Analyte sensors and methods with reduced interferent signals

Abstract

An analyte sensor includes: a substrate having an upper surface, the upper surface including a first portion and a second exposed portion; an electrode layer disposed on the first portion and having an elongated body including a proximal end and a distal end, the electrode layer including an active working electrode region having a surface area between 0.15 mm2 and 0.25 mm2; and at least one sensing point having at least one analyte-responsive enzyme disposed on the active working electrode region. Additional analyte sensors are disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 390,518, filed on July 19, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The subject matter described herein relates generally to systems, devices, and methods for monitoring analyte levels in vivo. Background Art

[0004] The detection of various analytes in individuals is sometimes essential for monitoring their health conditions. Deviations from normal analyte levels can usually indicate many physiological conditions. For example, it may be particularly important to detect and monitor blood glucose levels in diabetic individuals. By monitoring blood glucose levels with sufficient regularity, diabetic individuals can take corrective measures (e.g., lower blood glucose levels by injecting insulin or raise blood glucose levels by eating) before significant physiological damage occurs. For various other physiological conditions, it may be necessary to monitor other analytes. In some cases, it may also be necessary to monitor multiple analytes, particularly for complications that cause two or more analytes to combine with each other and become unbalanced at the same time.

[0005] Many analytes represent interesting targets for physiological analysis, provided that suitable detection chemicals can be identified. To this end, in vivo analyte sensors configured to analyze various physiological analytes have been developed and improved in recent years, many of which utilize enzyme-based detection strategies to promote detection specificity. In fact, in vivo analyte sensors that utilize blood glucose responsive enzymes to monitor blood glucose levels are now commonly used in diabetic individuals. In vivo analyte sensors for other analytes are at different stages of development, including in vivo analyte sensors that can monitor multiple analytes. For some analyte sensors, low sensitivity may be particularly problematic, particularly due to background signals generated by the interaction of interferents with the working electrode or other analyte sensing chemistry. Summary of the invention

[0006] The objects and advantages of the disclosed subject matter will be set forth and apparent in the following description, and will be learned by the practice of the disclosed subject matter. Additional advantages of the disclosed subject matter will be realized and obtained by the methods and systems particularly pointed out in the written description and claims as well as the drawings.

[0007] To achieve these and other advantages and in accordance with the purposes of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter is directed to an analyte sensor comprising: a substrate having an upper surface including a first portion and a second exposed portion, and an electrode layer disposed on the first portion and having an elongated body, the elongated body including a proximal end and a distal end. The electrode layer includes a surface area of ​​0.15 mm 2 Up to 0.25mm 2 an active working electrode area therebetween and configured to reduce a signal indicative of an interfering substance, and at least one sensing site on which at least one analyte-responsive enzyme is disposed. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The following drawings are included to illustrate certain aspects of the present disclosure and should not be considered to be exclusive embodiments. The disclosed subject matter is capable of considerable modification, alteration, combination, and equivalents in form and function without departing from the scope of the present disclosure.

[0009] Figure 1 A diagram of an exemplary sensing system that may incorporate the analyte sensors of the present disclosure is shown.

[0010] FIG. 2A to FIG. 2C A cross-sectional view of an analyte sensor including a single active area is shown.

[0011] FIG. 3A to FIG. 3C A cross-sectional view of an analyte sensor including two active regions is shown.

[0012] Figure 4 A cross-sectional view of an analyte sensor including two working electrodes, each having an active area thereon is shown.

[0013] Figure 5 is a top view showing a conventional carbon working electrode with an active area thereon.

[0014] Fig. 6A A photograph showing a top view of a working electrode with no membrane disposed thereon. Figure 6B is along Fig. 6A Depth profile of the line shown.

[0015] Fig. 7A A photograph showing a top view of the working electrode with the membrane disposed thereon. Figure 7B is along Fig. 7A Depth profile of the line shown.

[0016] Figure 8 is a photograph showing a 3D view of a laser planed working electrode according to one or more aspects of the present disclosure.

[0017] Fig. 9Ais a depiction of a conventional sensor without the incorporation of an interferent-reactive substance. Fig. 9B is an interfering substance-reacting substance incorporating one or more aspects of the present disclosure Fig. 9B Depiction of the sensor.

[0018] Fig.10 is a depiction of a sensor electrode configuration including a purge electrode according to one or more aspects of the present disclosure.

[0019] Fig.11 is a depiction of a sensor electrode configuration including a permeable purge electrode according to one or more aspects of the present disclosure.

[0020] Fig.12 is a depiction of a sensor electrode configuration including an impermeable purge electrode and a permeable purge electrode according to one or more aspects of the present disclosure.

[0021] Fig.13A A photograph showing a top view of a working electrode without a membrane and active area disposed thereon. Fig. 13B is along Fig.13A Depth profile of the line shown. Fig. 13C FIG. 1 shows a laser planed surface according to one or more aspects of the present disclosure. Fig.13A Photograph of the top view of the working electrode. Fig.13D is along Fig. 13C Depth profile of the line shown.

[0022] Fig.14A A photograph showing a top view of a working electrode after laser planing without a membrane and active area disposed thereon according to one or more aspects of the present disclosure. Fig. 14B is along Fig.14A Depth profile of the line shown.

[0023] Fig.15 is a graph of paired difference tests comparing shaved and unshaved working electrodes with or without active areas in response to the interferent ascorbic acid.

[0024] FIG. 16A to FIG. 16F A photograph of the working electrode is shown. Fig.16A and Fig. 16C There is no laser planing. Fig. 16B , Fig.16D , Fig.16E and Fig.16F is laser planed according to one or more aspects of the present disclosure.

[0025] Fig.17 is a sensor configuration including an interferent-reagent material layer according to one or more embodiments of the present disclosure.

[0026] Fig.18 According to one or more aspects of the present disclosure, a method for preparing a material comprising an interferer-reactant material layer Fig.17 Ascorbic acid calibration curve of the analyte sensor.

[0027] Fig.19 According to one or more aspects of the present disclosure, the Fig.17 Blood glucose calibration curve for the analyte sensor.

[0028] Fig. 20 A sensor configuration including an interferent-reagent material layer according to one or more aspects of the present disclosure.

[0029] Figure 21-24 is a sensor current trace of a sensor including a wiped electrode according to one or more aspects of the present disclosure.

[0030] Fig.25 A sensor configuration including a permeable purge electrode according to one or more aspects of the present disclosure.

[0031] Fig.26 According to one or more aspects of the present disclosure Fig.25 Sensor current trace of the sensor.

[0032] Fig.27A is a top view of an electrode layer having a U-shaped gap thereon according to one or more aspects of the present disclosure.

[0033] Fig.27B is a top view of an electrode layer having a wavy U-shaped gap thereon according to one or more aspects of the present disclosure.

[0034] Fig.27C is a top view of an electrode layer having laterally spaced-apart gaps thereon according to one or more aspects of the present disclosure.

[0035] Fig.28 is a top view of an electrode layer having a second electrode portion connected to a second wipe electrode trace according to one or more aspects of the present disclosure.

[0036] Fig.29A A cross-sectional view of an active area grid configuration of a carbon working electrode suitable for use in an analyte sensor of the present disclosure is shown. Fig.29B Another active area grid configuration is shown.

[0037] Fig. 30A is a top view of a working electrode with an active area thereon. FIG. 30B to FIG. 30G A diagram is shown of an illustrative process whereby a carbon working electrode and active area thereon can be enhanced to reduce interferent signals.

[0038] Fig. 30H The ratio of the surface area of ​​a standard sensor to the surface area of ​​a sensor according to an embodiment of the disclosed subject matter is shown.

[0039] Fig.31A is a diagram illustrating a top view of a working electrode having an active area thereon including first and second adjacent sensing sites in an overlapping configuration. FIG. 31B to FIG. 31F Depicted is a top view of an example embodiment including a configuration of first and second adjacent sensing points in an overlapping configuration in accordance with one or more aspects of the present disclosure.

[0040] FIG. 32A to FIG. 32D Depicted is a top view of an example embodiment including a configuration of at least two adjacent sensing points in an overlapping configuration in accordance with one or more aspects of the present disclosure.

[0041] Fig.33 Depicted is a top view of an example embodiment including at least three adjacent sensing points in an overlapping configuration according to one or more aspects of the present disclosure.

[0042] Fig.34A is a cross-sectional view of an example embodiment according to one or more aspects of the present disclosure, the example embodiment including a substrate having a conventional working electrode and a membrane disposed thereon.

[0043] Fig.34B yes Fig.34A A top view of an embodiment of the present invention is shown, showing a substrate having a roughened second exposed portion.

[0044] Fig.35A and Fig.35B A sensor configuration including an interferent blocking film layer according to one or more aspects of the present disclosure.

[0045] Fig.36A and Fig.36B is a graph of signal versus time for an analyte sensor and a control analyte sensor according to one or more aspects of the present disclosure. DETAILED DESCRIPTION

[0046] The present disclosure generally describes analyte sensors suitable for in vivo use, and more particularly, describes analyte sensors featuring one or more enhancements for reducing or eliminating signals indicative of interfering species to facilitate increased detection sensitivity and methods of making and using the same.

[0047] Such enhancements may include reducing the availability of the working electrode surface on the sensor tail (the portion of the sensor for insertion into the tissue), particularly the availability of the carbon working electrode on the sensor tail, where the interferents may react and contribute to signals unrelated to the analyte. Other components of the analyte sensor may also react with the interferents and contribute to the signal at the carbon working electrode. Aspects of the present disclosure include planing the roughness of the carbon working electrode, either alone or in combination, including compounds that react with the interferents to prevent them from interacting with the carbon working electrode, and / or adding a cleaning electrode that reacts with the interferents to prevent them from interacting with the carbon working electrode. In one or more aspects, the enhancements described herein may reduce the sensitivity of the sensor to the interferents (e.g., by prohibiting or reducing the interferents from generating signals at the working electrode, e.g., by using sensing chemicals and / or membranes to eliminate excess carbon electrode surface), and / or reduce the local concentration of the interferents at the working electrode (e.g., by "pre-reacting" the interferents so that they do not or substantially do not reach the working electrode). Although not required, when the signal of the analyte of interest is not compromised, one or all of the enhancements described herein may be used in conjunction with a working electrode having a low working potential lower than the oxidation potential of the interferent of interest. In some cases, an analyte sensor comprising a low potential working electrode may further comprise a low potential redox mediator to enhance detection of an analyte signal of interest at such a low working potential. Additionally, aspects of the present disclosure include treating the substrate layer below the working electrode, either alone or in combination, to securely attach the membrane to the substrate.

[0048] In some embodiments, the analyte sensor includes: a substrate having an upper surface, an electrode layer disposed on the upper surface and having an elongated body, the elongated body including a proximal end and a distal end, the electrode layer including a surface area of ​​0.15 mm 2 Up to 0.25mm 2 An active working electrode area between the electrodes, wherein the active working electrode area is configured to reduce signals indicative of interfering substances, and at least one sensing point disposed on the active working electrode area, wherein the at least one sensing point includes at least one analyte-responsive enzyme.

[0049] In other embodiments, a method of manufacturing an analyte sensor includes: providing a substrate having an upper surface; providing an electrode layer disposed on the upper surface and having an elongated body, the elongated body including a proximal end and a distal end, the electrode layer including an active working electrode area; providing at least one sensing point on the active working electrode area, wherein the at least one sensing point includes at least one analyte-responsive enzyme; and reducing the surface area of ​​the active working electrode area to 0.15 mm 2 With 0.25mm 2 Between, wherein the active working electrode area is configured to reduce the signal indicative of the interfering substance.

[0050] In other embodiments, a method of using an analyte sensor includes providing an analyte sensor having: a substrate having an upper surface; an electrode layer disposed on the upper surface and having an elongated body, the elongated body including a proximal end and a distal end, the electrode layer including a surface area of ​​0.15 mm 2 Up to 0.25mm 2 An active working electrode area between the electrodes, wherein the active working electrode area is configured to reduce signals indicative of interfering substances, and at least one sensing point is arranged on the active working electrode area, wherein the at least one sensing point includes at least one analyte-responsive enzyme; and an analyte responsive to the at least one analyte-responsive enzyme is sensed using the at least one sensing point.

[0051] The analyte sensor described herein includes a sensor tail, which includes at least one working electrode (particularly a carbon working electrode) and an active area disposed thereon. The mass transport limiting membrane is then disposed on the carbon working electrode (i.e., disposed on the active area and any external carbon working electrode lacking the active area forming the sensor tail). Various aspects of the present disclosure include analyte sensors as described herein, wherein the analyte sensor includes a substrate having an upper surface and a second exposed surface. Specifically, the substrate includes at least one carbon electrode disposed on a first portion of the upper surface of the substrate. Various aspects of the present disclosure include a film disposed on at least a portion of the second exposed surface of the carbon working electrode and the substrate, either individually or in combination.

[0052] Various carbon electrode roughness may be present along the edge of the carbon working electrode, wherein these carbon electrode roughness may not be sufficiently coated or not coated at all with a mass transfer limiting film, thereby providing a carbon surface for interferents to react and contribute to the measurement signal at the working electrode. As used herein, the term "roughness" and its grammatical variants refer to a rough edge along a surface (e.g., along a working electrode). The roughness may have the form of ridges along the edge of the working electrode, resulting in insufficient coating of the mass transfer limiting film at this location. In order to reduce or eliminate such interferent signals, the present disclosure provides planing of one or more edges of the carbon working electrode to remove carbon roughness therefrom, thereby providing a more uniform profile of the working electrode surface. In the case where the working electrode is formed of a material other than carbon, such roughness may also be present in the composition of the particular working electrode ("electrode roughness").

[0053] Separately or in combination with planing one or more edges of a carbon working electrode to remove carbon roughness, the present disclosure further provides an analyte sensor comprising one or more devices for preventing or reducing the proximity of interferents to the working electrode. Specifically, one or more enzymes or chemical compounds may be included in the analyte sensor, which react with the interferent of interest to render the interferent of interest inactive so that it cannot contribute to the measurement signal at the working electrode. Alternatively, or in combination again, a cleaning electrode may be included in the analyte sensor, which reacts with the interferent of interest to render the interferent of interest inactive so that it cannot contribute to the measurement signal at the working electrode.

[0054] Additionally, either individually or in combination, one or more physical features, such as at least one gap in the electrode layer, can be incorporated into the analyte sensor, which allows an inactive portion of the electrode layer to pre-react with the interferent of interest to inactivate the interferent of interest so that it cannot contribute to the measurement signal at the active working electrode area. In addition, either individually or in combination, the present disclosure provides an analyte sensor comprising a reduced available area of ​​the active working electrode surface on the sensor tail (the portion of the sensor used to insert into the tissue), particularly the availability of a carbon working electrode on the sensor tail, on which the interferent can react and contribute a signal unrelated to the analyte by providing a configuration of the working electrode described below.

[0055] Specific details and further advantages of each type of enhancement are described in greater detail herein. Depending on particular needs, the analyte sensors of the present disclosure may be configured to detect one analyte or multiple analytes simultaneously or nearly simultaneously.

[0056] Analyte sensors using enzyme-based detection are generally used to analyze a single analyte, for example, blood sugar, because enzymes are often specific to a particular substrate or substrate class. Analyte sensors using a single enzyme and enzyme systems comprising multiple enzymes acting in unison can be used for this purpose. As used herein, the term "unison" and its grammatical variants refer to a coupled enzymatic reaction, wherein the product of the first enzymatic reaction becomes a substrate for a second enzymatic reaction, and the second enzymatic reaction or subsequent enzymatic reactions are used as the basis for measuring analyte concentrations. In addition, a combination of enzymes and / or enzyme systems can be used to detect more than one analyte type. In order to facilitate detection, the use of an in vivo analyte sensor characterized by an enzyme or an enzyme system may be particularly beneficial to avoid frequent extraction of body fluids, which may otherwise require frequent extraction of body fluids for analyte monitoring.

[0057] In vivo analyte sensors monitor one or more analytes in a biological fluid of interest, such as dermal fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, etc. Such fluids may include one or more interferents that may react with the working electrode of the analyte sensor, react directly on the working electrode itself (e.g., a carbon working electrode), or react with one or more sensing chemical components disposed thereon. As used herein, the term "interferant" and its grammatical variants refer to any electroactive substance present in a body fluid (e.g., interstitial fluid, etc.) that is not an analyte of interest (e.g., an in vivo electroactive substance that is not an analyte of interest). Examples include, but are not limited to, ascorbic acid (vitamin C), glutathione, uric acid, paracetamol (acetaminophen), isoniazid, salicylates, etc., and any combination thereof. The reaction of these interferents with the working electrode may produce an electrochemical signal that is inseparable or not easily separable from the signal derived from the analyte of interest, which may complicate the accurate detection of such analytes, particularly those analytes of low abundance (e.g., as low as sub-millimolar concentrations). Electrochemical signals generated by interferents can be particularly problematic because the signal from the interferent becomes closer in amplitude to the signal from the target analyte. This can occur, for example, when the concentration of the interferent approaches or exceeds the concentration of the analyte of interest. Some interferents are ubiquitous in vivo and are not easily avoided. Therefore, techniques to minimize their effects during in vivo analysis can be highly desirable.

[0058] The present disclosure provides analyte sensor enhancement, which can improve the detection sensitivity of single analyte and multiple analyte combinations with each other, alone or in combination with other enhancements, as explained in further detail below. That is, the present disclosure provides an analyte sensor with reduced carbon working electrode edge roughness and / or admixed compounds or washed electrodes, which can provide a reduced background signal caused by in vivo interferents. Although some aspects of the present disclosure relate to the enhancement of carbon working electrodes, it should be understood that, according to the disclosure herein, other types of electrodes can also be enhanced similarly. Electrode types that can be enhanced by using the disclosure herein also include gold, platinum, PEDOT, etc.

[0059] Before describing the analyte sensors and enhancements thereof of the present disclosure in further detail, a brief overview of suitable in vivo analyte sensor configurations and sensor systems employing analyte sensors will first be provided so that embodiments of the present disclosure may be better understood. Figure 1A diagram of an exemplary sensing system that can be combined with an analyte sensor of the present disclosure is shown. As shown, the sensing system 100 includes a sensor control device 102 and a reader device 120, which are configured to communicate with each other through a local communication path or link 140, and the local communication path or link can be wired or wireless, unidirectional or bidirectional and encrypted or unencrypted. According to some embodiments, the reader device 120 can constitute an output medium for observing the analyte concentration and alarm or notification determined by the sensor 104 or a processor associated therewith, and allowing one or more user inputs. The reader device 120 can be a multi-purpose smart phone or a dedicated electronic reader instrument. Although only one reader device 120 is shown, multiple reader devices 120 can exist in some cases.

[0060] The reader device 120 may also communicate with the remote terminal 170 and / or the trusted computer system 180 via communication paths / links 141 and / or 142, respectively, which may also be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. The reader device 120 may also or alternatively communicate with a network 150 (e.g., a mobile phone network, the Internet, or a cloud server) via communication path / link 151. The network 150 may also be communicatively coupled to the remote terminal 170 via communication path / link 152, and / or communicatively coupled to the trusted computer system 180 via communication path / link 153. Alternatively, the sensor 104 may communicate directly with the remote terminal 170 and / or the trusted computer system 180 without the presence of an intermediate reader device 120. For example, according to some embodiments, sensor 104 may communicate with remote terminal 170 and / or trusted computer system 180 via a direct communication link to network 150, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is incorporated herein by reference in its entirety.

[0061] Any suitable electronic communication protocol may be used for each communication path or link, such as near field communication (NFC), radio frequency identification (RFID), or Low energy protocols, WiFi, etc. According to some embodiments, in addition to the primary user, individuals interested in the user's analyte level may also access the remote terminal 170 and / or the trusted computer system 180. The reader device 120 may include a display 122 and an optional input component 121. According to some embodiments, the display 122 may include a touch screen interface.

[0062] 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) is communicatively coupled to the sensor 104, the processor being physically located within 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, which is suitable for adhering the sensor housing 103 to a tissue surface, such as skin.

[0063] The sensor 104 is adapted to be at least partially inserted into a tissue of interest, for example, within the dermis or subcutaneous layer of the skin. Alternatively, the sensor 104 may be adapted to penetrate the epidermis. Further alternatively, the sensor 104 may be disposed on the surface layer and not penetrate the tissue, for example, when analyzing one or more analytes in sweat on the skin. The sensor 104 may include a sensor tail of sufficient length for inserting a desired depth into a given tissue. The sensor tail may include at least one working electrode and an active region, the active region including an enzyme or enzyme system configured to analyze one or more analytes of interest.

[0064] The counter electrode may be present in combination with at least one working electrode, and optionally further combined with a reference electrode. Figures 2A to 4 The specific electrode configuration on the tail of the sensor is described in more detail. According to different embodiments, one or more enzymes in the active region can be covalently bonded to the polymer comprising the active region. Alternatively, the enzyme can be non-covalently bonded in the active region, for example, by encapsulation or physical entrainment. One or more analytes can 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 a specific embodiment, the analyte sensor of the present disclosure can be suitable for analyzing skin fluid or interstitial fluid to determine the analyte concentration in the body. However, it should be understood that the entire sensor control device 102 can have one or more different configurations, allowing complete implantation in one or more body fluids under the organization for analyzing one or more analytes of interest without departing from the scope of the present disclosure.

[0065] Reference again Figure 1 , the sensor 104 can automatically forward the data to the reader device 120. For example, the analyte concentration data can be automatically and periodically transmitted, for example, when the data is obtained or at a certain frequency after a certain period of time has passed, and the data is stored in the memory until transmission (for example, every minute, five minutes or other predetermined period of time), for example, by or Low energy protocol. Data associated with different analytes can be forwarded at the same or different frequencies and / or using the same or different communication protocols. 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, when the sensor electronics enter the communication range of the reader device 120, RFID technology can be used to transmit data from the sensor 104. Before being transmitted to the reader device 120, the data can remain stored in the memory of the sensor 104. Therefore, the user does not have to maintain close proximity to the reader device 120 at all times, but can automatically or non-automatically upload data when it is convenient. In other embodiments, a combination of automatic and non-automatic data transmission can be implemented. For example, data transmission can continue automatically until the reader device 120 is no longer within the communication range of the sensor 104.

[0066] The introducer may be temporarily present to facilitate the introduction of the sensor 104 into the tissue. In an illustrative embodiment, the introducer may include a needle or similar sharp object or a combination thereof. It should be appreciated that in alternative embodiments, other types of introducers may be present, such as a sheath or a blade. More specifically, a needle or other introducer may temporarily reside near the sensor 104 before the tissue is inserted and then withdrawn. When present, the needle or other introducer may facilitate the insertion of the sensor 104 into the tissue by opening an access path followed by the sensor 104. For example, according to one or more embodiments, the needle may facilitate penetration of the epidermis as an access path to the dermis to allow the implantation of the sensor 104. After the access path is opened, the needle or other introducer may be withdrawn without the danger of sharp objects. In an illustrative embodiment, a suitable needle may be solid or hollow, beveled or non-beveled, and / or have a circular or non-circular cross section. In a more specific embodiment, a suitable needle may be comparable to an acupuncture needle in cross-sectional diameter and / or tip design, and an acupuncture needle may have a cross-sectional diameter of approximately 250 microns. However, it should be appreciated that suitable needles may have larger or smaller cross-sectional diameters if desired for a particular application. For example, needles having cross-sectional diameters ranging from about 300 microns to about 400 microns may be used.

[0067] In some embodiments, the tip of the needle (when present) may be angled above the end of the sensor 104 so that the needle first penetrates the tissue and opens an access path for the sensor 104. In other illustrative embodiments, the sensor 104 may be located within a lumen or groove of the needle, which similarly opens an access path for the sensor 104. In either case, after facilitating sensor insertion, the needle may then be withdrawn.

[0068] Sensor configurations featuring a single active area configured to detect a corresponding single analyte may employ a two-electrode or three-electrode detection pattern, as described herein with reference to Figure 2A-2C Further described below. Figure 3A-Figure 4 Sensor configurations are described that feature two distinct active areas for detecting separate analytes on separate working electrodes or on the same working electrode. Sensor configurations with multiple working electrodes may be particularly advantageous for incorporating two distinct active areas on the same sensor tail, since the signal contribution from each active area can be more easily determined by interrogating each working electrode individually. Each active area may be coated with a mass transport limiting membrane of the same or different composition.

[0069] When there is a single working electrode in the analyte sensor, the three-electrode sensor configuration may include a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration may include a working electrode and a second electrode, wherein the second electrode may be used as a counter electrode and a reference electrode (i.e., a counter / reference electrode). The various electrodes may be at least partially stacked (layered) on each other and / or laterally spaced apart from each other on the sensor tail. In any sensor configuration disclosed herein, the various electrodes may be electrically isolated from each other by dielectric materials or similar insulators.

[0070] An analyte sensor featuring multiple working electrodes may similarly include at least one additional electrode. When one additional electrode is present, the one additional electrode may serve as a counter / reference electrode for each of the multiple working electrodes. When 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 additional electrode may serve as a reference electrode for each of the multiple working electrodes.

[0071] Any of the working electrode configurations described below may benefit from the further disclosure below directed to reducing the availability of edge roughness on the working electrode on the sensor tail.

[0072] Figure 2A A diagram of an illustrative dual electrode analyte sensor configuration is shown, which is applicable to the disclosure herein. As shown, the analyte sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter / reference electrode 216. Alternatively, the working electrode 214 and the counter / reference electrode 216 can be located on the same side of the substrate 212, with a dielectric material (configuration not shown) inserted therebetween. An active region 218 is disposed as at least one layer on at least a portion of the working electrode 214. The active region 218 may include a plurality of points or a single point, which is configured for detection of an analyte, as further discussed herein.

[0073] Still reference Figure 2AAccording to some embodiments, the membrane 220 covers at least the active area 218, and may optionally cover some or all of the working electrode 214 and / or the counter / reference electrode 216 or cover 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 flow of analytes to the active area 218 (i.e., the membrane 220 is a mass transport limiting membrane with a certain permeability to the analyte of interest). The composition and thickness of the membrane 220 may vary to facilitate the desired analyte flow to the active area 218, thereby providing the desired signal strength and stability. The analyte sensor 200 can be used to analyze analytes by any of coulometric, amperometric, voltammetric or potentiometric electrochemical detection techniques.

[0074] Figure 2B and Figure 2C 201 and 202 ( Figure 2B and Figure 2C ), the three-electrode analyte sensor configuration may be similar to Figure 2A . With the additional electrode 217, the counter / reference electrode 216 can be used as a counter electrode or a reference electrode, and the additional electrode 217 performs another electrode function not otherwise described. The working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed on the working electrode 214 or the electrode 216 with a separation layer of dielectric material therebetween. For example, as Figure 2B As shown, 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 an opposite side of substrate 212, such as Figure 2C Therefore, in some embodiments, electrode 214 (working electrode) and electrode 216 (counter electrode) may be located on opposite sides of substrate 212, and electrode 217 (reference electrode) may be located on one of electrodes 214 or 216 and separated therefrom by a dielectric material. Reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, and the location of reference material layer 230 is not limited to Figure 2B and Figure 2C As shown in the position. Figure 2A As shown in sensor 200, active area 218 in analyte sensors 201 and 202 may include multiple spots or a single spot. Additionally, analyte sensors 201 and 202 may also be used to analyze analytes using any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

[0075] Like analyte sensor 200, membrane 220 can also cover active area 218 and other sensor components in analyte sensors 201 and 202, thereby acting as a mass transport limiting membrane. In some embodiments, additional electrode 217 can be coated with membrane 220. Membrane 220 can also be produced by dip coating or in-situ photopolymerization and have different or identical compositions at different locations. Figure 2B and Figure 2C All electrodes 214, 216, and 217 have been depicted as being coated with membrane 220, but it should be appreciated that in some embodiments, only the working electrode 214 or the active area 218 may be coated. In addition, the thickness of the membrane 220 at each electrode 214, 216, and 217 may be the same or different. As in the dual electrode analyte sensor configuration ( Figure 2A ), in Figure 2B and Figure 2C In the sensor configuration of FIG. 2 , one or both faces of the analyte sensors 201 and 202 may be coated with the membrane 220 , or the entirety of the analyte sensors 201 and 202 may be coated. Figure 2B and Figure 2C The three-electrode sensor configuration shown in should be understood as not limiting the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of the present disclosure.

[0076] Figure 3A An exemplary configuration of a sensor 203 having a single working electrode having two distinct active areas disposed thereon is shown. Figure 3A Similar to Figure 2A , except that there are two active areas on the working electrode 214: a first active area 218a and a second active area 218b, which respond to different analytes and are laterally spaced from each other on the surface of the working electrode 214. The active areas 218a and 218b may include multiple points or a single point configured to detect each analyte. At the active areas 218a and 218b, the composition of the membrane 220 may vary or be the same. 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 discussed further below.

[0077] Figure 3B and Figure 3C Cross-sectional views of exemplary three-electrode sensor configurations are shown for sensors 204 and 205, respectively, each featuring a first active area 218a and a second active area 218b disposed on a single working electrode. Figure 3B and Figure 3C In other respects similar to Figure 2B and Figure 2C , which can be better understood by referring to them. Figure 3A Likewise, the composition of the film 220 may vary or be the same at the active regions 218a and 218b.

[0078] Figure 4 A cross-sectional view of an exemplary analyte sensor configuration having two working electrodes, a reference electrode and a counter electrode is shown, which is applicable to the disclosure herein. As shown, the analyte sensor 400 includes working electrodes 404 and 406 disposed on opposite sides of a substrate 402. A first active area 410a is disposed on the surface of the working electrode 404, and a second active area 410b is disposed on the surface of the working electrode 406. The counter electrode 420 is electrically isolated from the working electrode 404 by a dielectric layer 422, and the reference electrode 421 is electrically isolated from the working electrode 406 by a dielectric layer 423. External dielectric layers 430 and 432 are located on the reference electrode 421 and the counter electrode 420, respectively. According to various embodiments, a membrane 440 may cover at least the active areas 410a and 410b, and other components of the analyte sensor 400 or the entire analyte sensor 400 may also be optionally coated with a membrane 440. Likewise, if desired, membrane 440 may vary in composition at active regions 410a and 410b to provide appropriate permeability values ​​for differentially regulating analyte flux at each location.

[0079] Having multiple working electrodes and being different from Figure 4 Alternative sensor configurations to the configuration shown may feature counter / reference electrodes rather than separate counter and reference electrodes 420, 421, and / or feature layer and / or film arrangements different from those explicitly described. For example, the locations of counter and reference electrodes 420, 421 may be different from those shown. Figure 4 In addition, working electrodes 404 and 406 do not need to be Figure 4 The illustrated approach is on opposite sides of the substrate 302 .

[0080] The carbon working electrode may suitably include a working electrode in any analyte sensor disclosed herein. Although carbon working electrodes are very commonly used in electrochemical detection, it is not without difficulties to use carbon working electrodes in electrochemical detection. Specifically, only when the active area interacts with the analyte and transfers electrons to the carbon working electrode portion adjacent to the active area, a current associated with the analyte of interest will be generated. Body fluids containing analytes of interest also interact with the carbon surface of the carbon working electrode that is not coated with the active area, and do not contribute to the analyte signal because there is no enzyme or enzyme system at these locations to promote the transfer of electrons from the analyte to the working electrode. However, interferents may be oxidized in the working electrode portion lacking the active area and contribute background to the entire signal. Therefore, a carbon working electrode with an in vitro (or "exposed") carbon area on the electrode surface has no meaningful contribution to the analyte signal and may result in a contributing background signal in some cases. Other electrodes with excessively large surface areas that do not directly detect analytes of interest may experience similar background signals and may be enhanced by modifying the disclosure herein.

[0081] Although various interferents can interact with the working electrode of the analyte sensor described herein, ascorbic acid is an example of an interferent commonly present in biological fluids that can produce a background signal on the carbon working electrode. For example, ascorbic acid is oxidized on the working electrode to produce dehydroascorbic acid. Various embodiments of the present disclosure will be described herein with reference to the interferent as ascorbic acid. However, it should be understood that the embodiments and analyte sensor configurations described herein are equally applicable to other interferents (electroactive substances in body fluids with analytes of interest).

[0082] As mentioned above, the active region described herein can be a single sensing layer or a sensing layer having multiple sensing points. Figure 5, shows a top view of a conventional carbon working electrode 500 having an active area 504 including a plurality of sensing sites 518 disposed thereon. When an analyte interacts with the active area 504, only the portion of the carbon working electrode 500 including the sensing sites 518 contributes a signal associated with the analyte of interest. Although the carbon working electrode 500 shows six sensing sites 518 within the active area 504, it should be understood that less than or more than six sensing sites 518 may be included on the carbon working electrode 500 without departing from the scope of the present disclosure. The in vitro carbon area 510 is not directly covered by the sensing sites 518 and does not contribute a signal associated with the analyte, but may generate background signals associated with one or more interferents. Therefore, the oxidation of interferents at the carbon working electrode 500 is proportional to the area of ​​the in vitro carbon area 510 available for interaction with the interferent. In fact, the oxidation of ascorbic acid on the carbon working electrode 500 is approximately linearly proportional to the area of ​​the available in vitro carbon area 510.

[0083] As shown, active area 504 is discontinuous and in the form of a plurality of sensing points 518. As defined herein, the term "discontinuous" and its grammatical variations mean that any single point (sensing element) does not share an edge or boundary with an adjacent point (e.g., does not touch).

[0084] The sensor tail including the carbon working electrode 500 described in the present disclosure can be prepared on a template substrate material (see Figure 2A-2B , Figure 3A-3C , Figure 4 ). During the sensor manufacturing process, the sensor tail including the carbon working electrode 500 is subsequently segmented by one or more means. The segmentation can be achieved by one or more cutting or separation schemes, including but not limited to laser segmentation, cutting, shearing, stamping, etc. The segmentation of the sensor tail can be performed before or after the active area is coated on the carbon working electrode 500 toward the distal end of the sensor tail (i.e., the portion of the sensor tail that will be inserted deepest into the group). As used herein, the distal "tip" of the sensor tail refers to the distalmost edge of the sensor tail or the portion that is inserted deepest into the tissue.

[0085] One or more portions of the sensor tail are laser segmented, typically requiring multiple laser passes to cut the sensor tail into a desired shape. The tip of the sensor tail includes the working electrode and at least a portion of the active area. Typically, the laser segmented sensor tail has a width in the range of about 100 μm to about 500 μm and a length of about 3 mm to about 10 mm, including any values ​​and subsets therebetween. Typically, the distal portion of the sensor tail occupies a distal length of about 0.5 mm to about 5 mm, including any values ​​and subsets therebetween. After laser segmentation, a mass transport limiting film is deposited on at least the sensor tip including the active area.

[0086] In one or more aspects of the present disclosure, carbon roughness may exist along the edge of the carbon electrode due to the laser segmentation process before the mass transport limiting membrane is provided. These carbon roughness may provide a surface on which interferents may react and contribute background signals to the analyte sensor.

[0087] Laser segmentation of the carbon working electrode may result in the formation of carbon roughness having a width of about 50 μm or less, for example, in the range of about 5 μm to about 50 μm, or about 10 μm to about 30 μm, including any values ​​and subsets therebetween. In addition, these carbon roughnesses may have a height of about 20 μm or less, for example, in the range of about 1 μm to about 20 μm, or about 2 μm to about 10 μm, as described in more detail below, including any values ​​and subsets therebetween. Therefore, these carbon roughnesses can provide a considerable area with which interferents can interact. In addition, the roughnesses can cause inconsistent coverage (thickness) of the mass transport limiting membrane. As described below, these carbon roughnesses can be reduced or removed by one or more laser planing methods.

[0088] First reference Fig. 6A , a close-up of an example of a laser segmented carbon working electrode for use as at least a portion of a sensor tail is shown, prior to any laser planing to reduce or remove carbon asperities in accordance with the present disclosure, wherein the carbon working electrode has no mass transport limiting film deposited thereon. Electrodes cut to a desired shape by other means may have asperities of similar appearance and size. Carbon asperities are evident along the edges of the working electrode with which interferents may react. Figure 6B Shown along Fig. 6A Depth profile of the line indicated in , evaluated along the identified 430.71 μm profile width. ZEGAGE, Inc. (Middlefield, Connecticut) TM The 3D optical profiler obtains the 3D optical profile. Figure 6BAs shown, the carbon asperities along the segmented (ablated) edge of the example segmented sensor tail are up to about 30 μm wide and up to about 10 μm high.

[0089] Mass transfer limiting membranes can reduce or prevent interfering substances from entering the in vitro carbon region (e.g. Figure 5 When disposed on a laser segmented carbon working electrode (and active region thereon), the thickness of the film varies across the width of the working electrode, particularly where significant asperities are present. Typically, the film is thinnest at the electrode edge, where carbon asperities are located. Thus, even if a film is present, the carbon asperities may not be sufficiently coated by the film to reduce or prevent interferents from interacting therewith.

[0090] refer to Fig. 7A , showing a close-up of an example laser segmented carbon working electrode having a mass transport limiting film deposited thereon, prior to any laser planing to reduce or remove carbon asperities in accordance with one or more aspects of the present disclosure. Figure 7B Shown along Fig. 7A The depth profile of the line shown in was evaluated along the identified 345.53 μm profile width. ZEGAGE, Inc. (Middlefield, Connecticut) TM The 3D optical profiler obtains the 3D optical profile. Figure 7B As shown, the film is quite thin along the segmentation ridges of the carbon working electrode.

[0091] In various aspects, the present disclosure provides methods and analyte sensors in which a carbon working electrode used to form the tail of the sensor is planed by one or more single or multiple pass laser planing cuts, alone or in combination with the additional enhancements described herein. In some embodiments, a single pass laser planing method is used, in which the laser depth is set to be less than the thickness of the working electrode. For example, laser planing can remove the top of the carbon layer, for example, 50% of the top of the carbon layer. The carbon layer is typically in the range of 10 μm (without asperities); in some embodiments, about 5 μm (or about 50%) can be removed therefrom (e.g., see FIG. 9C ). Laser planing according to the present disclosure can remove or reduce the protrusion of asperities.

[0092] In some embodiments, more than one (e.g., less than about 10) single-pass laser planing cuts may be made, each progressively closer to the midline length of the working electrode, to reduce or eliminate carbon asperities. In this manner, an initial laser planing cut may be made at the outermost location of any single carbon asperity, and seven subsequent laser planing cuts may be made toward the midline length of the working electrode to produce a milled edge, which may be a step edge of approximately 90° or a beveled edge (i.e., an edge that is not perpendicular to the electrode surface) if, for example, the nearest laser planing cut toward the midline of the electrode does not produce a true 90° angle (see Figure 8 , the laser planed cuts (edges) 810 are shown as beveled edges rather than sheared 90° angled edges). For example, in one embodiment, about 2 to about 10 single-pass laser planed cuts may be made, each having a separation distance of about 1 μm to about 100 μm, including any values ​​and subsets therebetween. The selection of the specific number of laser planed passes and their spacing may be based on many factors, including, but not limited to, the shape and size of the carbon asperities, the length and width of the working electrode, the coverage profile of any films disposed thereon, and the like, and any combination thereof.

[0093] Laser planing can be preferably used to remove at least about 5% to about 95% of the total carbon asperity area from a single sensor tail comprising a carbon working electrode, including any values ​​and subsets therebetween. In some embodiments, up to 100% of the carbon asperity is removed, or about 5% to about 50% is removed, including any values ​​and subsets therebetween. In a preferred embodiment, at least about 50% of the carbon asperity is removed. The specific amount of carbon asperity removal can be based on a variety of factors, including but not limited to the density, shape and size of the carbon asperity, the concentration of the analyte of interest compared to the concentration of interferents available in the body fluid being analyzed, and the like, and any combination thereof.

[0094] Figure 8 A photograph of the edge of a sensor tail 800 is shown, showing a laser cut cut (ridge) 805 recessed from the edge of the sensor tail and a laser planed cut (edge) 810 to remove a portion of the edge of a carbon working electrode (carbon or electrode layer) in accordance with one or more embodiments of the present disclosure. That is, the laser planed cut 810 is intended to reduce carbon asperities along the upper or top portion of the carbon electrode (e.g., where the active region is located), while a thinner portion of the working electrode remains along the periphery (and at the opposite portion of the electrode that does not include the active region).

[0095] In one or more aspects of the present disclosure, an analyte sensor including an interferent-reactant substance is provided, either alone or in combination with any other enhancement to reduce or eliminate an analyte sensor signal associated with an interferent. As used herein, the term "interferent-reactant substance" and its grammatical variants refer to any compound, whether biological or non-biological, that is capable of reacting with an interferent and deactivating it so that it cannot contribute to the measurement signal at the working electrode. That is, an interferent-reactant substance can be included as part of an analyte sensor so that the interferent can be "pre-reacted" before it can react on the working electrode of the analyte sensor. Therefore, the interferent-reactant substance can eliminate or reduce the local concentration of the interferent present at or accessible to the working electrode, thereby eliminating or reducing the signal attributed to such an interferent because the interferent never reaches the excess area of ​​the working electrode.

[0096] The various aspects of the method and analyte sensor in conjunction with the interferor-reactant material are described with reference to the interferor-reactant material for ascorbic acid elimination or removal, and it should be understood that the enhancement described herein is applicable to other potential interferors without limitation. Such interferors may include, for example, ascorbic acid (vitamin C), glutathione, uric acid, paracetamol (acetaminophen), isoniazid, salicylates, etc. and any combination thereof. In a non-limiting example, the interferor-reactant material of the present disclosure may be ascorbic acid oxidase (reacting with ascorbic acid), glutathione peroxidase (reacting with glutathione), xanthine oxidase (reacting with uric acid), urate oxidase (reacting with uric acid), cytochrome P450 (reacting with paracetamol), eosinophil peroxidase (reacting with isoniazid), salicylic acid oxidase (reacting with salicylate), other enzymes capable of oxidizing, reducing or otherwise reacting and decomposing the interferor of interest, etc. and any combination thereof. In alternative or combined embodiments, the interferor-reactant material may be a non-enzyme material. For example, various metal oxides (e.g., manganese oxide (MnO 2 ) or iron oxide (Fe 2 CO 3 )) can oxidize or otherwise react and decompose ascorbic acid and serve as one or more interferer-reagent species of the present disclosure.

[0097] refer to Fig. 9A , depiction of a conventional sensor is shown demonstrating that ascorbic acid reacts with excess working electrode and potential interferents of the sensing chemistry to produce a signal attributable to the ascorbic acid. Fig. 9A The sensor does not incorporate an interferent-reactant species. As shown, the ascorbic acid interferent diffuses from the interstitial fluid toward the sensor working electrode where it can be oxidized at least on the excess working electrode 908 and / or otherwise on the sensing chemistry.

[0098] According to various aspects of the present disclosure, Fig. 9B Shown is a Fig. 9A , the interferent-reactant species is specifically an interferent-reactant species of ascorbate oxidase (AOx). As shown, the ascorbate oxidase reacts with ascorbic acid before contacting the working electrode or sensing chemical, thereby preventing the reacted ascorbic acid from contributing to the analyte signal. It is noted that Fig. 9B The sensors depicted in can have any configuration and / or components of the sensors described herein, without limitation.

[0099] The specific location of one or more interfering substances-reacting substances included in the analyte sensor of the present disclosure is not considered to be particularly limited. For example, an interfering substance-reacting substance is provided as part of the analyte sensing active area; a film coating the analyte sensing active area; a layer thereof on any one of the working electrode, the analyte sensing active area and / or the membrane coating; and so on; and any combination thereof. When provided as a part of an active layer, a membrane or its own layer, it can be free floating or otherwise fixed (e.g., covalently or non-covalently bound) in a polymer matrix. The specific concentration of the interfering substance-reacting substance contained in the analyte sensor (at any one or more locations) can depend on many factors, including but not limited to, the specific analyte of interest, the specific interfering substance of interest, the in vivo location of the analyte sensor, etc. and any combination thereof. In some cases, when the interfering substance-reacting substance is an enzyme, the total amount of the interfering substance-reacting substance can be in the range of about 0.01 units to about 100 units of activity per sensor, including any value and subset therebetween. For example, a sensor with an interfering substance-reacting substance of ascorbic acid oxidase can have an activity of about 0.5 units per sensor. In other cases, when the interferent-reactant substance is a non-enzymatic compound (e.g., a metal oxide), the total amount of the interferent-reactant substance can be in the range of about 0.1 μg to about 100 μg per sensor, including any values ​​and subsets therebetween. 2 The interferent-reactant substance to the sensor may be present in an amount of approximately 1 μg per sensor.

[0100] As described above, typically, the interferent-reactant materials described herein, whether present as a layer itself, present in a membrane, or present in an active region, will be present in a polymer matrix, whether mobile or fixed. The polymer matrix can be composed of any polymer, cross-linker and / or additive compatible with the interferent-reactant material selected for the analyte sensor, which interferent-reactant material does not interfere with the sensing chemical substance. Each polymer, cross-linker and / or additive can be selected from any one described herein without limitation. For example, non-limiting examples of such polymers include poly (4-vinyl pyridine) and poly (N-vinyl imidazole) (PVI) or copolymers thereof, sulfonated tetrafluoroethylene fluoropolymer copolymers (e.g., NAFION TM , Komu Corporation, Wilmington, DE), polyvinyl alcohol, and any combination thereof; non-limiting examples of cross-linking agents include triglycidyl ether (gly3) and / or PEDGE and / or polydimethylsiloxane diglycidyl ether (PDMS-DGE); non-limiting examples of additives include stabilizers, e.g., albumin, and / or any other stabilizer described herein.

[0101] In one or more aspects of the present disclosure, an analyte sensor including a cleaning electrode (e.g., with or without the integration of an interferent-reactant substance and / or roughness planing) is provided, either alone or in combination with any other enhancement to reduce or eliminate an analyte sensor signal associated with an interferent. As described herein, the term "cleaning electrode" and its grammatical variants refer to an electrode that is capable of reacting with an interferent to inactivate the interferent so that it cannot contribute to the measurement signal at the working electrode. That is, a cleaning electrode can be included as part of an analyte sensor so as to "pre-react" with the interferent before the interferent can react on the working electrode of the analyte sensor. Therefore, similar to the presence of an interferent-reactant substance, the cleaning electrode can eliminate or reduce the local concentration of an interferent present or accessible on the working electrode, thereby eliminating or reducing the signal due to such an interferent because the interferent never reaches the excess area of ​​the working electrode.

[0102] In one or more aspects, the cleaning electrode can be positioned in a facing relationship and spatially offset from the working electrode. That is, the active area of ​​the working electrode and the active area of ​​the cleaning electrode may or may not be disposed on a substrate, facing each other, and separated by a gap. Preferably, the gap is a thin layer between the two electrodes, allowing body fluid to pass therebetween, including the analyte of interest and any interferents therein. The configuration of the cleaning electrode relative to the working electrode desirably allows the body fluid to contact the cleaning electrode for a sufficient period of time to react with any interferents before the body fluid reaches the working electrode. The cleaning electrode does not include any sensing chemicals, and therefore, the analyte of interest does not react with it. In this way, the body fluid has been free of or substantially free of interferents, and the signal obtained at the working electrode is entirely or primarily attributed to the analyte of interest.

[0103] Various electrode configurations can be used to ensure that body fluid contacts the wipe electrode before the working electrode. Fig.10 One such non-limiting configuration is shown in FIG. As shown, the wipe electrode and the working electrode are in a facing relationship, and the working electrode is recessed, or has a smaller width than the wipe electrode. Although Fig.10 The particular configuration of working and cleaning electrodes shown in FIG. 1 has a rectangular shape, but other configurations are equally applicable to the embodiments described herein, for example, square, circular, spiral, etc. In general, the length of the working and cleaning electrodes can be greater than their width.

[0104] In one or more aspects, the width of the cleaning electrode to the working electrode can be in the range of about 2:1 to about 50:1, including any values ​​and subsets therebetween. For example, in some cases, the cleaning electrode can have a width in the range of about 300 μm to about 5000 μm, and the working electrode can have a width in the range of about 100 μm to about 1000 μm, including any values ​​and subsets therebetween. These dimensions include the direction in which the thin layer can extend the length of the sensor tail, with a linear or non-linear shape, so as to increase the ratio between the size of the cleaning electrode and the size of the working electrode without making the sensor tail too wide for practical in vivo use (insertion).

[0105] A thin layer is formed between the purging electrode and the working electrode. The thin layer can be in the range of about 10 μm to about 100 μm, including any values ​​and subsets therebetween. In some cases, the thin layer can be about 50 μm. The thin layer is typically formed by sealing fluid channels (e.g., thin layer "cells") along two opposing edges of the purging electrode so that body fluids can enter the space between the unsealed thin layer spaces in a controlled manner to ensure that they reach the purging electrode before the working electrode. In general, a larger ratio between the purging electrode surface area and the thin layer volume may be preferred to maximize the opportunity for solutes (e.g., interferents) to interact with the purging electrode. For example, refer to Fig.10 , the thin layer between the cleaning electrode and the working electrode can be formed by applying an adhesive, separator or other non-restrictive separation means along the width edge of the electrode. In this way, the body fluid is guided through the edge along the length. Therefore, when the body fluid including the interferent and the analyte of interest diffuses through the thin layer, it has sufficient interaction with the cleaning electrode before reaching the working electrode. Thus, an analyte sensor including such a cleaning electrode does not need to (although it may) rely on a membrane to limit the interaction of the interferent with the working electrode, which can provide manufacturing and cost advantages.

[0106] In various embodiments, the thin layer may be modified with surfactants, hydrogels, membranes, or other materials that aid in the introduction of body fluids into the thin layer, to aid in biocompatibility, to provide microbicidal or microstatic properties, etc., and any combination thereof.

[0107] In one or more aspects, the purge electrode can be independently controlled, for example, by adjusting the purge electrode potential in order to fine-tune its reaction efficiency with a particular interferent. Typically, the efficiency of the purge electrode in reacting with the interferent increases with increasing potential. The purge electrode potential can be in the range of about -1000 mV to about +1000 mV, including any values ​​and subsets therebetween. Typically, the purge electrode potential can be any operating potential within the potential window of water; that is, the potential at which the relevant solvent water of the body fluid itself is not oxidized or reduced. In some embodiments, the purge electrode potential can be relative to an included reference electrode (e.g., an Ag / AgCl reference electrode), which can be shared by the purge electrode and the working electrode. In addition, operating the purge electrode at a generally negative potential can achieve additional purge of oxidants (e.g., oxygen), which may be beneficial depending on the analyte of interest. That is, the purge electrode can be used to remove oxygen to reduce its contribution to the analyte signal.

[0108] The composition of the cleaning electrode is not considered to be particularly limited and can be made of known electrode materials and can be the same or different from the composition of the working electrode. Examples of suitable materials include, but are not limited to, carbon, gold, platinum, PEDOT, etc. In some cases, among other advantages, the composition of the cleaning electrode can be modified or supplemented with materials that are specific to react with the interferent of interest, or the surface area of ​​the cleaning electrode can be increased. It should also be understood that the interferent-reactant material can be coated on the cleaning electrode in any manner, as described above, to further enhance the elimination or reduction of the interferent reaching the working electrode.

[0109] In some embodiments, instead of having a thin layer configuration containing a cleaning electrode, the cleaning electrode composition can be selected so that it is permeable to the analyte of interest. In this way, the cleaning electrode can be stacked above the working electrode, with an analyte permeable membrane or dielectric layer therebetween to avoid sensor short circuits, and without a thin layer. That is, an insulating material that is permeable to the analyte of interest is disposed between the permeable cleaning electrode and the working electrode containing the analyte detection material. In this way, and based on the same basic principles as the above-mentioned thin layer cleaning electrode configuration, a body fluid including an analyte of interest and an interfering substance will contact the permeable cleaning electrode, and before the body fluid (including the analyte of interest and no or little interfering substance) contacts the working electrode, the interfering substance reacts at the permeable cleaning electrode and is eliminated or otherwise reduces the concentration. Therefore, the cleaning electrode can eliminate or reduce the local concentration of the interfering substance present or accessible at the working electrode, thereby eliminating or reducing the signal attributed to such an interfering substance, because the interfering substance will not reach the excess area of ​​the working electrode.

[0110] Fig.11 One such non-limiting configuration of an analyte sensor including a permeable wash electrode is shown in . As shown, the working electrode includes a sensing chemical disposed thereon to form an active area (either as a single area or including multiple discrete points). On the active area is an analyte permeable insulating material, for example, any polymer described herein, so long as the analyte of interest can diffuse through it. For example, the analyte permeable insulating layer can be a diffusion limiting membrane. The analyte permeable wash electrode is disposed on the diffusion limiting membrane. Although the analyte permeable wash electrode needs to be the same size as the basic working electrode, in a preferred embodiment, the analyte permeable wash electrode has a shape and size that contacts the body fluid before the insulating layer or working electrode. An outer membrane may be included to provide additional diffusion limiting properties, biocompatibility properties, microbicidal or microstatic properties, protection for the permeable wash electrode, and the like, and any combination thereof. As Fig.11 As shown, the interferent can diffuse through the outer membrane to the permeable cleaning electrode, where the interferent reacts and becomes inactive so that the interferent cannot contribute to the measurement signal at the working electrode. In contrast, the analyte of interest does not react with the cleaning electrode (which has no analyte sensing chemistry), and the analyte diffuses through the outer membrane, cleaning electrode, and insulating material to the sensing layer on the working electrode. As discussed below Fig.25 As shown, another non-limiting configuration may employ a "well" structure with an analyte permeable purge electrode.

[0111] Fig.12Another non-limiting configuration of an analyte sensor including a permeable purge electrode is shown. In this configuration, the permeable purge electrode is combined with an impermeable purge electrode trace to provide electrical contact so that an electric potential can be applied to the permeable purge electrode. The impermeable purge electrode can be printed on a dielectric material and sense the chemical substances distributed on the exposed portion of the working electrode. Portions of sensor A can be produced and segmented. Thereafter, dip coating can be performed to apply an inner polymer film and solidify, then dip coating to apply a permeable purge membrane and solidify, and then finally dip coating to apply an outer polymer film. This configuration can provide manufacturing and cost advantages.

[0112] Fig.27A Shows Figure 5 2700. In this example embodiment, the electrode layer 2700 includes an elongated body including a proximal end 2701 and a distal end 2702. The electrode layer 2700 can have a first active working electrode area 2704 disposed thereon, which includes at least one sensing point 2718 having at least one analyte-responsive enzyme disposed thereon. When the analyte interacts with the active area 2704, only the portion of the electrode layer 2700 including the sensing point 2718 contributes to the signal associated with the analyte of interest. Although the electrode layer 2700 shows six sensing points 2718 within the active area 2704, it should be understood that less than or more than six sensing points 2718 can be included on the electrode layer 2700 without departing from the scope of the present disclosure. In fact, the sensing points 2718 can have any configuration described herein without limitation.

[0113] The electrode layer 2700 also includes a second electrode portion 2710 and at least one gap 2719 that separates the active area 2704 from the second electrode portion 2710. In the illustrated embodiment, the U-shaped gap 2719 extends from the proximal end 2701 of the elongated body on the first side of the first active working electrode region to near the distal end 2702 of the elongated body of the electrode layer, and returns to the proximal end of the elongated body on the second side of the first active working electrode region 2704. The gap 2719 and the second electrode portion 2710 do not include any sensing chemicals, so the analyte of interest does not react with it. In addition, since the gap 2719 electrically separates the active area 2704 from the second electrode portion 2710, any interferents in contact with the second battery portion 2710, such as ascorbic acid in body fluids, will not generate interferent signals to the sensor. Therefore, the effective electrode area affected by potential interferents is reduced, so the overall interference with the sensor signal is reduced. In some embodiments, the second electrode portion 2710 is not connected to the sensor current conductive trace. Alternatively, the second electrode portion 2710 can be connected to a conductive trace, as further described below.

[0114] According to the disclosed subject matter, Fig.27A The U-shaped gap 2719 of the electrode layer 2700, or any gap disclosed herein, can have a linear configuration or a non-linear configuration. For purposes of illustration and not limitation, Fig.27B Shows something like Fig.27A A top view of an electrode layer of a device 2700, wherein the gap 2720 has a non-linear configuration. As shown, the gap includes a wavy pattern, and in some embodiments, the wavy pattern can be designed to closely surround the sensing point, which can further reduce the amount of active area 2704 to reduce the amount of sensor interference. Non-limiting examples of other non-linear configurations include spiral patterns, curved patterns, undulating patterns, curled patterns, etc. As used herein, the term "U-shaped" includes ends that can be circular, non-circular, or have any suitable shape (e.g., rectangular, etc.).

[0115] As Fig.27A and Fig.27B A suitable alternative to the U-shaped embodiment, Fig.27C An example embodiment is depicted showing a top view of an electrode layer 2700 including at least two laterally spaced gaps 2721a and 2721b thereon. The gaps extend from a proximal end to a distal end of the elongated body of the electrode layer on opposite sides of the first active working electrode area. Fig.27C The clearance provided is the same as above for Figure 3 and Fig.27A The same functions as described include electrically separating the active area of ​​the electrode region from the second electrode portion, thereby reducing the effective size of the working electrode area that is subject to potential interference to reduce signal interference.

[0116] As used herein, the term "gap" and its grammatical variations refer to a channel or well in an electrode layer formed by removing an electrode layer to electrically insulate a portion thereof. In addition, at least one gap may be formed in the electrode layer during or after manufacturing the electrode layer by various non-limiting techniques, such as photolithography or screen printing. At least one gap in the electrode layer has a width of about 1 μm to about 100 μm.

[0117] Another non-limiting configuration of the disclosed subject matter includes a purge electrode that can be connected to a purge electrode sensor current conductive trace, such as Fig.28 In particular, for purposes of illustration and not limitation, Fig.28 2800, the electrode layer having a first active working electrode area 2804 disposed thereon, and the first active working electrode area can be connected to a first sensor current conductive trace 2805. Although similar in other respects to Fig.27A, but in this configuration, the second electrode portion 2810 separated from the working electrode area via a gap can be configured as a purge electrode and can be connected to the second sensor current conductive trace 2806 so that a potential can be applied to the purge electrode. Thus, the purge electrode 2810 can be configured to oxidize or pre-react with one or more interferents, such as but not limited to ascorbic acid, before the interferents are able to react on the active working electrode area of ​​the analyte sensor. Thus, the purge electrode can eliminate or reduce the local concentration of interferents present at or accessible to the active working electrode, thereby eliminating or reducing the signal due to such interferents because the interferents never reach the active area of ​​the working electrode.

[0118] In one or more aspects, the purge electrode 2810 can be independently controlled, for example by adjusting the purge electrode potential, so as to fine-tune its reaction efficiency with a particular interferent. Typically, the efficiency of the purge electrode 2810 in reacting with interferents increases as the potential increases. The purge electrode potential can be in the range of about -1000mV to about +1000mV, including any values ​​and subsets therebetween. Typically, the purge electrode potential can be any working potential within the potential window of water; that is, the potential at which the relevant solvent water of the body fluid itself is not oxidized or reduced. In some embodiments, the purge electrode potential can be relative to an included reference electrode (e.g., an Ag / AgCl reference electrode), which can be shared by the purge electrode and the working electrode. In addition, operating the purge electrode at a generally negative potential can achieve additional purge of oxidants (e.g., oxygen), which may be beneficial depending on the analyte of interest. That is, the purge electrode can be used to remove oxygen to reduce its contribution to the analyte signal.

[0119] In yet another non-limiting configuration of the present disclosure, it is shown how to reduce the extracorporeal carbon region 510 in a carbon working electrode 500, such as Figure 5 As shown, while still retaining the functionality for generating a signal associated with the analyte of interest and minimizing or eliminating interferent signals. Specifically, the spacing and diameter of the discrete sensing points 518 of the conventional carbon working electrode 500 and the configuration of the discrete sensing points 518 relative to each other can be reduced to reduce the area of ​​the in vitro carbon region 510. As used herein, the term "grid" and its grammatical variants refer to a grid along the length of the working electrode (along the sensor tail 104 ( Figure 1 )'s axis length to the width of the working electrode.

[0120] To illustrate various grid configurations, the active area of ​​the present disclosure may have the form of a 1×n grid, where n is an integer greater than 1, for example, in the range of 2 to about 20 or 2 to about 10, including any values ​​and subsets therebetween. In some embodiments, for example, Figure 5 As shown, the active area may include discrete sensing points in a 1×6 grid format. In the embodiments described herein, other grid configurations of the active area may be employed, for example, FIG. 29A to FIG. 29B Those shown in , ref. Figure 5 It is best understood that like elements retain like labels. For example, in some embodiments, the active area may include discrete sensing points in a 2×n grid, where n is an integer from 2 to about 10 or 2 to about 5, including any values ​​and subsets therebetween. Fig.29A A carbon working electrode 600 is depicted having a 2×3 grid of sensing points 518 and an in vitro carbon region 510. In other embodiments, the active region may include discrete sensing points in a 3×n grid, where n is an integer from 2 to about 6 or 2 to about 3, including any values ​​and subsets therebetween. Fig.29B A carbon working electrode 610 is depicted with a 3×2 grid of sensing points 518 and an in vitro carbon region 510. Of note, Figure 5 , Fig.29A and Fig.29B Although each of the diagrams shows various different grid configurations for the embodiments described herein, each retains the same area of ​​the in vitro carbon region 510 because the areas of the carbon electrodes 500, 600, and 610 have not been reduced in the diagrams, respectively. It can be appreciated that Fig.29A and Fig.29B The grid configuration ratio in Figure 5 The grid configuration in is arranged over a shorter longitudinal distance, thereby providing the possibility of reducing the sensor area with exposed working electrodes.

[0121] Embodiments of the present disclosure utilize grid configuration, spacing, active area and / or sensing spot size reduction, and active area location on the sensor tail to minimize the in vitro carbon area, thereby minimizing signals associated with interferents, such as FIG. 30A to FIG. 30E , top views of carbon electrodes with various active area configurations are shown. Fig. 30A Represents something like Figure 5 A control (or conventional) 1 x 6 active area configuration is shown. Fig. 30A The in vitro carbon area is represented as the shaded working electrode surface with no active area. FIG. 30B to FIG. 30F Each of these is a reference Fig. 30A Embodiments of the present disclosure are made and shown.

[0122] FIG. 30B to FIG. 30FEach of the sensing points is reduced to reduce the in vitro carbon area by reducing the distance between the sensing points, and in some embodiments, merged together so that each sensing point is no longer discontinuous. In addition, Fig. 30B Also shown is the reduction in spacing between adjacent sensing spots, allowing for a reduction in the in vitro carbon area without sensing spots, represented by the shaded working electrode surface (below the double line). Fig. 30C Shows Fig. 30B The reduction in spacing, combined with the shift of the active area toward the tip of the sensor tail, allows for a further reduction in the in vitro carbon area without a sensing site, represented by the shaded working electrode surface (below the double line). Fig.30D Representation and Fig. 30C The comparison spacing is further reduced, allowing for a further reduction in the in vitro carbon area without sensing sites, represented by the shaded working electrode surface (below the double line). Fig.30E express Fig.30D The spacing is reduced and Fig. 30C The sensor tail shift, combined with the 2 × 3 active area grid configuration, allows for further reduction of the in vitro carbon area in the absence of active areas, represented by the shaded working electrode surface (below the double line). Fig.30F express Fig.30D The spacing is reduced and Fig. 30C The sensor tail shift, combined with the 3 × 3 active area grid configuration, allows for further reduction of the in vitro carbon area in the absence of active areas, represented by the shaded working electrode surface (below the double line). FIG. 30D to FIG. 30F It is shown that as the pitch decreases, the sensing points become more difficult to distinguish, and in some embodiments, may be represented as a single active area lacking discrete sensing points.

[0123] For illustrative purposes, Table 1 compares the percentage of in vitro carbon reduction based on the estimated reduction in the signal of an interferent (e.g., ascorbic acid). Fig. 30A , Fig. 30B and FIG. 30D to FIG. 30F The interference is measured relative to the signal intensity based on the tested analyte concentration and the known interferent concentration.

[0124] Table 1

[0125]

[0126] As shown in Table 1, as the in vitro carbon area decreased, the reduction of the interferent signal also decreased, almost linearly.

[0127] Embodiments of the present disclosure allow for a reduction in interferent signal (e.g., ascorbic acid) of at least in the range of greater than about 20% or more, such as between about 20% and about 60% or more, preferably at least about 40% or more, at least about 45% or more, or at least about 50%, including any values ​​and subsets therebetween.

[0128] In another embodiment, and with reference to Figure 30B-Figure 30D and Figure 30G , the sensing point spacing can be reduced, and the sensing point spacing can have a 1×6 configuration, thereby also reducing foreign carbon, resulting in favorable sensing performance and reduced ascorbic acid interference. In particular, in an embodiment, the sensing point spacing can be in the range of about 200 μm to 250 μm, or any spacing therebetween. Fig. 30A This reduction in spacing can result in a reduction in extraneous carbon surface area of ​​approximately 40% compared to the configuration depicted in . Moreover, and with reference to Example 4 described herein, when combined with a 20-40-60-80 planing configuration, the extraneous carbon surface area can be further advantageously reduced. For example, in known embodiments, the analyte sensor can have a spacing of approximately 0.71 mm 2 However, according to embodiments disclosed herein, the analyte sensor may have a carbon surface area of ​​about 0.15 mm 2 Up to 0.25mm 2 In an embodiment, the surface area can be reduced to 0.23 mm 2 That is, the carbon surface area may be reduced by about 60-70% (eg, but not limited to, 68%).

[0129] Fig. 30H The ratio of the surface area of ​​a standard sensor (FSL sensor) to the surface area of ​​a sensor according to an embodiment of the disclosed subject matter (FSL LVC sensor) is shown. Fig. 30H As shown in , by shaving the carbon electrode and reducing the inter-point spacing, the sensing points of the FSL LVC sensor can occupy a larger proportion of the analyte sensor surface area compared to foreign carbon. For example, in known embodiments represented by FSL sensors, the ratio of the area of ​​the sensing point to the area of ​​the carbon electrode can be 4:96, 11:89, or 17:83. In contrast, according to embodiments disclosed herein, the ratio of the area of ​​the sensing point to the area of ​​the carbon electrode can be (as an example but by no means limiting) 13:87, 33:67, or 52:48.

[0130] like Fig. 30H As shown in , reducing the spacing between sensing sites and laser planing the working electrode allows for increasing the ratio of the surface area of ​​active sensing sites to the exposed portion of the working electrode. By increasing the ratio of active sensing sites to exposed working electrode, a reduction in interfering signals relative to the sensor signal of the analyte of interest can be achieved.

[0131] The present disclosure provides a working electrode (e.g., a carbon electrode) having a reduced area on which one or more active regions are disposed. In some embodiments, a plurality of discontinuous active regions are disposed on the working electrode. Typically, the width (diameter) of the discontinuous active regions of the present disclosure is in the range from about 50 μm to about 300 μm, including any values ​​and subsets therebetween. The area range of the non-circular active region (not shown) can be equivalent to the area range of the circular feature having the aforementioned width (diameter) range. The spacing between each discontinuous active region (the distance between adjacent active regions) can be about 50 μm to about 500 μm, including any values ​​and subsets therebetween. Typically, the most distal active region is located at least about 200 μm from the tip of the working electrode (which can be the same as the tip of the sensor tail) to be located at the most distal end in the body fluid, including in the range of about 50 μm to about 500 μm, including any values ​​and subsets therebetween.

[0132] In general, the area of ​​the working electrode including the active area (single active area or multiple discrete active areas) can be about 0.1 mm 2 To about 2mm 2 In general, the in vitro working electrode area (smaller than any active area) can be about 0.01 mm 2 To about 1.8mm 2 , including any values ​​and subsets therebetween.

[0133] In order to achieve a reduced in vitro working electrode area to reduce interferent signals while maintaining sensitivity to one or more analytes of interest, the ratio of the area of ​​the in vitro working electrode to the area of ​​the active area can be in the range of about 1:10 to about 10:1, including any values ​​and subsets therebetween. This ratio is maintained regardless of the grid configuration or spacing of the analyte sensor described herein; that is, the ratio range of the area of ​​the in vitro working electrode to the area of ​​the active area is always within this range to achieve the desired benefits described herein.

[0134] Thus, the analyte sensor of the present disclosure may include: a working electrode having a sensing portion and an exposed electrode portion, wherein the sensing portion includes an active region having an analyte-responsive enzyme disposed thereon, and the exposed electrode portion does not include the active region, and wherein the ratio of the exposed electrode portion to the sensing portion is in the range of about 1:10 to about 10:1. The working electrode may be a carbon electrode. At least the sensing portion may have a mass transport limiting membrane coated thereon.

[0135] In addition, the method of the present disclosure may include: exposing an analyte sensor to a body fluid, the analyte sensor including a working electrode having a sensing portion and an exposed electrode portion, wherein the sensing portion includes an active area having an analyte-responsive enzyme disposed thereon, and the exposed electrode portion does not include the active area, and wherein the ratio of the exposed electrode portion to the sensing portion is in a range of about 1:10 to about 10:1. The working electrode may be a carbon electrode. At least the sensing portion may have a mass transport limiting membrane coated thereon.

[0136] Thus, the analyte sensor of the present disclosure may include: a working electrode having a sensing portion and an exposed electrode portion, wherein the sensing portion includes an active region having an analyte-responsive enzyme disposed thereon, and the exposed electrode portion does not include the active region, and wherein the ratio of the exposed electrode portion to the sensing portion is in the range of about 1:10 to about 10:1. The working electrode may be a carbon electrode. At least the sensing portion may have a mass transport limiting membrane coated thereon.

[0137] In addition, the method of the present disclosure may include: exposing an analyte sensor to a body fluid, the analyte sensor including a working electrode having a sensing portion and an exposed electrode portion, wherein the sensing portion includes an active area having an analyte-responsive enzyme disposed thereon, and the exposed electrode portion does not include the active area, and wherein the ratio of the exposed electrode portion to the sensing portion is in a range of about 1:10 to about 10:1. The working electrode may be a carbon electrode. At least the sensing portion may have a mass transport limiting membrane coated thereon.

[0138] In addition, the non-limiting configuration of the present disclosure demonstrates how to reduce the Figure 5 The in vitro carbon region 510 shown can be reduced while still retaining the functionality for generating a signal associated with the analyte of interest and minimizing or eliminating interferent signals. Specifically, the spacing and / or diameter of the sensing points 518 of the carbon working electrode 500, as well as the configuration of the sensing points 518 relative to each other, can be reduced to reduce the area of ​​the in vitro carbon region 510.

[0139] For example, the analyte sensor includes an electrode layer having an elongated body including a proximal end and a distal end. The electrode layer includes a first active working electrode region having a plurality of sensing sites, and at least one analyte-responsive enzyme is disposed on the sensing sites. First and second adjacent sensing sites in the first active working electrode region are in an overlapping configuration, such as FIG. 31A to FIG. 31F As shown. As shown, each embodiment takes advantage of reducing the spacing between adjacent sensing points to reduce the in vitro carbon area by merging at least first and second adjacent sensing points together to form an overlapping configuration. As defined herein, "overlap" means that the boundaries of at least two adjacent sensing points at least touch each other, so that the adjacent sensing points are no longer discontinuous. For example, Fig.31A The reduction in spacing between first and second adjacent sensing points is shown so that the first and second sensing points can be in an overlapping configuration, thereby allowing the in vitro carbon area of ​​the electrode layer to be reduced, for example by reducing the length of the electrode required to contain the sensing points. Fig.31B The reduction in spacing between first and second adjacent sensing points arranged in a 3×2 grid configuration is shown so that the first and second adjacent sensing points can be in an overlapping configuration. Fig.31C The reduction in spacing between first and second adjacent sensing points arranged in a 2×3 grid configuration is shown so that the first and second adjacent sensing points can be in an overlapping configuration. FIG. 31D to FIG. 31F Further illustrated is a reduction in spacing between first and second adjacent sensing points arranged in a non-linear configuration such that the first and second adjacent sensing points may be in an overlapping configuration.Other suitable configurations utilizing overlapping sensing points are also contemplated and within the scope of the disclosed subject matter.

[0140] In some embodiments, the third and fourth adjacent sensing points in the first active working electrode region also adopt an overlapping configuration. Fig.32A An example embodiment of a reduction in the spacing between third and fourth adjacent sensing points in a linear configuration is shown to allow for a reduction in the extracorporeal carbon area of ​​the electrode layer, wherein the third and fourth sensing points may be in an overlapping configuration.

[0141] In some embodiments, the fifth and sixth adjacent sensing points in the first active working electrode region also adopt an overlapping configuration. Fig.32B An embodiment is shown in which the spacing between three pairs of adjacent points in a linear configuration is reduced to allow for a reduction in the extracorporeal carbon area of ​​the electrode layer, wherein each pair of adjacent sensing points can be in an overlapping configuration. In addition, Fig.32C An example embodiment of a reduction in spacing between three pairs of adjacent points in a grid configuration is shown, whereby each pair of adjacent sensing points is in an overlapping configuration. In some embodiments, the plurality of sensing points in the first active working electrode region may all be in an overlapping configuration.

[0142] Fig.32D An example embodiment of a reduction in spacing between at least two adjacent sensing points is shown, wherein the sensing points are arranged in a non-linear configuration having alternating single and double points along their length.

[0143] Fig.33 An example embodiment of a reduction in the spacing between at least three adjacent sensing points is shown to allow for a reduction in the in vitro carbon area of ​​the electrode layer, wherein the sensing points may be in a linear configuration and at least three adjacent sensing points may be in an overlapping configuration. It should be noted that the at least three sensing points may have any configuration described herein without limitation.

[0144] Although in Figure 32A-Figure 32Dand Fig.33 The shape of the middle sensing point is illustrated as a circle, but any other suitable shape may be used, including substantially spherical, circular, square, rectangular, triangular, conical, or elliptical, or combinations thereof.

[0145] Fig.34A Another non-limiting example embodiment is shown in . In particular, the analyte sensor 3400 includes a substrate 3412 having an upper surface including a first portion 3413 and a second exposed portion 3414. The working electrode layer 3416 can be disposed only on the first portion 3413 of the upper surface of the substrate, so that the second exposed portion of the substrate is not covered by the electrode layer. The working electrode can have a first active electrode area having a single or multiple sensing points 3417 disposed thereon, which are configured to detect analytes, as further discussed herein. The membrane 3420 can cover at least a portion of the electrode layer 3416 and the second exposed portion of the substrate 3414. The membrane can directly cover and contact the electrode layer and the second exposed portion of the substrate. In this way, the membrane 3420 is attached to the second exposed portion of the substrate 3414.

[0146] like Fig.34B As shown, in some embodiments, the surface of the second exposed portion of the substrate 3414 can have a rough surface to facilitate the film 3420 to be firmly attached to the second exposed portion of the substrate 3414. As used herein, a rough surface refers to having irregularities on the surface to provide an increased surface area for attaching the film 3420. In one or more aspects of the present disclosure, the second exposed portion of the substrate can be roughened using a physical or chemical treatment technique. In a non-limiting example, the substrate surface can be roughened by etching the substrate surface, bombarding the substrate surface with ions, embossing the substrate surface, or using a laser. The roughened second exposed portion of the substrate can have any suitable roughness value.

[0147] In one or more aspects of the present disclosure, the substrate may include a material that matches the material of the working electrode. In a non-limiting example, the substrate may include a polymer material, such as polyester, polyimide, and a combination thereof. The membrane may include a material that matches the material of the substrate 3412. In a specific embodiment of the present disclosure, the membrane covering one or more active regions may include a cross-linked polyvinylpyridine homopolymer or copolymer. In certain embodiments, the mass transport limiting membrane is a membrane composed of a cross-linked polymer containing a heterocyclic nitrogen group, for example, a polymer of polyvinylpyridine and polyvinyl imidazole. Embodiments also include membranes made of polyurethane or polyether urethane or chemically related materials, or membranes made of silicone resins and the like. In addition, the membrane can be formed by in-situ cross-linking in a buffer solution (e.g., an alcohol buffer solution) with a zwitterionic portion, a non-pyridine copolymer component, and an optional hydrophilic or hydrophobic and / or another partially modified polymer with other desired properties (including those described above). The modified polymer may be made of a precursor polymer containing a heterocyclic nitrogen group. For example, the precursor polymer may be polyvinylpyridine or polyvinyl imidazole. Alternatively, hydrophilic or hydrophobic modifiers can be used to "fine tune" the permeability of the resulting membrane to the analyte of interest. Optional hydrophilic modifiers (e.g., polyethylene glycol, hydroxyl or polyhydroxyl modifiers, etc. and any combination thereof) can be used to enhance the biocompatibility of the polymer or resulting membrane. In some embodiments, the membrane can include compounds including, but not limited to, poly(styrene-co-maleic anhydride), dodecylamine, and poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol) (2-aminopropyl ether) crosslinked with poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol) bis(2-aminopropyl ether); poly(N-isopropylacrylamide); copolymers of poly(ethylene oxide) and poly(propylene oxide); polyvinyl pyridine; derivatives of polyvinyl pyridine; polyvinylimidazole; derivatives of polyvinylimidazole; polyvinylpyrrolidone (PVP), etc.; and any combination thereof. In some embodiments, the membrane may be composed of a polyvinylpyridine-co-styrene polymer in which a portion of the pyridinic nitrogen atoms are functionalized with non-crosslinked poly(ethylene glycol) tails and a portion of the pyridinic nitrogen atoms are functionalized with alkyl sulfonic acid groups. Other membrane compounds, alone or in combination with any of the foregoing membrane compounds, may include suitable copolymers of 4-vinylpyridine and styrene and amine-free polyether arms.

[0148] The membrane compounds described herein may be further cross-linked with one or more cross-linking agents, including those listed herein with reference to enzymes described herein. For example, suitable cross-linking agents may include, but are not limited to, polyethylene glycol diglycidyl ether (PEGDGE), glycerol triglycidyl ether (Gly3), polydimethylsiloxane diglycidyl ether (PDMS-DGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, iminoesters, epichlorohydrin or its derivative variants and any combination thereof. Branched versions with similar terminal chemistry are also applicable to the present disclosure. For example, in some embodiments, the cross-linking agent may be triglycidyl ether and / or PEDGE and / or polydimethylsiloxane diglycidyl ether (PDMS-DGE).

[0149] In some embodiments, the membrane components used as the mass transfer limiting layer of the present disclosure may include polydimethylsiloxane (PDMS), polydimethylsiloxane diglycidyl ether (PDMS-DGE), aminopropyl-terminated polydimethylsiloxane, and the like, and any combination thereof, used as leveling agents (e.g., to reduce the contact angle of the membrane component or active area component). Branched versions with similar terminal chemistry are also suitable for use in the present disclosure. Certain leveling agents may also be included, such as those found in U.S. Pat. No. 8,983,568, the disclosure of which is incorporated herein by reference in its entirety.

[0150] Fig.35A and Fig.35B In the figure, an additional non-limiting configuration of an analyte sensor is shown, the analyte sensor includes an interfering substance blocking film layer to substantially reduce or eliminate the interfering substance signal of at least one interfering substance. As shown in the figure, the electrode layer is arranged on a substrate (not shown), and may have an elongated body, the elongated body includes a proximal end and a distal end and a first active working area of ​​an electrode, the first active working area having at least one sensing point on which at least one analyte response enzyme is arranged, as described above. The first active working electrode area of ​​the electrode is connected to the sensor current conductive trace. The analyte response enzyme arranged on at least one sensing point of the first active working electrode area can sense analytes, such as: glucose, acetylcholine, amylase, bilirubin, cholesterol, chorionic gonadotropin, creatine kinase (e.g., CK-MB), creatine, DNA, fructosamine, glucose, glutamine, growth hormone, hormone, ketone (e.g., ketone body), lactate, oxygen, peroxide, prostate-specific antigen, prothrombin, RNA, thyroid stimulating hormone, troponin, alcohols, aspartic acid, asparagine and potassium, or creatinine reaction enzyme.

[0151] exist Fig.35AIn the embodiment of the invention, the interfering substance blocking film layer covers the active area. In some embodiments, the interfering substance blocking layer may only cover the first active working area of ​​the electrode. In other embodiments, the interfering substance blocking layer may cover the entire working electrode, i.e., the first active working area and the second electrode portion of the electrode. The interfering substance blocking film layer may take the form of a sheet or a membrane, and may be made of a material that provides a barrier to one or more interfering substances, as long as the analyte of interest can diffuse through. The interfering substance blocking film may be made of a suitable polymer, such as, but not limited to, an ion exchange membrane, and the ion exchange membrane is selected from a perfluorosulfonic acid polymer composed of a polytetrafluoroethylene (PTFE) skeleton. Specifically, the interfering substance blocking film may include one or more sulfonated tetrafluoroethylene-based fluoropolymer copolymers (e.g., NAFION of Komu Corporation) TM , Wilmington, Germany). Alternatively, the sulfonated tetrafluoroethylene-based fluoropolymer copolymer may include Flemion TM (Asahi Glass Co., Ltd.), (Asahi Chemical) or (Fumatic), Aquivion TM (Solvay Solexis) or FS (Fumatek) or a combination thereof. One or more of these polymers may also be used with In some embodiments, a mixture of aliphatic alcohol and water containing The perfluororesin solution may contain 1 to 10 wt % of The interferent blocking film layer may have a thickness in the range of about 5 μm to about 30 μm.

[0152] In some embodiments, the analyte sensor may have one or more membrane layers in addition to the interferent blocking membrane layer. In some embodiments, for example, a membrane such as a diffusion limiting membrane may be included. Fig.35B As shown, for illustration, the sensor includes a second membrane layer (e.g., a diffusion limiting membrane) disposed on the electrode layer, and the interferent blocking membrane layer is disposed (e.g., coated) on the second membrane layer. In alternative embodiments, the second membrane may be disposed on the interferent blocking layer. The interferent blocking layer may be composed of the same material as described above with respect to Fig.35A The diffusion limiting membrane may be made of any material, such as any polymer described herein, as long as the analyte of interest can diffuse through it. For example, the diffusion limiting membrane may include a polyvinyl pyridine homopolymer or copolymer.

[0153] like Fig.35A and Fig.35BAs shown, interferor (right arrow) can not diffuse through interferor blocking film layer, and interferor reacts here and becomes inactive, so that it can not contribute to the measurement signal at working electrode.On the contrary, analyte of interest (left arrow) does not react with the interference blocking film layer, and analyte diffuses to the sensing layer (that is, the first active working area) arranged on the working electrode by the interference blocking film layer.Interferor can be any interferor as described above, such as ascorbic acid, glutathione, uric acid, acetaminophen, isoniazid, salicylate and combination thereof.When electrode potential is in the range of about -100mV to about +100mV, interferor blocking film can reduce interferor signal to about 10%, 5%, 2.5% or 1% lower than total signal.

[0154] Each of the various components of the common layers and elements of the sensors described herein can be equally included in embodiments including an analyte-permeable purge electrode. The components of the analyte-permeable purge electrode are not considered to be particularly limited, as long as they are conductive, capable of reacting with interferents (e.g., oxidized ascorbic acid), and permeable to the specific analyte of interest. In some cases, the permeable electrode can be composed of carbon nanotube materials. Other formulations can include, but are not limited to, conductive nanoparticles, conductive nanowires, and the like, and any combination thereof. The permeable purge electrode can be further supplemented with other conductive inks or polymers to enhance conductivity, enhance permeability, enhance the physical properties of the permeable electrode, and the like, and any combination thereof. For example, PEDOT:PSS can be combined or impregnated with a carbon nanotube permeable purge electrode component to increase its viscosity, thereby enhancing dip coating. In one or more aspects, an electron transfer agent (e.g., those described herein) can be included in or otherwise impregnated into the porous structure of the analyte-permeable purge electrode to improve the efficiency of the purge of interferents.

[0155] The thickness of the analyte permeable electrode is not considered to be particularly limited and can be in the range of about 1 μm to about 50 μm, including any values ​​and subsets therebetween. Without being bound by theory, the thickness of the permeable scrubbing electrode can be increased to increase scrubbing efficiency because interferents will be exposed to a larger surface area of ​​the scrubbing electrode, as long as the thickness does not adversely interfere with the diffusion of the analyte of interest.

[0156] Without wishing to be bound by theory, in some embodiments, purging the electrode (whether permeable or not) may additionally serve to regenerate the product of the analyte detection system, thereby increasing the concentration of the analyte and effectively amplifying the analyte signal.

[0157] The various layers of any of the foregoing components of the analyte sensors described herein may be deposited by any suitable means, such as, but not limited to, automated dispensing or dip coating. For example, electrodes may be screen printed and traces provided to make appropriate electrical connections.

[0158] The active region within any analyte sensor disclosed herein can include one or more analyte-responsive enzymes acting alone or in concert within an enzyme system. The one or more enzymes can be covalently bonded to a polymer comprising the active region, as can one or more electron transfer agents located within the active region.

[0159] Examples of suitable polymers in each active region may include poly(4-vinylpyridine) and poly(N-vinylimidazole) or copolymers thereof, for example, where the quaternized pyridine and imidazole groups serve as attachment points for electron transfer agents or enzymes. Other suitable polymers that may be present in the active region include, but are not limited to, those described in U.S. Pat. No. 6,605,200, which is incorporated herein by reference in its entirety, for example, 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).

[0160] The enzymes that facilitate analyte detection covalently bonded to the polymer in the active region are considered to be not particularly limited. Suitable enzymes may include enzymes that can detect blood glucose, lactate, ketones, creatinine, etc. In analyte sensors that can detect multiple analytes, any of these analytes can be combined with each other for detection. Suitable enzymes and enzyme systems for detecting these analytes are described below.

[0161] In some embodiments, the analyte sensor may include a blood glucose response active region, which includes a blood glucose response enzyme disposed on the tail of the sensor. Suitable blood glucose response enzymes may include, for example, blood glucose oxidase or blood glucose dehydrogenase (e.g., pyrroloquinoline quinone (PQQ) or cofactor-dependent blood glucose dehydrogenase, for example, flavin adenine dinucleotide (FAD)-dependent blood glucose dehydrogenase or nicotinamide adenine dinucleotide (NAD)-dependent blood glucose dehydrogenase). The difference between blood glucose oxidase and blood glucose dehydrogenase is their ability to use oxygen as an electron acceptor when oxidizing blood glucose; blood glucose oxidase can use oxygen as an electron acceptor, while blood glucose dehydrogenase transfers electrons to a natural or artificial electron acceptor, for example, an enzyme cofactor. Blood glucose oxidase or blood glucose dehydrogenase can be used to facilitate detection. Both blood glucose oxidase and blood glucose dehydrogenase can be covalently bonded to a polymer comprising a blood glucose response active region and exchange electrons with an electron transfer agent (e.g., an osmium (Os) complex or a similar transition metal complex), which can also be covalently bonded to a polymer. Suitable electron transfer agents will be described in further detail below. Glucose oxidase can directly exchange electrons with electron transfer agents, while glucose dehydrogenase can utilize cofactors to promote electron exchange with electron transfer agents. FAD cofactor can directly exchange electrons with electron transfer agents. In contrast, NAD cofactor can utilize diaphorase to promote electron transfer from cofactor to electron transfer agent. For more details about glucose responsive active regions comprising glucose oxidase or glucose dehydrogenase and using them for blood glucose detection, see, for example, co-owned U.S. Patent No. 8,268,143.

[0162] In some embodiments, the active regions of the present disclosure may be configured to detect ketones. Additional details about enzyme systems that respond to ketones may be found in co-owned U.S. Patent Application No. 16 / 774,835, filed on January 28, 2020, entitled “Analyte Sensors and Sensing Methods Featuring Dual Detection of Glucose and Ketones” and published as U.S. Patent Application Publication No. 2020 / 0237275, the entire contents of which are incorporated herein. In such a system, β-hydroxybutyrate is used as a substitute for ketones formed in vivo, which undergoes a reaction with an enzyme system comprising β-hydroxybutyrate dehydrogenase (HBDH) and diaphorase to facilitate ketone detection within a ketone-responsive active region disposed on the surface of at least one working electrode, as further described herein. Within the ketone-responsive active region, β-hydroxybutyrate dehydrogenase can convert β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD +) are converted into acetoacetate and reduced nicotinamide adenine dinucleotide (NADH), respectively. It should be understood that the term "nicotinamide adenine dinucleotide (NAD)" includes the phosphate-bound form of the aforementioned enzyme cofactor. That is, the term "NAD" as used herein refers to NAD + Phosphate and NADH phosphate, particularly a diphosphate that links two nucleotides, one containing an adenine nucleobase and the other containing a nicotinamide nucleobase. NAD + / NADH enzyme cofactor helps promote the consistent enzymatic reactions disclosed in this article. Once formed, NADH can be oxidized under the mediation of diaphorase, and the electrons transferred in this process provide the basis for the detection of ketones at the working electrode. Therefore, there is a 1:1 molar correspondence between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted. The transfer of electrons to the working electrode can be further mediated by an electron transfer agent (e.g., an osmium (Os) compound or a similar transition metal complex), as described in more detail below. Albumin can also be present in the active region as a stabilizer. β-Hydroxybutyrate dehydrogenase and diaphorase can be covalently bonded to a polymer comprising a ketone-responsive active region. NAD + may or may not be covalently bonded to the polymer, but if NAD + If not covalently bonded, the ketone responsive active region may be physically retained within the ketone responsive active region, for example, by covering the ketone responsive active region with a mass transport limiting membrane, wherein the mass transport limiting membrane is also permeable to the ketone.

[0163] According to embodiments of the present disclosure, other suitable chemicals for enzymatic detection of ketones may be utilized. For example, β-hydroxybutyrate dehydrogenase (HBDH) may again convert β-hydroxybutyrate and NAD + Converted to acetoacetate and NADH. Instead of completing the electron transfer to the working electrode via diaphorase and a suitable redox mediator, the reduced form of NADH oxidase (NADHOx(Red)) undergoes a reaction to form the corresponding oxidized form (NADHOx(Ox)). NADHOx(Red) can then be reformed by reaction with molecular oxygen to produce superoxide, which can then be converted to hydrogen peroxide under the mediator of superoxide dismutase (SOD). The hydrogen peroxide can then be oxidized at the working electrode to provide a signal that can be related to the amount of ketone initially present. According to various embodiments, SOD can be covalently bonded to a polymer in the ketone-responsive active region. β-Hydroxybutyrate dehydrogenase and NADH oxidase can be covalently bonded to a 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, they can be physically retained within the ketone-responsive active region, where the membrane polymer promotes NAD + Remaining in the ketone-responsive active region. There is also a 1:1 molar correspondence between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted, thus providing a basis for the detection of ketones.

[0164] Another enzyme detection chemistry for ketones can utilize β-hydroxybutyrate dehydrogenase (HBDH) to convert β-hydroxybutyrate to NAD + In this case, the electron transfer cycle oxidizes NADH to reform NAD via 1,10-phenanthroline-5,6-dione. + to complete, wherein 1,10-phenanthroline-5,6-dione then transfers electrons to the working electrode. 1,10-phenanthroline-5,6-dione may or may not be covalently bonded to the polymer in the ketone-responsive active region. β-Hydroxybutyrate dehydrogenase may be covalently bonded to the polymer in the ketone-responsive active region, NAD + The polymer may or may not be covalently bonded to the ketone-responsive active region. Inclusion of albumin in the active region can provide dramatic improvements in response stability. Suitable membrane polymers can promote NAD + Remaining in the ketone-responsive active region. There is also a 1:1 molar correspondence between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted, thus providing a basis for the detection of ketones.

[0165] In some embodiments, the analyte sensor may further include a creatinine responsive active region, which includes an enzyme system that operates in unison to promote creatinine detection. In the presence of creatinine amidohydrolase (CNH), creatinine can reversibly and hydrolytically react to form creatine. Creatine can also be catalytically hydrolyzed in the presence of creatine amidohydrolase (CRH) to form sarcosine. None of these reactions produces an electron flow (e.g., oxidation or reduction) to provide a basis for the electrochemical detection of creatinine. Sarcosine produced by creatine hydrolysis can be oxidized in the presence of an oxidized form of sarcosine oxidase (SOX-ox) to form glycine and formaldehyde, thereby generating a reduced form of sarcosine oxidase (SOX-red) in the process. Hydrogen peroxide can also be generated in the presence of oxygen. The reduced form of sarcosine oxidase can then undergo reoxidation in the presence of an oxidized form of an electron transfer agent (e.g., Os (III) complex), thereby generating a corresponding reduced form of an electron transfer agent (e.g., Os (II) complex) and transmitting the electron flow to the working electrode.

[0166] According to the above disclosure, oxygen may interfere with the consistent reaction sequence for detecting creatinine. Specifically, the reduced form of sarcosine oxidase can react with oxygen to reform the corresponding oxidized form of the enzyme, but without exchanging electrons with the electron transfer agent. Although all enzymes remain active when reacting with oxygen, no electrons flow to the working electrode. Without being bound by theory or mechanism, the competitive reaction with oxygen is believed to be caused by kinetic effects. That is, the rate at which the reduced form of sarcosine oxidase is oxidized by oxygen is believed to be faster than the oxidation promoted by the electron transfer agent. Hydrogen peroxide is also formed in the presence of oxygen.

[0167] By including an oxygen scavenger near the enzyme system, the desired reaction pathway for promoting creatinine detection can be supported. Various oxygen scavengers and configurations thereof may be suitable, including oxidases, for example, glucose oxidase. Small molecule oxygen scavengers may also be suitable, but may be completely consumed before the sensor life is completely exhausted. In contrast, enzymes can undergo reversible oxidation and reduction, thereby providing a longer sensor life. By preventing the reduced form of sarcosine oxidase from being oxidized by oxygen, a slower electron exchange reaction with an electron transfer agent can occur, allowing current to be generated at the working electrode. The magnitude of the current generated is proportional to the amount of creatinine initially reacted.

[0168] In any embodiment of the present disclosure, the oxygen scavenger used to promote the desired reaction can be an oxidase. Any oxidase can be used to promote oxygen scavenging near the enzyme system, as long as there is also a suitable substrate for the enzyme, thereby providing a reagent that reacts with oxygen in the presence of the oxidase. Oxidases that may be suitable for oxygen scavenging in the present disclosure include, but are not limited to, glucose oxidase, lactate oxidase, xanthine oxidase, and the like. Since blood glucose is readily available in various body fluids, glucose oxidase may be a particularly ideal oxidase for promoting oxygen scavenging. Reaction 1 below shows an enzymatic reaction promoted by glucose oxidase to provide oxygen scavenging.

[0169] β-D-glucose+O 2 --→D-Glucono-1,5-lactone+H 2 O 2 Reaction 1

[0170] The concentration of available lactate in the body is lower than that of blood glucose, but still sufficient to promote oxygen clearance.

[0171] The oxidase (e.g., glucose oxidase) can be located at any position suitable for promoting oxygen scavenging in the analyte sensor disclosed herein. For example, the glucose oxidase can be located at the tail of the sensor so that the glucose oxidase is effective and / or ineffective in promoting blood glucose detection. When it is ineffective in promoting blood glucose detection, the glucose oxidase can be located at the tail of the sensor so that the electrons generated during the oxidation of glucose cannot reach the working electrode, for example, by electrically isolating the glucose oxidase from the working electrode.

[0172] Additional details regarding enzyme systems responsive to creatinine can be found in U.S. Patent Application No. 16 / 774,835, filed on September 25, 2019, entitled “Analyte Sensors and Sensing Methods for Detecting Creatinine,” and commonly owned U.S. Patent Application Publication No. 2020 / 0237275, the contents of which are incorporated herein by reference.

[0173] In some embodiments, the analyte sensor may include an active region of lactate response arranged on the sensor tail, the active region including a lactate response enzyme. Suitable lactate response enzymes may include, for example, lactate oxidase. Lactate oxidase or other lactate response enzymes may be covalently bonded to a polymer comprising a lactate response active region, and exchange electrons with an electron transfer agent (e.g., osmium (Os) complex or similar transition metal complex), which may also be covalently bonded to a polymer. Suitable electron transfer agents will be described in further detail below. Albumin (e.g., human serum albumin) may be present in the lactate response active region to stabilize the sensor response, as described in more detail in co-owned U.S. Patent Application Publication 20190320947, which is incorporated herein by reference as a whole. Lactate levels may vary in response to many environmental or physiological factors, including, for example, the presence of eating, stress, exercise, sepsis or septic shock, infection, hypoxia, cancerous tissue, etc.

[0174] In some embodiments, the analyte sensor may include an active area responsive to a pH value. Suitable analyte sensors configured to determine pH are described in co-owned U.S. Patent Application Publication 20200060592, which is incorporated herein by reference. Such an analyte sensor may include a sensor tail, the sensor tail including a first working electrode and a second working electrode, wherein the first active area located on the first working electrode includes a substance having a redox chemical property that depends on pH, and the second active area located on the second working electrode includes a substance having a redox chemical property that does not substantially change with pH. By obtaining a difference between the first signal and the second signal, the difference can be related to the pH of the fluid to which the analyte sensor is exposed.

[0175] Two different types of active regions can be located on a single working electrode, for example, the carbon working electrode discussed above, and are separated from each other. Each active region can have a redox potential, wherein the redox potential of the first active region is sufficiently separated from the redox potential of the second active region to allow a signal to be independently generated from an active region. As a non-limiting example, the redox potential can differ by at least about 100mV, or at least about 150mV, or at least about 200mV. The upper limit of the interval between the redox potentials is determined by the working electrochemical window in vivo. By making the redox potentials of the two active regions fully separated from each other in magnitude, an electrochemical reaction can occur in one of the two active regions (i.e., in the first active region or the second active region), and an electrochemical reaction will not be substantially triggered in another active region. Therefore, a signal from one of the first active region or the second active region can be independently generated at its corresponding redox potential (lower redox potential) or higher than the potential but lower than the redox potential of another active region. Different signals can allow the signal contribution from each analyte to be resolved.

[0176] Some or all embodiments of the analyte sensors disclosed herein may feature one or more active areas located on the surface of at least one working electrode, wherein the active areas detect the same or different analytes. The membrane may cover at least the active area (comprising the analyte-responsive enzyme) and may further cover all or a portion of the working electrode lacking the active area (the exposed or in vitro portion of the working electrode). The membrane may be a mass transport limiting membrane and may be a monolayer membrane, a bilayer membrane of two different membrane polymers, or a mixture of two different membrane polymers.

[0177] An electron transfer agent may be present in any active region disclosed herein. After one or more analytes undergo an enzymatic redox reaction in the corresponding active region, a suitable electron transfer agent may promote the transmission of electrons to an adjacent working electrode, thereby generating an electron flow indicating the presence of a specific analyte. The amount of current generated is proportional to the amount of analyte present. Depending on the sensor configuration used, the electron transfer agents in the active regions in response to different analytes may be the same or different. For example, when two different active regions are disposed on the same working electrode, the electron transfer agents in each active region may be different (e.g., different chemical properties, such that the electron transfer agents exhibit different redox potentials). When multiple working electrodes are present, the electron transfer agents in each active region may be the same or different, because each working electrode may be individually interrogated.

[0178] Suitable electron transfer agents may include electroreducible and electrooxidizable ions, complexes or molecules (e.g., quinones) whose redox potential is higher or lower than the redox potential of the standard calomel electrode (SCE) by a few hundred millivolts. According to some embodiments, suitable electron transfer agents may include low potential osmium complexes, such as those described in U.S. Pat. Nos. 6,134,461 and 6,605,200, which are incorporated herein by reference in their entirety. Additional examples of suitable electron transfer agents include those described in U.S. Pat. Nos. 6,736,957, 7,501,053 and 7,754,093, the disclosures of each of which are incorporated herein by reference in their entirety. Other suitable electron transfer agents may include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyethylene ferrocene or hexacyanoferrate), or cobalt, including, for example, metallocene compounds thereof. Suitable ligands for metal complexes can also include, for example, bidentate or higher dentate ligands, for example, bipyridine, biimidazole, phenanthroline or pyridyl (imidazole). Other suitable bidentate ligands can include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkane or ortho-diaminoarene. Any combination of monodentate, bidentate, tridentate, tetradentate or higher dentate ligands can be present in the metal complex to achieve a complete coordination layer.

[0179] Active regions suitable for detecting any analyte disclosed herein may include polymers covalently bonded to electron transfer agents. Any electron transfer agents disclosed herein may include suitable functions to promote covalent bonding of polymers in active regions. Suitable examples of polymer-bound electron transfer agents may include those described in U.S. Patents 8,444,834, 8,268,143 and 6,605,201, the disclosures of which are all incorporated herein by reference. Suitable polymers included in active regions 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 suitable for inclusion in active regions include copolymers containing monomeric units, e.g., styrene, acrylamide, methacrylamide or acrylonitrile. When there are two or more different active regions, the polymers in each active region may be the same or different.

[0180] Covalent bonding of the electron transfer agent to the polymer in the active region can be performed by polymerizing a monomer unit with a covalently bonded electron transfer agent, or the electron transfer agent can be reacted separately with the polymer after the polymer has been synthesized. A bifunctional spacer can covalently bond the electron transfer agent to the polymer in the active region, with a first functional group that reacts with the polymer (e.g., a functional group that is capable of quaternizing a pyridinium nitrogen atom or an imidazole nitrogen atom) and a second functional group that reacts with the electron transfer agent (e.g., a functional group that reacts with a ligand that coordinates a metal ion).

[0181] Similarly, one or more enzymes in the active region can be covalently bonded to the polymer comprising the active region.When the enzyme system comprising multiple enzymes is present in a given active region, in some embodiments, all multiple enzymes can be covalently bonded to the polymer, and in other embodiments, only a part of the multiple enzymes can be covalently bonded to the polymer.For example, one or more enzymes comprising the enzyme system can be covalently bonded to the polymer, and at least one enzyme can be non-covalently associated with the polymer so that the non-covalently bonded enzyme is physically entrained in the polymer.In a given active region, the covalent bonding of the enzyme to the polymer can be carried out via the cross-linking agent introduced together with a suitable cross-linking agent.Suitable cross-linking agents for reacting with the free amino groups in the enzyme (for example, with the free side chain amines in lysine) can include cross-linking agents, for example, polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imino esters, epichlorohydrin or its derivative variants.Suitable cross-linking agents for reacting with free carboxylic acid groups in the enzyme can include, for example, carbodiimide. The cross-linking of the enzyme to the polymer is typically intermolecular, but may be intramolecular in some embodiments. In certain embodiments, all enzymes within a given active region may be covalently bonded to the polymer.

[0182] The electron transfer agent and / or enzyme may also be bound to the polymer in the active region by means other than covalent bonds. In some embodiments, the electron transfer agent and / or enzyme may be associated with a polymer ion or coordination. For example, a charged polymer may be associated with an electron transfer agent or enzyme ion with an opposite charge. In other embodiments, the electron transfer agent and / or enzyme may be physically entrained in the polymer without being bonded thereto. Physically entrained electron transfer agents and / or enzymes may still appropriately interact with the fluid to facilitate analyte detection without being substantially leached from the active region.

[0183] The polymer in the active region can be selected so that NAD + The outward diffusion of another cofactor that is not covalently bonded to the polymer is limited. Limited outward diffusion of the cofactor can promote reasonable sensor lifetimes (days to weeks) while still allowing sufficient inward diffusion of the analyte to facilitate detection.

[0184] In some embodiments, stabilizers can be incorporated into the active region of the analyte sensor described herein to improve the function of the sensor and achieve the desired sensitivity and stability. For example, such stabilizers can include antioxidants and / or chaperone proteins to stabilize the enzyme. Examples of suitable stabilizers can include, but are not limited to, serum albumin (e.g., human serum albumin or bovine serum albumin or other compatible albumins), catalase, other enzyme antioxidants, etc. and any combination thereof. Stabilizers can be conjugated or non-conjugated.

[0185] In a particular embodiment of the present disclosure, the mass transport limiting membrane covering one or more active areas may include a cross-linked polyvinyl pyridine homopolymer or copolymer. When the mass transport limiting membrane covers different types of active areas, the composition of the mass transport limiting membrane may be the same or different. When the membrane composition changes at two different positions, the membrane may include a double-layer membrane or a uniform mixture of two different membrane polymers, one of which may be a cross-linked polyvinyl pyridine or polyvinyl imidazole homopolymer or copolymer. Suitable techniques for depositing the mass transport limiting membrane on the active area may include, for example, spraying, painting, inkjet printing, screen printing, stencil printing, roller coating, dip coating, etc. and any combination thereof. Dip coating techniques may be particularly ideal for polyvinyl pyridine and polyvinyl imidazole polymers and copolymers.

[0186] In certain embodiments, the mass transport limiting membrane is a membrane composed of a cross-linked polymer containing heterocyclic nitrogen groups, such as a polymer of polyvinyl pyridine and polyvinyl imidazole. Embodiments also include membranes made of polyurethane or polyether urethane or chemically related materials, or membranes made of silicone resins, etc.

[0187] In some embodiments, the film can be formed by in situ cross-linking in a buffer solution (e.g., an alcohol buffer solution) with a zwitterionic portion, a non-pyridine copolymer component, and an optional hydrophilic or hydrophobic and / or another partially modified polymer with other desired properties (including those described above). The modified polymer can be made of a precursor polymer containing a heterocyclic nitrogen group. For example, the precursor polymer can be polyvinyl pyridine or polyvinyl imidazole. Alternatively, a hydrophilic or hydrophobic modifier can be used to "fine tune" the permeability of the resulting film to an analyte of interest. Optional hydrophilic modifiers (e.g., polyethylene glycol, hydroxyl or polyhydroxy modifiers, etc., and any combination thereof) can be used to enhance the biocompatibility of the polymer or resulting film.

[0188] In some embodiments, the membrane may include compounds including, but not limited to, poly(styrene-co-maleic anhydride), dodecylamine, and poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol) (2-aminopropyl ether) crosslinked with poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol) bis(2-aminopropyl ether); poly(N-isopropylacrylamide); copolymers of poly(ethylene oxide) and poly(propylene oxide); polyvinyl pyridine; derivatives of polyvinyl pyridine; polyvinylimidazole; derivatives of polyvinylimidazole; polyvinyl pyrrolidone (PVP), etc.; and any combination thereof. In some embodiments, the membrane may be composed of polyvinyl pyridine-co-styrene polymers in which a portion of the pyridine nitrogen atoms are functionalized with non-crosslinked poly(ethylene glycol) tails and a portion of the pyridine nitrogen atoms are functionalized with alkyl sulfonic acid groups. Other membrane compounds, alone or in combination with any of the foregoing membrane compounds, may include suitable copolymers of 4-vinyl pyridine and styrene and polyether arms that do not contain amines.

[0189] The membrane compounds described herein can be further cross-linked with one or more cross-linking agents, including those listed above with reference to enzymes described herein. For example, suitable cross-linking agents can include but are not limited to polyethylene glycol diglycidyl ether (PEGDGE), glycerol triglycidyl ether (Gly3), polydimethylsiloxane diglycidyl ether (PDMS-DGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, iminoesters, epichlorohydrin or its derivative variants and any combination thereof. Branched versions with similar terminal chemistry are also applicable to the present disclosure. For example, in some embodiments, the cross-linking agent can be triglycidyl ether and / or PEDGE and / or polydimethylsiloxane diglycidyl ether (PDMS-DGE).

[0190] The film can be formed in situ by coating an alcohol buffer solution of a crosslinker and a modified polymer on the active area and any additional compounds (e.g., electron transfer agents) contained in the active area, and allowing the solution to solidify for about one to two days or other suitable time periods. The crosslinker-polymer solution can be applied to the active area by placing one or more droplets of the film solution on at least one or more sensor elements of the sensor tail, by immersing the sensor tail in the film solution, by spraying the film solution on the sensor, by hot pressing or melting the film in any size layer (e.g., discrete or fully covered), and before or after segmentation, vapor deposition of the film, powder coating of the film, etc., and any combination thereof. In order to coat the distal end and side of the sensor, the film material can be applied after applying (e.g., segmenting) the sensor electronic precursor (e.g., electrode). In some embodiments, after applying the electronic precursor to coat one or more films, the analyte sensor is dip-coated. Alternatively, the analyte sensor can be slot-die coated, wherein each side of the analyte sensor is coated separately. Membranes coated in the manner described above may have any of a variety of functions including, but not limited to, mass transport limitation (i.e., reducing or eliminating the flux of one or more analytes and / or compounds to the active area), biocompatibility enhancement, interference reduction, etc., and any combination thereof.

[0191] Typically, the thickness of the film is controlled by the concentration of the film solution, the number of droplets of the film solution applied, the number of times the sensor is immersed in the film solution, the volume of the film solution sprayed on the sensor, and any combination of these factors. In some embodiments, the film described herein can have a thickness ranging from about 0.1 microns (μm) to about 1000 μm, including any value and subset therebetween. As described above, the film can cover one or more active areas, and in some embodiments, the active area can have a thickness from about 0.1 μm to about 50 μm, including any value and subset therebetween. In some embodiments, a series of droplets can be applied on top of each other to achieve the desired thickness of the active area and / or film without substantially increasing the diameter of the applied droplets (i.e., maintaining its desired diameter or range). For example, each single droplet can be applied, then cooled or dried, and then one or more additional droplets are applied. The active area and the film can but need not have the same thickness or composition as a whole.

[0192] In some embodiments, the membrane components used as the mass transfer limiting layer of the present disclosure may include polydimethylsiloxane (PDMS), polydimethylsiloxane diglycidyl ether (PDMS-DGE), aminopropyl-terminated polydimethylsiloxane, and the like, and any combination thereof, used as leveling agents (e.g., to reduce the contact angle of the membrane component or active area component). Branched versions with similar terminal chemistry are also suitable for use in the present disclosure. Certain leveling agents may also be included, such as those found in U.S. Pat. No. 8,983,568, the disclosure of which is incorporated herein by reference in its entirety.

[0193] In some cases, the membrane can form one or more bonds with the active region. As used herein, the term "bond" and its grammatical variants refer to any type of interaction between atoms or molecules that allows chemical compounds to form associations with each other, such as but not limited to covalent bonds, ionic bonds, dipole-dipole interactions, hydrogen bonds, London dispersion forces, etc. and any combination thereof. For example, in situ polymerization of the membrane can form crosslinks between the polymer of the membrane and the polymer in the active region. In some embodiments, crosslinking of the membrane to the active region helps reduce the occurrence of peeling of the membrane from the sensor.

[0194] Embodiments disclosed herein include:

[0195] The analyte sensor includes an electrode layer having an elongated body including a proximal end and a distal end. The electrode layer includes a first active working electrode region, a second electrode portion, and at least one gap electrically separating the first active working electrode portion and the second electrode portion. The first active working electrode region includes at least one sensing site having at least one analyte responsive enzyme disposed thereon. Other analyte sensors are disclosed.

[0196] Various aspects of the invention are set out in the independent claims, and preferred and optional features are set out in the dependent claims thereof. Preferred and optional features may be provided in combination within a single analyte sensor. Furthermore, an analyte sensor may be provided which combines the features of the independent claims with any of the features of the dependent claims.

[0197] In order to facilitate a better understanding of the embodiments described herein, the following examples of various representative embodiments are given. The following examples should not be construed as limiting or defining the scope of the present invention.

[0198] Example 1: In this example, Fig.13A The example laser segmentation shown is performed on a working electrode after laser planing. Fig.13A The active area disposed thereon is not included. Fig. 13B shows the width evaluation along the identified contours Fig.13A 3D optical profile of a segmented portion of the working electrode. ZEGAGE, Inc. (Middlefield, Connecticut) TM The 3D optical profiler obtains the 3D optical profile. Fig. 13B As shown, the electrode asperities at the trailing edge of the segmented sensor exhibit a height of approximately 5 μm.

[0199] Laser planing was performed using three single-pass laser lines located at the edge of the carbon asperities and gradually separated by 10 μm toward the centerline of the electrode at 10% laser power. In the examples described herein, a UV laser was used, but it should be understood that any laser may be used to perform laser planing without departing from the scope of the present disclosure. Fig. 13C is a photograph of a planed sensor tail showing the angled edge of the working electrode on the sensor tail. Fig.13D is a 3D optical profile (obtained as described above) along the identified contour line, showing the removed electrode asperities.

[0200] Example 2: In this example, refer to Fig.14A , a laser-shaved carbon working electrode 1000 was prepared according to Example 1, the carbon electrode including an active region 1010 dispensed thereon. An unshaved carbon electrode including the active region 1010 is not shown, but will be referred to as an "unshaved, dispensed" electrode. Fig. 14B is a 3D optical profile (obtained as described previously) along the identified contour line, showing minimal electrode asperities as a result of planing.

[0201] Example 3: Paired difference test was performed. Using the "unshaved, assigned" electrodes from Example 2 to check Fig.13A The unshaved electrode and Fig. 13CShaved electrodes without active areas (“unshaved, unassigned” and “shaved, unassigned”, respectively) were evaluated with multiple sensing points in 50 mg / dL blood glucose and 2 mg / dL ascorbate in 100 mM PBS at 37°C. Fig.14A The results are provided in Table 1 below and in Fig.15 Medium picture.

[0202] Table 1

[0203]

[0204] *Background corrected; **Laser planing relative to control

[0205] As shown, paired difference tests indicated that the laser-shaved electrodes exhibited a reduction of about 24% to about 29% in 2 mg / dL ascorbate compared to their unshaved counterparts.

[0206] Example 4: Paired differential testing was performed on laser-segmented working electrodes prepared as follows. An unshaved "control" working electrode included active areas at multiple sensing points. An electrode described as "compressed" included the same concentration of active areas, but the multiple sensing points were closer together and / or closer to the tip of the electrode. The total amount of analyte-responsive enzyme was the same for all samples, whether compressed or not. Laser shaving is described with reference to three separate single-pass laser lines, each at a specific distance from the edge of the initial unshaved electrode (a "shaving scheme"). For example, "20-40-60" refers to a first single-pass laser line 20 μm from the edge of the unshaved electrode, a second single-pass laser line 40 μm from the edge of the unshaved electrode, and a third single-pass laser line 60 μm from the edge of the unshaved electrode.

[0207] Table 2

[0208] Fig.16A Fig. 16B Fig. 16C Fig.16D Fig.16E compression? no no yes yes yes Planing solution Unplaned 20-40-60 Unplaned 20-40-60 15-25-40

[0209] In some cases, the electrodes 16A-16E described in Table 2 were coated with a mass transport limiting membrane having a thickness as shown in Table 3 below. Paired differential tests were performed in 50 mg / dL blood glucose and 2 mg / dL ascorbate at 37°C in 100 mM PBS (average n=6 / condition). The results are shown in Table 3 below.

[0210] Table 3

[0211]

[0212]

[0213] As shown in Table 3, paired difference tests indicated that the laser-shaved electrodes showed a reduction of about 30% to about 65% at 2 mg / dL ascorbate compared to their unshaved counterparts. The difference between a 40 μm shaved distance and a 60 μm distance toward the midline of the electrode did not appear to produce a significant difference in the anti-interferent signal, suggesting that a relatively small amount of laser shaving is effective.

[0214] In another example, and with reference to Fig.16F , a sensor with a "20-40-60-80" planing configuration was also tested. "20-40-60-80" refers to the first single-pass laser line at 20μm from the non-planed electrode edge, the second single-pass laser line at 40μm from the non-planed electrode edge, the third single-pass laser line at 60μm from the non-planed electrode edge, and the fourth single-pass laser line at 80μm from the non-planed electrode edge.

[0215] Example 5: In this example, the effectiveness of including an enzyme interferer-reactant species into an analyte sensor to eliminate or reduce the interferer signal at the working electrode was evaluated. Fig.17 As shown, a blood glucose sensor with an interferent-reactant layer for reacting with ascorbic acid is prepared. The blood glucose active area sensing layer is coated on the carbon working electrode in the form of six discontinuous sensing points and contains blood glucose oxidase sensing chemicals. The diffusion limiting membrane is coated on the entire working electrode, covering each sensing point, and includes a cross-linked polyvinyl pyridine-co-styrene polymer (called "10Q5"). 50mL of the interferent-reactant material layer is coated on the top of the diffusion limiting membrane, covering the sensing layer and the excess (exposed) carbon working electrode. The interferent-reactant material layer contains ascorbate oxidase (24.6mg / ml) in a matrix of PVI polymer (9.2mg / ml), PEDGE-400 crosslinker (6.2mg / ml) and albumin stabilizer (24.6mg / ml) (solution prepared in 10mM MES buffer, pH5.5). Two types of ascorbate oxidase, ASO-301 and ASO-311, were evaluated, both available from TOYOBO, headquartered in Osaka, Japan. A thin outer layer of membrane consisting of PVP cross-linked with PEGDGE400 was coated on top of the entire interferent-reagent material layer. These sensors were named "GOx / 10Q5+AscOx301 / PVP" and "GOx / 10Q5+AscOx311 / PVP" according to the ascorbate oxidase used.

[0216] The sensor was tested in 100 mM PBS at a temperature of 33°C, pH 7.4 and a working potential of +40 mV, along with two controls, in quadruplicate, tested in ascorbic acid and blood glucose, respectively. The first control ("Gox / 10Q5 control") included a carbon working electrode, sensing points and a sensing membrane (without the interferent-reactant material layer and the outer layer), as described above. The second control ("Gox / 10Q5+PVP control") included a carbon working electrode, sensing points, a sensing membrane and an outer layer coated thereon. The sensor was calibrated in ascorbic acid, as described above. Fig.18 As shown, calibration was performed in 30 mM blood glucose, as Fig.19 shown.

[0217] like Fig. 20 As shown, compared with the control sensor with and without PVP film, the sensor with interfering substance-reactant layer (comprising ascorbate oxidase) shows very little response to the addition of ascorbic acid. In addition, compared with the control sensor with and without PVP film, the response of blood glucose is not significantly affected by the inclusion of interfering substance-reactant layer. In addition, even if the interfering substance-reactant layer has affected blood glucose sensing, any such influence can be easily resolved as long as the linearity and stability of blood glucose are maintained. Therefore, the inclusion of enzyme interfering substance-reactant material layer is a feasible method to eliminate or reduce the signal attributable to the interfering substance on the working electrode.

[0218] Example 6: In this example, the effectiveness of including a metal oxide interferent-reactant species into an analyte sensor to eliminate or reduce the interferent signal at the working electrode was evaluated. Fig. 20 As shown, a blood glucose sensor having an interferent-reactant layer for reacting with ascorbic acid was prepared. The blood glucose active area sensing layer was coated onto a carbon working electrode disposed on a substrate. The active area sensing layer had the form of six discrete sensing points containing blood glucose oxidase sensing chemicals. The total area of ​​the active area was 0.1 mm 2 The working electrode containing the sensing point was immersed in a diffusion limiting membrane containing either a control composition or an experimental composition. The control diffusion limiting membrane included 4 ml of 140 mg / ml 10Q5, 0.4 ml of 100 mg / ml gly3 in a solvent consisting of 80% ethanol and 20% 10 mM HEPES buffer, pH = 8.1. The experimental diffusion limiting membrane was the same as the control, additionally containing 10 mg / ml MnO 2 (Catalog #217646, available from Sigma-Aldrich, headquartered in St. Louis, MO.) Control and experimental diffusion limiting films were cured.

[0219] The sensor was tested in beaker assay in 100 mM PBS at 30°C, along with two controls, in quadruplicate, in 1 mg / ml ascorbic acid and 5 mM blood glucose. The sensor current results are shown in Table 5.

[0220] Table 5

[0221]

[0222] As shown, the experimental sensor containing the interferent-reactant species within the diffusion limiting membrane showed a reduction in the ascorbic acid interferent. Therefore, the inclusion of metal oxide interferent-reactant species is a viable method to eliminate or reduce the signal attributable to interferents at the working electrode.

[0223] Example 7: In this example, the effectiveness of incorporating a washout electrode into an analyte sensor to eliminate or reduce interferent signals at the working electrode was evaluated. A blood glucose sensor was prepared by applying a blood glucose sensing active region of a blood glucose oxidase chemistry to a working electrode. The width of the working electrode was approximately 170 μm. The washout electrode was incorporated by applying a layer of adhesive to create a thin layer of approximately 50 μm. The width of the washout electrode was approximately 2500 μm. No diffusion limiting membrane was included in the sensor.

[0224] The sensors were beaker tested in 1 mM blood glucose in 100 mM PBS at a temperature of approximately 33°C. Fig. 20 The currents of the working electrode and the purged electrode are shown. As shown, the working electrode maintains a substantially stable glucose response, while the purged electrode has no response to glucose, as expected, since it does not contain glucose sensing chemistry, for more than two weeks, even without the diffusion limiting membrane. Therefore, the purged electrode can be used to achieve the diffusion limiting function of the membrane.

[0225] Example 8. In this example, the effectiveness of including a purge electrode into an analyte sensor to eliminate or reduce the interferent signal at the working electrode was evaluated. A blood glucose sensor including the purge electrode of Example 7 was tested in the presence of blood glucose and ascorbic acid, with a potential applied or removed from the purge electrode. When the potential was applied to the working electrode or the purge electrode, the potential was +40 mV. Fig. 22As shown in A, the sensor was tested in a beaker in 100 mM PBS at a temperature of about 33°C. After about 24 hours, 250 μM blood glucose was added and the working electrode and the washed electrode were observed. As shown, the working electrode reached a steady state when blood glucose was detected, while the washed electrode remained essentially unaffected. After about 25 hours, 2 mg / dL (114 μM) of ascorbic acid was added, and as shown, the response of the working electrode remained stable (detecting blood glucose) and the response current of the washed electrode increased momentarily (detecting ascorbic acid). Thereafter, the potential of the washed electrode was turned off and on again, and the relative increase between these effects of the blood glucose signal can be attributed to the ascorbic acid. Fig.23 The sensor is shown after approximately 18 days, indicating its stability at least for this period of time. Thus, it is clear that washing the electrode is effective in removing ascorbic acid from the working electrode.

[0226] Example 9. In this example, the effectiveness of incorporating a purge electrode into an analyte sensor to eliminate or reduce the interferent signal at the working electrode was evaluated. Two blood glucose sensors including a purge electrode were prepared according to Example 7, each sensor having a different carbon ink type and a different screen printing location. The purge electrode was purchased from Steven Label, Inc. (Santa Fe, CA) (in Fig.24 marked "C1", black line) and internally (in Fig.24 Commercial compositions of carbon inks vary (e.g., different carbon particles, different binders, and / or different ratios of carbon to binder), but the exact composition is unknown. Additionally, the locations of screen printing vary, likely due to proprietary printing processes, temperatures, curing times, etc. Beaker tests were performed on two different sensors in 100 mM PBS in 2.1 mg / dL ascorbic acid at 33 °C. Fig.24 The sensor current of each cleaning electrode is shown in FIG. 1 . Obviously, the cleaning electrode composition material, position and potential applied to the cleaning electrode will affect its cleaning efficiency. Therefore, the cleaning electrode can be optimized taking into account the interference of interest and / or its concentration in the body fluid, etc. and any combination thereof.

[0227] Example 10: In this example, the effectiveness of incorporating an analyte permeable purge electrode into an analyte sensor to eliminate or reduce interferent signals at the working electrode was evaluated. Fig.25 A blood glucose sensor comprising a carbon nanotube analyte permeable electrode is prepared as shown. The working electrode is screen printed onto a plastic substrate with a surrounding well to allow for the deposition of solutions of additional components of the sensor to be tested. The well is indicated as Fig.25As described above, this well configuration and variations thereof may be used in embodiments of the present disclosure. The active area of ​​the ketone sensing chemical is automatically liquid dispensed into the well and on top of the working electrode. The sensing chemical covers a portion of the working electrode, but excess (exposed) working electrode remains. Thereafter, an initial diffusion limiting film of 10Q5 is manually deposited into the well on top of the sensing chemical and into the excess working electrode portion. A carbon nanotube analyte permeable scrubber electrode is deposited into the well on top of the initial 10Q5 film, followed by a dip coating of the entire sensor in a second coating of 10Q5.

[0228] like Fig.26 As shown, in 100 mM PBS, Fig.25 The blood glucose sensor with the carbon nanotube analyte permeable cleaning electrode shown in the beaker test was performed. After about 1 hour, 5 mg / dL ascorbate was added and the working electrode (labeled as "substrate electrode") and the cleaning electrode (labeled as "CNT electrode") were observed. As shown in the figure, the addition of ascorbate resulted in the interference signal from the working electrode. After applying a potential of +40mV on the cleaning electrode, the interference signal of the working electrode was reduced by about 85%. Disconnect the cleaning electrode and the interference signal on the substrate electrode returned to the previous level. The cleaning electrode was connected again, and the applied potential was adjusted to +40, +200 and +600mV, and the cleaning efficiency was moderately improved at higher potentials. Although not shown, it was also observed that various analytes of interest (including blood glucose and β-hydroxybutyrate) were easily diffused through the cleaning electrode, thereby generating signals on the working electrode below.

[0229] Example 11: In this example, the membrane comprising a sulfonated tetrafluoroethylene-based fluoropolymer copolymer (i.e. ) is bonded to the analyte sensor to eliminate or reduce the effectiveness of the interferent signal at the working electrode. Fig.35B As shown, a method for reacting with ascorbic acid is prepared. A glucose sensor is provided with an interferent blocking membrane layer. A glucose sensing layer is generated on a carbon electrode by distributing six discrete points including a glucose oxidase-based sensing chemical on the carbon electrode. A composition including polyvinyl pyridine and a crosslinking agent is deposited on the glucose sensing layer and the electrode to generate a mass transport limiting membrane. Then, a 2-hydroxy- ... The sensor was dip coated with a perfluororesin solution of (commercially available from Sigma-Aldrich, 274704). The experimental interferent blocking film layer was cured. A control sensor was prepared in the same material but without the dip coated interferent blocking film layer.

[0230] In 100 mM PBS buffer, 5 mM glucose and 1 mg / dL ascorbic acid, the cells were tested with and without The sensor with the interference barrier film layer is tested. Fig.36A The +80mV potential shown and Fig.36B The experiments were performed at -80 mV potential as shown. Fig.36A and Fig.36B , after about 20 minutes, 5 mM glucose was added. As shown in the figure, the addition of glucose resulted in an analyte signal from the working electrode. Then, 1 mg / dL ascorbic acid was added between 35 minutes and 55 minutes. As shown in the figure, the addition of ascorbic acid resulted in an analyte signal from the working electrode. Interferor signals were generated in both the sensor with the interferent blocking film layer and the control sensor. However, as shown in the figure, the interferent signal of the sensor including the interferent blocking film layer was significantly lower than that of the control. The sensor current results showing the percentage change after adding 1 mg / dL ascorbic acid are shown in Table 6.

[0231] Table 6

[0232]

[0233] As shown in the figure, at a potential of +80 mV, the amount of interference in the signal is reduced from 16% of the total signal to only 3%. Similarly, at a potential of -80 mV, the amount of interference in the signal is reduced from 13% of the total signal to only 3%. Therefore, the incorporation of sulfonated tetrafluoroethylene-based fluoropolymer copolymers (e.g., ) can significantly reduce the interferent signal at the working electrode.

[0234] Other aspects of the apparatus, systems, and methods may be found in applicant's patent application publications, including US20220007978A1, US20210386340A1, US20210386339A1, and US20220192550A1, each of which is incorporated herein by reference in its entirety.

[0235] Unless otherwise indicated, all numbers expressing quantities and the like in this specification and the associated claims are to be understood in all cases as being modified by the term "about". Therefore, unless otherwise indicated, the numerical parameters set forth in the specification and the attached claims are approximate values ​​that may vary depending on the desired properties sought to be obtained by the embodiments of the present disclosure. At the very least, and not intending to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0236] One or more illustrative embodiments in combination with various features are presented herein. For the sake of clarity, all features of physical implementation are not described or shown in this application. It should be understood that in the development of physical embodiments in combination with embodiments of the present disclosure, many implementation-specific decisions must be made to achieve the developer's goals, for example, to meet system-related, business-related, government-related and other constraints, which vary with implementation and time. Although the developer's efforts may be time-consuming, such efforts will be routine tasks for those of ordinary skill in the art who benefit from the present disclosure.

[0237] Although various systems, tools, and methods are described herein as “comprising” various components or steps, these systems, tools, and methods may also “consist essentially of” or “consist of” the various components and steps.

[0238] As used herein, the phrase "at least one of" preceding a list of items and the terms "and" or "or" used to separate any items modify the list as a whole, rather than each member of the list (i.e., each item). The phrase "at least one of" allows for a meaning that includes at least one of any one item and / or at least one of any combination of items and / or at least one of each item. For example, the phrase "at least one of A, B, and C" or "at least one of A, B, or C" each refers to only A, B, or C; any combination of A, B, and C; and / or at least one of A, B, and C.

[0239] Therefore, the disclosed systems, tools and methods are well suited for achieving the mentioned purposes and advantages and those inherent therein. The specific embodiments disclosed above are illustrative only, as the teachings of the present disclosure can be modified and practiced in different but equivalent ways, which are obvious to those skilled in the art who benefit from the teachings herein. In addition, except as described in the claims below, it is intended that there is no limitation on the details of the construction or design shown herein. Therefore, it is obvious that the specific illustrative embodiments disclosed above can be changed, combined or modified, and all such changes are considered to be within the scope of the present disclosure. The systems, tools and methods disclosed herein illustratively can be appropriately implemented in the absence of any element not specifically disclosed herein and / or any optional element disclosed herein. Although the systems, tools and methods are described in terms of "including", "comprising" or "containing" various components or steps, the systems, tools and methods can also be "essentially composed of various components and steps" or "composed of various components and steps". All numbers and ranges disclosed above can have a certain amount of variation. Whenever a numerical range with a lower limit and an upper limit is disclosed, any number and any included range falling within the range are specifically disclosed. Specifically, each numerical range disclosed herein (in the form of "from about a to about b", or equivalently, "from about a to b", or equivalently, "from about ab") should be understood to set forth each numerical value and range contained in the broader numerical range. In addition, the terms in the claims have their plain, ordinary meaning unless otherwise clearly 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 elements that they introduce. If there is any conflict in the use of a word or term in this specification and one or more patents or other documents, the definition consistent with this specification should be adopted.

[0240] The invention may also be described according to the following numbered items:

[0241] Item 1. An analyte sensor comprising:

[0242] a substrate having an upper surface;

[0243] The electrode layer is disposed on the upper surface and has an elongated body, the elongated body including a proximal end and a distal end, the electrode layer including a surface area of ​​0.15 mm 2 Up to 0.25mm 2 wherein the active working electrode area is configured to reduce a signal indicative of an interfering substance; and

[0244] At least one sensing site is disposed on the active working electrode area, wherein the at least one sensing site comprises at least one analyte-responsive enzyme.

[0245] Item 2. The analyte sensor according to Item 1, wherein the active working electrode area has a diameter of 0.23 mm 2 surface area.

[0246] Item 3. The analyte sensor of Item 1, wherein the at least one sensing site comprises a plurality of sensing sites.

[0247] Item 4. The analyte sensor of Item 3, wherein the plurality of sensing points comprises six sensing points disposed along a longitudinal axis of the substrate.

[0248] Item 5. The analyte sensor of Item 4, wherein the sensing spots have a pitch ranging between approximately 200 μm and 250 μm.

[0249] Item 6. The analyte sensor of Item 1, wherein at least a portion of the electrode layer is shaved.

[0250] Item 7. The analyte sensor of Item 6, wherein the electrode layer is planed using laser planing.

[0251] Item 8. The analyte sensor of Item 7, wherein the laser planing comprises a plurality of single-pass laser planed cuts, wherein the plurality of single-pass laser planed cuts are spaced apart by a distance in a range of 1 μm to 100 μm.

[0252] Item 9. An analyte sensor according to Item 7, wherein the laser planing includes at least one initial cut made at an outermost location of a single carbon asperity and at least one cut made between the initial cut and a midline length of the electrode.

[0253] Item 10. An analyte sensor according to Item 1, wherein the ratio of the cumulative surface area of ​​each of the one or more sensing points to the surface area of ​​the active working electrode is at least one of 13:87, 33:67 and 52:48.

[0254] Item 11. The analyte sensor of Item 1, wherein the at least one analyte-responsive enzyme is responsive to an analyte comprising at least one of glucose, ketones, or lactate.

[0255] Item 12. A method of manufacturing an analyte sensor, comprising:

[0256] providing a substrate having an upper surface;

[0257] providing an electrode layer disposed on the upper surface and having an elongated body, the elongated body including a proximal end and a distal end, the electrode layer including an active working electrode region;

[0258] providing at least one sensing site on the active working electrode area, wherein the at least one sensing site comprises at least one analyte-responsive enzyme; and

[0259] The surface area of ​​the active working electrode area was reduced to 0.15 mm 2 With 0.25mm 2 Between, wherein the active working electrode area is configured to reduce the signal indicative of interfering substances.

[0260] Item 13. The method according to Item 12 further comprises providing a 2 Surface area of ​​the working electrode.

[0261] Item 14. The method of Item 12, further comprising setting six sensing points along the longitudinal axis of the substrate.

[0262] Item 15. The method of Item 14, further comprising arranging the sensing points on the active working electrode area at a spacing between approximately 200 μm and 250 μm.

[0263] Item 16. The method according to Item 12 further includes planing at least a portion of the electrode layer.

[0264] Item 17. The method of Item 16, further comprising making a plurality of single-pass planing cuts, wherein the plurality of single-pass planing cuts are spaced apart by a distance in the range of 1 μm to 100 μm.

[0265] Item 18. The method of Item 16, further comprising planing including at least one initial cut made at an outermost position of a single carbon asperity and at least one cut made between the initial cut and a centerline length of the electrode.

[0266] Item 19. The method according to Item 12 further includes planing at least a portion of the electrode layer so that the ratio of the cumulative surface area of ​​each of the one or more sensing points to the surface area of ​​the active working electrode area is one of 13:87, 33:67 and 52:48.

[0267] Item 20. A method of using an analyte sensor, comprising:

[0268] An analyte sensor is provided, the analyte sensor comprising: a substrate having an upper surface, an electrode layer disposed on the upper surface and having an elongated body, the elongated body comprising a proximal end and a distal end, the electrode layer comprising a surface area of ​​0.15 mm 2 Up to 0.25mm 2an active working electrode region between the electrodes, wherein the active working electrode region is configured to reduce a signal indicative of an interfering substance, and at least one sensing site disposed on the active working electrode region, wherein the at least one sensing site comprises at least one analyte-responsive enzyme; and

[0269] An analyte responsive to the at least one analyte responsive enzyme is sensed using the at least one sensing site.

Claims

1. An analyte sensor, comprising: a substrate having an upper surface; The electrode layer is disposed on the upper surface and has an elongated body, the elongated body including a proximal end and a distal end, the electrode layer including a surface area of ​​0.15 mm 2 Up to 0.25mm 2 wherein the active working electrode area is configured to reduce a signal indicative of an interfering substance; and At least one sensing site is disposed on the active working electrode region, wherein the at least one sensing site comprises at least one analyte-responsive enzyme.

2. The analyte sensor according to claim 1, wherein The active working electrode area has a size of 0.23 mm 2 surface area.

3. The analyte sensor according to claim 1, wherein: The at least one sensing point includes a plurality of sensing points.

4. The analyte sensor according to claim 3, wherein: The plurality of sensing points includes six sensing points disposed along a longitudinal axis of the substrate.

5. The analyte sensor according to claim 4, wherein: The sensing points have a pitch ranging between approximately 200 μm and 250 μm.

6. The analyte sensor according to claim 1, wherein: At least a portion of the electrode layer is shaved.

7. The analyte sensor according to claim 6, wherein: The electrode layer is planed using laser planing.

8. The analyte sensor according to claim 7, wherein: The laser planing includes a plurality of single-pass laser planing cuts, wherein the plurality of single-pass laser planing cuts are spaced apart by a distance in a range of 1 μm to 100 μm.

9. The analyte sensor according to claim 7, wherein: The laser planing includes at least one initial cut made at an outermost position of a single carbon asperity and at least one cut made between the initial cut and a centerline length of the electrode.

10. The analyte sensor according to claim 1, wherein The ratio of the cumulative surface area of ​​each of the one or more sensing points to the surface area of ​​the active working electrode is at least one of 13:87, 33:67, and 52:

48.

11. The analyte sensor according to claim 1, wherein: The at least one analyte-responsive enzyme is responsive to an analyte comprising at least one of glucose, ketones, or lactate.

12. A method of manufacturing an analyte sensor, comprising: providing a substrate having an upper surface; providing an electrode layer disposed on the upper surface and having an elongated body, the elongated body including a proximal end and a distal end, the electrode layer including an active working electrode region; providing at least one sensing site on the active working electrode area, wherein the at least one sensing site comprises at least one analyte-responsive enzyme; and The surface area of ​​the active working electrode area was reduced to 0.15 mm 2 With 0.25mm 2 Between, wherein the active working electrode area is configured to reduce the signal indicative of interfering substances.

13. The method according to claim 12, further comprising providing a 0.23 mm 2 Surface area of ​​the working electrode.

14. The method of claim 12, further comprising providing six sensing points along a longitudinal axis of the substrate.

15. The method of claim 14, further comprising arranging the sensing points on the active working electrode area at a spacing between about 200 μm and 250 μm.

16. The method of claim 12, further comprising shaving at least a portion of the electrode layer.

17. The method of claim 16, further comprising making a plurality of single-pass planing cuts, wherein: The plurality of single-pass planing cuts are spaced apart by a distance in the range of 1 μm to 100 μm.

18. The method of claim 16, further comprising planing, the planing comprising at least one initial cut made at an outermost location of a single carbon asperity and at least one cut made between the initial cut and a midline length of the electrode.

19. The method of claim 12, further comprising shaving at least a portion of the electrode layer so that the ratio of the cumulative surface area of ​​each of the one or more sensing points to the surface area of ​​the active working electrode area is one of 13:87, 33:67 and 52:

48.

20. A method of using an analyte sensor, comprising: An analyte sensor is provided, the analyte sensor having: a substrate having an upper surface; The electrode layer is disposed on the upper surface and has an elongated body, the elongated body including a proximal end and a distal end, the electrode layer including a surface area of ​​0.15 mm 2 Up to 0.25mm 2 an active working electrode region between the electrodes, wherein the active working electrode region is configured to reduce a signal indicative of an interfering substance; and at least one sensing site; disposed on the active working electrode region, wherein the at least one sensing site comprises at least one analyte-responsive enzyme; and An analyte responsive to the at least one analyte responsive enzyme is sensed using the at least one sensing site.

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