Analyte sensor and sensing method characterized by low potential detection

Abstract

An analyte sensor that responds at a low working electrode potential can include an active area on the working electrode surface, wherein the active area includes 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 that responds at a low potential can have the structure of (I), wherein G is a linker group that covalently bonds the redox mediator to the polymer. An analyte-permeable mass transport limiting membrane can coat the active area. In certain sensor configurations, the mass transport limiting membrane can include a membrane polymer cross-linked with a branched cross-linking agent, wherein the branched cross-linking agent includes 3 or more cross-linkable groups, such as polyethylene glycol tetraglycidyl ether.

Description

Background Art

[0001] Detecting various analytes in an individual can sometimes be crucial to monitoring their health status. Deviations from relatively normal analyte levels can often indicate many physiological conditions. For example, glucose levels can be particularly important for the detection and monitoring of diabetic individuals. By monitoring glucose levels regularly enough, diabetic individuals can take corrective measures (such as injecting insulin to reduce glucose levels or eating to improve glucose levels) before significant physiological hazards occur. In order to monitor other physiological conditions, other analytes may be desirable. In some cases, monitoring multiple analytes may also be desirable, especially for the comorbid conditions that cause two or more analytes combined with each other to be simultaneously unregulated.

[0002] Many analytes represent interesting targets for physiological analysis, provided that suitable detection chemistry can be identified. For this reason, current sensors configured for continuous testing of glucose in vivo have been developed and improved in recent years to help monitor the health of diabetic individuals. Other analytes that are generally out of balance with glucose in diabetic individuals include, for example, lactic acid, oxygen, pH, A1c, ketones, etc. It is also possible to monitor these and other analytes independently of glucose imbalance. Analyte sensors configured for in vivo detection of analytes other than glucose are known but not currently significantly improved. Poor sensitivity for low abundance analytes can be particularly problematic.

[0003] Analyte monitoring in individuals can occur periodically or continuously over a period of time. Periodic analyte monitoring can be performed as follows: extract sample body fluids such as blood or urine at set time intervals and analyze in vitro. Periodic in vitro analyte monitoring can be sufficient to determine the physiological conditions of many individuals. However, in some cases in vitro analyte monitoring can be inconvenient or painful. In addition, if the analyte measurement is not obtained at the appropriate time, there is no way to recover the lost data. Continuous analyte monitoring can be performed with one or more sensors, which remain at least partially implanted (such as skin, subcutaneous or intravenous) in the tissue of the individual so that it can be analyzed in vivo. Implanted sensors can collect analyte data on demand, with a set plan or continuously, depending on the individual's specific health needs and / or pre-measured analyte levels. For individuals with severe analyte imbalance and / or rapidly fluctuating analyte levels, analyte monitoring with sensors implanted in vivo can be a more promising approach, but it can also be beneficial to other individuals. Since implanted analyte sensors often remain within an individual's tissue for an extended period of time, it can be highly desirable that the analyte sensors be prepared from stable materials that exhibit a high degree of biocompatibility.

[0004] In order to improve biocompatibility, the analyte sensor may include a membrane located on the implanted portion of the sensor, particularly a membrane coating at least the active region of the sensor. In addition to promoting biocompatibility, the membrane may also be permeable or semi-permeable to the analyte concerned and limit the total flux of the analyte to the active region of the sensor. The mass transport limiting membrane may help avoid overloading (saturation) of the sensing components within the active region, thereby improving sensor performance and accuracy. For example, in the case where the sensor uses enzyme-based detection, limiting the mass transport of the analyte to the active region can make the chemical kinetics of the sensing process analyte-limited rather than enzyme-limited, thereby allowing the sensor output to be easily related to the amount of analyte present.

[0005] A problem related to adding a film on an analyte sensor is that the analyte flux across the membrane can vary significantly with temperature and / or the length of time that the analyte sensor has been implanted in tissue. Compared with others, some membrane materials are more difficult to cause temperature-dependent analyte flux. If necessary, the variability of the analyte flux with temperature can be explained with a calibration factor or formula, but doing so can add significant complexity to the use of the sensor. The composition change of the membrane (particularly due to the leaching loss of each membrane component and / or due to membrane degradation or metabolism) can occur over time, which can cause the difference in analyte permeability values, and the value depends on the degree of composition change that has occurred. The composition change that causes the difference in membrane permeability values ​​can be difficult to deal with quantitatively, and sometimes it can be difficult to determine when membrane permeability is sufficiently stable so as to allow measurement of accurate analyte concentration. Generally, several hours or longer equilibrium time periods can be used to stabilize the analyte flux through the membrane in the case of implanting a new analyte sensor in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0007] Figure 1 A diagram showing an exemplary sensing system into which an analyte sensor of the present disclosure may be inserted.

[0008] Figure 2A-2C A schematic cross-section of an analyte sensor is shown, including a single active area.

[0009] Figure 3A-Figure 4 A schematic cross-section of an analyte sensor is shown, including two active areas.

[0010] Figure 5A-5C A perspective view of an analyte sensor is shown, including two active areas on separate working electrodes.

[0011] Figure 6A-6C A diagram showing an enzyme system that can be used to detect ketones in an analyte sensor.

[0012] Figure 7 Cyclic voltammograms showing the low potential redox mediator in free and polymer bound form.

[0013] Figure 8 Current vs. time plots at various working electrode potentials are shown for a ketone sensor incorporating a low potential redox mediator.

[0014] Fig. 9 A plot showing the current versus ketone concentration at various working electrode potentials for a ketone sensor incorporating a low potential redox mediator.

[0015] Fig.10 and 11 Shown are UV-VIS absorbance data for extracts from membrane polymers cross-linked with polyethylene glycol tetraglycidyl ether, compared to membrane polymers cross-linked with polyethylene glycol diglycidyl ether.

[0016] Fig.12 and 13 A graph showing the 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 ether. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present disclosure generally describes analyte sensors suitable for use in vivo, and more particularly describes analyte sensors comprising components, the components comprising low potential operating capabilities and / or stabilized membrane material features. Depending on the sensor configuration, the analyte sensors of the present disclosure can be configured to detect one analyte or multiple analytes simultaneously or nearly simultaneously. Sequential immersion coating operations can be performed to introduce different membrane compositions at specified locations on the analyte sensor.

[0019] Various analyte sensor components can cause certain difficulties during monitoring of certain analytes or analyte combinations. Redox mediators used to promote electron transfer to the working electrode can make it necessary to operate the analyte sensor at relatively high potentials, which can lead to electrochemical side reactions that can complicate the detection of certain low-abundance analytes. In the case of analyte sensor implantation in vivo, especially during long-term sensor wear, compositional changes in the mass transport limiting membrane in the analyte sensor can lead to undesirable analyte permeability differences. In addition, in the case of analyzing multiple analytes with a single analyte sensor, different analyte permeability characteristics may require the use of different mass transport limiting membranes at each location.

[0020] In order to cope with the aforementioned needs, the present disclosure provides redox mediators for promoting electron transfer at lower working electrode potentials than are usually used. Using this "low potential" redox mediator can reduce the presence of electrochemical side reactions by allowing analyte detection to occur at a potential that is lower than otherwise possible. By reducing the presence of electrochemical side reactions and the signal noise associated therewith, the detection of low abundance analytes such as ketones can occur more easily than at a higher working electrode potential. The low potential redox mediator can also be advantageous in the case of being used in conjunction with detecting multiple analytes, as discussed further below. The redox mediators that can promote analyte detection at low working electrode potentials are further described below.

[0021] Various cross-linked polyvinyl imidazole and polyvinyl pyridine membrane polymers can be used in analyte sensors to promote biocompatibility and provide mass transport limiting properties. Functionalization on the membrane material can be selected to modify the analyte permeability and limit the temperature difference of the analyte permeability. The membrane polymer can be cross-linked with a linear glycidyl ether such as polyethylene glycol diglycidyl ether (PEGDGE) with two cross-linkable groups, but the equilibrium time period may still need to exist after the sensor is implanted to allow the analyte flux to stabilize. Not limited by theory or mechanism, it is believed that the membrane composition can change during the equilibrium time period because a small amount of extractable substances is released from the membrane, which may change the analyte permeability. Surprisingly, the present disclosure shows that branched cross-linking agents such as branched polyethylene glycol glycidyl ethers, more particularly polyethylene glycol tetraglycidyl ethers, containing 3 or more cross-linkable groups can reduce the amount of extractable substances released from the membrane after the sensor is implanted. Even in the case where the crosslink density and amount (mass) of the crosslinking agent are substantially the same as those provided by a linear glycidyl ether having 2 crosslinkable groups, a reduction in the production of extractable substances from the membrane can be achieved. That is, a given mass of crosslinkable groups from a branched crosslinker such as polyethylene glycol tetraglycidyl ether can cause a reduction in extractables for a given membrane material, which is compared to the amount of extractables caused by a substantially similar mass of crosslinkable groups from a linear glycidyl ether crosslinker such as polyethylene glycol diglycidyl ether. Advantageously, membrane materials crosslinked with the branched crosslinkers disclosed herein can result in reduced extractables, thereby providing an improved toxicological profile. In addition, the reduced compositional changes provided by the membrane materials disclosed herein can provide a shorter sensor equilibrium time after sensor implantation, which can potentially extend the wear life of the sensor.

[0022] Before describing the analyte sensors of the present disclosure and their components in further detail, a brief overview of suitable in vivo analyte sensor configurations and sensor systems using analyte sensors will first be provided so that embodiments of the present disclosure may be better understood. Figure 1Show can be inserted into the diagram of the exemplary sensing system of the analyte sensor of the present disclosure.As shown, the sensing system 100 includes a sensor control device 102 and a reader device 120 configured to communicate with each other through a local communication path or link, which can be wired or wireless, unidirectional or bidirectional and encrypted or unencrypted. The reader device 120 can constitute an output medium for observing the analyte concentration and warning or notification determined by the sensor 104 or a processor associated therewith, and allowing one or more user inputs according to certain embodiments. The reader device 120 can be a multi-purpose smart phone or a dedicated electronic reader device. Although only one reader device 120 is shown, multiple reader devices 120 can exist in some cases. The reader device 120 can also communicate with a remote terminal 170 and / or a trusted computer system 180 via communication paths / links 141 and / or 142, respectively, which can also be wired or wireless, unidirectional or bidirectional and encrypted or unencrypted. Reader device 120 may also or alternatively communicate with network 150 (e.g., a mobile phone network, the Internet, or a cloud server) via communication path / link 151. Network 150 may be further 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 an intermediary reader device 120. For example, according to certain embodiments, sensor 104 may communicate with remote terminal 170 and / or trusted computer system 180 via a direct communication link with network 150, as described in US Patent Application Publication 2011 / 0213225 and incorporated herein by reference in its entirety. Any suitable electronic communication scheme may be used for each communication path or link, such as near field communication (NFC), radio frequency identification (RFID), or Low energy solutions, WiFi, etc. According to some embodiments, the remote terminal 170 and / or the trusted computer system 180 can be accessed by an individual of interest to the user's analyte level other than the primary user. The reader device 120 can include a display 122 and an optional input component 121. According to some embodiments, the display 122 can include a touch screen interface.

[0023] The sensor control device 102 includes a sensor housing 103, which can house circuitry and a power source for operating the sensor 104. Optionally, the power source and / or active circuitry can be omitted. A processor (not shown) can be communicatively coupled to the sensor 104, wherein the processor is physically located in the sensor housing 103 or the reader device 120. According to some embodiments, the sensor 104 protrudes from the underside of the sensor housing 103 and extends through an adhesive layer 105 suitable for attaching 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 relevant tissue, such as the dermis or subcutaneous layer of the skin. The sensor 104 may include a sensor tail of sufficient length for insertion into a given tissue to a desired depth. The sensor tail may include at least one working electrode. In certain configurations, the sensor tail may include a ketone-responsive active region, which in certain cases may include a low potential redox mediator, as further discussed herein. A counter electrode may exist in combination with at least one working electrode. The particular electrode configuration on the sensor tail is described in more detail below.

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

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

[0027] Also refer to Figure 1, the sensor 104 can automatically forward the data to the reader device 120. For example, the analyte concentration data (i.e., glucose and / or ketone concentration) can be communicated automatically and periodically, such as at a certain frequency to obtain the data or after a certain time interval, wherein the data is stored in the memory until it is sent (e.g., every minute, five minutes, or other predetermined time intervals). In other embodiments, the sensor 104 can communicate with the reader device 120 in a non-automatic manner and not according to a set schedule. For example, in the case where the sensor electronic device enters the communication range of the reader device 120, the data can be communicated from the sensor 104 using RFID technology. Until the communication to the reader device 120, the data can remain stored in the memory of the sensor 104. Thus, the user does not have to remain in proximity to the reader device 120 at all times, but can upload data at a convenient time. In other embodiments, a combination of automatic and non-automatic data transmission can be implemented. For example, the data transmission can continue automatically until the reader device 120 is no longer located in the communication range of the sensor 104.

[0028] The introducer may be present briefly to facilitate the introduction of the sensor 104 into the tissue. In an exemplary embodiment, the introducer may include a needle or similar sharp object. It should be recognized that other types of introducers such as sheaths or blades may exist in alternative embodiments. More particularly, a needle or other introducer may be briefly located near the sensor 104 before the tissue is inserted, and then withdrawn thereafter. When 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 epidermal penetration because the access path to the dermis allows the sensor 104 to be implanted. After opening the access path, the needle or other introducer may be withdrawn so that it does not bring a sharp object hazard. In an exemplary embodiment, a suitable needle may be solid or hollow, beveled or non-beveled, and / or circular or non-circular in cross section. In a more specific embodiment, a suitable needle may be comparable to an acupuncture needle in terms of cross-sectional diameter and / or tip design, which may have a cross-sectional diameter of about 250 microns. It will be appreciated, however, that a suitable needle may have a larger or smaller cross-sectional diameter if desired for a particular application.

[0029] In certain embodiments, the tip of the needle (when 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 be positioned within a lumen or groove of the needle, where the needle similarly opens an access path for the sensor 104. In either case, the needle is subsequently retracted after facilitating sensor insertion.

[0030] A sensor configuration featuring a single active area configured to detect a corresponding single analyte may use a two-electrode or three-electrode detection format, as described herein with reference to Figures 2A-2C Further described. A sensor configuration characterized by two different active areas for detecting separate analytes on separate working electrodes or on the same working electrode is hereinafter referred to as Figures 3A-5C The sensor configuration with multiple working electrodes can be particularly advantageous for incorporating two different active regions in the same sensor tail, since the signal contribution from each active region can be more easily determined. Furthermore, in the case where the active region is present on the second working electrode, deposition of different film compositions on each active region can be easily performed by sequential dip coating operations.

[0031] In the case where 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, wherein the second electrode may serve as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). Various electrodes may be at least partially stacked (laminated) on the sensor tail and / or spaced apart from each other laterally. Suitable sensor configurations may be substantially flat or substantially cylindrical. In any sensor configuration disclosed herein, various electrodes may be electrically isolated from each other by dielectric substances or similar insulators.

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

[0033] Figure 2A A diagram showing an exemplary two-electrode analyte sensor configuration compatible with use in the present disclosure. As shown, the analyte sensor 200 includes a substrate 212 located between a working electrode 214 and a counter / reference electrode 216. Alternatively, the working electrode 214 and the counter / reference electrode 216 can be located on the same side of the substrate 212 with a dielectric substance therebetween (configuration not shown). The active area 218 is arranged as at least one layer on at least a portion of the working electrode 214. The active area 218 may include multiple points or a single point configured to detect an analyte at a low working electrode potential, as further discussed herein.

[0034] Also refer to Figure 2AAccording to certain embodiments, the membrane 220 coats at least the active area 218 and may optionally coat some or all of the working electrode 214 and / or the counter / reference electrode 216, or the entire analyte sensor 200. One or both sides of the analyte sensor 200 may be coated with the membrane 220. The membrane 220 may include one or more polymer membrane materials having the ability to limit the analyte flux reaching the active area 218 (i.e., the membrane 220 is a mass transport limiting membrane having some permeability to the analyte concerned). According to the disclosure herein, the membrane 220 may be cross-linked with a branched cross-linking agent in a specific sensor configuration. The composition and thickness of the membrane 220 may vary to facilitate the desired analyte flux reaching the active area 218, thereby providing the desired signal strength and stability. The analyte sensor 200 may be used to test the analyte by any of the coulometric, amperometric, voltammetric or potentiometric electrochemical detection techniques.

[0035] Figure 2B and 2C A diagram showing an exemplary three-electrode analyte sensor configuration that is also compatible with use in the present disclosure. The three-electrode analyte sensor configuration may be similar to Figure 2A 200, with the exception of the inclusion of additional electrodes 217 ( Figure 2B and 2C ). With the additional electrode 217, the counter / reference electrode 216 can act as a counter electrode or a reference electrode, and the additional electrode 217 performs other electrode functions not explained otherwise. The working electrode 214 continues to perform its original function. The additional electrode 217 can be located on the working electrode 214 or the electrode 216 with a dielectric material separating layer therebetween. For example, Figure 2B As described above, dielectric layers 219a, 219b, and 219c separate electrodes 214, 216, and 217 from each other and provide electrical isolation. Alternatively, at least one of electrodes 214, 216, and 217 may be located on the opposite side of substrate 212, such as Figure 2C Thus, in some embodiments, electrode 214 (working electrode) and electrode 216 (counter electrode) may be located on opposite sides of substrate 212, wherein electrode 217 (reference electrode) is located on one of electrodes 214 or 216 and is separated from the electrode by a dielectric substance. Reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, wherein the location of reference material layer 230 is not limited to Figure 2B and 2C As shown in Figure 2AFor use with the sensor 200, the active area 218 of the analyte sensors 201 and 202 may include multiple points or a single point. Additionally, the analyte sensors 201 and 202 may be used to test for analytes by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

[0036] Similar to analyte sensor 200, membrane 220 may also encapsulate active region 218 and other sensor components in analyte sensors 201 and 202, thereby acting as a mass transport limiting membrane. In certain embodiments, additional electrode 217 may be encapsulated by membrane 220. Although Figure 3B and 2C While electrodes 214, 216, and 217 have all been described as being coated with film 220, it should be appreciated that in certain embodiments only working electrode 214 may be coated. Furthermore, the thickness of film 220 at each electrode 214, 216, and 217 may be the same or different and / or the film composition may vary locally. As in a two-electrode analyte sensor configuration ( Figure 2A ),exist Figure 2B and 2C In the sensor structure, one or both sides of the analyte sensors 201 and 202 may be covered with the membrane 220, or the entire analyte sensors 201 and 202 may be covered. Figure 2B and 2C The three-electrode sensor configuration should be understood as not limiting the embodiments disclosed herein, wherein alternative electrode and / or layer configurations also fall within the scope of the present disclosure.

[0037] Figure 3A An exemplary configuration of sensor 203 is shown, having a single working electrode with two distinct active areas disposed thereon. Figure 3A similar Figure 2A , with the exception 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 laterally spaced from each other on the surface of the working electrode 214. The active areas 218a and 218b can include multiple points or a single point, which are configured to detect respective analytes. The composition of the membrane 220 can vary or be identical in composition in the active areas 218a and 218b. The first active area 218a and the second active area 218b can be configured to detect their respective analytes at different working electrode potentials from each other, as further discussed below.

[0038] Figure 3B and 3C Cross-sectional illustrations of exemplary three-electrode sensor configurations are shown for sensors 204 and 205, respectively, each featuring a single working electrode having a first active area 218a and a second active area 218b disposed thereon. Figure 3B and 3CSimilar in other respects Figure 2B and 2C and may be better understood by reference to them. Figure 3A , the composition of the film 220 in the active regions 218a and 218b may vary or be compositionally identical.

[0039] An exemplary sensor configuration with multiple working electrodes, in particular two working electrodes, is shown in FIG. Figure 4-5C Further detailed description. Although the following description is mainly related to the sensor configuration with two working electrodes, it should be recognized that more than two working electrodes can be incorporated by expanding the disclosure of this article. Additional working electrodes can be used to impart additional sensing capabilities to the analyte sensor, beyond only the first analyte and the second analyte.

[0040] Figure 4 A cross-sectional illustration of an exemplary analyte sensor configuration having two working electrodes (a reference electrode and a counter electrode) is shown, which is compatible with use in the disclosure herein. As shown, the analyte sensor 300 includes working electrodes 304 and 306 located on opposite sides of a substrate 302. A first active area 310a is located on the surface of the working electrode 304, while a second active area 310b is located on the surface of the working electrode 306. The counter electrode 320 is electrically isolated from the working electrode 304 by a dielectric layer 322, while the reference electrode 321 is electrically isolated from the working electrode 306 by a dielectric layer 323. Outer dielectric layers 330 and 332 are located on the reference electrode 321 and the counter electrode 320, respectively. According to various embodiments, a membrane 340 can be coated with at least the active areas 310a and 310b, wherein other components of the analyte sensor 300 or the analyte sensor 300 as a whole are also optionally coated with the membrane 340. Likewise, if desired, membrane 340 may vary in composition at active regions 310a and 310b to provide suitable permeability values ​​for differentially modulating analyte flux at each location. For example, membrane 340 may be homogeneous at coating active region 310a and heterogeneous at coating active region 310b.

[0041] Having multiple working electrodes and different from Figure 4 Alternative sensor configurations to the configuration shown may feature counter / reference electrodes rather than separate counter and reference electrodes 320, 321, and / or layer and / or membrane arrangements other than those explicitly described. For example, the positioning of the counter electrode 320 and the reference electrode 321 may be relative to each other. Figure 4 In addition, working electrodes 304 and 306 do not necessarily need to be as shown in Figure 4304 and 306 are located on the opposite side of the substrate 302. On the contrary, the working electrodes 304 and 306 can be located on the same side of the substrate 302 and spaced from each other with a gap therebetween. In particular, the working electrode 304 can be positioned more toward the distal end (tip) of the analyte sensor compared to the working electrode 306 that can be positioned more toward the proximal end. In the case where the interval between the working electrodes 304 and 306 is large enough, the film 340 can be deposited on the working electrodes 304 and 306 by successive dip coating operations, wherein the composition of the film 340 can be locally varied. In particular, the first dip coating operation can deposit the first film polymer on both the working electrodes 304 and 306, while the second dip coating operation can deposit the second film polymer with different compositions on only the working electrode 304, thereby defining a double layer on the working electrode 304 and leaving a homogeneous film on the working electrode 306. Thus, the lower layer of the double-layer film and the homogeneous film can contain the same film polymer. Alternatively, a first dip coating operation may deposit a first membrane polymer on working electrode 304 and a second dip coating operation may deposit a second membrane polymer having a different composition on both working electrodes 304 and 306, thereby defining a bilayer membrane and a homogeneous membrane. In this case, the upper layer of the bilayer membrane and the homogeneous membrane may comprise the same membrane polymer.

[0042] The size of the gap between working electrodes 304 and 306 is at least sufficient to provide electrical isolation between the two electrodes, and more generally sufficient to facilitate sequential dip coating operations (e.g., by allowing the analyte sensor to be dipped to different depths so as to preferentially coat one of the working electrodes in at least one dipping step). In other words, the size of the gap can provide a margin of error for lowering the analyte sensor to a specific dip coating formulation to a specified depth, thereby facilitating film deposition on one of the working electrodes in preference to the other.

[0043] Although suitable sensor configurations may feature electrodes of substantially planar nature, including planar sensor configurations with spaced working electrodes, it will be appreciated that sensor configurations featuring non-planar electrodes may be advantageous and particularly suitable for use in the disclosure herein. In particular, cylindrical electrodes that are concentrically arranged relative to one another and spaced along the length of the sensor tail may facilitate deposition of mass transport limiting films having compositional variations, as described below. In particular, concentric working electrodes spaced along the length of the sensor tail may facilitate film deposition by sequential dip coating operations in a manner similar to that described above for substantially planar sensor configurations. Figures 5A-5C A perspective view of an analyte sensor is shown featuring two working electrodes arranged concentrically relative to one another. It should be appreciated that sensor configurations having a concentric electrode arrangement but lacking a second working electrode are also possible in the present disclosure.

[0044] Figure 5A A perspective view showing an exemplary sensor configuration in which a plurality of electrodes are substantially cylindrical and are arranged concentrically relative to one another around a central substrate. As shown, an analyte sensor 400 includes a central substrate 402 around which all electrodes and dielectric layers are arranged concentrically relative to one another. In particular, a working electrode 410 is located on a surface of the central substrate 402, and a dielectric layer 412 is located on a portion of the working electrode 410 at the distal end of the sensor tip 404. A working electrode 420 is located on the dielectric layer 412, and a dielectric layer 422 is located on a portion of the working electrode 420 at the distal end of the sensor tip 404. A counter electrode 430 is located on the dielectric layer 422, and a dielectric layer 432 is located on a portion of the counter electrode 430 at the distal end of the sensor tip 404. A reference electrode 440 is located on the dielectric layer 432, and a dielectric layer 442 is located on a portion of the reference electrode 440 at the distal end of the sensor tip 404. As such, the exposed surfaces of working electrode 410, working electrode 420, counter electrode 430, and reference electrode 440 are spaced apart from one another along longitudinal axis B of analyte sensor 400. This electrode arrangement can allow deposition of film 450 by sequential dip coating operations to place a bilayer membrane portion on working electrode 410 and a homogeneous membrane portion on working electrode 420, as discussed below. The bilayer membrane portion and the homogeneous membrane portion can be adjacent to one another.

[0045] Also refer to Figure 5A , first active regions 414a and second active regions 414b that respond to different analytes are located on the exposed surfaces of working electrodes 410 and 420, respectively, thereby allowing fluid contact for sensing. Figure 5A While described as three discrete points in FIG. 1 , it should be appreciated that in alternative sensor configurations there may be fewer or more than three points, including a continuous layer of active areas.

[0046] exist Figure 5A In FIG. 4 , sensor 400 is partially covered with a membrane 450 over working electrodes 410 and 420 and active areas 414a and 414b located thereon. Figure 5B An alternative sensor configuration is shown in which the substantial entirety of sensor 401 is coated with film 450. Film 450 may be identical or vary in composition at active regions 414a and 414b. For example, film 450 may include a bilayer film portion coating active region 414a and a homogeneous film portion coating active region 414b. Film 450 may be deposited by sequential dip coating operations such that the bilayer film portion is deposited on working electrode 410 and active region 414a and the homogeneous film portion is deposited on working electrode 420 and active region 414b.

[0047] It should also be recognized that various electrodes Figure 5A and 5BThe positioning in the embodiment may be different from the explicit description. For example, the positions of the counter electrode 430 and the reference electrode 440 may be relative to each other. Figure 5A and 5B Similarly, the positions of the working electrodes 410 and 420 are not limited to Figure 5A and 5B Those explicitly described in . Figure 5C Show relative Figure 5B An alternative sensor configuration is shown, wherein sensor 405 includes a pair of electrodes 430 and reference electrode 440 positioned closer to sensor tip 404, and working electrodes 410 and 420 positioned further away from sensor tip 404. A sensor configuration in which working electrodes 410 and 420 are positioned further away from sensor tip 404 may be advantageous because a greater surface area is provided for deposition of active regions 414a and 414b (illustratively shown in Figure 5C 404), thereby promoting increased signal strength in some cases. Again, with working electrodes 410 and 420 concentric to each other and spaced apart along the sensor tail, the bilayer membrane portion can be deposited on the working electrode positioned closer to the sensor tip 404 by sequential dip coating operations while the homogeneous membrane portion can be deposited on the working electrode further away from the sensor tip 404. Similarly, the central substrate 402 can be omitted in any concentric sensor configuration disclosed herein, where the innermost electrode can instead support subsequently deposited layers.

[0048] It should be appreciated that an analyte sensor capable of low potential operation according to the disclosure below may also be characterized by a mass transport limiting membrane having a double membrane portion and a homogeneous membrane portion. Successive immersion coating operations may be advantageous for depositing the mass transport limiting membrane. However, the present disclosure also contemplates an analyte sensor that does not necessarily need to be detected at a low potential, but still incorporates a mass transport limiting membrane comprising a double membrane portion and a homogeneous membrane portion.

[0049] Accordingly, an analyte sensor capable of low potential operation according to the present disclosure may include a sensor tail, which includes at least a first working electrode; a first active region located on the surface of the first working electrode and responding to a first analyte at a low potential, the first active region including a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme that responds to the first analyte covalently bonded to the first polymer; and a mass transport limiting membrane permeable to the first analyte, which coats at least the first active region. As used herein, the term "low potential" refers to a potential that is higher than the redox potential of the first redox mediator and is measured relative to an Ag / AgCl reference and is lower than about +200mV (including lower than about +100mV, lower than about -50mV, lower than about -80mV, or lower than about -100mV). Exemplary first redox mediators that can facilitate operation at the working electrode potential can have redox potentials below about -200 mV, such as about -400 mV to about -200 mV, or about -350 mV to about -250 mV, or about -300 mV to about -250 mV, as measured relative to a Ag / AgCl reference.

[0050] A suitable example of the first redox mediator capable of facilitating low potential operation may have a structure represented by Formula 1

[0051]

[0052] Wherein M is osmium, ruthenium, vanadium, cobalt or iron, L 1 -L 6 L is independently a heteroaromatic ligand coordinatively bound to M, wherein two or more may optionally be bound together to form a bidentate, tridentate or higher dentate ligand, 1 -L 6 At least one of the first redox mediators contains a linking group that bonds the first redox mediator to the first polymer and L 1 -L 6 At least one of the two is functionalized with an electron donating group. The electron donating group is separate and distinct from the linking group.

[0053] Particularly suitable bidentate ligands that are characterized as heteroaromatic ligands for inclusion in low potential redox mediators include optionally substituted 2,2'-biimidazole, 2-(2-pyridyl)imidazole and 2,2'-bipyridine ligands.

[0054] Examples of 2,2′-biimidazole ligands are shown in Formula 2

[0055]

[0056] Where R 1 and R 2is independently selected from optionally substituted alkyl, alkenyl or aryl. 1 and R 2 can be independently selected from unsubstituted C 1 To C 12 Alkyl or unsubstituted C 1 To C 4 In certain embodiments, R 1 and R 2 All are methyl. Q is an optional substitution (n=0, 1 or 2) connected to one or more of the imidazole ring carbon atoms, wherein the optional Q substitution may be an electron donating group or a linking group in certain embodiments. Suitable electron donating groups that may constitute one or more Q include, for example, alkyl, alkoxy, hydroxy, amino, alkylamino, dialkylamino, etc. If Q is absent, the carbon atom carries an H atom.

[0057] Examples of 2-(2-pyridyl)imidazole ligands may have a structure represented by Formula 3

[0058]

[0059] Where R 1’ is an optionally substituted alkyl, alkenyl or aryl group, such as an unsubstituted C 1 To C 12 Alkyl, or C 1 To C 4 Alkyl, particularly methyl. Q is an optional substitution (n=0, 1 or 2 for imidazole and p=0, 1, 2, 3 or 4 for pyridine) attached to one or more carbon atoms of the imidazole or pyridine ring, wherein the optional Q substitution may be an electron-donating group or a linking group in certain embodiments. Suitable electron-donating groups that may constitute one or more Q include, for example, alkyl, alkoxy, hydroxy, amino, alkylamino, dialkylamino, etc. If Q is absent, the carbon atom carries an H atom.

[0060] Examples of 2,2′-bipyridine ligands may have a structure represented by Formula 4

[0061]

[0062] Wherein Q is an optional substitution (p=0, 1, 2, 3 or 4) connected to one or more of the pyridine ring carbon atoms, wherein the optional Q substitution may be an electron donating group or a linking group in certain embodiments. Suitable electron donating groups that may constitute one or more Q include, for example, alkyl, alkoxy, hydroxy, amino, alkylamino, dialkylamino, etc. If Q is absent, the carbon atom carries an H atom.

[0063] A particularly suitable example of a redox mediator capable of promoting electron transfer at a low potential may have a structure represented by Formula 5, wherein L 1 and L 2 , L 3 and L 4 and L 5 and L 6 Each is linked to form a bidentate ligand, one of the bidentate ligands carries a linker group G that covalently bonds the redox mediator to the polymer, and L 1 -L 2 , L 3 -L 4 and L 5 -L 6 At least one of them carries an electron donating group. The electron donating group is separate and different from the linking group. The electron donating group can be on the same bidentate ligand containing the linking group G or on a different bidentate ligand. For example, L 1 -L 2 , L 3 -L 4 and L 5 -L 6 A bidentate ligand represented by one or more of Formulae 2-4 may be included.

[0064]

[0065] In certain embodiments, the redox mediator can be positively charged (e.g., with a charge range of +1 to +5). The redox mediator can alternatively be negatively charged (e.g., with a charge range of -1 to -5), such as in the case of a ligand or framework derivatized with a sufficient number of negatively charged functional groups such as carboxylate, phosphate, or sulfonate functional groups. One or more counterions can be used to charge. Examples of suitable counterions include anions such as halide (e.g., fluoride, chloride, bromide, or iodide) anions, sulfate anions, phosphate anions, hexafluorophosphate anions, and tetrafluoroborate anions, as well as cations, particularly monovalent cations such as lithium, sodium, potassium, tetraalkylammonium, and ammonium cations.

[0066] In a more specific example, the redox mediator in the first active region may have a structure represented by Formula 6

[0067]

[0068] Wherein G is a linking group that covalently bonds the redox mediator to the active site polymer, and D is an electron donating group. Specific examples of suitable electron donating groups include, for example, hydroxyl, alkoxy (e.g., methoxy or ethoxy), amino, or alkyl or dialkylamino (e.g., methylamino, ethylamino, dimethylamino or diethylamino). In more specific examples, the electron donating group can be located at the 4-position of the pyridine ring, as shown in Formula 7.

[0069]

[0070] In any of the embodiments herein, the redox mediator capable of promoting electron transfer at a low potential may have a structure represented by Formula 8.

[0071]

[0072] In at least one embodiment, the linking group G may contain a reactive group that promotes covalent bonding with the polymer. The reactive group can react with a complementary reactive group located on the polymer or in a polymer precursor to promote covalent bonding with it. In certain embodiments, an amide group may be present in the linking group G.

[0073] Any suitable polymer backbone may be present in the active region for promoting low potential detection of analytes by covalently bonding redox mediators and enzymes thereto. Examples of suitable polymers in the active region include poly(4-vinylpyridine) and poly(N-vinylimidazole) or copolymers thereof, for example wherein quaternized pyridine and imidazole groups serve as attachment points for redox mediators or enzymes thereto. Other suitable polymers that may be present in the active region include, but are not limited to, those described in US Pat. No. 6,605,200 (incorporated herein in their entirety by reference), such as poly(acrylic acid), styrene / maleic anhydride copolymers, methyl vinyl ether / maleic anhydride copolymers (GANTREZ polymers), poly(vinylbenzyl chloride), poly(allylamine), polylysine, poly(4-vinylpyridine) quaternized with carboxypentyl groups, and poly(sodium 4-styrenesulfonate).

[0074] The enzyme covalently bound to the polymer in the first active region that can promote low potential detection is believed to be not particularly limited. Suitable enzymes may include those that can detect glucose, lactic acid, ketones, creatinine, etc. In some cases, at least one enzyme covalently bound to the polymer in the first active region may include multiple enzymes that respond to analytes at low potentials. For detecting ketones and creatinine, an enzyme system may be particularly desirable.

[0075] In more specific embodiments, the first active region may comprise an enzyme system capable of detecting ketones. As previously described, ketones are generally present at low biological abundance and may benefit from detection at low potentials according to the disclosure herein. Figures 6A-6C , a particular enzyme system that can be used to detect ketones will be described in further detail. In the described enzyme reaction, β-hydroxybutyrate acts as a surrogate for ketones formed in vivo. Fig. 6A As shown, a pair of synergistic enzymes that can be used to detect ketones according to the disclosure herein are β-hydroxybutyrate dehydrogenase (HBDH) and diaphorase, which can be deposited in a ketone-responsive active region on the surface of at least one working electrode, as further described herein. In the case where the ketone-responsive active region contains the synergistic enzyme pair, β-hydroxybutyrate dehydrogenase can convert β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD + ) are converted into acetoacetate and reduced nicotinamide adenine dinucleotide (NADH), respectively. Enzyme cofactor NAD + and NADH help promote the synergistic enzyme reaction disclosed herein. NADH can then be oxidized under the mediation of diaphorase, wherein the electron transfer during this process provides the basis for detecting ketones at the working electrode. Thus, there is a 1:1 molar correspondence between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted, thereby providing a basis for detecting and quantifying ketones based on the amount of current measured at the working electrode. The transfer of electrons generated by the oxidation of NADH to the working electrode can occur through a redox mediator that can promote operation at a low potential. Albumin can serve as a stabilizer for the synergistic enzyme pair. According to a specific embodiment, β-hydroxybutyrate dehydrogenase and diaphorase can be covalently bonded to a polymer in the ketone-responsive active region of the analyte sensor. NAD + may or may not be covalently bonded to the polymer, but if NAD + Not covalently bonded, it can be physically retained in the ketone-responsive active region. The membrane covering the ketone-responsive active region can help to transfer NAD + Remaining in the ketone-responsive active zone while still allowing enough ketone to diffuse inwardly to allow its detection.

[0076] Other suitable chemistries for enzymatic detection of ketones are shown in Figure 6B and 6C In both cases, there is again a 1:1 molar correspondence between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted, thereby providing the basis for ketone detection.

[0077] like Figure 6B As shown, β-hydroxybutyrate dehydrogenase (HBDH) can convert β-hydroxybutyrate and NAD + acetoacetate and NADH, respectively. Fig. 6A) and a suitable redox mediator, the reduced form of NADH oxidase (NADHOx(Red)) reacts to form the corresponding oxidized form (NADHOx(Ox)). NADHOx(Red) can then be reformed by reacting with molecular oxygen to produce superoxide, which can then be converted to hydrogen peroxide under the mediation of superoxide dismutase (SOD). The hydrogen peroxide can then be oxidized at the working electrode to provide a signal that can be correlated to the amount of ketones initially present. According to various embodiments, SOD can be covalently bonded to the polymer in the ketone-responsive active region. As shown in Fig. 6A The enzyme system, β-hydroxybutyrate dehydrogenase and NADH oxidase may be covalently bonded to the polymer in the ketone-responsive active region, and NAD may or may not be covalently bonded to the polymer in the ketone-responsive active region. + If not covalently bonded, it can be physically retained in the ketone-responsive active region, where the membrane polymer promotes NAD + Retention in the keto-responsive active region.

[0078] like Figure 6C As shown, another enzymatic detection chemistry for ketones can use β-hydroxybutyrate dehydrogenase (HBDH) to convert β-hydroxybutyrate and NAD + are converted to acetoacetate and NADH, respectively. The electron transfer cycle in this case is completed by the oxidation of 1,10-phenanthroline-5,6-dione at the working electrode to reform NAD. 1,10-phenanthroline-5,6-dione may or may not be covalently bonded to the polymer in the ketone-responsive active region. As shown in Fig. 6A In an enzyme system, β-hydroxybutyrate dehydrogenase may be covalently bonded to a polymer in the ketone-responsive active region, while NAD may or may not be covalently bonded to a polymer in the ketone-responsive active region. Inclusion of albumin in the active region may provide unexpected improvements in response stability. Suitable membrane polymers may promote NAD + Retention in the keto-responsive active region.

[0079] In addition to the analyte detectable at low potential in the first active region, the analyte sensor of the present disclosure can also be further configured to analyze a second or subsequent analyte. In order to promote the detection of the second analyte, the analyte sensor of the present disclosure may also include a second working electrode, and a second active region located on the surface of the second working electrode and responding to a second analyte different from the first analyte, wherein the second active region includes 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 that responds to the second analyte covalently bonded to the second polymer. The second portion of the mass transport limiting membrane can coat the second active region. The at least one enzyme that responds to the second analyte can include an enzyme system, and the enzyme system includes multiple enzymes that respond to the second analyte together. The second redox mediator in the second active region does not necessarily need to be able to promote electron transfer at low potential, but it may be able to promote electron transfer at low potential.

[0080] Suitable redox mediators for inclusion in the second active region may include, but are not limited to, osmium complexes and other transition metal complexes, such as those described in US Patents 6,134,461 and 6,605,200, which are incorporated herein by reference in their entirety. Additional examples of suitable redox mediators include those described in US Patents 6,736,957, 7,501,053 and 7,754,093, which are also incorporated herein by reference in their entirety. Other suitable redox mediators for inclusion in the second active region may include, for example, metal compounds or complexes of ruthenium, osmium, iron (e.g., polyethylene ferrocene or hexacyanoferrate) or cobalt, including metallocene compounds thereof. Suitable ligands for metal complexes may also include, for example, bidentate or higher dentate ligands such as bipyridine, biimidazole, phenanthroline or pyridyl (imidazole). Other suitable bidentate ligands may include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes or o-diaminoarenes.Any combination of monodentate, bidentate, tridentate, tetradentate or higher dentate ligands may be present in the metal complex to achieve a complete coordination sphere.

[0081] According to the disclosure herein for promoting the active area of ​​analyte detection, a redox mediator may be included to which the polymer is covalently bound. Suitable examples of polymer-bound redox mediators may include those described in US Patents 8,444,834, 8,268,143 and 6,605,201, which are all disclosed and incorporated herein by reference. 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, for example, those containing monomeric units such as styrene, acrylamide, methacrylamide or acrylonitrile. The polymer in each active area may be the same or different.

[0082] In a particular example, the second active area can be configured to detect glucose and an analyte detectable at a low potential in the first active area in combination therewith. Thus, in a particular embodiment of the present disclosure, the second enzyme can be glucose oxidase. In a more particular example, the first analyte can be a ketone, which is detectable by an enzyme system as described herein, and the second analyte can be glucose, which is detectable by glucose oxidase.

[0083] Detection of each analyte may include applying a potential separately to each working electrode, thereby obtaining a separate signal from each analyte. The signal from each analyte may then be associated with the analyte concentration using a calibration curve or function or by using a lookup table. In a specific example, the association of the analyte signal with the analyte concentration may be performed using a processor.

[0084] In other analyte sensor configurations, the first active area and the second active area can be located on a single working electrode. A first signal can be obtained from the first active area at a low potential, and a second signal containing signal contributions from both active areas can be obtained at a higher potential. Subtracting the first signal from the second signal can then allow the signal contribution caused by the second analyte to be determined. The signal contribution from each analyte can then be associated with the analyte concentration in a manner similar to those described for sensor configurations with multiple working electrodes.

[0085] Accordingly, the present disclosure provides a method, which includes: providing an analyte sensor, the analyte sensor including: a sensor tail including at least a first working electrode; a first active area located on the surface of the first working electrode and responding to a first analyte at a low potential, the first active area including a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme responsive to the first analyte covalently bonded to the first polymer, wherein the redox mediator has a structure represented by any one of Formulas 1-8 described above, wherein G is a linking group that covalently bonds the first redox mediator to the first polymer; and a first analyte-permeable mass transport limiting membrane covering at least the first active area; applying a low potential to the first working electrode; obtaining a first signal at a redox potential located at or above the first active area, the first signal being proportional to the concentration of the first analyte in a fluid contacting the first active area; and correlating the first signal with the concentration of the first analyte in the fluid.

[0086] As measured relative to Ag / AgCl reference, the low potential can be less than about +200mV, including less than about +100mV, less than about -50mV, less than about -80mV or less than about -100mV. The low potential can also be higher than the redox potential of the first redox mediator. Exemplary redox potentials of the first redox mediator that can facilitate operation at a low working electrode potential can be less than about -200mV, such as about -400mV to about -200mV, or about -350mV to about -250mV, or about -300mV to about -250mV, as measured relative to Ag / AgCl reference.

[0087] The analyte sensor disclosed herein also includes a mass transport limiting membrane that is permeable to the analyte of at least the first active area. In the presence of multiple active areas, the mass transport limiting membrane can coat each active area, including options with composition changes in different active areas, which can be achieved, for example, by successive dip coating operations, which produce a double-layer membrane portion on a working electrode positioned closer to the sensor tip. The mass transport limiting membrane can include a membrane polymer, such as a polyvinyl pyridine or polyvinyl imidazole homopolymer or copolymer, which can be further cross-linked with a suitable cross-linking agent. In a specific embodiment, the membrane polymer can include a copolymer of vinyl pyridine and styrene. In a more particular example, the membrane polymer coating one or more active areas can be cross-linked with a branched cross-linking agent such as polyethylene glycol tetraglycidyl ether containing 3 or more cross-linkable groups, which can surprisingly reduce the amount of extractables available from the mass transport limiting membrane, as described above. Still more particularly, the mass transport limiting membrane may comprise a copolymer of polyvinyl pyridine or vinyl pyridine and styrene, crosslinked with a branched glycidyl ether crosslinking agent such as polyethylene glycol tetraglycidyl ether comprising three crosslinkable groups. In particular, the epoxide groups of polyethylene glycol tetraglycidyl ether may react with pyridine nitrogen atoms or imidazole nitrogen atoms to promote covalent bonding of the crosslinking agent via epoxide ring opening. Hydroxyalkyl groups bridging the crosslinking agent body to the membrane polymer heterocycle may be produced.

[0088] Suitable copolymers of vinylpyridine and styrene can have a styrene content ranging from about 0.01% to about 50% mole percent, or from about 0.05% to about 45% mole percent, or from about 0.1% to about 40% mole percent, or from about 0.5% to about 35% mole percent, or from about 1% to about 30% mole percent, or from about 2% to about 25% mole percent, or from about 5% to about 20% mole percent. Substituted styrene can be used similarly and in similar amounts. Suitable copolymers of vinylpyridine and styrene can have a molecular weight of 5kDa or more, or about 10kDa or more, or about 15kDa or more, or about 20kDa or more, or about 25kDa or more, or about 30kDa or more, or about 40kDa or more, or about 50kDa or more, or about 75kDa or more, or about 90kDa or more, or about 100kDa or more. In non-limiting examples, suitable copolymers of vinylpyridine and styrene may 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.

[0089] Accordingly, at least some of the analyte sensors described herein may include a sensor tail (the sensor tail includes at least a first working electrode), a first active region located on a surface of the first working electrode, and a first analyte permeable mass transport limiting membrane covering at least the first active region. The first active region includes a first polymer and at least one enzyme covalently bonded to the first polymer and responsive to the first analyte. The mass transport limiting membrane comprises a membrane polymer crosslinked with a branched glycidyl ether crosslinker comprising 3 or more crosslinkable groups, such as polyethylene glycol tetraglycidyl ether.

[0090] In more specific embodiments, the crosslinking can be intermolecular. 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 with a single molecule of the polyethylene glycol tetraglycidyl ether crosslinking agent. 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 generally vary within a certain range in a given sample to produce an average molecular weight. Prior to crosslinking, the structure of the polyethylene glycol tetraglycidyl ether can be represented by the following formula 9

[0091]

[0092] Wherein n1, n2, n3 and n4 are each greater than or equal to 0, typically an integer of 1 or higher, and n1, n2, n3 and n4 can be the same or different. The sum of n1, n2, n3 and n4 can be selected so that the molecular weight of polyethylene glycol tetraglycidyl ether falls within the aforementioned range. In other words, in order to produce polyethylene glycol tetraglycidyl ether with a molecular weight within the aforementioned range, the sum of n1, n2, n3 and n4 can range from about 14 to about 110, or from about 15 to about 104, including any sub-ranges between these values, wherein n1, n2, n3 and n4 can independently be greater than or equal to 0 or any integer greater than or equal to 1.

[0093] Crosslink density refers to the number of membrane polymer side chains that have a crosslinker attached thereto. Membrane polymers crosslinked with branched glycidyl ethers such as polyethylene glycol tetraglycidyl ether or similar polyethylene oxide crosslinkers having 3 or more crosslinkable groups can have crosslink densities that vary over a wide range. In specific examples, the proportion of side chains that can have a crosslinker attached thereto can be about 0.1% or more of the available heterocycles in the membrane polymer, or about 0.2% or more of the available heterocycles in the membrane polymer, or about 0.3% or more of the available heterocycles in the membrane polymer, or about 0.4% or more of the available heterocycles in the membrane polymer, or about 0.5% or more of the available heterocycles in the membrane polymer, or about 0.6% or more of the available heterocycles in the membrane polymer, or about 0.7% or more of the available heterocycles in the membrane polymer, or about 0.8% or more of the available heterocycles in the membrane polymer. about 0.8% or more of the available heterocycles in the membrane polymer, or about 0.9% or more of the available heterocycles in the membrane polymer, or about 1.0% or more of the available heterocycles in the membrane polymer, or about 1.2% or more of the available heterocycles in the membrane polymer, or about 1.4% or more of the available heterocycles in the membrane polymer, or about 1.6% or more of the available heterocycles in the membrane polymer, or about 1.8% or more of the available heterocycles in the membrane polymer, or about 2.0% or more of the available heterocycles in the membrane polymer, or about 2.2 ...2% or more of the available heterocycles in the membrane polymer, or about 1.4% or more of the available heterocycles in the membrane polymer, or about 1.6% or more of the available heterocycles in the membrane polymer, or about 1.8% or more of the available heterocycles in the membrane polymer, or about 2.0% or more of the available heterocycles in the membrane polymer, or about 2.2% or more of the available heterocycles in the membrane polymer The invention relates to about 2.4% or more of the available heterocycles in the membrane polymer, or about 2.6% or more of the available heterocycles in the membrane polymer, or about 2.8% or more of the available heterocycles in the membrane polymer, or about 3.0% or more of the available heterocycles in the membrane polymer, or about 3.5% or more of the available heterocycles in the membrane polymer, or about 4.0% or more of the available heterocycles in the membrane polymer, or about 4.5% or more of the available heterocycles in the membrane polymer, or about 5.0% or more of the available heterocycles in the membrane polymer, or about 5.5% or more of the available heterocycles in the membrane polymer, or about 6.0% or more of the available heterocycles in the membrane polymer, or about 6.5% or more of the available heterocycles in the membrane polymer, or about 7.0% or more of the available heterocycles in the membrane polymer, or about 7.5% or more of the available heterocycles in the membrane polymer, or about 8.0% or more of the available heterocycles in the membrane polymer, or about 8.5 .... In some embodiments, the crosslinker can be attached to about 6.0% or more of the available heterocycles in the membrane polymer, or about 6.5% or more of the available heterocycles in the membrane polymer, or about 7.0% or more of the available heterocycles in the membrane polymer, or about 7.5% or more of the available heterocycles in the membrane polymer, or about 8.0% or more of the available heterocycles in the membrane polymer, or about 8.5% or more of the available heterocycles in the membrane polymer, or about 9.0% or more of the available heterocycles in the membrane polymer, or about 9.5% or more of the available heterocycles in the membrane polymer, or about 10% or more of the available heterocycles in the heterocyclic polymer. In more specific embodiments, the crosslinker can be attached to about 1% to about 20% of the available heterocycles in the membrane polymer, or about 2% to about 10% of the available heterocycles in the membrane polymer, or about 3% to about 8% of the available heterocycles in the membrane polymer, or about 4% to about 9% of the available heterocycles in the membrane polymer, or about 5% to about 12% of the available heterocycles in the membrane polymer.

[0094] Suitable membrane polymers may also include one or more polyether arms (side chains) bonded to the nitrogen atoms of the pyridine or imidazole monomer units. Any membrane polymer disclosed herein may also include one or more polyether arms. The difference between the polyether arms and the crosslinking groups formed from polyethylene glycol tetraglycidyl ether or similar crosslinking agents is that the polyether arms do not extend between separate polymer chains or terminate intramolecularly in a single polymer chain. Thus, the polyether arms are separate and different from the crosslinking groups formed from the crosslinking agent. The polyether arms may include polyethylene oxide blocks and polypropylene oxide blocks, and in particular the polyether arms have polypropylene oxide blocks inserted between two polyethylene oxide blocks. The bonding of the polyether arms to the heterocyclic nitrogen atoms may occur through any reactive functional group capable of bonding to the heterocyclic nitrogen atoms in the membrane polymer. The bonding of the polyether arms to the heterocyclic nitrogen atoms may be carried out by alkyl, hydroxyl-functionalized alkyl, or carbonyl. The polyether arms may also contain amine groups away from the heterocyclic nitrogen atoms or do not contain amines in other special cases.

[0095] The polyether arm of the membrane polymer may comprise at least one polyethylene oxide block and at least one polypropylene oxide block, thereby providing a diblock arrangement of at least polyethylene oxide and polypropylene oxide monomer units, which are bonded to the heterocyclic nitrogen atom via a spacer. The polyethylene oxide block or the polypropylene oxide block may be bonded to the spacer. In other more specific embodiments, the polyether arm may comprise a spacer, a first polyethylene oxide block, a polypropylene oxide block, and a second polyethylene oxide block in sequence (i.e., an ABA repeating pattern) or a spacer, a first polypropylene oxide block, a polyethylene oxide block, and a second polypropylene oxide block in sequence (i.e., a BAB repeating pattern). The amine group may be between the polyethylene oxide block and the polypropylene oxide block in the amine-containing polyether arm. Thus, the polyether arm in the membrane polymer described herein may have a structure generally defined by Formulas 10-13 below,

[0096]

[0097] Wherein PE represents polyethylene oxide block, PP represents polypropylene oxide block, A is an amine group, and J is a spacer group. The spacer group J can become bonded to the heterocyclic ring of the membrane polymer. Suitable spacer groups J may include, but are not limited to, alkyl, hydroxy-functionalized alkyl, carbonyl, carboxylate, formamide, etc. The variables q, r, s, and t are positive integers defining the number of monomer units in each block and the number of times the block is repeated, provided that in a diblock arrangement, the variable t can be 0 and the variable s can be 1. According to certain 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 certain or other various embodiments, the variable s is an integer ranging from 1 to about 20 or from 1 to about 10. In certain embodiments, the variable s is equal to 1.

[0098] In a more specific embodiment of the present disclosure, the amine-free polyether arm having a triblock arrangement of polyethylene oxide, polypropylene oxide, and polyethylene oxide (corresponding to Formula 10) arms may have a structure defined by Formula 14,

[0099]

[0100] 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 can range from about 8 to about 16 or from about 9 to about 12, the variable y can 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 can range from about 10 to about 20 or from about 14 to about 18. In certain embodiments, the variable x can be less than the variable z, so that the second polyethylene oxide block is longer (larger) than the first polyethylene oxide block. If the variable w is 0, the polyether arm without an amine is directly bonded to the membrane polymer through a two-carbon alkyl group, but the present disclosure also contemplates longer alkyl groups.

[0101] In other specific embodiments of the present disclosure, the polyether arm having a triblock arrangement of polyethylene oxide, polypropylene oxide, and polyethylene oxide and having an amine group between the polyethylene oxide block and the polypropylene oxide block (corresponding to Formula 12) may have a structure defined by Formula 15,

[0102]

[0103] wherein w, x, y, z and R are as defined above for Formula 14. If the variable w is 0, the polyether arm is directly bonded to the membrane polymer through the two carbon alkyl group, although longer alkyl groups are also contemplated by the present disclosure.

[0104] The polyether arms described herein can become bonded to heterocyclic nitrogen atoms via reactive functions in polyether arm precursors. Suitable reactive functions can include, for example, halogens or epoxides. For example, epoxides result in the formation of hydroxyalkyl spacer groups that connect the polyether arms to the heterocyclic nitrogen atoms of the membrane polymer, as illustrated in Formulas 14 and 15 above (n=1 in Formulas 14 and 15). In contrast, halogen-functionalized polyether arm precursors can result in alkyl spacer moieties (n=0 in Formulas 14 and 15), wherein suitable alkyl groups can be linear or branched and contain 2 to about 20 carbon atoms.

[0105] In certain embodiments, the arm containing sulfonate / ester can be attached to at least a portion of the membrane polymer disclosed herein as a side chain. The arm containing sulfonate / ester can be present in any suitable ratio combination with polyether arms and / or cross-linking agents. Any membrane polymer disclosed herein can include a polyether arm or a cross-linking group with a higher amount than the arm containing sulfonate / ester. The arm containing sulfonate / ester can be attached to the membrane polymer via an alkyl group. According to various embodiments, the alkyl group can contain 1 to about 6 carbon atoms, or 2 to about 4 carbon atoms. Suitable reagents for introducing the arm containing sulfonate / ester into the membrane polymer disclosed herein can include halogenated sulfonic acid compounds such as chloromethanesulfonic acid, bromoethylsulfonic acid, etc., or cyclic sulfonates (sultones).

[0106] Polydimethylsiloxane (PDMS) may be incorporated into any of the mass transport limiting membranes disclosed herein.

[0107] In the case where the first active area and the second active area configured for testing different analytes are located on separate working electrodes, the mass transport limiting membrane can have different permeability values ​​for the first analyte and the second analyte. Although the film thickness and / or active area size of each working electrode can be changed to level the sensitivity to each analyte, this approach can significantly complicate the preparation of the analyte sensor. As a solution, the mass transport limiting membrane coating at least one active area can include a mixture of a first membrane polymer and a second membrane polymer or a double layer of a first membrane polymer and a second membrane polymer. A homogeneous membrane can coat an active area that is not coated with a mixture or a double layer, wherein the homogeneous membrane includes only one of the first membrane polymer or the second membrane polymer. Advantageously, the analyte sensor construction disclosed herein conveniently makes it possible to have a continuous membrane, which has a uniform membrane portion to be placed on the first active area of ​​the analyte sensor and a multi-component membrane portion to be placed in the second active area, thereby simultaneously leveling the permeability values ​​for each analyte, thereby providing improved sensitivity and detection accuracy. In a specific embodiment, continuous film deposition can occur by successive immersion coating operations.

[0108] Embodiments disclosed herein include:

[0109] A. An analyte sensor capable of low-potential detection of an analyte. The analyte sensor comprises: a sensor tail comprising at least a first working electrode; a first active region located on the surface of the first working electrode and responding to a first analyte at a low potential, the first active region comprising a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme that responds to the first analyte covalently bonded to the first polymer; wherein the first redox mediator has the following structure:

[0110]

[0111] wherein G is a linker group that covalently bonds the first redox mediator to the first polymer; and a first analyte-permeable mass transport limiting membrane that covers at least the first active region.

[0112] B. A method for detecting an analyte using an analyte sensor capable of low potential detection. The method comprises: providing an analyte sensor, the analyte sensor comprising: a sensor tail comprising at least a first working electrode; a first active region located on the surface of the first working electrode and responding to a first analyte at a low potential, the first active region comprising a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme that responds to the first analyte covalently bonded to the first polymer; wherein the first redox mediator has the following structure:

[0113]

[0114] wherein G is a linking group that covalently bonds the first redox mediator to the first polymer; and a mass transport limiting membrane permeable to the first analyte that covers at least the first active area; applying a low potential to the first working electrode; obtaining a first signal at a redox potential at or above the first active area, the first signal being proportional to the concentration of the first analyte in a fluid contacting the first active area; and correlating the first signal to the concentration of the first analyte in the fluid.

[0115] B1. A method for detecting an analyte using an analyte sensor capable of low potential detection. The method comprises: exposing the analyte sensor to a fluid containing a first analyte; wherein the analyte sensor comprises: a sensor tail comprising at least a first working electrode; a first active region located on the surface of the first working electrode and responding to the first analyte at a low potential, the first active region comprising a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme that responds to the first analyte covalently bonded to the first polymer; wherein the first redox mediator has the following structure:

[0116]

[0117] wherein G is a linker group that covalently bonds the first redox mediator to the first polymer; and a mass transport limiting membrane permeable to the first analyte that covers at least the first active region; applying a low potential to the first working electrode; obtaining a first signal at a redox potential at or above the first active region, the first signal being proportional to the concentration of the first analyte in the fluid; and correlating the first signal to the concentration of the first analyte in the fluid.

[0118] C. An analyte sensor comprising a mass transport limiting membrane cross-linked with a branched glycidyl ether cross-linking agent. The analyte sensor comprises: a sensor tail comprising at least a first working electrode; a first active region located on a surface of the first working electrode, the first active region comprising a first polymer and at least one enzyme covalently bonded to the first polymer and responsive to a first analyte; and a first analyte permeable mass transport limiting membrane coating at least the first active region, the mass transport limiting membrane comprising a membrane polymer cross-linked with a branched cross-linking agent, the branched cross-linking agent comprising 3 or more cross-linkable groups.

[0119] D. A method for detecting an analyte using an analyte sensor, the analyte sensor comprising a mass transport limiting membrane cross-linked with a branched glycidyl ether cross-linking agent. The method comprises: providing an analyte sensor, the analyte sensor comprising: a sensor tail comprising at least a first working electrode; a first active region located on a surface of the first working electrode, the first active region comprising a first polymer and at least one enzyme covalently bonded to the first polymer and responsive to a first analyte; and a first analyte permeable mass transport limiting membrane coating at least the first active region, the mass transport limiting membrane comprising a membrane polymer cross-linked with a branched cross-linking agent, the branched cross-linking agent comprising 3 or more cross-linkable groups; applying a potential to the first working electrode; obtaining a first signal at a redox potential at or above the first active region, the first signal being proportional to the concentration of the first analyte in a fluid contacting the first active region; and correlating the first signal with the concentration of the first analyte in the fluid.

[0120] Embodiments AD may have one or more of the following elements in any combination.

[0121] Element 1: wherein the at least one enzyme comprises an enzyme system comprising a plurality of enzymes that collectively respond to a first analyte.

[0122] Element 2: wherein the first analyte comprises one or more ketones.

[0123] Element 3: wherein the mass transport limiting membrane comprises a membrane polymer cross-linked with a branched cross-linking agent, the branched cross-linking agent comprising 3 or more cross-linkable groups.

[0124] Element 4: wherein the membrane polymer comprises polyvinyl pyridine or polyvinyl imidazole.

[0125] Element 5: wherein the membrane polymer comprises a copolymer of vinylpyridine and styrene.

[0126] Element 6: wherein the branched cross-linking agent comprises polyethylene glycol tetraglycidyl ether.

[0127] Element 7: wherein the analyte sensor also includes a second working electrode; and a second active area located 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 different from the first redox mediator covalently bonded to the second polymer, and at least one enzyme responsive to the second analyte covalently bonded to the second polymer; wherein the second portion of the mass transport limiting membrane covers the second active area.

[0128] Element 8: wherein the at least one enzyme responsive to the second analyte comprises an enzyme system comprising a plurality of enzymes that collectively respond to the second analyte.

[0129] Element 9: wherein the second analyte comprises glucose.

[0130] Element 10: wherein the low potential is above the redox potential of the first redox mediator and is below about -80 mV relative to a Ag / AgCl reference.

[0131] Element 10A: wherein the redox potential of the first redox mediator ranges from about -200 mV to about -400 mV relative to a Ag / AgCl reference.

[0132] Element 11: wherein the polyethylene glycol tetraglycidyl ether has a molecular weight ranging from about 1000 g / mol to about 5000 g / mol.

[0133] By way of non-limiting example, possible exemplary combinations for A and B may include, but are not limited to, 1 and 2; 1-3; 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. By way of further non-limiting example, possible exemplary combinations for C and D may include, but are not limited to, 6 and 11; 4 and 6; 5 and 6; 6 and 7; 6-8; 4, 6 and 11; 5, 6 and 11; 6, 7 and 11; and 6-8 and 11.

[0134] Additional embodiments disclosed herein include:

[0135] A': A method for forming a mass transport limiting membrane by dip coating. The method comprises: providing an analyte sensor, the analyte sensor comprising: a sensor tail, which comprises at least a first working electrode and a second working electrode spaced apart from each other along the length of the sensor tail; and a first active region located on the surface of the first working electrode and a second active region located on the surface of the second working electrode, the first active region and the second active region responding to different analytes; and depositing a mass transport limiting membrane on the first active region and the second active region by successive dip coating operations; wherein the mass transport limiting membrane comprises a double-layer membrane portion coating the first active region and a homogeneous membrane portion coating the second active region.

[0136] B': An analyte sensor with an impregnated coated mass transport limiting membrane. The analyte sensor comprises: a sensor tail comprising at least a first working electrode and a second working electrode spaced apart from each other along the length of the sensor tail; a first active region located on the surface of the first working electrode and a second active region located on the surface of the second working electrode, the first active region and the second active region being responsive to different analytes; and an impregnated coated mass transport limiting membrane on the first active region and the second active region, the impregnated coated mass transport limiting membrane comprising an impregnated coated double layer membrane portion coating the first active region and an impregnated coated homogeneous membrane portion coating the second active region.

[0137] Embodiments A' and B' may have one or more of the following elements in any combination:

[0138] Element 1': wherein the bilayer membrane portion and the homogeneous membrane portion are adjacent to each other.

[0139] Element 2': wherein the first working electrode and the first active area are positioned closer to the tip of the analyte sensor than the second working electrode and the second active area.

[0140] Element 3': wherein the upper layer of the bilayer membrane portion and the homogeneous membrane portion comprise the same membrane polymer.

[0141] Element 4': wherein a first dip coating operation deposits a first membrane polymer on a first active area and a second dip coating operation deposits a second membrane polymer on both the first active area and the second active area thereby defining a bilayer membrane portion on the first active area and a homogeneous membrane portion on the second active area, the first membrane polymer and the second membrane polymer being different from each other.

[0142] Element 5': wherein the lower layer of the bilayer membrane portion and the homogeneous membrane portion comprise the same membrane polymer.

[0143] Element 6': wherein a first dip coating operation deposits a first membrane polymer in both the first active area and the second active area and a second dip coating operation deposits a second membrane polymer on the first active area thereby defining a bilayer membrane portion on the first active area, the first membrane polymer and the second membrane polymer being different from each other.

[0144] Element 7': wherein the first active area is responsive to glucose, lactate, ketones or creatinine.

[0145] Element 8': wherein the first active region is responsive to a ketone.

[0146] Element 9': wherein the second active region is responsive to glucose.

[0147] Element 10': wherein at least a portion of the mass transport limiting membrane comprises a cross-linked polyvinylpyridine homopolymer or copolymer.

[0148] Element 11 ': wherein at least a portion of the mass transport limiting membrane comprises a membrane polymer cross-linked with a branched cross-linking agent comprising 3 or more cross-linkable groups.

[0149] Element 12': wherein the branched cross-linking agent comprises polyethylene glycol tetraglycidyl ether.

[0150] Element 13': wherein the dip-coated bilayer membrane portion and the dip-coated homogeneous membrane portion are adjacent to each other.

[0151] Element 14': wherein the first working electrode and the first active area are positioned closer to the tip of the analyte sensor than the second working electrode and the second active area.

[0152] Element 15': wherein the upper layer of the dip-coated bilayer membrane portion and the dip-coated homogeneous membrane portion comprise the same membrane polymer.

[0153] Element 16': wherein the lower layer of the dip-coated bilayer membrane portion and the dip-coated homogeneous membrane portion comprise the same membrane polymer.

[0154] Element 17': wherein the first active region is responsive to a ketone.

[0155] Element 18': wherein the second active area is responsive to glucose.

[0156] By way of non-limiting example, exemplary combinations possible for A' include, but are not limited to: 1' and 2'; 1'-3'; 1'-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'-4'; 2'-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 possible combinations for 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'.

[0157] In order to facilitate a better understanding of the disclosure herein, the following examples of various representative embodiments are provided.The following examples should not be construed as limiting or defining the scope of the invention. Example

[0158] Example 1: Detection of ketones at low potential using an analyte sensor having diaphorase and β-hydroxybutyrate dehydrogenase acting in concert. For this example, Fig. 6A The detection of ketones was facilitated by an enzyme system using the active zone formulations specified in Tables 1 and 2 below, using either unbound transition metal complexes of formula 8 (Table 1) or transition metal complexes of formula 8 bound to polyvinylpyridine-co-styrene (Table 2) as redox mediators (HBHD = β-hydroxybutyrate dehydrogenase; HSA = human serum albumin; PEGDGE400 = polyethylene glycol diglycidyl ether). By depositing approximately 0.2 mm 2 The sensing active area formulations shown in Tables 1 and 2 were coated onto the carbon-coated working electrode at one point of the area. The active area was cured at 25°C for 24 hours. After curing, a polyvinylpyridine film with the formulation specified in Table 3 was applied to the active area via a total of four dip coatings. After applying the film, the sensor was cured at 25°C for 24 hours and then at 56°C for 48 hours.

[0159] Table 1

[0160]

[0161] Table 2

[0162]

[0163] Table 3

[0164] Components Concentration (mg / mL) Solvents Volume added (mL) PVP 100 80 / 20 EtOH / HEPES 4 PDMS 100 EtOH 0.0132 PEGDGE400 100 80 / 20 EtOH / HEPES 0.2

[0165] Figure 7 Cyclic voltammograms of a transition metal complex of Formula 8 or a polymer-bound transition metal complex of Formula 8 are shown. The cyclic voltammograms were obtained in a 100 mM PBS solution at pH 7.4, which was deoxygenated via nitrogen bubbling and maintained at a temperature of 33°C. Scanning was performed from -0.5 V to 0.1 V at a scan rate of 5 mV / s with a carbon counter electrode and an Ag / AgCl reference electrode. From the cyclic voltammograms, the E1 / 2 of the transition metal complex of Formula 8 was measured to be -0.29 V vs. Ag / AgCl and the E1 / 2 of the polymer-bound transition metal complex of Formula 8 was measured to be -0.24 V vs. Ag / AgCl. The comparative redox mediator lacking N,N-dimethylamino substitution exhibited a significantly less negative E 1 / 2 : -0.08 V vs. Ag / AgCl (data not shown).

[0166] Figure 8 A current vs. time plot of a ketone sensor comprising a polymer-bound transition metal complex of formula 8 at each working electrode potential is shown. The sensor was maintained at one of four potentials ranging from +40 mV to -200 mV in 100 mM PBS at pH 7.4 and titrated with varying amounts of β-hydroxybutyrate at 33°C to a final ketone concentration of 8 mM. As shown, the sensor response stabilized rapidly after the addition of ketone at each potential. A linear response to ketone concentration was observed for each of the four potentials measured (see Fig. 9 ).

[0167] Example 2: Extractables from membranes cross-linked with polyethylene glycol tetraglycidyl ether. Polymer membrane samples were prepared that were cross-linked with polyethylene glycol tetraglycidyl ether (molecular weight ~2500). The base membrane polymer was a copolymer of vinyl pyridine and styrene, containing amine-free polyether arms bonded to at least a portion of the pyridine moieties in the base membrane polymer. Comparative polymer membrane samples were prepared using the same membrane polymer, which was cross-linked with polyethylene glycol diglycidyl ether (molecular weight ~1000). The comparative polymer membrane samples were cross-linked with polyethylene glycol diglycidyl ether, and the mass of the cross-linker was similar to the amount of polyethylene glycol tetraglycidyl ether used in the other samples, thereby providing similar cross-linking density.

[0168] The film polymer was cast as a film at the bottom of the sample vial with 0.5 mL of a solution containing the film polymer. The solvent was evaporated and then the film was cured at 25°C for 48 hours and at 56°C for 56 hours. Water or 95% ethanol (3 mL) was added to cast the polymer film and the extraction was carried out at room temperature for 72 hours (water) or 144 hours (ethanol). The vial was agitated on a shaker during the extraction period. The water extract was analyzed neat by UV-Vis spectrophotometry ( Fig.10 ), while the ethanol extract was diluted 1:9 before UV-Vis analysis ( Fig.11 ). The sample cross-linked with polyethylene glycol tetraglycidyl ether exhibited lower amounts of extractable material as shown by the UV-Vis absorbance intensity in both cases.

[0169] Example 3: Equilibration time of sensors with polyethylene glycol tetraglycidyl ether cross-linked membranes. Glucose responsive analyte sensors were coated with a membrane polymer cross-linked with polyethylene glycol tetraglycidyl ether (see Example 2). Comparative sensors were prepared with a comparable membrane polymer cross-linked with 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. Fig.12 A comparison of sensor output vs. time over 10 hours is shown, comparing an analyte sensor coated with a membrane polymer crosslinked with polyethylene glycol tetraglycidyl ether to an analyte sensor coated with a membrane polymer crosslinked with polyethylene glycol diglycidyl ether. As shown, both membranes provided stable sensor output after approximately 1 hour of equilibration. Fig.13 A magnified image of the first hour of sensor equilibration is shown, where it can be seen that the polyethylene glycol tetraglycidyl ether cross-linked membrane provides a stable response faster than the polyethylene glycol diglycidyl ether cross-linked membrane.

[0170] Unless otherwise specified, all numerical values ​​expressing quantities and the like in this specification and the related claims should be understood as being modified in all cases by the term "about". Accordingly, unless otherwise indicated, the numerical parameters in this specification and the appended claims are approximate values, which may vary depending on the desired characteristics that the embodiments of the present invention are intended to obtain. At least, and without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be interpreted with reference to the number of reported significant figures and by applying ordinary rounding methods.

[0171] One or more exemplary embodiments incorporating various features are shown herein. For the sake of clarity, all features of physical implementations are not described or shown in this application. It should be understood that in developing physical implementations incorporating embodiments of the present invention, many implementation-specific decisions must be made to achieve the developer's goals, such as complying with system dependencies, transaction dependencies, government dependencies, and other restrictions that vary depending on implementation and time. Although the developer's efforts may be time-consuming, the efforts will still be a common practice for those of ordinary skill in the art and benefit from this disclosure.

[0172] Although various systems, tools and methods are described herein as "comprising" various components or steps, the systems, tools and methods can also "consist essentially of" or "consist of" the various components and steps.

[0173] As used herein, where the phrase "at least one" is used with respect to a list of items separated by the terms "and," "or," or "or," any of these items, it modifies the entire list, not the individual members of the list (i.e., each item). Permissible meanings of the phrase "at least one" include at least one of any of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. For example, the phrase "at least one of A, B, and C" or "at least one of A, B, or C" refers to only A, only B, or only C, respectively; any combination of A, B, and C; and / or at least one of each of A, B, and C.

[0174] Therefore, the disclosed systems, tools and methods are fully suitable for obtaining the mentioned purposes and advantages and those inherent therein. The specific embodiments disclosed above are only exemplary, because the teachings of the present disclosure can be modified and implemented in different but equivalent ways that are obvious to those skilled in the art, which have the benefits of the teachings herein. In addition, except for the description of the attached claims, it is not expected to limit the details of the construction or design shown herein. Therefore, it is obvious that the specific exemplary embodiments disclosed above can be changed, combined or modified, and all of the changes are considered to belong to the scope of the present disclosure. In the absence of any elements not specifically disclosed herein and / or any optional elements disclosed herein, the systems, tools and methods disclosed exemplarily herein can be suitably implemented. Although systems, tools and methods are described as "comprising", "containing" or "including" various components or steps, the systems, tools and methods can also be "essentially composed of" or "composed of" the various components and steps. All numbers and ranges disclosed above can vary by a certain amount. In the case of a public numerical range with a lower limit and an upper limit, any number belonging to the range and any included range are specifically disclosed. In particular, each range value disclosed herein (in the form of "about a to about b" or equivalently "about a to b" or equivalently "about ab") should be understood to describe each number and range encompassed within the broader range of values. In addition, the terms in the claims have their clear, ordinary meaning unless otherwise explicitly and clearly defined by the patentee. In addition, the indefinite articles "a" or "an" used in the claims are defined herein to mean one or more than one of the elements introduced by the word. If there is any conflict in the use of words or terms in this specification and in one or more patents or other documents that may be incorporated by reference herein, the definition consistent with this specification shall be adopted.

Claims

1. Analyte sensor, comprising: Ag / AgCl reference electrode; at least a first working electrode; a first active region located on the surface of the first working electrode and responsive to one or more ketones at a low potential, the first active region comprising a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme responsive to the one or more ketones covalently bonded to the first polymer; wherein the low potential is higher than the redox potential of the first redox mediator and lower than -80 mV relative to a Ag / AgCl reference electrode; wherein the redox potential of the first redox mediator relative to the Ag / AgCl reference electrode ranges from -200 mV to -400 mV; Wherein the first redox mediator has the following structure: wherein G is a linker group that covalently bonds the first redox mediator to the first polymer; one or more ketone permeable mass transport limiting membranes covering at least the first active region; a second working electrode; a second active region located on the surface of the second working electrode and responsive to a second analyte, the second active region comprising a second polymer, a second redox mediator different from the first redox mediator covalently bonded to the second polymer, and at least one enzyme responsive to the second analyte covalently bonded to the second polymer; wherein a second portion of the mass transport limiting membrane covers the second active region; and The at least one enzyme responsive to the second analyte comprises an enzyme system comprising a plurality of enzymes that collectively respond to the second analyte.

2. The analyte sensor of claim 1, wherein the at least one enzyme responsive to one or more ketones comprises an enzyme system comprising a plurality of enzymes that collectively respond to one or more ketones.

3. The analyte sensor of claim 1, wherein the mass transport limiting membrane comprises a membrane polymer cross-linked with a branched cross-linking agent comprising 3 or more cross-linkable groups.

4. The analyte sensor of claim 3, wherein the membrane polymer comprises polyvinylpyridine or polyvinylimidazole.

5. The analyte sensor of claim 3, wherein the branched crosslinking agent comprises polyethylene glycol tetraglycidyl ether.

6. A method comprising: Providing the analyte sensor of claim 1; applying the low potential to the first working electrode; obtaining a first signal at or above the redox potential of the first active region, the first signal being proportional to a concentration of one or more ketones in a fluid contacting the first active region; and The first signal is correlated to a concentration of one or more ketones in the fluid.

7. The method of claim 6, wherein the at least one enzyme responsive to one or more ketones comprises an enzyme system comprising a plurality of enzymes that collectively respond to one or more ketones.

8. The method of claim 6, wherein the mass transport limiting membrane comprises a membrane polymer cross-linked with a branched cross-linking agent comprising 3 or more cross-linkable groups.

9. The method of claim 8, wherein the membrane polymer comprises polyvinylpyridine or polyvinylimidazole.

10. The method of claim 8, wherein the branched crosslinking agent comprises polyethylene glycol tetraglycidyl ether.

Images