting an analyte using an analyte sensor capable of low potential detection, comprising a sensor tail including at least a first working electrode and a first active area disposed on a surface of the first working electrode and responsive to the first analyte at low potential, the first active area including a first polymer, a first redox mediator covalently bound to the first polymer, and at least one enzyme responsive to the first analyte covalently bound to the first polymer, the first redox mediator comprising: [ka] wherein G is a linking group covalently bonding the first redox mediator to the first polymer; and a mass transport limiting membrane permeable to the first analyte covering at least the first active area; applying a low potential to the first working electrode; obtaining a first signal proportional to the concentration of the first analyte in a fluid in contact with the first active area and equal to or greater than the redox potential of the first active area; and correlating the first signal with the concentration of the first analyte in the fluid.

[0089] B1. A method for detecting an analyte using an analyte sensor capable of low potential detection, comprising: a sensor tail including at least a first working electrode; and a first active area disposed on a surface of the first working electrode and responsive to the first analyte at low potential, the first active area including a first polymer, a first redox mediator covalently bound to the first polymer, and at least one enzyme responsive to the first analyte covalently bound to the first polymer, the first redox mediator comprising: [ka] wherein G is a linking group covalently bonding the first redox mediator to the first polymer; and a mass transport limiting membrane covering at least the first active area, the mass transport limiting membrane being permeable to the first analyte, to a fluid containing the first analyte; applying a low potential to the first working electrode; obtaining a first signal proportional to the concentration of the first analyte in the fluid and equal to or greater than the redox potential of the first active area; and correlating the first signal with the concentration of the first analyte in the fluid.

[0090] C. Analyte Sensor Comprising a Mass Transport Limiting Membrane Crosslinked with a Branched Glycidyl Ether Crosslinker: An analyte sensor comprising: a sensor tail including at least a first working electrode; a first active area disposed on a surface of the first working electrode, the first active area comprising a first polymer and at least one enzyme covalently bonded to the first polymer and responsive to a first analyte; and a mass transport limiting membrane permeable to the first analyte covering at least the first active area, the mass transport limiting membrane comprising a membrane polymer crosslinked with a branched crosslinker comprising three or more crosslinking groups.

[0091] D. A method for detecting an analyte using an analyte sensor comprising a mass transport limiting membrane crosslinked by a branched glycidyl ether crosslinker, the method comprising: providing an analyte sensor comprising: a sensor tail including at least a first working electrode; a first active area disposed on a surface of the first working electrode, the first active area comprising a first polymer and at least one enzyme covalently bound to the first polymer and responsive to a first analyte; and a mass transport limiting membrane covering at least the first active area and permeable to the first analyte, the mass transport limiting membrane comprising a membrane polymer crosslinked by a branched crosslinker comprising three or more crosslinking groups; applying a potential to the first working electrode; obtaining a first signal proportional to the concentration of the first analyte in a fluid in contact with the first active area and equal to or greater than the redox potential of the first active area; and correlating the first signal with the concentration of the first analyte in the fluid.

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

[0093] Element 1: The at least one enzyme comprises 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 comprises one or more ketones.

[0095] Element 3: The mass transport limiting membrane comprises a membrane polymer crosslinked by a branched crosslinker containing three or more crosslinking groups.

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

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

[0098] Element 6: The branched crosslinker comprises polyethylene glycol tetraglycidyl ether.

[0099] Element 7: The analyte sensor further includes a second working electrode; a second active area disposed on a surface of the second working electrode and responsive to a second analyte different from the first analyte, the second active area 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 transport limiting membrane covering the second active area.

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

[0101] Element 9: The second test substance 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 versus an Ag / AgCl reference electrode.

[0103] Element 10A: The redox potential of the first redox mediator ranges from about −200 mV to about −400 mV versus an Ag / AgCl reference electrode.

[0104] Element 11: The polyethylene glycol tetraglycidyl ether has a molecular weight ranging from about 1000 g / mol to about 5000 g / mol.

[0105] As a non-limiting example, typical combinations applicable to A and B include, but are not limited to: 1 and 2; 1 through 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. As a further 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 through 8; 4, 6 and 11; 5, 6 and 11; 6, 7 and 11; and 6 through 8 and 11.

[0106] Further embodiments disclosed herein include the following.

[0107] A': A method for forming a mass transport limiting membrane by dip coating, the method comprising: providing an analyte sensor including a sensor tail including first and second working electrodes spaced apart from one another at least along the length of the sensor tail, and a first active area disposed on a surface of the first working electrode and a second active area disposed on a surface of the second working electrode, the first active area and the second active area responsive to different analytes; and depositing a mass transport limiting membrane on the first and second active areas by a series of dip coating operations, the mass transport limiting membrane including a bilayer membrane portion covering the first active area and a uniform membrane portion covering the second active area.

[0108] B': Analyte sensor with dip-coated mass transport limiting membrane. An analyte sensor comprising: a sensor tail including first and second working electrodes spaced apart from one another at least along the length of the sensor tail; a first active area disposed on a surface of the first working electrode; and a second active area disposed on a surface of the second working electrode, the first and second active areas responsive to different analytes; and a mass transport limiting membrane dip-coated on the first and second active areas, the dip-coated mass transport limiting membrane including a dip-coated bilayer membrane portion covering the first active area and a dip-coated uniform membrane portion covering the second active area.

[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 area are located closer to the tip of the analyte sensor than the second working electrode and the second active area.

[0112] Element 3': The bilayer membrane portion and the upper layer of the homogeneous membrane portion contain the same membrane polymer.

[0113] Element 4': A first dip-coating operation deposits a first film polymer on the first active area, and a second dip-coating operation deposits a second film polymer on both the first active area and the second active area, defining the bilayer film portion on the first active area and the uniform film portion on the second active area, and the first film polymer and the second film polymer are different from each other.

[0114] Element 5': The bilayer membrane portion and the lower layer of the homogeneous membrane portion contain the same membrane polymer.

[0115] Element 6': A first dip-coating operation deposits a first film polymer on both the first active area and the second active area, and a second dip-coating operation deposits a second film polymer on the first active area, defining the bilayer film portion on the first active area, and the first film polymer and the second film polymer are different from each other.

[0116] Element 7': The first active area responds to glucose, lactate, ketone, or creatinine.

[0117] Element 8': The first active area responds to ketones.

[0118] Element 9': The second active area responds to glucose.

[0119] Element 10': At least a portion of the mass transport limiting membrane comprises a crosslinked polyvinylpyridine homopolymer or copolymer.

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

[0121] Element 12': The branched crosslinker comprises polyethylene glycol tetraglycidyl ether.

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

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

[0124] Element 15': The dip-coated bilayer membrane portion and the top layer of the dip-coated uniform membrane portion comprise the same membrane polymer.

[0125] Element 16': The dip-coated bilayer membrane portion and the bottom layer of the dip-coated uniform membrane portion comprise the same membrane polymer.

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

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

[0128] By way of non-limiting example, typical combinations that are 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'. Exemplary 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] In order to facilitate understanding of the disclosure of this specification, examples of various representative embodiments are given below, which should not be read to limit or define the scope of the invention.

[0130] Example Example 1: Detection of ketones at low potentials using an analyte sensor with diaphorase and β-hydroxybutyrate dehydrogenase working together. For this example, the enzyme system of Figure 6A is used to facilitate the detection of ketones using either a free transition metal complex of Formula 8 (Table 1) or a transition metal complex of Formula 8 bound to polyvinylpyridine-co-styrene (Table 2) as the redox mediator, with the active zone formulations described in Tables 1 and 2 below (HBHD = β-hydroxybutyrate dehydrogenase; HSA = human serum albumin; PEGDGE400 = polyethylene glycol diglycidyl ether). The sensing active zone formulations shown in Tables 1 and 2 are approximately 0.2 mm 2 The active area was cured at 25°C for 24 hours. After curing, a polyvinylpyridine film having the formulation described in Table 3 was applied onto the active area by dip coating a total of four times. After film application, 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] FIG. 7 shows cyclic voltammograms for the transition metal complex of Formula 8 or the polymer-bound transition metal complex of Formula 8. The cyclic voltammograms were obtained in 100 mM PBS solution at pH 7.4, deoxygenated by bubbling nitrogen, maintained at a temperature of 33° C. The probes were performed from −0.5 V to 0.1 V at a probe rate of 5 mV / s using a carbon counter electrode and an Ag / AgCl reference electrode. The cyclic voltammograms reveal 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 formula 8 transition metal complex 1 / 2 was measured to be −0.24 V vs. Ag / AgCl. A comparative redox mediator lacking N,N-dimethylamino substitution exhibited a much less negative E of −0.08 V vs. Ag / AgCl. 1 / 2 The values are shown (data not shown).

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

[0133] Example 2: Extractables from Membranes Crosslinked with Polyethylene Glycol Tetraglycidyl Ether. A polymeric membrane sample crosslinked with polyethylene glycol tetraglycidyl ether (molecular weight ∼2500) was prepared. The base membrane polymer was a copolymer of vinylpyridine and styrene containing amine-free polyether arms attached to at least a portion of the pyridine moieties in the base membrane polymer. A comparative polymeric membrane sample was prepared using the same membrane polymer crosslinked with polyethylene glycol diglycidyl ether (molecular weight ∼1000). The comparative polymeric membrane sample was crosslinked with polyethylene glycol diglycidyl ether in an amount similar to the polyethylene glycol tetraglycidyl ether crosslinker used in the other samples, thereby providing a similar crosslink density.

[0134] The membrane polymer was cast as a film onto the bottom of a sample vial using 0.5 mL of a solution containing the membrane polymer. The solvent was evaporated, and then the film was cured at 25°C for 48 hours and 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 neat by UV-Vis spectrophotometry (Figure 10), while the ethanol extract was diluted 1:9 before UV-Vis analysis (Figure 11). In both cases, the polyethylene glycol tetraglycidyl ether crosslinked sample showed a lower amount of extractable material, as indicated by the intensity of the UV-Vis absorbance.

[0135] Example 3: Equilibration Time for Sensors with Membranes Cross-Linked by Polyethylene Glycol Tetraglycidyl Ether. Glucose-responsive analyte sensors were coated with a membrane polymer cross-linked by polyethylene glycol tetraglycidyl ether (as described in Example 2). Comparative sensors were prepared using a comparative membrane polymer cross-linked by polyethylene glycol diglycidyl ether. Each membrane-coated sensor 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 cross-linked by polyethylene glycol tetraglycidyl ether compared to an analyte sensor coated with a membrane polymer cross-linked by polyethylene glycol diglycidyl ether. As shown, both membranes provided stable sensor outputs after approximately 1 hour of equilibration. Figure 13 shows a zoomed-in plot of sensor equilibration over 1 hour, showing that the membrane cross-linked by polyethylene glycol tetraglycidyl ether provided a faster and more stable response than the membrane cross-linked by polyethylene glycol diglycidyl ether.

[0136] Unless otherwise indicated, all numbers expressing quantities and the like in the specification and the associated claims are understood to be modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by embodiments of the invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0137] One or more exemplary embodiments incorporating various features are presented herein. For clarity, not all features of a physical implementation are described or shown herein. It is understood that in developing a physical embodiment incorporating embodiments of the present invention, numerous implementation-specific decisions must be made to achieve the developer's goals, such as compliance with system, business, governmental, and other constraints, and will vary from implementation to implementation and case to case. While the developer's efforts may be time-consuming, such efforts are nonetheless routine undertakings for those skilled in the art who will have the benefit 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 said to "consist essentially of" or "consist of" various components or steps.

[0139] As used herein, the phrase "at least one of," along with the word "and" or "or" preceding a series of items and separating some of the items, modifies the list as a whole rather than each member (i.e., each item) of the list. The phrase "at least one of" allows for the inclusion of 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" all refer to A only, B only, or C only; any combination of A, B, and C; and / or at least one of each of A, B, and C.

[0140] Thus, the disclosed systems, tools, and methods are fully applicable to achieve the objectives and advantages stated as well as those inherent therein. The specific embodiments disclosed above are illustrative only, as the teachings of the present disclosure may be modified and implemented in different but equivalent manners, as will be apparent to those skilled in the art having the benefit of the teachings herein. Moreover, no limitations are intended to the details of construction or design shown herein, except as set forth in the claims below. It will therefore be apparent that certain specific embodiments disclosed above may be altered, combined, or modified, and all such variations are considered within the scope of the present disclosure. The systems, tools, and methods illustratively disclosed herein may suitably be practiced in the absence of any element not specifically disclosed herein and / or any element disclosed herein. Although systems, tools, and methods are described in terms of "comprising," "containing," or "including" various components or steps, the systems, tools, and methods can also "consist essentially of" or "consist of" various components or steps. All of the numbers and ranges disclosed above are subject to some variation. Whenever a numerical range with a lower and upper limit is disclosed, any number within that range and any included range is specifically disclosed. 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, "approximately a to b") are understood to describe all numbers and ranges within the broader range of values. Also, in the claims, terms have their plain and ordinary meaning unless expressly and unambiguously defined otherwise by the patent owner. Furthermore, the indefinite article "a" or "an," as used in the claims, is hereby defined to mean one or more than one of the element it introduces. In the event of any discrepancy in the usage of a word or term in this specification and one or more patents or other documents incorporated herein by reference, the definition consistent with this specification should prevail.

Claims

1. An analyte sensor, i) an 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 higher than the 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 ranging from −200 mV to −400 mV versus the Ag / AgCl reference electrode, the first redox mediator having the following structure: 【Chemical 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 the 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. 2. The analyte sensor according to claim 1, wherein the enzyme system comprises β-hydroxybutyrate dehydrogenase (HBDH) and diaphorase.

3. The analyte sensor according to claim 1 , wherein the second analyte is glucose.

4. 4. The analyte sensor of claim 3, wherein the at least one enzyme responsive to glucose is glucose oxidase.

5. 5. The analyte sensor according to claim 1, wherein the mass transport limiting membrane is a two-layer membrane consisting of a lower layer and an upper layer.

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

7. 7. The analyte sensor of claim 5, wherein the upper layer of the bilayer membrane covers the second active area.

8. The analyte sensor of claim 5 , wherein the underlayer comprises a first membrane polymer.

9. 9. The analyte sensor of claim 8, wherein the first membrane polymer comprises polyvinylpyridine.

10. 10. The analyte sensor according to claim 8, wherein the lower layer includes a first cross-linking agent.

11. 11. The analyte sensor of claim 10, wherein the first crosslinker comprises two or more crosslinkable groups.

12. 12. The analyte sensor according to claim 10, wherein the first cross-linking agent is polyethylene glycol diglycidyl ether.

13. 12. The analyte sensor according to claim 10, wherein the first cross-linking agent is polyethylene glycol tetraglycidyl ether.

14. 14. The analyte sensor of claim 5, wherein the top layer comprises a second membrane polymer.

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

16. The analyte sensor of claim 14 or 15, wherein the upper layer comprises a second cross-linking agent.

17. 17. The analyte sensor of claim 16, wherein the second crosslinker comprises two or more crosslinkable groups.

18. 18. The analyte sensor according to claim 16, wherein the second cross-linking agent is polyethylene glycol diglycidyl ether.

19. 18. The analyte sensor according to claim 16, wherein the second cross-linking agent is polyethylene glycol tetraglycidyl ether.

20. 20. A method of controlling an analyte sensor according to any one of claims 1 to 19 that has already been introduced into tissue, comprising: applying the lower potential to the first working electrode; obtaining a first signal proportional to a ketone concentration in a fluid contacting the first active area at or above the redox potential of the first active area; correlating the first signal to a ketone concentration in the fluid.

1. A method of controlling an analyte sensor, comprising:

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