Analyte sensors featuring enhancements directed to reducing interfering signals

Abstract

Analyte sensors are increasingly used to monitor various analytes in the body. Analyte sensors can feature enhancements to address signals obtained from interfering substances. Some analyte sensors can include a working electrode that includes an active area disposed thereon and electrode roughness laser planed therefrom. Some analyte sensors can include interfering-substance-reactant species incorporated therein. Some analyte sensors can include an interfering-substance scrubbing electrode. Combinations of these enhancements can additionally be employed.

Description

[0001] Cross-referencing related applications

[0002] This application claims priority and benefit to U.S. Provisional Application Serial No. 63 / 049,210, filed July 8, 2020, which is incorporated herein by reference in its entirety. Background Technology

[0003] The detection of various analytes in an individual's body is sometimes crucial for monitoring their health. Deviations from normal analyte levels can often indicate a number of physiological conditions. For example, detecting and monitoring blood glucose levels can be particularly important in individuals with diabetes. By monitoring blood glucose levels with sufficient regularity, individuals with diabetes are able to take corrective measures (e.g., lowering blood glucose levels by injecting insulin or raising blood glucose levels by eating) before significant physiological damage occurs. For various other physiological conditions, monitoring of other analytes may be necessary. In some cases, monitoring of multiple analytes may also be required, especially for complications that cause simultaneous imbalances in two or more analytes.

[0004] Many analytes represent the focus of physiological analysis, provided that suitable detection chemicals can be identified. To this end, in vivo analyte sensors configured to analyze a wide variety of physiological analytes have been developed and improved in recent years, many of which utilize enzyme-based detection strategies to enhance detection specificity. In fact, in vivo analyte sensors that utilize glucose-responsive enzymes to monitor blood glucose levels are now widely used in individuals with diabetes. In vivo analyte sensors for other analytes are at different stages of development, including those capable of monitoring multiple analytes. For some analyte sensors, low sensitivity to low-abundance analytes can be particularly problematic, especially due to background signals generated by the interaction of interfering substances with the working electrode or other analyte-sensing chemicals. Attached Figure Description

[0005] The following figures are included to illustrate certain aspects of this disclosure and should not be considered as exclusive embodiments. The disclosed subject matter is capable of considerable modifications, alterations, combinations, and equivalents in form and function without departing from the scope of this disclosure.

[0006] Figure 1 A diagram of an exemplary sensing system that can be incorporated into the analyte sensor of this disclosure is shown.

[0007] Figures 2A to 2C A cross-sectional view of an analyte sensor including a single active region is shown.

[0008] Figures 3A to 3C A cross-sectional view of an analyte sensor comprising two active regions is shown.

[0009] Figure 4 A cross-sectional view of an analyte sensor including two working electrodes, each with an active region, is shown.

[0010] Figure 5 This is a top view showing a conventional carbon working electrode with active regions on it.

[0011] Figure 6A A photograph shows a top view of the working electrode without a membrane. Figure 6B It is along Figure 6A The depth profile of the line shown.

[0012] Figure 7A A photograph shows a top view of the working electrode on which the membrane is disposed. Figure 7B It is along Figure 7A The depth profile of the line shown.

[0013] Figure 8 It is a photograph showing a 3D view of a working electrode for laser planing according to one or more aspects of this disclosure.

[0014] Figure 9A This is a description of a conventional sensor that does not incorporate interfering-reactive substances. Figure 9B It is based on one or more aspects of this disclosure that incorporates interfering-reactant substances. Figure 9B Description of the sensor.

[0015] Figure 10 This is a depiction of a sensor electrode configuration including a scrubbing electrode according to one or more aspects of this disclosure.

[0016] Figure 11 This is a depiction of a sensor electrode configuration including a permeable scrubbing electrode according to one or more aspects of this disclosure.

[0017] Figure 12 This is a depiction of the configuration of sensor electrodes, including impermeable and permeable scrubbing electrodes, according to one or more aspects of this disclosure.

[0018] Figure 13A A photograph shows a top view of the working electrode without a membrane and active region. Figure 13B It is along Figure 13A The depth profile of the line shown. Figure 13C The following is illustrated after laser planing, according to one or more aspects of this disclosure. Figure 13A A top-view photograph of the working electrode. Figure 13D It is along Figure 13C The depth profile of the line shown.

[0019] Figure 14AA photograph of a top view of a working electrode after laser planing, on which no film and active region are disposed, according to one or more aspects of this disclosure. Figure 14B It is along Figure 14A The depth profile of the line shown.

[0020] Figure 15 This is a graph showing the paired differences between planed and unplaned working electrodes that have active or inactive regions in response to the interfering substance ascorbic acid.

[0021] Figures 16A to 16E A photograph of the working electrode is shown. Figure 16A and Figure 16C It was laser-cut. Figure 16B , Figure 16D and Figure 16E Laser planing is performed according to one or more aspects of this disclosure.

[0022] Figure 17 It is a sensor configuration including a layer of interfering-reactant material according to one or more embodiments of the present disclosure.

[0023] Figure 18 It is according to one or more aspects of this disclosure for use in a layer of interfering-reactant material. Figure 17 The ascorbic acid calibration curve of the analyte sensor.

[0024] Figure 19 It is based on one or more aspects of this disclosure, including a layer of interfering-reactant substances. Figure 17 The blood glucose calibration curve of the analyte sensor.

[0025] Figure 20 , Figure 21A , Figure 21B and Figure 22 The sensor current trace of a sensor including a wiping electrode, according to one or more aspects of this disclosure.

[0026] Figure 23 It is a sensor configuration including a permeable scrubbing electrode according to one or more aspects of this disclosure.

[0027] Figure 24 Based on one or more aspects of this disclosure Figure 24 The sensor current trace of the sensor. Detailed Implementation

[0028] This disclosure generally describes an analyte sensor suitable for in vivo use, and more specifically, describes an analyte sensor characterized by one or more enhancements for reducing or eliminating signals indicating interfering substances to promote improved detection sensitivity, and methods for manufacturing and using the same.

[0029] Such enhancements may include reducing the availability of the working electrode surface on the sensor tail (the part of the sensor for insertion into tissue), particularly the availability of the carbon working electrode on the sensor tail, where interfering substances can react and contribute a signal unrelated to the analyte. Other components of the analyte sensor may also react with interfering substances and contribute a signal at the carbon working electrode. Aspects of this disclosure include, individually or in combination, roughening the carbon working electrode, including compounds that react with interfering substances to prevent their interaction with the carbon working electrode, and / or adding a scrubbing electrode that reacts with interfering substances to prevent their interaction with the carbon working electrode. In one or more aspects, the enhancements described herein may reduce the sensor’s sensitivity to interfering substances (e.g., by inhibiting or reducing the signal generated by interfering substances at the working electrode, e.g., by eliminating excess carbon electrode surface through the use of sensing chemicals and / or membranes), and / or reduce the local concentration of interfering substances at the working electrode (e.g., by “pre-reacting” the interfering substances so that they do not or substantially do not reach the working electrode). While not strictly necessary, 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 interfering substance of interest, provided that the signal of the analyte of interest is not impaired. In some cases, analyte sensors that include a low-potential working electrode may further include a low-potential redox mediator to enhance the detection of signals from such low-potential analytes.

[0030] Generally, but not limitingly, embodiments of this disclosure including one or more interfering agent enhancements can allow for at least a reduction in the interfering agent signal (e.g., ascorbic acid interfering agent signal) in the range greater than about 20%, potentially up to 100%, or, for example, in the range of about 20% to about 70% or greater, preferably at least about 40%, at least about 45%, or at least about 50%, including any values ​​and subsets therebetween, wherein the upper and lower limits are separable, as detailed herein. The amount of interfering agent reduction can depend on a number of factors, including but not limited to the specific configuration of the sensor (e.g., which one or more enhancements are selected), the concentration of the interfering agent in the body fluid, and any combination thereof.

[0031] The analyte sensor described herein includes a sensor tail comprising at least one working electrode (specifically, a carbon working electrode) and an active region disposed thereon. A mass transfer limiting membrane is then disposed on the carbon working electrode (i.e., on the active region and any external carbon working electrode lacking the active region forming the sensor tail).

[0032] Various carbon electrode roughnesses may be present along the edge of a carbon working electrode, where these roughnesses may not be adequately coated or not coated at all with a mass transfer limiting film, thus providing a carbon surface for interference to react and contribute to the measurement signal at the working electrode. As used herein, the term "roughness" and its grammatical variations refer to a rough edge along a surface (e.g., along the working electrode). Roughnesses may take the form of ridges along the edge of the working electrode, resulting in insufficient coating of the mass transfer limiting film at that location. To reduce or eliminate such interfering signals, this disclosure provides planing of one or more edges of the carbon working electrode to remove the carbon roughness therefrom, thereby providing a more uniform profile of the working electrode surface. In cases where the working electrode is formed of a material other than carbon, such roughnesses may also be present in the composition of a particular working electrode ("electrode asperity").

[0033] Separated from or integrated with the planing of one or more edges of the carbon working electrode to remove carbon roughness, this disclosure further provides an analyte sensor including one or more means for preventing or reducing the proximity of interfering substances to the working electrode. Specifically, one or more enzymes or chemical compounds may be incorporated into the analyte sensor, which reacts with the interfering substance of interest, deactivating it so that it cannot contribute to the measurement signal at the working electrode. Alternatively, or again in combination, a scrubbing electrode may be incorporated into the analyte sensor, which reacts with the interfering substance of interest, deactivating it so that it cannot contribute to the measurement signal at the working electrode.

[0034] This document will describe in more detail the specific features and further advantages of each type of enhancement. Depending on specific needs, the analyte sensor of this disclosure can be configured to detect one or more analytes simultaneously or nearly simultaneously.

[0035] Enzyme-based analyte sensors are typically used to analyze single analytes, such as blood glucose, because enzymes often exhibit specificity for particular substrates or substrate classes. Analyte sensors employing single enzymes and enzyme systems containing multiple enzymes with synergistic effects can be used for this purpose. As used herein, the term "consistent" and its grammatical variations refer to coupled enzymatic reactions in which the product of a first enzymatic reaction becomes the substrate of a second enzymatic reaction, which, or a subsequent enzymatic reaction, serves as the basis for measuring analyte concentration. Furthermore, combinations of enzymes and / or enzyme systems can be used to detect more than one type of analyte. Using in vivo analyte sensors characterized by enzymes or enzyme systems to facilitate detection may be particularly advantageous in avoiding frequent aspirations of bodily fluids that would otherwise require analyte monitoring.

[0036] In vivo analyte sensors monitor one or more analytes in biological fluids of interest, such as dermal fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, etc. These fluids may include one or more interfering substances that can react with the working electrode of the analyte sensor, either directly on the working electrode itself (e.g., a carbon working electrode) or with one or more sensing chemical components disposed thereon (e.g., redox polymers described below). As used herein, the term "interfering substance" and its grammatical variations refer to any electroactive substance present that is not part of the analyte of interest (e.g., an in vivo electroactive substance of the analyte of interest that is not present in body fluids (e.g., interstitial fluid, etc.)). Examples include, but are not limited to, ascorbic acid (vitamin C, also known as ascorbate), glutathione, uric acid, acetaminophen (paracetamol), isoniazid, salicylates, etc., and any combination thereof. The reaction of these interfering substances with the working electrode can generate an electrochemical signal that is inseparable from or difficult to separate from the signal originating from the analyte of interest. This can complicate the accurate detection of such analytes, especially those with low abundance (e.g., sub-millimolar concentrations). The electrochemical signal generated by interfering substances can be particularly problematic because the signal from the interfering substance becomes more similar in amplitude to that from the target analyte. This can occur, for example, when the concentration of the interfering substance is close to or exceeds that of the analyte of interest. Some interfering substances are ubiquitous in vivo and difficult to avoid. Therefore, techniques to minimize their influence during in vivo analysis are likely to be highly desirable.

[0037] This disclosure provides analyte sensor enhancements, which, alone or in combination with other enhancements, can improve the detection sensitivity of a single analyte and multiple analytes combined, as explained in further detail below. Specifically, this disclosure provides analyte sensors with reduced carbon working electrode edge roughness and / or contained compounds or scrubbing electrodes, which can provide reduced background signal due to in-vivo interferences. While some aspects of this disclosure relate to the enhancement of carbon working electrodes, it should be understood that other types of electrodes can be similarly enhanced according to the disclosure herein. Electrode types that can be enhanced using the disclosure herein include gold, platinum, PEDOT, etc.

[0038] Before proceeding with a more detailed description of the analyte sensors and enhancements thereof, a brief overview of suitable in vivo analyte sensor configurations and sensor systems employing analyte sensors will first be provided to facilitate a better understanding of the embodiments of this disclosure. Figure 1A diagram of an exemplary sensing system that can be incorporated into the analyte sensor of this disclosure is shown. As illustrated, the sensing system 100 includes a sensor control device 102 and a reader device 120, which are configured to communicate with each other via a local communication path or link 140, which may be wired or wireless, one-way or two-way, and encrypted or unencrypted. According to some embodiments, the reader device 120 may constitute an output medium for observing analyte concentrations and alarms or notifications determined by the sensor 104 or its associated processor, and for allowing input from one or more users. The reader device 120 may be a multipurpose smartphone or a dedicated electronic reader instrument. Although only one reader device 120 is shown, multiple reader devices 120 may be present in some cases.

[0039] The reader device 120 can also communicate with the remote terminal 170 and / or the trusted computer system 180 via communication paths / links 141 and / or 142, respectively. These communication paths / links can be wired or wireless, one-way or two-way, and encrypted or unencrypted. The reader device 120 can also, or alternatively, communicate with the network 150 (e.g., a mobile phone network, the Internet, or a cloud server) via communication path / link 151. The network 150 can 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 can communicate directly with the remote terminal 170 and / or the trusted computer system 180 without the 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 2011 / 0213225, which is incorporated herein by reference in its entirety.

[0040] Any suitable electronic communication protocol can be used for each communication path or link, such as Near Field Communication (NFC), Radio Frequency Identification (RFID), Bluetooth. or Bluetooth Low-energy protocols, WiFi, etc. According to some embodiments, in addition to the primary user, individuals with a focus on the user's analytics level can also access remote terminal 170 and / or trusted computer system 180. Reader device 120 may include display 122 and optional input components 121. According to some embodiments, display 122 may include a touchscreen interface.

[0041] Sensor control device 102 includes a sensor housing 103 that can house circuitry and power supply for operating sensor 104. Optionally, the power supply and / or active circuitry may be omitted. A processor (not shown) is communicatively coupled to sensor 104, physically located within sensor housing 103 or reader device 120. According to some embodiments, sensor 104 protrudes from the underside of sensor housing 103 and extends through adhesive layer 105, which is adapted to adhere sensor housing 103 to a tissue surface, such as skin.

[0042] Sensor 104 is adapted to be at least partially inserted into tissue of interest, such as the dermis or subcutaneous layer of the skin. Alternatively, sensor 104 may be adapted to penetrate the epidermis. Further alternatively, sensor 104 may be disposed on a surface and not penetrate tissue, for example, when analyzing one or more analytes in sweat on the skin. Sensor 104 may include a sensor tail of sufficient length for insertion to a desired depth in a given tissue. The sensor tail may include at least one working electrode and an active region comprising an enzyme or enzyme system configured for analyzing one or more analytes of interest.

[0043] The counter electrode can exist in combination with at least one working electrode, and optionally in further combination with a reference electrode. See below for reference. Figures 2A to 4 The specific electrode configuration at the sensor tail is described in more detail. According to different embodiments, one or more enzymes in the active region may be covalently bonded to the polymer containing the active region. Alternatively, the enzyme may be non-covalently bonded to 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 particular embodiment, the analyte sensor of this disclosure may be adapted to analyze skin fluid or interstitial fluid to determine the concentration of the analyte in vivo. However, it should be understood that the entire sensor control device 102 may have one or more different configurations, allowing for complete subtissue transplantation into one or more bodily fluids for the analysis of one or more analytes of interest without departing from the scope of this disclosure.

[0044] Refer again Figure 1 Sensor 104 can automatically forward data to reader device 120. For example, analyte concentration data can be transmitted automatically and periodically, such as upon data acquisition or at a specific frequency after a specific time period has elapsed. The data is stored in memory until transmission (e.g., every minute, five minutes, or other predetermined time intervals), for example, via... or Low-energy protocols. 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, sensor 104 can communicate with reader device 120 in a non-automatic manner and not according to a set schedule. For example, data can be transmitted from sensor 104 using RFID technology when the sensor electronics enter the communication range of reader device 120. Data can remain stored in the memory of sensor 104 before being transmitted to reader device 120. Therefore, the user does not need to maintain close proximity to reader device 120 at all times, but can upload data automatically or non-automatically at convenient times. In other embodiments, a combination of automatic and non-automatic data transmission can be implemented. For example, data transmission can continue automatically until reader device 120 is no longer within the communication range of sensor 104.

[0045] An introducer may be temporarily present to facilitate the introduction of sensor 104 into the tissue. In illustrative embodiments, 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 sheaths or blades. More specifically, the needle or other introducer may temporarily reside near sensor 104 before tissue insertion and then be withdrawn. When present, the needle or other introducer may facilitate insertion of sensor 104 into the tissue by opening the access path followed by 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 implantation of sensor 104. After opening the access path, the needle or other introducer may be withdrawn without the risk of sharp objects. In illustrative embodiments, suitable needles may be solid or hollow, beveled or non-beveled, and / or have a circular or non-circular cross-section. In more specific embodiments, suitable needles may be comparable to acupuncture needles in cross-sectional diameter and / or tip design, with acupuncture needles having a cross-sectional diameter of approximately 250 micrometers. However, it should be recognized that a suitable needle can have a larger or smaller cross-sectional diameter if required by a specific application. For example, needles with cross-sectional diameters ranging from approximately 300 micrometers to approximately 400 micrometers can be used.

[0046] In some embodiments, the tip of the needle (when present) may be angled above the end of the sensor 104, such that the needle first penetrates the tissue and opens the access path for the sensor 104. In other illustrative embodiments, the sensor 104 may be located within the lumen or recess of the needle, with the needle similarly opening the access path for the sensor 104. In either case, the needle can be subsequently withdrawn after facilitating sensor insertion.

[0047] A sensor configuration characterized by a single active region configured to detect a corresponding single analyte can employ a dual-electrode or triple-electrode detection pattern, as referenced herein. Figures 2A to 2C Further details are provided below. Figures 3A to 4 Sensor configurations are described separately, characterized by two distinct active regions for detecting separated analytes on separate working electrodes or on the same working electrode. Sensor configurations with multiple working electrodes may be particularly advantageous for combining two distinct active regions at the same sensor tail, as the signal contribution from each active region can be more easily determined by individually interrogating each working electrode. Each active region may be coated with a mass transport limiting film of the same or different composition.

[0048] When a single working electrode is present in the analyte sensor, a three-electrode sensor configuration may include a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration may include a working electrode and a second electrode, wherein the second electrode can serve as both a counter electrode and a reference electrode (i.e., counter electrode / reference electrode). The various electrodes may be at least partially stacked (layered) on top of each other and / or laterally spaced apart at the tail of the sensor. In any sensor configuration disclosed herein, the various electrodes may be electrically isolated from each other by a dielectric material or similar insulator.

[0049] An analyte sensor characterized by multiple working electrodes may similarly include at least one additional electrode. When one additional electrode is present, it can serve as the counter electrode / reference electrode for each of the multiple working electrodes. When two additional electrodes are present, one additional electrode can serve as the counter electrode for each of the multiple working electrodes, while the other additional electrode can serve as the reference electrode for each of the multiple working electrodes.

[0050] Any working electrode configuration described below can benefit from the further disclosure below regarding the availability of reducing the edge roughness of the working electrode at the tail of the sensor.

[0051] Figure 2A A diagram illustrating an illustrative dual-electrode analyte sensor configuration applicable to the disclosure herein is shown. As shown, the analyte sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter electrode / reference electrode 216. Alternatively, the working electrode 214 and the counter electrode / reference electrode 216 may be located on the same side of the substrate 212, with a dielectric material (configuration not shown) inserted between them. 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 discontinuous points or a single continuous point configured for the detection of the analyte, as further discussed herein.

[0052] Still referencing Figure 2AAccording to some embodiments, membrane 220 at least covers the active region 218 and optionally covers some or all, or the entire, of the working electrode 214 and / or the counter electrode / reference electrode 216. One or both sides of the analyte sensor 200 may be coated with membrane 220. Membrane 220 may comprise one or more polymer membrane materials capable of restricting analyte flow to the active region 218 (i.e., membrane 220 is a mass transport restriction membrane with a certain degree of permeability to the analyte of interest). The composition and thickness of membrane 220 may be varied to facilitate desired analyte flow to the active region 218, thereby providing desired signal strength and stability. The analyte sensor 200 can be used to analyze analytes using any of coulometric, current, voltammetric, or potentiochemical detection techniques.

[0053] Figure 2B and Figure 2C A diagram illustrating an illustrative three-electrode analyte sensor configuration is shown, which also applies to the disclosure herein. Except for analyte sensors 201 and 202 (… Figure 2B and Figure 2C Apart from the additional electrode 217, the three-electrode analyte sensor configuration can be similar to... Figure 2A The configuration of the analyte sensor 200 is shown. The counter electrode / reference electrode 216 can be used as a counter electrode or reference electrode via the additional electrode 217, and the additional electrode 217 performs other electrode functions unless otherwise stated. The working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed on the working electrode 214 or electrode 216, with a dielectric material separation layer between them. 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 insulation. Alternatively, at least one of electrodes 214, 216, and 217 may be located on opposite surfaces of substrate 212, as shown. Figure 2C As shown. Therefore, in some embodiments, electrode 214 (working electrode) and electrode 216 (counter electrode) may be located on opposite surfaces of substrate 212, and electrode 217 (reference electrode) may be located on one of electrodes 214 or 216 and separated from it by a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, and the location of the reference material layer 230 is not limited to... Figure 2B and 2C The location shown. As indicated. Figure 2A The active region 218 in the illustrated sensor 200, analyte sensors 201 and 202 may include multiple points or a single point. Furthermore, analyte sensors 201 and 202 can also be used to analyze analytes by any of coulometric, current, voltammetric, or potentiochemical detection techniques.

[0054] Like analyte sensor 200, membrane 220 can also cover the active region 218 and other sensor components in analyte sensors 201 and 202, thereby serving as a mass transfer limiting membrane. In some embodiments, additional electrode 217 may be coated with membrane 220. Membrane 220 can also be produced by dip coating or in-situ photopolymerization, and may have different or the same composition at different locations. Although Figure 2B and Figure 2C All electrodes 214, 216, and 217 have been depicted as being coated with film 220; however, it should be understood that in some embodiments, only the working electrode 214 or the active region 218 may be coated. Furthermore, the thickness of film 220 at each electrode 214, 216, and 217 may be the same or different. This is similar to a dual-electrode analyte sensor configuration (…). Figure 2A As in ) Figure 2B and Figure 2C In the sensor configuration, one or two surfaces of the analyte sensors 201 and 202 can be coated with film 220, or the entire surface of the analyte sensors 201 and 202 can be coated. Therefore, Figure 2B and Figure 2C The three-electrode sensor configuration shown should be understood as not limiting the embodiments disclosed herein, and alternative electrode and / or layer configurations are still within the scope of this disclosure.

[0055] Figure 3A An exemplary configuration of a sensor 203 with a single working electrode is shown, on which two different active regions are provided. Figure 3A Similar to Figure 2A In addition to two active regions on the working electrode 214—a first active region 218a and a second active region 218b—which respond to different analytes and are laterally spaced from each other on the surface of the working electrode 214, the active regions 218a and 218b may comprise multiple points or a single point configured to detect each analyte. At the active regions 218a and 218b, the composition of the membrane 220 may vary or be identical. The first active region 218a and the second active region 218b may be configured to detect their respective analytes at different working electrode potentials, as discussed further below.

[0056] Figure 3B and Figure 3C Cross-sectional views of exemplary three-electrode sensor configurations of sensors 204 and 205 are shown, each sensor configuration characterized by having a single working electrode with a first active region 218a and a second active region 218b disposed thereon. Figure 3B and Figure 3C Similar to in other aspects Figure 2B and Figure 2C By referring to them, one can gain a better understanding. Figure 3A Similarly, the composition of membrane 220 may vary or remain the same in active regions 218a and 218b.

[0057] 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 surfaces of a substrate 402. A first active region 410a is disposed on the surface of the working electrode 404, and a second active region 410b is disposed on the surface of the working electrode 406. A counter electrode 420 is electrically isolated from the working electrode 404 by a dielectric layer 422, and a reference electrode 431 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 431 and the counter electrode 420, respectively. According to various embodiments, a membrane 440 may at least cover the active regions 410a and 410b, and other components of the analyte sensor 400 or the entire analyte sensor 400 may optionally be coated with the membrane 440. Similarly, if needed, membrane 440 can change its composition at active regions 410a and 410b to provide suitable permeability values ​​for differentially adjusting analyte flow rates at each location.

[0058] It has multiple working electrodes and is different from Figure 4 Alternative sensor configurations to the illustrated configuration may feature a counter electrode / reference electrode instead of separate counter electrode and reference electrode 420, 431, and / or feature a layer and / or film arrangement different from those explicitly described. For example, the positions of the counter electrode 420 and reference electrode 431 may be... Figure 4 The opposite is shown. Furthermore, working electrodes 404 and 406 do not necessarily need to be... Figure 4 The arrangement shown is located on the opposite surface of substrate 402.

[0059] Carbon working electrodes can suitably include the working electrodes in any analyte sensor disclosed herein. While carbon working electrodes are very common in electrochemical detection, their use is not without its challenges. Specifically, a current associated with the analyte of interest is generated only when the active region interacts with the analyte and transfers electrons to the carbon working electrode portion adjacent to the active region. Body fluids containing the analyte of interest also interact with the carbon surface of a carbon working electrode that is not coated with an active region and do not contribute to the analyte signal because there are no enzymes or enzyme systems at these locations to facilitate electron transfer from the analyte to the working electrode. However, interfering substances may oxidize in the working electrode portion lacking an active region and contribute background to the overall signal. Therefore, carbon working electrodes with foreign (or “exposed” carbon regions on the electrode surface do not contribute meaningfully to the analyte signal and may, in some cases, result in a contributing background signal. Other electrodes with excessively large surface areas that do not directly detect the analyte of interest may experience similar background signals and can be enhanced by modifying the disclosure herein.

[0060] While various interfering substances can interact with the working electrode of the analyte sensor described herein, ascorbic acid is one example of an interfering substance commonly found in biological fluids that can generate background signals on the carbon working electrode. For example, ascorbic acid can be oxidized on the working electrode to produce dehydroascorbic acid. Various embodiments of this disclosure will be described herein with reference to ascorbic acid as an interfering substance; however, it should be understood that the embodiments and analyte sensor configurations described herein are equally applicable to other interfering substances (electroactive substances in body fluids containing the analyte of interest).

[0061] As described above, the active region described herein can be a single sensing layer, a sensing layer with multiple sensing points, or a sensing layer with multiple sensing points compressed together and thus essentially representing a single sensing layer. Now refer to... Figure 5The diagram shows a top view of a conventional carbon working electrode 500, on which an active region 504 including a plurality of sensing points 518 is disposed. When an analyte interacts with the active region 504, only a portion of the carbon working electrode 500 including the sensing points 518 contributes a signal related to the analyte of interest. Although the carbon working electrode 500 displays six sensing points 518 within the active region 504, it should be understood that the carbon working electrode 500 may include fewer or more than six sensing points 518 without departing from the scope of this disclosure. Foreign carbon regions 510 are not directly covered by the sensing points 518 and do not contribute a signal related to the analyte, but may generate background signals related to one or more interfering substances. Therefore, the oxidation of interfering substances at the carbon working electrode 500 is proportional to the area of ​​the foreign carbon regions 510 available for interaction with the interfering substances. In fact, the oxidation of ascorbic acid on the carbon working electrode 500 is approximately linearly proportional to the area of ​​the available foreign carbon regions 510.

[0062] As shown, the active region 504 is discontinuous and has the form of multiple sensing points 518. As defined herein, the term "discontinuous" and its grammatical variations mean that any individual point (sensing element) does not share the edges or boundaries of adjacent points (e.g., does not contact).

[0063] The sensor tail, including the carbon working electrode 500, described in this disclosure can be fabricated on a template substrate material together with additional layered elements of the sensor tail (e.g., dielectric material, other electrodes, etc.) (see [link to relevant documentation]). Figures 2A to 2B , Figures 3A to 3C , Figure 4 During sensor manufacturing, the sensor tail, including the carbon working electrode 500, is subsequently segmented in one or more ways. Segmentation can be achieved through one or more cutting or separating methods, including but not limited to laser segmentation, cutting, shearing, stamping, etc. The segmentation of the sensor tail can be performed before or after applying a segmentation to the active region on the carbon working electrode 500 toward the distal end of the sensor tail (i.e., the portion where the sensor tail will be inserted deepest into the tissue). As used herein, the distal “tip” of the sensor tail refers to the most distal edge of the sensor tail or the portion inserted deepest into the tissue.

[0064] One or more portions of the sensor tail are segmented, typically requiring multiple laser passes to cut the sensor tail into the desired shape. The tip of the sensor tail includes at least a portion of the working electrode and the active region. Typically, the laser-segmented sensor tail has a width ranging from about 50 μm to about 800 μm and a length ranging from about 1 mm to about 20 mm, for example, a width ranging from about 100 μm to about 500 μm and a length ranging from about 3 mm to about 10 mm, including any values ​​and subsets therebetween, and wherein the upper and lower limits are separable. Typically, the distal portion of the sensor tail occupies a distal length of about 0.1 mm to about 10 mm, for example, about 0.1 mm to about 5 mm, including any values ​​and subsets therebetween, and wherein the upper and lower limits are separable. After laser segmentation, a mass transport confinement film is deposited at least on the sensor tip including the active region.

[0065] In one or more aspects of this disclosure, carbon roughness may be present along the edges of the carbon electrode due to the laser segmentation process prior to the application of the mass transfer limiting membrane. This carbon roughness can provide a surface on which interfering substances can react and contribute background signals to the analyte sensor.

[0066] Laser segmentation of the carbon working electrode can result in carbon roughness bodies with widths of approximately 75 μm or less, for example, ranging from approximately 1 μm to approximately 75 μm, or from approximately 5 μm to approximately 50 μm, or from approximately 10 μm to approximately 30 μm, including any values ​​and subsets thereof, wherein the upper and lower limits are separable. Furthermore, these carbon roughness bodies can have heights of approximately 50 μm or less, or from approximately 20 μm or less, for example, ranging from approximately 1 μm to approximately 50 μm, or from approximately 1 μm to approximately 30 μm, or from approximately 1 μm to approximately 20 μm, or from approximately 2 μm to approximately 10 μm, as described in more detail below, including any values ​​and subsets thereof, wherein the upper and lower limits are separable. Therefore, these carbon roughness bodies can provide a considerable area over which interfering elements can interact. In addition, the roughness bodies can lead to inconsistent coverage (thickness) of the mass transport limiting film. These carbon roughness bodies can be reduced or removed by one or more laser-planed methods, as described below.

[0067] First refer to Figure 6A Before any laser planing is performed according to this disclosure to reduce or remove carbon roughness, a close-up of an example of a laser-segmented carbon working electrode used as at least a portion of the sensor tail is shown, wherein the carbon working electrode has no mass transport limiting film deposited thereon. Electrodes cut into the desired shape by other means may have roughness of similar appearance and size. The carbon roughness is noticeable along the edges of the working electrode with which interfering substances may react. Figure 6B It shows along Figure 6AThe depth profile of the line indicated in the image is evaluated along the identified 430.71 μm profile width. Using... ZEGAGE (Middlefield, Connecticut) TM A 3D optical profilometer obtains a 3D optical profile. For example... Figure 6B As shown, the carbon roughness along the segmented (ablation) edge of the example segmented sensor tail is about 30 μm wide and up to about 10 μm high.

[0068] Mass transport limiting membranes can reduce or prevent interfering substances from entering foreign carbon regions (e.g., Figure 5 The foreign carbon region 510). When applied to a laser-segmented carbon working electrode (and its active region), the film thickness varies across the width of the working electrode, particularly where significant roughness exists. Typically, the film is thinnest at the electrode edges, which is also where the carbon roughness is located. Therefore, even in the presence of the film, the carbon roughness may not be adequately coated to reduce or prevent its interaction with interfering substances.

[0069] refer to Figure 7A Before any laser planing is performed according to one or more aspects of this disclosure to reduce or remove carbon roughness, a close-up of an example laser-segmented carbon working electrode on which a mass transport limiting film is deposited is shown. Figure 7B It shows along Figure 7A The depth profile of the line shown is evaluated along the identified 345.53 μm profile width. Using... ZEGAGE (Middlefield, Connecticut) TM A 3D optical profilometer obtains a 3D optical profile. For example... Figure 7B As shown, the film is quite thin along the ridges of the carbon working electrode.

[0070] In various aspects, this disclosure provides methods and analyte sensors in which a carbon working electrode for forming the tail of the sensor is planed via one or more single-pass or multi-pass laser planing cuts, alone or in combination with additional enhancements described herein. In some embodiments, a single-pass laser planing method is used, wherein 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, about 50% of the top of the carbon layer. The carbon layer is typically in the range of 5 μm to about 20 μm (without roughness); in some embodiments, about 5 μm to about 10 μm (e.g., about 20%, 30%, 40%, 50%, 60%, or 70%, up to 100%) can be removed therefrom (e.g., see [link to relevant documentation]). Figure 13C Laser planing according to this disclosure can remove or reduce the protrusions of rough bodies.

[0071] In some embodiments, more than one single-pass laser gouging kerf can be performed (e.g., less than about 20 (or about 15, or about 10) kerfs, each kerf gradually approaching the centerline length of the working electrode to reduce or eliminate carbon roughness. In this way, an initial laser gouging kerf can be made at the outermost position of any single carbon roughness, and subsequent laser gouging kerfs can be made towards the centerline length of the working electrode to produce a ground edge, which can be a stepped edge or bevel (i.e., an edge not perpendicular to the electrode surface) of about 90°, if, for example, the nearest laser gouging kerf towards the electrode centerline does not produce a true 90° angle (see...). Figure 8 The laser-plaining kerf (edge) 810 is shown as a bevel rather than a 90° angled sheared edge. For example, in one embodiment, about 1 to about 20 (e.g., about 2 to about 10) single-pass laser-plaining kerfs can be made, each kerf having a distance between about 0.1 μm and about 200 μm, for example, about 1 μm to about 100 μm, including any values ​​and subsets therein, and wherein the upper and lower limits are separable. The specific number of laser plaining passes and the selection of their spacing can be based on a number of factors, including but not limited to the shape and size of the carbon roughening body, the length and width of the working electrode, the coverage profile of any film disposed thereon, and any combination thereof.

[0072] Laser planing is preferably used to remove at least about 5% to about 100% of the total carbon roughness area from a single sensor tail containing a carbon working electrode, including any values ​​and subsets therebetween, wherein the upper and lower limits are separable. In some embodiments, up to 100% of the carbon roughness is removed, or about 5% to about 75%, or about 5% to about 50%, or about 5% to about 25% of the total carbon roughness area is removed, including any values ​​and subsets therebetween, wherein the upper and lower limits are separable. In a preferred embodiment, at least about 50% of the total carbon roughness area is removed. The specific amount of carbon roughness removed can be based on a variety of factors, including but not limited to the density, shape and size of the carbon roughness, the concentration of the analyte of interest compared to the concentration of interfering substances available in the body fluid being analyzed, and any combination thereof.

[0073] Figure 8 A photograph of the edge of a sensor tail 800 according to one or more embodiments of the present disclosure is shown, illustrating a laser-cut ridge 805 and a laser-gouging ridge 810 recessed from the edge of the sensor tail to remove a portion of the edge of the carbon working electrode (carbon or electrode layer). That is, the laser-gouging ridge 810 is designed to reduce carbon roughness 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 is retained along the outer periphery (and in the opposite portion of the electrode, which does not include the active region).

[0074] In one or more aspects of this disclosure, alone or in combination with any other enhancements to reduce or eliminate analyte sensor signals associated with interfering substances, an analyte sensor comprising an interfering-reactant substance is provided. As used herein, the term "interfering-reactant substance" and its grammatical variations refer to any compound, whether biological or non-biological, capable of reacting with and inactivating an interfering substance, such that it cannot contribute to the measurement signal at the working electrode. That is, an interfering-reactant substance may be included as part of the analyte sensor to "pre-react" to the interfering substance before it can react (contact) at the working electrode of the analyte sensor. Thus, the interfering-reactant substance can eliminate or reduce the local concentration of interfering substances present at or near the working electrode, thereby eliminating or reducing the signal attributed to such interfering substances, since the interfering substances never reach an excessive area of ​​the working electrode.

[0075] The methods and aspects of the interfering-reactant substances combined with the analyte sensor have been described with reference to interfering-reactant substances for the elimination or removal of ascorbic acid; it should be understood that the enhancements described herein are applicable to other potential interferences without limitation. Such interfering substances may include, for example, ascorbic acid (vitamin C), glutathione, uric acid, acetaminophen (paracetamol), isoniazid, salicylates, etc., and any combination thereof. In a non-limiting example, the interfering-reactant substances of this disclosure may be ascorbic acid oxidase (reacting with ascorbic acid), glutathione peroxidase (reacting with glutathione), xanthine oxidase (reacting with uric acid), uricase oxidase (reacting with uric acid), cytochrome P450 (reacting with acetaminophen), eosinophil peroxidase (reacting with isoniazid), salicylate oxidase (reacting with salicylates), other enzymes capable of oxidizing, reducing, or otherwise reacting with and decomposing the interfering substance of interest, etc., and any combination thereof. In alternative or combined embodiments, the interfering-reactant substance may be a non-enzymatic substance. For example, various metal oxides (e.g., manganese oxide (MnO2) or iron oxide (Fe2CO3)) can oxidize or otherwise react with and decompose ascorbic acid, and can be used as one or more interfering-reactant substances of this disclosure.

[0076] refer to Figure 9A The illustration shows a description of a conventional sensor 900, demonstrating the potential interference reaction of ascorbic acid 902 with an excess of the working electrode and potential sensing chemicals, thereby generating a signal attributed to ascorbic acid. Figure 9AThe sensor does not incorporate interfering reactant substances. Ascorbic acid 902 encounters the sensor 900 via body fluid 904 (e.g., interstitial fluid) and contacts the sensing chemical 906 and an excess area of ​​the working electrode 908 (e.g., an electrode excluding the sensing chemical 906) disposed on the substrate 910. Upon encountering the sensing chemical 906 and the working electrode 908, ascorbic acid 902 can be oxidized at least on the excess working electrode 908 and / or additionally on the sensing chemical 908, and converted into dehydroascorbic acid 912.

[0077] According to various aspects of this disclosure, Figure 9B The following is shown: containing interfering-reactant substance 914 Figure 9A The sensor is described, particularly regarding the interfering reactant substances of ascorbic acid oxidase (AOx). As shown, ascorbic acid oxidase 914 reacts with ascorbic acid 902 before contacting the working electrode 906 or the sensing chemical 906, thereby preventing the ascorbic acid 902 from contributing to the analyte signal. It should be noted that... Figure 9B The sensors depicted herein may have any configuration and / or components of the sensors described herein, without limitation.

[0078] The specific location of one or more interfering-reactant substances included in the analyte sensor of this disclosure is not considered particularly limited. For example, interfering-reactant substances may be provided as part of an analyte-sensing active region; a membrane covering the analyte-sensing active region; a layer thereof over any of the working electrode, the analyte-sensing active region, and / or a membrane coating; and so on; and any combination thereof. When provided as part of an active layer, a membrane, or its own layer, it may be free-floating within a polymer matrix or otherwise fixed (e.g., covalently or non-covalently bound). The specific concentration of the interfering-reactant substance included in the analyte sensor (at any one or more locations) may depend on a number of 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, and any combination thereof. In some cases, when the interfering-reactant substance is an enzyme, the total amount of the interfering-reactant substance may range from about 0.01 units to about 200 units of activity per sensor, for example, from about 0.01 units to about 100 units of activity per sensor, including any values ​​and subsets therebetween, and wherein the upper and lower limits are separable. For example, a sensor containing an interfering-reactant substance of ascorbic acid oxidase may have an activity of about 0.01 units to about 10 units, or about 0.01 units to about 5 units, or about 0.1 units to about 1 unit per sensor. In other cases, when the interfering-reactant substance is a non-enzymatic compound (e.g., a metal oxide), the total amount of the interfering-reactant substance may range from about 0.01 μg to about 200 μg per sensor, or from about 0.1 μg to about 100 μg per sensor, including any values ​​and subsets therein, wherein the upper and lower limits are separable. For example, a sensor containing an interfering-reactant substance of MnO2 may be present in an amount of about 0.1 μg to about 10 μg per sensor, wherein the upper and lower limits are separable.

[0079] As described above, typically, the interfering-reactant substances described herein, whether present as a layer itself, within the membrane, or within the active region, will be situated within a polymer matrix, whether fluid or stationary. This polymer matrix can consist of any polymer, crosslinker, and / or additive compatible with the interfering-reactant substance selected for the analyte sensor, which will not interfere with the sensing chemical. Each polymer, crosslinker, and / or additive can be selected from any of those described herein without limitation. For example, non-limiting examples of such polymers include poly(4-vinylpyridine) and poly(N-vinylimidazole) (PVI) or copolymers thereof, and sulfonated tetrafluoroethylene vinyl fluoropolymer copolymers (e.g., NAFION). TMChemours Company, Wilmington, DE (The Chemours Company, Wilmington, DE), polyvinyl alcohol and any combination thereof; non-limiting examples of crosslinking agents include triglycidyl ether (gly3) and / or PEDGE and / or polydimethylsiloxane diglycidyl ether (PDMS-DGE); non-limiting examples of additives include stabilizers, such as albumin, and / or any other stabilizers described herein.

[0080] In one or more aspects of this disclosure, individually or in combination with any other enhancements to reduce or eliminate analyte sensor signals associated with interfering substances, an analyte sensor including a scrubbing electrode is provided (e.g., with or without interfering-reactant substances and / or roughness planing). As described herein, the term "scrubbing electrode" and its grammatical variations refer to an electrode capable of reacting with interfering substances to deactivate them, preventing them from contributing to the measurement signal at the working electrode. That is, a scrubbing electrode can be included as part of the analyte sensor to "pre-react" to the interfering substances before they can react on the working electrode of the analyte sensor. Thus, similar to the presence of interfering-reactant substances, a scrubbing electrode can eliminate or reduce the local concentration of interfering substances present or accessible on the working electrode, thereby eliminating or reducing signals attributed to such interfering substances because the interfering substances never reach an excessive area of ​​the working electrode.

[0081] In one or more aspects, the scrubbing electrode may be positioned facing each other and spatially offset from the working electrode. That is, the active regions of the working electrode and the scrubbing 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, including the analyte and any interfering substances therein, to pass through. The configuration of the scrubbing electrode relative to the working electrode is ideally such that the body fluid is in contact with the scrubbing electrode for a sufficient time to react with any interfering substances before the body fluid reaches the working electrode. The scrubbing electrode does not contain any sensing chemicals, and therefore, the analyte does not react with it. In this way, the body fluid has removed or substantially removed interfering substances, and the signal obtained at the working electrode is entirely or primarily attributable to the analyte.

[0082] Various electrode configurations can be used to ensure that bodily fluids contact the scrubbing electrode before the working electrode. Figure 10 A non-limiting configuration 1000 is shown. As illustrated, the scrubbing electrode 1006 and the working electrode 1008 face each other, and the working electrode 1008 is recessed or has a smaller width compared to the scrubbing electrode 1006. The working electrode also includes a sensing chemical substance (not shown) disposed thereon. Although Figure 10The specific configuration of the working electrode 1008 and the cleaning electrode 1006 shown herein has a rectangular shape, but other configurations are equally applicable to the embodiments described herein, such as square, circular, spiral, etc. Typically, the working electrode 1008 and the cleaning electrode 1006 may have a length 1004 greater than their width 1002.

[0083] In one or more aspects, the width of the scrubbing electrode relative to the working electrode can range from about 2:1 to about 50:1, including any values ​​and subsets therebetween, and wherein the upper and lower limits are separable. For example, in some cases, the scrubbing electrode 1006 can have a width ranging from about 200 μm to about 8000 μm, for example, from about 300 μm to about 5000 μm, and the working electrode 1008 can have a width ranging from about 50 μm to about 2000 μm, for example, from about 50 μm to about 1000 μm, including any values ​​and subsets therebetween, and wherein the upper and lower limits are separable. These dimensions include the thin layer 1010 extending in the direction of the length of the sensor tail, having a linear or non-linear shape, in order to increase the ratio between the dimensions of the scrubbing electrode 1006 and the working electrode 1008 without making the sensor tail too wide for actual in vivo use (insertion).

[0084] A thin layer 1010 is formed between the scrubbing electrode 1006 and the working electrode 1008. This thin layer can range from about 1 μm to about 200 μm, for example, from about 10 μm to about 100 μm, including any values ​​and subsets therebetween, and wherein the upper and lower limits are separable. In some cases, the thin layer can be from about 10 μm to about 50 μm, or about 10 μm, about 20 μm, about 30 μm, about 40 μm, or about 50 μm, and wherein the upper and lower limits are separable. The thin layer 1010 is typically formed by sealing fluid channels (e.g., thin layer “cells”) along two opposite edges of the scrubbing electrode 1006, allowing body fluid to enter the space between the unsealed spaces of the thin layer 1010 in a controlled manner to ensure that it reaches the scrubbing electrode 1006 before the working electrode 1008. Typically, a larger ratio between the surface area of ​​the scrubbing electrode 1006 and the volume of the thin layer 1010 is preferred to maximize the chance of interaction between the solute (e.g., interfering substances) and the scrubbing electrode 1006. For example, refer to Figure 10The thin layer 1010 between the scrubbing electrode 1006 and the working electrode 1008 can be formed by applying an adhesive, separator, or other non-limiting separation method along the width edge of the electrode. In this way, bodily fluid is guided along its length through the edge and into the thin layer 1010. Therefore, when bodily fluid containing interfering substances and analytes of interest diffuses through the thin layer 1010, it interacts sufficiently with the scrubbing electrode 1006 before reaching the working electrode 1008. Thus, the analyte sensor including this scrubbing electrode 1006 does not (although it may) rely on a membrane to limit the interaction between interfering substances and the working electrode 1008, which can provide manufacturing and cost advantages.

[0085] In various embodiments, the thin layer 1010 may be modified with surfactants, hydrogels, membranes or other materials that facilitate the introduction of bodily fluids into the thin layer 1010 to improve biocompatibility, provide antimicrobial or microstatic properties, and any combination thereof.

[0086] In one or more aspects, various configurations can be adjusted to allow independent control of the scrubbing electrode, for example, by adjusting the scrubbing electrode potential to fine-tune its reaction efficiency with a specific interfering substance. Typically, the efficiency of the scrubbing electrode in reacting with interfering substances increases with increasing potential. The scrubbing electrode potential can be in the range of about -2000 mV to about +2000 mV, for example, about -1000 mV to about +1000 mV, including any values ​​and subsets therebetween, and wherein the upper and lower limits are separable. Typically, the scrubbing electrode potential can be any working potential within the potential window of water; that is, the potential at which the relevant solvent water in the body fluid is not oxidized or reduced. In some embodiments, the scrubbing electrode potential can be relative to a included reference electrode (e.g., an Ag / AgCl reference electrode), which can be shared by both the scrubbing electrode and the working electrode. Furthermore, operating the scrubbing electrode at a generally negative potential can enable additional scrubbing of oxidants (e.g., oxygen), which may be beneficial depending on the analyte of interest. In other words, wiping the electrode can be used to remove oxygen, thereby reducing its contribution to the analyte signal.

[0087] The composition of the scrubbing electrode is not considered particularly limited and can be made of known electrode materials, and can be the same as 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 scrubbing electrode can be modified or supplemented with materials specific to react with the interfering substances of concern, or the surface area of ​​the scrubbing electrode can be increased. It should also be understood that interfering-reactant substances can be coated on the scrubbing electrode in any way, as described above, to further enhance the elimination or reduction of interfering substances reaching the working electrode.

[0088] In some embodiments, instead of having a thin-layer configuration for containing the scrubbing electrode, the scrubbing electrode composition can be selected such that it is permeable to the analyte of interest. In this way, the scrubbing electrode can be stacked over the working electrode, with an analyte-permeable membrane or dielectric layer in between to prevent sensor short circuits, and without a thin layer. That is, an insulating material that is itself permeable to the analyte of interest is disposed between the permeable scrubbing electrode and the working electrode containing the analyte detection material. In this way, and based on the same basic principle as the thin-layer scrubbing electrode configuration described above, a bodily fluid containing the analyte of interest and interfering substances will come into contact with the permeable scrubbing electrode. Before the bodily fluid (containing the analyte of interest and with little or no interfering substances) comes into contact with the working electrode, the interfering substances react at the permeable scrubbing electrode and are eliminated or otherwise reduced in concentration. Therefore, the scrubbing electrode can eliminate or reduce the local concentration of interfering substances present or accessible on the working electrode, thereby eliminating or reducing the signal attributed to such interfering substances, because the interfering substances do not reach excessive areas of the working electrode.

[0089] Figure 11 This document illustrates one such non-limiting configuration of an analyte sensor 1100 including a permeable scrubbing electrode. As shown, the working electrode 1102 includes a sensing chemical substance disposed thereon to form an active region 1104 (either as a single region or comprising multiple discontinuous points). On the active region 1104 is an analyte-permeable insulating layer 1106, which can be of any material, such as any polymer described herein, as long as one or more analytes of interest 1112 can diffuse through it. For example, the analyte-permeable insulating layer 1106 can be a diffusion-limiting membrane. An analyte-permeable scrubbing electrode 1108 is disposed on the analyte-permeable insulating layer 1106. While the analyte-permeable scrubbing electrode 1108 can have the same dimensions as the base working electrode 1102, in a preferred embodiment, the analyte-permeable scrubbing electrode 1108 has a shape and size that contacts the body fluid before the insulating layer 1106 or the working electrode 1102. It may include an outer membrane 1110 to provide additional diffusion-limiting properties, biocompatibility properties, antimicrobial or microstatic properties, protection of the permeable scrubbing electrode 1108, and any combination thereof. Figure 11 As shown, interfering agent 1114 can diffuse through the outer membrane 1110 to the permeable scrubbing electrode 1106, where it reacts and loses its activity, preventing it from contributing to the measurement signal at the working electrode 1102. In contrast, analyte 1112 does not react with the scrubbing electrode 1106 (which does not have analyte-sensing chemicals), and diffuses through the outer membrane 1110, scrubbing electrode 1108, and insulating layer 1106 to the sensing layer 1104 on the working electrode 1102. This will be discussed below. Figure 23As shown, another non-limiting configuration can employ a "trap" structure with a scrubbing electrode that is permeable to the analyte.

[0090] Figure 12 Another non-limiting configuration of the analyte sensor 1200, including a permeable scrubbing electrode, is shown. In this configuration, the permeable scrubbing electrode 1202 is coupled with traces of an impermeable scrubbing electrode 1204 to provide electrical contact, allowing a potential to be applied to the permeable scrubbing electrode 1202. The impermeable scrubbing electrode 1204 can be traced on a dielectric material 1206, disposed on a working electrode 1208, and itself disposed on a substrate 1210 (e.g., a plastic substrate). A second dielectric can be further disposed on the impermeable scrubbing electrode 1204. Sensing chemical substances 1204 can be dispensed onto exposed portions of the working electrode 1208. Portions of the sensor A can be generated and segmented. Subsequently, dip coating can be performed to apply and cure an inner polymer film 1214, followed by dip coating to apply and cure a permeable scrubbing film 1202, and then final dip coating to apply an outer polymer film 1216. This configuration can provide manufacturing and cost advantages.

[0091] Each of the various components of the common layer and elements of the sensor described herein can be equivalently included in any or all embodiments including an analyte-permeable scrubbing electrode. The composition of the analyte-permeable scrubbing electrode is not considered particularly limited, as long as it is conductive, capable of reacting with interfering substances (e.g., oxidized ascorbic acid), and permeable to the specific analyte of interest. In some cases, the permeable electrode may be composed of carbon nanotube materials. Other formulations may include, but are not limited to, conductive nanoparticles, conductive nanowires, etc., and any combination thereof. The permeable scrubbing electrode may be further supplemented with other conductive inks or polymers to enhance conductivity, permeability, physical properties of the permeable electrode, etc., and any combination thereof. For example, poly(3,4-ethylenedioxythiophene)polystyrene sulfonate (PEDOT:PSS) can be combined with or impregnated with the carbon nanotube-permeable scrubbing electrode composition to increase its viscosity, thereby enhancing dip coating. In one or more aspects, an electron transfer agent (such as those described herein) may be contained in or otherwise impregnated into the porous structure of an analyte-permeable scrubbing electrode to improve the efficiency of interfering substance scrubbing.

[0092] The thickness of the analyte-permeable scrubbing electrode is not considered particularly limited and can range from about 0.5 μm to about 100 μm, for example, from about 1 μm to about 50 μm, including any values ​​and subsets therein, wherein the upper and lower limits are separable. Unbound by theory, the thickness of the permeable scrubbing electrode can be increased to improve scrubbing efficiency, as interfering substances will be exposed to a larger surface area of ​​the scrubbing electrode, provided that the thickness does not adversely interfere with the diffusion of the analyte of interest.

[0093] Unbound by theory, in some embodiments, the scrubbing electrode (whether permeable or not) can be additionally used to regenerate the products of the analyte detection system, thereby increasing the concentration of the analyte and effectively amplifying the analyte signal.

[0094] The various layers of any of the aforementioned components of the analyte sensor described herein can be deposited by any suitable method, such as, but not limited to, automated dispensing or dip coating. For example, electrodes can be screen-printed and provided with traces for appropriate electrical connections.

[0095] The active region within any analyte sensor disclosed herein may include one or more analyte-responsive enzymes, which act individually or synergistically within the enzyme system. One or more enzymes may be covalently bonded to the polymer containing the active region, as may one or more electron transfer agents located within the active region.

[0096] Examples of suitable polymers for each active region may include poly(4-vinylpyridine) and poly(N-vinylimidazole) or copolymers thereof, for example, wherein the quaternized pyridine and imidazole groups serve as electron transfer agents or enzyme linking points. Other suitable polymers that may be present in the active regions include, but are not limited to, those described in U.S. Patent 6,605,200, which is incorporated herein by reference in its entirety, such as poly(acrylic acid), styrene / maleic anhydride copolymer, methyl vinyl ether / maleic anhydride copolymer (GANTREZ polymer), poly(vinyl benzyl chloride), poly(allylamine), polylysine, poly(4-vinylpyridine) quaternized with carboxypentyl, and poly(sodium 4-styrene sulfonate).

[0097] There are no particular limitations on enzymes that are covalently bonded to the polymer at their active sites to facilitate analyte detection. Suitable enzymes may include those capable of detecting blood glucose, lactate, ketones, creatinine, etc. In analyte sensors capable of detecting multiple analytes, any of these analytes can bind to each other for detection. Suitable enzymes and enzyme systems for detecting these analytes are described below.

[0098] In some embodiments, the analyte sensor may include a glucose-responsive active region comprising a glucose-responsive enzyme disposed at the tail of the sensor. Suitable glucose-responsive enzymes may include, for example, glucose oxidases or glucose dehydrogenases (e.g., pyrroloquinoline quinone (PQQ) or cofactor-dependent glucose dehydrogenases, such as flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase or nicotinamide adenine dinucleotide (NAD)-dependent glucose dehydrogenase). The difference between glucose oxidases and glucose dehydrogenases lies in their ability to utilize oxygen as an electron acceptor when oxidizing glucose; glucose oxidases can utilize oxygen as an electron acceptor, while glucose dehydrogenases transfer electrons to natural or artificial electron acceptors, such as enzyme cofactors. Glucose oxidases or glucose dehydrogenases may be used to facilitate detection. Both glucose oxidases and glucose dehydrogenases may be covalently bonded to a polymer containing the glucose-responsive active region and exchange electrons with an electron transfer agent (e.g., an osmium (Os) complex or a similar transition metal complex), which may also be covalently bonded to the polymer. Suitable electron transfer agents will be described in further detail below. Glucose oxidases can directly exchange electrons with electron transfer agents, while glucose dehydrogenases can utilize cofactors to facilitate electron exchange with electron transfer agents. FAD cofactors can directly exchange electrons with electron transfer agents. Conversely, NAD cofactors can utilize flavoprotein transferases to facilitate electron transfer from the cofactor to the electron transfer agent. Further details regarding glucose-responsive active regions containing glucose oxidases or glucose dehydrogenases and their use in glucose detection can be found, for example, in commonly owned U.S. Patent 8,268,143, the entire contents of which are incorporated herein by reference.

[0099] In some embodiments, the active region of this disclosure can be configured for the detection of ketones. Further details regarding ketone-responsive enzyme systems can be found in U.S. Patent Application 16 / 774,835, co-owned and filed January 28, 2020, entitled “Analyte Sensors and Sensing Methods Featuring Dual Detection of Glucose and Ketones,” published as U.S. Patent Application Publication 2020 / 0237275, the entire contents of which are incorporated herein by reference. In such a system, β-hydroxybutyrate is used as a substitute for ketones formed in vivo, undergoing a reaction with an enzyme system comprising β-hydroxybutyrate dehydrogenase (HBDH) and a flavin transactivator to facilitate the detection of ketones in 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 react β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD)... +The enzyme is converted into acetoacetic acid 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. In other words, the term "NAD" used in this article refers to NAD+. + Phosphate and NADH phosphate, especially diphosphates linking two nucleotides, one containing an adenine nucleotide and the other containing a nicotinamide nucleotide. NAD + The NADH enzyme cofactor facilitates the synergistic enzymatic reaction disclosed herein. Once formed, NADH can be oxidized under the mediation of a flavin transducer, 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. Electron transfer to the working electrode can be further mediated by electron transfer agents (e.g., osmium (Os) compounds or similar transition metal complexes), as described in more detail below. Albumin can also be present as a stabilizer within the active region. β-hydroxybutyrate dehydrogenase and flavin transducer can be covalently bonded to the polymer containing the ketone-responsive active region. + It can be covalently or non-covalently bonded to polymers, but if NAD... + If they are not covalently bonded, they can be physically retained within the ketone-responsive region, for example, by covering the ketone-responsive region with a mass transport restriction membrane, wherein the mass transport restriction membrane is also permeable to ketones.

[0100] According to embodiments of this disclosure, other suitable chemicals for enzymatic detection of ketones can be utilized. For example, β-hydroxybutyrate dehydrogenase (HBDH) can again separate β-hydroxybutyrate and NAD+. + The ketone is converted to acetoacetic acid and NADH. Instead of electron transfer to the working electrode via a flavotransferase 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 subsequently be converted to hydrogen peroxide under the mediation of superoxide dismutase (SOD). Hydrogen peroxide can then be oxidized at the working electrode to provide a signal that can be correlated with the amount of ketone initially present. According to different embodiments, SOD can be covalently bonded to the polymer in the ketone-responsive active region. β-hydroxybutyrate dehydrogenase and NADH oxidase can be covalently bonded to the polymer in the ketone-responsive active region, NAD... + It may or may not be covalently bonded to the polymer in the ketone-responsive active region. If NAD +Non-covalently bonded membrane polymers can be physically retained within the ketone-responsive region, promoting NAD50 absorption. + It remains within 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 ketone detection.

[0101] Another enzyme-based detection chemistry for ketones can utilize β-hydroxybutyrate dehydrogenase (HBDH) to react β-hydroxybutyrate and NAD+. + It is converted into acetoacetic acid and NADH, respectively. In this case, the electron transfer cycle involves the oxidation of NADH with 1,10-phenanthroline-5,6-dione to reform NAD. + This process is completed, in which 1,10-phenanthroline-5,6-dione subsequently transfers electrons to the working electrode. 1,10-phenanthroline-5,6-dione may or may not be covalently bonded to the polymer within the ketone-responsive active region. β-hydroxybutyrate dehydrogenase may be covalently bonded to the polymer within the ketone-responsive active region, NAD... + Albumin can be covalently or non-covalently bonded to polymers that respond to ketones. Including albumin in the active region can provide a significant improvement in response stability. Suitable membrane polymers can promote NAD+ response. + It remains within 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 ketone detection.

[0102] In some embodiments, the analyte sensor may further include a creatinine-responsive active region comprising an enzyme system that works synergistically to facilitate creatinine detection. In the presence of creatinine amide hydrolase (CNH), creatinine can reversibly and hydrolyze to form creatine. Creatine can then be catalytically hydrolyzed in the presence of creatine aminohydrolase (CRH) to form sarcosine. Neither of these reactions generates an electron flow (e.g., oxidation or reduction) to provide a basis for the electrochemical detection of creatinine. Sarcosine generated via 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 re-oxidation in the presence of an oxidized form of an electron transfer agent (e.g., an Os(III) complex) to generate a corresponding reduced form of the electron transfer agent (e.g., an Os(II) complex) and deliver an electron flow to the working electrode.

[0103] According to the aforementioned disclosure, oxygen may interfere with the concerted reaction sequence used to detect creatinine. Specifically, the reduced form of sarcosine oxidase can react with oxygen to reform the corresponding oxidized form of the enzyme, but does not exchange electrons with the electron transfer agent. Although all enzymes remain active when reacting with oxygen, no electrons flow to the working electrode. Unbound by theory or mechanism, the competitive reaction with oxygen is considered to be caused by kinetic effects. That is, the reduced form of sarcosine oxidase is thought to be oxidized by oxygen faster than by oxidation promoted by the electron transfer agent. Hydrogen peroxide is also formed in the presence of oxygen.

[0104] By including an oxygen scavenger near the enzyme system, the desired reaction pathway for facilitating creatinine detection can be encouraged. Various oxygen scavengers and their configurations may be suitable, including oxidases, such as glucose oxidase. Small molecule oxygen scavengers may also be suitable, but may be completely consumed before the sensor lifetime is fully exhausted. In contrast, enzymes can undergo reversible oxidation and reduction, thus providing a longer sensor lifetime. 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 a current to be generated at the working electrode. The magnitude of the generated current is proportional to the amount of creatinine initially reacted.

[0105] In any embodiment of this 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, provided that a suitable substrate for the enzyme is also present, thus providing a reagent for the reaction with oxygen in the presence of the oxidase. Oxidases that may be suitable for oxygen scavenging in this disclosure include, but are not limited to, glucose oxidase, lactate oxidase, xanthine oxidase, etc. Glucose oxidase may be a particularly desirable oxidase for promoting oxygen scavenging due to the readily available availability of glucose in various body fluids. Reaction 1 below illustrates an enzymatic reaction that provides oxygen scavenging, promoted by glucose oxidase.

[0106] β-D-glucose + O2 → D-gluconic acid-1,5-lactone + H2O2

[0107] Reaction 1

[0108] The concentration of available lactate in the body is lower than that of blood glucose, but it is still sufficient to promote oxygen clearance.

[0109] Oxidases (e.g., glucose oxidases) can be located anywhere suitable for facilitating oxygen removal in the analyte sensors disclosed herein. For example, glucose oxidases can be located at the tail of the sensor, such that the glucose oxidase is functional and / or non-functional for facilitating glucose detection. When non-functional for facilitating glucose detection, the glucose oxidase can be located at the tail of the sensor such that electrons generated during glucose oxidation cannot reach the working electrode, for example, by electrically isolating the glucose oxidase from the working electrode.

[0110] Additional details regarding enzyme systems responsive to creatinine can be found in co-owned U.S. Patent Application 16 / 774,835, filed September 25, 2019, entitled “Analyte Sensors and Sensing Methods for Detecting Creatinine,” published as U.S. Patent Application Publication 2020 / 0241015, the entire contents of which are incorporated herein by reference.

[0111] In some embodiments, the analyte sensor may include a lactate-responsive active region disposed at the tail of the sensor, the active region comprising a lactate-responsive enzyme. Suitable lactate-responsive enzymes may include, for example, lactate oxidase. Lactate oxidase or other lactate-responsive enzymes may be covalently bonded to a polymer comprising the lactate-responsive active region and exchange electrons with an electron transfer agent (e.g., an osmium (Os) complex or a similar transition metal complex), which may also be covalently bonded to the polymer. Suitable electron transfer agents will be described in further detail below. Albumin (e.g., human serum albumin) may be present in the lactate-responsive active region to stabilize the sensor response, as described in more detail in commonly owned U.S. Patent Application Publication 2019 / 0320947, which is incorporated herein by reference in its entirety. Lactate levels may vary in response to a number of environmental or physiological factors, including, for example, food intake, stress, exercise, sepsis or septic shock, infection, hypoxia, the presence of cancerous tissue, etc.

[0112] In some embodiments, the analyte sensor may include an active region responsive to a pH value. A suitable analyte sensor configured to determine pH is described in commonly owned U.S. Patent Application Publication 2020 / 0060592, which is incorporated herein by reference in its entirety. Such an analyte sensor may include a sensor tail comprising a first working electrode and a second working electrode, wherein a first active region on the first working electrode comprises a substance having pH-dependent redox chemistry, and a second active region on the second working electrode comprises a substance having redox chemistry substantially independent of pH. By obtaining a difference between the first and second signals, this difference can be correlated with the pH of the fluid to which the analyte sensor is exposed.

[0113] Two different types of active regions can be located on a single working electrode, such as the carbon working electrode discussed above, and spaced apart from each other. Each active region can have a redox potential, wherein the redox potential of the first active region is sufficiently separated from that of the second active region to allow signal generation independently from one active region. As a non-limiting example, the redox potentials can differ by at least about 100 mV, or at least about 150 mV, or at least about 200 mV. The upper limit of the interval between redox potentials is determined by the working electrochemical window in the body. By making the redox potentials of the two active regions sufficiently separated from each other in magnitude, electrochemical reactions can occur within one of the two active regions (i.e., within the first or second active region) without substantially initiating electrochemical reactions in the other active region. Therefore, a signal from one of the first or second active regions can be generated independently at its corresponding redox potential (the lower redox potential) or above that potential but below the redox potential of the other active region. Different signals can allow for resolution of the signal contribution from each analyte.

[0114] Some or all embodiments of the analyte sensors disclosed herein may feature one or more active regions located on the surface of at least one working electrode, wherein the active regions detect the same or different analytes. A membrane may at least cover the active regions (containing analyte-responsive enzymes) and may further cover all or a portion of the working electrode lacking active regions (exposed or foreign portions of the working electrode). The membrane may be a mass transfer-limiting membrane and may be a monolayer membrane, a bilayer membrane of two different membrane polymers, or a mixture of two different membrane polymers.

[0115] Electron transfer agents can be present in any of the active regions disclosed herein. After one or more analytes undergo an enzymatic redox reaction in their respective active regions, a suitable electron transfer agent can facilitate the transfer of electrons to adjacent working electrodes, 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 responding to different analytes can 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 can be different (e.g., different chemical properties causing the electron transfer agents to exhibit different redox potentials). When multiple working electrodes are present, the electron transfer agents in each active region can be the same or different because each working electrode can be queried individually.

[0116] Suitable electron transfer agents may include electroreducible and electrooxidizable ions, complexes, or molecules (e.g., quinones) with redox potentials several hundred millivolts higher or lower than the redox potential of a standard calomel electrode (SCE). According to some embodiments, suitable electron transfer agents may include low-potential osmium complexes, such as those described in U.S. Patents 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. Patents 6,736,957, 7,501,053, and 7,754,093, the disclosure of each of which is incorporated herein by reference in its entirety. Other suitable electron transfer agents may include metal compounds or complexes of ruthenium, osmium, iron (e.g., ferrocene or hexacyanoferrate), or cobalt, including, for example, their metallocene compounds. Suitable ligands for metal complexes may also include, for example, bidentate or higher-dentent ligands, such as bipyridine, imidazole, phenanthroline, or pyridyl (imidazolium). Other suitable bidentate ligands may include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkane, or o-diaminoaromatic hydrocarbon. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher-dentate ligands may be present in metal complexes to achieve a complete coordination layer.

[0117] Active regions suitable for detecting any analyte disclosed herein may include polymers covalently bonded to an electron transfer agent. Any electron transfer agent disclosed herein may include suitable functions to facilitate covalent bonding of the polymer within the active region. Suitable examples of polymer-bonded 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 incorporated herein by reference in their entirety. Suitable polymers contained within the active region may include, but are not limited to, polyvinylpyridine (e.g., poly(4-vinylpyridine)), polyvinylimidazole (e.g., poly(1-vinylimidazole)), or any copolymers thereof. Exemplary copolymers suitable for inclusion within the active region include copolymers containing monomeric units, such as styrene, acrylamide, methacrylamide, or acrylonitrile. When two or more distinct active regions are present, the polymers within each active region may be the same or different.

[0118] Covalent bonding of electron transfer agents to polymers within the active region can be achieved by polymerizing monomer units containing covalently bonded electron transfer agents, or the electron transfer agent can react with the polymer independently after the polymer has been synthesized. Bifunctional spacers can covalently bond electron transfer agents to the polymer within the active region, where the first functional group reacts with the polymer (e.g., a functional group capable of quaternizing pyridine or imidazole nitrogen atoms), and the second functional group reacts with the electron transfer agent (e.g., a functional group that reacts with ligands of coordinating metal ions).

[0119] Similarly, one or more enzymes within the active region can be covalently bonded to the polymer containing the active region. When an enzyme system containing multiple enzymes is present in a given active region, in some embodiments, all of the multiple enzymes can be covalently bonded to the polymer, while in other embodiments, only a portion of the multiple enzymes can be covalently bonded to the polymer. For example, one or more enzymes containing the enzyme system can be covalently bonded to the polymer, and at least one enzyme can non-covalently associate with the polymer, such that the non-covalently bonded enzyme is physically entrained within the polymer. In a given active region, the covalent bonding of the enzyme to the polymer can be carried out via a cross-linking agent introduced together with a suitable cross-linking agent. Suitable cross-linking agents for reacting with the free amino group in the enzyme (e.g., with the free side chain amine in lysine) can include cross-linking agents such as polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imino esters, epichlorohydrin, or derivatives thereof. Suitable cross-linking agents for reacting with the free carboxylic acid group in the enzyme can include, for example, carbodiimide. Crosslinking between enzymes and polymers is typically intermolecular, but in some embodiments it can be intramolecular. In specific embodiments, all enzymes within a given active region can be covalently bonded to the polymer.

[0120] Electron transfer agents and / or enzymes can also bind to the polymer in the active region via means other than covalent bonding. In some embodiments, electron transfer agents and / or enzymes can bind to polymer ions or coordinates. For example, a charged polymer can associate with an electron transfer agent or enzyme ion of opposite charge. In other embodiments, electron transfer agents and / or enzymes can be physically entrained within the polymer without being bonded to it. Physically entrained electron transfer agents and / or enzymes can still appropriately interact with the fluid to facilitate analyte detection without substantially leaching from the active region.

[0121] Polymers within the active region can be selected to enable NAD. + Alternatively, the outward diffusion of another cofactor not covalently bonded to the polymer is limited. Limited outward diffusion of the cofactor can contribute to a reasonable sensor lifetime (days to weeks) while still allowing sufficient analyte inward diffusion to facilitate detection.

[0122] In some embodiments, stabilizers may be incorporated into the active region of the analyte sensor described herein to improve sensor function and achieve desired sensitivity and stability. For example, such stabilizers may include antioxidants and / or chaperone proteins to stabilize enzymes. Examples of suitable stabilizers may 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, and any combination thereof. Stabilizers may be conjugated or non-conjugated.

[0123] In specific embodiments of this disclosure, the mass transfer limiting film covering one or more active regions may comprise a crosslinked polyvinylpyridine homopolymer or copolymer. When the mass transfer limiting film covers different types of active regions, the composition of the mass transfer limiting film may be the same or different. When the film composition varies at two different locations, the film may comprise a bilayer film or a homopolymer or copolymer of two different film polymers, one of which may be a crosslinked polyvinylpyridine or a polyvinylimidazolium homopolymer or copolymer. Suitable techniques for depositing the mass transfer limiting film on the active regions may include, for example, spraying, painting, inkjet printing, screen printing, stencil printing, roll coating, dip coating, and any combination thereof. Dip coating techniques may be particularly desirable for polyvinylpyridine and polyvinylimidazolium polymers and copolymers.

[0124] In some embodiments, the mass transfer limiting membrane described above is a membrane composed of a crosslinked polymer containing heterocyclic nitrogen groups, such as a polymer of polyvinylpyridine and polyvinylimidazole. Embodiments also include membranes made of polyurethane or polyether urethane or chemically related materials, or membranes made of silicone resins, etc.

[0125] In some embodiments, the membrane can be formed by in situ crosslinking in a buffer solution (e.g., an alcohol buffer) a zwitterionic portion, a non-pyridine copolymer component, and optionally a hydrophilic or hydrophobic and / or other portion modified polymer (including those described above) having other desired properties. The modified polymer can be made from a precursor polymer containing heterocyclic nitrogen groups. For example, the precursor polymer can be polyvinylpyridine or polyvinylimidazole. Optionally, a hydrophilic or hydrophobic modifier 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, and any combination thereof) can be used to enhance the biocompatibility of the polymer or the resulting membrane.

[0126] 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); polyvinylpyridine; derivatives of polyvinylpyridine; polyvinylimidazole; derivatives of polyvinylimidazole; polyvinylpyrrolidone (PVP); and any combination thereof. In some embodiments, the membrane may be composed of a polyvinylpyridine-co-styrene polymer, wherein a portion of the pyridine nitrogen atoms are functionalized with a non-crosslinked poly(ethylene glycol) tail and a portion of the pyridine nitrogen atoms are functionalized with an alkyl sulfonic acid group. 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.

[0127] The membrane compounds described herein can be further crosslinked with one or more crosslinking agents, including those listed above with reference to the enzymes described herein. Suitable crosslinking agents may include, but are not limited to, polyethylene glycol diglycidyl ether (PEGDGE), glyceryl triglycidyl ether (Gly3), polydimethylsiloxane diglycidyl ether (PDMS-DGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imino esters, epichlorohydrin or their derivatives and any combinations thereof. Branched versions with similar terminal chemistry are also applicable to this disclosure. For example, in some embodiments, Formula 1 can be crosslinked with triglycidyl ether and / or PEDGE and / or polydimethylsiloxane diglycidyl ether (PDMS-DGE).

[0128] A membrane can be formed in situ by coating the active region and any additional compounds (e.g., electron transfer agents) contained within the active region with an alcohol-buffered solution of a crosslinking agent and a modified polymer, and allowing the solution to cure for approximately one to two days or other suitable time periods. The crosslinking agent-polymer solution can be applied to the active region by placing one or more droplets of the membrane solution onto at least one or more sensor elements at the sensor tail, by immersing the sensor tail in the membrane solution, by spraying the membrane solution onto the sensor, by hot-pressing or melting the membrane in any size layer (e.g., discrete or fully enclosed), and before or after segmentation, vapor deposition of the membrane, powder coating of the membrane, etc., and any combination thereof. To coat the distal end and sides of the sensor, the membrane material can be applied after applying (e.g., segmenting) the sensor electronic precursor (e.g., electrodes). In some embodiments, the analyte sensor is dip-coated after applying the electronic precursor to apply one or more membranes. Alternatively, the analyte sensor can be slit-coated, wherein each side of the analyte sensor is coated individually. The membrane applied in the manner described above can have any and all functions, including but not limited to mass transport restriction (i.e., reducing or eliminating the flow of one or more analytes and / or compounds to the active region), enhanced biocompatibility, reduction of interfering substances, and any combination thereof.

[0129] Typically, membrane thickness is controlled by the concentration of the membrane solution, the number of droplets of the applied membrane solution, the number of times the sensor is immersed in the membrane solution, the volume of the membrane solution sprayed onto the sensor, and any combination of these factors. In some embodiments, the membrane described herein may have a thickness ranging from about 0.1 μm to about 1000 μm, including any values ​​and subsets thereof, wherein the upper and lower limits are separable. As mentioned above, the membrane may cover one or more active regions, and in some embodiments, the active regions may have a thickness ranging from about 0.1 μm to about 50 μm, including any values ​​and subsets thereof, wherein the upper and lower limits are separable. In some embodiments, a series of droplets may be applied over each other to achieve the desired thickness of the active regions and / or the membrane without substantially increasing the diameter of the applied droplets (i.e., maintaining their desired diameter or range). For example, each individual droplet may be applied, followed by cooling or drying, and then one or more additional droplets may be applied. The active regions and the membrane may, but do not necessarily, have the same thickness or composition.

[0130] In some embodiments, the membrane component used as the mass transport limiting layer of this disclosure may include polydimethylsiloxane (PDMS), polydimethylsiloxane diglycidyl ether (PDMS-DGE), aminopropyl-terminated polydimethylsiloxane, and any combination thereof, used as a leveling agent (e.g., for reducing the contact angle of the membrane component or active region component). Branched versions having similar terminal chemistry are also applicable to this disclosure. Certain leveling agents may also be included, for example, those found in U.S. Patent 8,983,568, the entire disclosure of which is incorporated herein by reference.

[0131] In some cases, the membrane can form one or more bonds with the active region. As used herein, the term "bond" and its grammatical variations refer to any type of interaction between atoms or molecules that allows chemical compounds to associate with each other, such as, but not limited to, covalent bonds, ionic bonds, dipole-dipole interactions, hydrogen bonds, London dispersion forces, and any combination thereof. For example, in-situ polymerization of the membrane can form crosslinks between the polymer in the membrane and the polymer in the active region. In some embodiments, crosslinking of the membrane with the active region helps reduce the occurrence of membrane peeling from the sensor.

[0132] The embodiments disclosed herein include:

[0133] A. A method comprising: laser-segmenting a working electrode including an active region disposed thereon having an analyte-responsive enzyme, wherein the resulting laser-segmented working electrode includes an electrode roughness; and laser-gowing at least a portion of the electrode roughness, the laser gowing recessing from the edge of the laser-segmented working electrode to produce a laser-gowned working electrode.

[0134] B. An analyte sensor comprising: a working electrode including an active region disposed thereon having an analyte-responsive enzyme, wherein the working electrode is first laser-segmented to create an electrode roughness, and then the edges of the working electrode are laser-gouged to remove at least a portion of the electrode roughness thereon, thereby creating a laser-gouged working electrode.

[0135] C. A method comprising: laser-segmenting a working electrode including an active region disposed thereon having an analyte-responsive enzyme, wherein the resulting laser-segmented working electrode includes an electrode roughness; disposing a membrane on at least a portion of the active region; and laser-gouging at least a portion of the electrode roughness, the laser gouging recessing from the edge of the laser-segmented working electrode to produce a laser-gouged working electrode.

[0136] D. An analyte sensor, comprising: a laser-cut working electrode including an active region disposed thereon and an electrode roughened therefrom by laser cutting, the active region including an analyte-responsive enzyme.

[0137] E. A method comprising: exposing an analyte sensor to a body fluid, the analyte sensor including a laser-cut working electrode having an active region disposed thereon and an electrode roughening material laser-cut therefrom, the active region including an analyte-responsive enzyme.

[0138] F. An analyte sensor, comprising: a working electrode including an active region disposed thereon, the active region including an analyte-responsive enzyme; a membrane disposed on at least a portion of the working electrode including the active region; and an interfering-reactant substance contained in the analyte sensor.

[0139] G. A method comprising: exposing an analyte sensor to a bodily fluid, the analyte sensor comprising: a working electrode including an active region disposed thereon, the active region including an analyte-responsive enzyme; a membrane disposed on at least a portion of the working electrode including the active region; and an interfering-reactant substance contained in the analyte sensor.

[0140] H. An analyte sensor comprising: a working electrode including an active region disposed thereon, the active region including an analyte-responsive enzyme; and a scrubbing electrode, wherein the scrubbing electrode (1) is positioned facing the working electrode and the working electrode and the scrubbing electrode are spatially offset, or (2) is located above the working electrode and is permeable to the analyte of interest for diffusion of the analyte of interest to the active region.

[0141] I. A method comprising: exposing an analyte sensor to a body fluid, the analyte sensor comprising: a working electrode including an active region disposed thereon, the active region including an analyte-responsive enzyme; and a wiping electrode, wherein the wiping electrode (1) is positioned facing the working electrode and the working electrode and the wiping electrode are spatially offset, or (2) is positioned above the working electrode and is permeable to the analyte of interest for diffusion of the analyte of interest to the active region.

[0142] J. An analyte sensor, comprising: a working electrode including an active region disposed thereon, the active region including an analyte-responsive enzyme; and a scrubbing electrode positioned facing the working electrode, wherein the working electrode and the scrubbing electrode are spatially offset.

[0143] K. A method comprising: exposing an analyte sensor to a body fluid, the analyte sensor comprising: a working electrode including an active region disposed thereon, the active region including an analyte-responsive enzyme; and a wiping electrode positioned opposite to the working electrode, wherein the working electrode and the wiping electrode are spatially offset.

[0144] L. An analyte sensor comprising: a working electrode including an active region disposed thereon, the active region including an analyte-responsive enzyme; and a permeable scrubbing electrode located above the working electrode, wherein the permeable scrubbing electrode is permeable to the analyte of interest for diffusion of the analyte of interest into the active region.

[0145] M. A method comprising: exposing an analyte sensor to a body fluid, the analyte sensor comprising: a working electrode including an active region disposed thereon, the active region including an analyte-responsive enzyme; and a permeable scrubbing electrode positioned above the working electrode, wherein the permeable scrubbing electrode is permeable to the analyte of interest for diffusion of the analyte of interest to the active region.

[0146] Implementation A may have any combination of one or more of the following additional elements:

[0147] Component A1: Among them, the laser-cut working electrode shows a reduction in interference signals from interfering objects compared to the uncut working electrode.

[0148] Component A2: Among them, compared with the unplaned working electrode, the laser-planed working electrode shows a reduction in the interference signal of the interfering object, and the reduction in the interference signal of the interfering object is greater than about 20%.

[0149] Component A3: wherein, compared with an unplaned working electrode, a laser-planed working electrode exhibits a reduction in the interference signal of the interfering object, the reduction in the interference signal of the interfering object being in the range of about 20% to about 70%, including any values ​​and subsets therebetween, and wherein the upper and lower limits are separable.

[0150] Component A4: The laser-planed working electrode shows a reduction in interference signals from an interfering substance, ascorbic acid, compared to the unplaned working electrode.

[0151] Component A5: also includes at least about 5% of the total area of ​​the removed electrode roughness.

[0152] Component A6: also includes about 5% to about 75% of the total area of ​​the removed electrode roughness, including any values ​​and subsets therein, wherein the upper and lower limits are separable.

[0153] Component A7: Among them, laser planing includes multiple single-pass laser planing cuts.

[0154] Component A8: wherein the laser planing includes a plurality of single-pass laser planing kerfs, the plurality of single-pass laser planing kerfs being at least one of being perpendicular to the edge of the laser-segmented working electrode or inclined relative to the edge of the laser-segmented working electrode.

[0155] Component A9: wherein the width of the electrode roughness is in the range of 1 μm to about 75 μm, including any value and subset thereof, and wherein the upper and lower limits are separable.

[0156] Component A10: wherein the height of the electrode roughness is from about 1 μm to about 50 μm, including any values ​​and subsets therebetween, and wherein the upper and lower limits are separable.

[0157] Component A11: The active region consists of multiple discontinuous active regions or a single continuous active region.

[0158] Component A12: where the compression active region is located.

[0159] Component A13: Among them, the active responsive enzyme is a glucose responsive enzyme.

[0160] Element A14: wherein the membrane is disposed on at least a portion of the active region.

[0161] As a non-limiting example, exemplary combinations applicable to A include, but are not limited to, any combination of A with any one or more of A1-A14.

[0162] Embodiment B may have any combination of one or more of the following additional elements:

[0163] Component B1: Among them, the laser-cut working electrode shows a reduction in interference signals from interfering objects compared to the uncut working electrode.

[0164] Component B2: Among them, compared with the unplaned working electrode, the laser-planed working electrode shows a reduction in interference signals from interfering objects, with a reduction of more than about 20%.

[0165] Component B3: wherein, compared with an unplaned working electrode, a laser-planed working electrode exhibits a reduction in the interference signal of the interfering object, the reduction in the interference signal of the interfering object being in the range of approximately 20% to approximately 70%, including any values ​​and subsets therebetween, and wherein the upper and lower limits are separable.

[0166] Component B4: The laser-planed working electrode shows a reduction in interference signals from an interfering substance, ascorbic acid, compared to the unplaned working electrode.

[0167] Component B5: wherein at least about 5% of the total area of ​​the removed electrode roughness is removed.

[0168] Element B6: wherein approximately 5% to approximately 75% of the total area of ​​the removed electrode rough body is removed, including any values ​​and subsets therein, and wherein the upper and lower limits are separable.

[0169] Component B7: wherein the width of the electrode roughness is in the range of 1 μm to about 75 μm, including any value and subset thereof, and wherein the upper and lower limits are separable.

[0170] Component B8: wherein the height of the electrode roughness is from about 1 μm to about 50 μm, including any values ​​and subsets therebetween, and wherein the upper and lower limits are separable.

[0171] Component B9: The active region consists of multiple discontinuous active regions or a single continuous active region.

[0172] Component B10: where the compression active region is located.

[0173] Component B11: Among them, the active responsive enzyme is a glucose responsive enzyme.

[0174] Element B12: wherein the membrane is disposed on at least a portion of the active region.

[0175] As a non-limiting example, exemplary combinations applicable to B include, but are not limited to, any combination of B with any one or more of B1-B12.

[0176] Implementation C may have any combination of one or more of the following additional elements:

[0177] Component C1: Among them, the laser-cut working electrode shows a reduction in interference signals from interfering objects compared to the uncut working electrode.

[0178] Component C2: Among them, compared with the unplaned working electrode, the laser-planed working electrode shows a reduction in the interference signal of the interfering object, and the reduction in the interference signal of the interfering object is greater than about 20%.

[0179] Component C3: wherein, compared with an unplaned working electrode, a laser-planed working electrode exhibits a reduction in the interference signal of the interfering object, the reduction in the interference signal of the interfering object being in the range of approximately 20% to approximately 70%, including any values ​​and subsets therebetween, and wherein the upper and lower limits are separable.

[0180] Component C4: The laser-planed working electrode shows a reduction in interference signals from an interfering substance, ascorbic acid, compared to the unplaned working electrode.

[0181] Component C5: also includes at least about 5% of the total area of ​​the removed electrode roughness.

[0182] Component C6: also includes approximately 5% to approximately 75% of the total area of ​​the removed electrode roughness, including any values ​​and subsets therein, wherein the upper and lower limits are separable.

[0183] Component C7: Among them, laser planing includes multiple single-pass laser planing cuts.

[0184] Component C8: wherein the laser planing includes multiple single-pass laser planing cuts, and the multiple single-pass laser planing cuts are at least one of being perpendicular to the edge of the laser-segmented working electrode or inclined relative to the edge of the laser-segmented working electrode.

[0185] Component C9: wherein the width of the electrode roughness is in the range of 1 μm to about 75 μm, including any value and subset thereof, and wherein the upper and lower limits are separable.

[0186] Component C10: wherein the electrode roughness has a height of about 1 μm to about 50 μm, including any value and subset thereof, and wherein the upper and lower limits are separable.

[0187] Component C11: The active region consists of multiple discontinuous active regions or a single continuous active region.

[0188] Component C12: where the active region is compressed.

[0189] Component C13: Among them, the active responsive enzyme is a glucose responsive enzyme.

[0190] As a non-limiting example, exemplary combinations applicable to C include, but are not limited to, any combination of C with any one or more of C1-C13.

[0191] Embodiments D and E may have any combination of one or more of the following additional elements:

[0192] Component D / E1: Among them, the laser-planed working electrode shows a reduction in interference signals from interfering objects compared to the unplaned working electrode.

[0193] Component D / E2: Among them, compared with the unplaned working electrode, the laser-planed working electrode shows a reduction in interference signals from interfering objects, with a reduction of more than about 20%.

[0194] Component D / E3: wherein, compared with an unplaned working electrode, a laser-planed working electrode exhibits a reduction in the interference signal of the interfering object, the reduction in the interference signal of the interfering object being in the range of approximately 20% to approximately 70%, including any values ​​and subsets therebetween, and wherein the upper and lower limits are separable.

[0195] Component D / E4: Among them, the laser-planed working electrode shows a reduction in interference signals from an interfering substance, ascorbic acid, compared to the unplaned working electrode.

[0196] Component D / E5: wherein at least about 5% of the total area of ​​the removed electrode roughness is removed.

[0197] Component D / E6: wherein approximately 5% to approximately 75% of the total area of ​​the removed electrode roughness is removed, including any values ​​and subsets therein, and wherein the upper and lower limits are separable.

[0198] Component D / E7: wherein the width of the electrode roughness is in the range of 1 μm to about 75 μm, including any value and subset thereof, and wherein the upper and lower limits are separable.

[0199] Component D / E8: wherein the electrode roughness has a height of about 1 μm to about 50 μm, including any value and subset thereof, and wherein the upper and lower limits are separable.

[0200] Component D / E9: The active region consists of multiple discontinuous active regions or a single continuous active region.

[0201] Component D / E10: Wherein, the compression active region.

[0202] Component D / E11: Among them, the active responsive enzyme is a glucose responsive enzyme.

[0203] Element D / E12: wherein the membrane is disposed on at least a portion of the active region.

[0204] As a non-limiting example, exemplary combinations applicable to D / E include, but are not limited to, any combination of D / E with any one or more of D / E1-D / E12.

[0205] According to the configuration of the scrubbing electrodes described below, embodiments H to M may have any combination of one or more of the following additional elements:

[0206] Component H-M1: wherein the scrubbing electrode is positioned facing the working electrode, and the working electrode and the scrubbing electrode are spatially offset over a thin layer ranging from about 1 μm to about 200 μm, including any values ​​and subsets therebetween, and wherein the upper and lower limits are separable.

[0207] Element H-M2: wherein the scrubbing electrode has a width of approximately 200 μm to approximately 8000 μm, including any values ​​and subsets thereof, and wherein the upper and lower limits are separable.

[0208] Component H-M3: wherein the scrubbing electrode is positioned facing the working electrode, and the working electrode and the scrubbing electrode are spatially offset, and the width of the scrubbing electrode relative to the working electrode is in the range of about 2:1 to about 50:1, including any values ​​and subsets therebetween, and wherein the upper and lower limits are separable.

[0209] Component H-M4: wherein the scrubbing electrode has a potential in the range of approximately -2000mV to approximately +2000mV, including any values ​​and subsets thereof, and wherein the upper and lower limits are separable.

[0210] Component H-M5: The scrubbing electrode is located above the working electrode and is permeable to the analyte of interest (AIO) to diffuse the AIO into the active region. The AIO is ascorbic acid.

[0211] Component H-M6: The scrubbing electrode is located above the working electrode and is permeable to the analyte of interest, used to diffuse the analyte of interest into the active region. The scrubbing electrode is composed of carbon nanotube material.

[0212] Component H-M7: The scrubbing electrode is located above the working electrode and is permeable to the analyte of interest for diffusion of the analyte of interest into the active region. The scrubbing electrode is composed of PEDOT:PSS impregnated with carbon nanotube material.

[0213] Component H-M8: The scrubbing electrode is located above the working electrode and is permeable to the analyte of interest for diffusion into the active region. The scrubbing electrode is impregnated with an electron transfer agent.

[0214] Element H-M9: wherein the scrubbing electrode is located above the working electrode and is permeable to the analyte of interest for diffusion of the analyte of interest into the active region, the scrubbing electrode has a thickness of about 0.5 μm to about 100 μm, including any values ​​and subsets therein, and wherein the upper and lower limits are separable.

[0215] As a non-limiting example, exemplary combinations applicable to HM include, but are not limited to, any combination of HM with any one or more of H-M1-H-M9, depending on the configuration of the scrubbing electrodes (i.e., spatial offset or permeable).

[0216] To facilitate a better understanding of the embodiments described herein, the following examples of various representative embodiments are provided. These examples should not be construed as limiting or restricting the scope of the invention.

[0217] Example 1. In this example, in Figure 13A The example shown is laser planing performed on the working electrode of laser segmentation. Figure 13A Excluding the active regions set on it. Figure 13B The width of the identified contour is shown. Figure 13A A 3D optical profile of a segmented working electrode. Using ZEGAGE (Middlefield, Connecticut) TM A 3D optical profilometer obtains a 3D optical profile. For example... Figure 13B As shown, the electrode roughness at the edge of the segmented sensor tail section exhibits approximately 1 μm to approximately A height of 5μm.

[0218] Laser planing is performed using three single-pass laser lines located at the edge of the carbon roughened body, with the laser power at 10% gradually separated towards the centerline of the electrodes by approximately 5 μm to approximately 20 μm, for example, approximately 5 μm to approximately 10 μm. In the example described herein, a UV laser is used; however, it should be understood that any laser can be used to perform laser planing without departing from the scope of this disclosure. Figure 13C It is a photo of the planed sensor tail, showing the slanted edge of the working electrode at the sensor tail. Figure 13D The 3D optical profile (obtained as previously described) along the identified contour lines shows the removed electrode roughness.

[0219] Example 2. In this example, refer to Figure 14A A laser-planed carbon working electrode 1400 is prepared according to Example 1. The carbon electrode includes an active region 1402 distributed thereon. An unplaned carbon electrode containing the active region 1402 is not shown, but will be referred to as an "unplaned, distributed" electrode. Figure 14BThe 3D optical profile (obtained as described above) along the identified contour lines shows the minimum electrode roughness as a result of planing.

[0220] Example 3. A pairwise difference test was performed. The test was conducted using the "unplaned, distributed" electrodes from Example 2. Figure 13A Unmashed electrodes and Figure 13C Planed electrodes without active areas (referred to as "unplaned, unassigned" and "planed, unassigned") were evaluated in 50 mg / dL blood glucose and 2 mg / dL ascorbic acid at 37°C in 100 mM PBS, with multiple sensing points. Figure 14A The planed electrodes (“planed, distributed”). Results are provided in Table 1 below, and... Figure 15 The diagram is shown in the image.

[0221] Table 1

[0222]

[0223]

[0224] *Background corrected;** Laser planing compared to control.

[0225] As shown, paired difference tests indicate that, compared with the unplaned counterpart, the laser-planed electrode showed a reduction of approximately 25% to approximately 30% in ascorbic acid at 2 mg / dL.

[0226] Example 4. Paired difference tests were performed on laser-segmented working electrodes prepared as follows. The unplaned “control” working electrode comprises active regions with multiple sensing points. The electrode described as “compressed” comprises active regions of the same concentration (compressed active regions), but multiple sensing points are closer together and / or closer to the tip of the electrode. For example, the spacing between each discontinuous active region (the distance between adjacent sensing points) can be from about 50 μm to about 800 μm, for example, from about 50 μm to about 500 μm, including any values ​​and subsets therein, and wherein the upper and lower limits are separable. As used herein, the term “spacing” and its grammatical variations refer to the spacing between adjacent sensing points in the active region sensing layer, measured from the center of each adjacent sensing point. Typically, the most distal active region is located at least about 200 μm to 300 μm (measured from its sensing center) from the tip of the working electrode (which may be the same as the tip of the sensor tail) to be at the most distal point in the body fluid, but may be in the range of about 50 μm to about 500 μm, including any values ​​and subsets therein, and wherein the upper and lower limits are separable.

[0227] Regardless of compression, the total amount of analyte-responsive enzymes is the same for all samples. Laser planing is described using three separate single-pass laser lines, each at a specific distance from the edge of the initially unplaned electrode (“planing scheme”). For example, “20-40-60” refers to a first single-pass laser line 20 μm from the edge of the unplaned electrode, a second single-pass laser line 40 μm from the edge of the unplaned electrode, and a third single-pass laser line 60 μm from the edge of the unplaned electrode.

[0228] Table 2

[0229] compression? no no yes yes yes Planing scheme Unplaned 20-40-60 Unplaned 20-40-60 15-25-40

[0230] In some cases, electrodes 16A to 16E described in Table 2 are coated with a mass transport limiting film, the thickness of which is shown in Table 3 below. Paired difference tests were performed at 37°C in 100 mM PBS at 50 mg / dL blood glucose and 2 mg / dL ascorbic acid (mean n = 6 / condition). The results are shown in Table 3 below.

[0231] Table 3

[0232] 16A no ~35μm ～0 16A no ~50μm ～-20 16B 20-40-60 ~35μm ～-30 16B 20-40-60 ~50μm ～-45 16C no ~35μm ～-30 16C no ~50μm ～-50 16D 20-40-60 ~35μm ～-50 16D 20-40-60 ~50μm ～-65 16E 15-25-40 ~35μm ～-50 16E 15-25-40 ~50μm ～-60

[0233] As shown in Table 3, pairwise difference tests indicate that laser-planed electrodes exhibit a reduction in ascorbic acid signal interference of greater than about 20% compared to their unplaned counterparts. Therefore, embodiments of this disclosure allow for a reduction in interfering signals (e.g., ascorbic acid) of at least about 20%, which, depending on the configuration of the analyte sensor, can be as high as 100%, or, for example, in the range of about 20% to about 70% or greater, and preferably at least about 40%, at least about 45%, or at least about 50%, including any values ​​and subsets therebetween, wherein the upper and lower limits are separable. Furthermore, results show that even small amounts of laser planing are effective.

[0234] Example 5. In this example, the effectiveness of incorporating enzyme-interfering reactant substances into the analyte sensor to eliminate or reduce interference signals at the working electrode was evaluated. Figure 17As shown, a blood glucose sensor 1700 with an interfering-reactant layer for reacting with ascorbic acid is fabricated. A blood glucose active area sensing layer 1704 is coated onto a carbon working electrode 1702 (a carbon electrode disposed on a substrate (not shown)) in the form of six discontinuous sensing points and contains a blood glucose oxidase sensing chemical. A diffusion-limiting membrane 1706 is coated over the entire working electrode 1702, covering each sensing point 1702. 50 nL of an interfering-reactant material layer 1708 is coated on top of the diffusion-limiting membrane 1706, covering the sensing layer 1704 and any excess (exposed) carbon working electrode 1702. The interfering-reactant material layer 1708 contains ascorbic acid oxidase (~25 mg / ml) in a matrix of PVI polymer (~9 mg / ml), PEDGE-400 crosslinking agent (~6 mg / ml), and albumin stabilizer (~25 mg / ml) (prepared in 10 mM MES buffer, pH 5.5).

[0235] Two types of ascorbic acid oxidases, ASO-301 and ASO-311, both available from TOYOBO, headquartered in Osaka, Japan, were evaluated. A thin layer of cross-linked outer membrane 1710 was coated on top of the entire interferant-reactant material layer 1708. Depending on the ascorbic acid oxidase used, these sensors are referred to as “GOx / membrane / cross-linker / AscOx301” and “GOx / membrane / cross-linker / AscOx311”.

[0236] The sensor and two controls were tested in quadruplicate in 100 mM PBS at a temperature of ~30°C, a pH of ~7.5, and an operating potential of +40 mV, in ascorbic acid and glucose, respectively. The first control (“Gox / membrane control”) consisted of a carbon working electrode 1702, a sensing point 1703, and a diffusion-limiting membrane 1704 (without interfering reactant layer 1708 and an outer membrane layer). The second control (“Gox / membrane / crosslinker”) consisted of a carbon working electrode 1702, a sensing point 1704, a diffusion-limiting membrane 1706, and an outer membrane layer 1710 coated thereon (without interfering reactant layer 1708). The sensor was calibrated in ascorbic acid, as... Figure 18 As shown, calibration is performed in 30mM blood glucose, as... Figure 19 As shown.

[0237] As shown, compared to control sensors with and without PVP membranes, the sensor with an interfering-reactant layer (containing ascorbic acid oxidase) exhibited a very small response to the addition of ascorbic acid. Furthermore, the presence of the interfering-reactant layer had no significant effect on the glucose response compared to control sensors with and without PVP membranes. Moreover, even if the interfering-reactant layer did affect glucose sensing, any such effect could be easily explained as long as the linearity and stability of glucose levels were maintained. Therefore, containing an enzyme-interfering-reactant layer is a feasible method to eliminate or reduce signals at the working electrode attributable to interfering substances.

[0238] Example 6. In this example, the effectiveness of incorporating metal oxide interfering reactant substances into the analyte sensor to eliminate or reduce interfering signals at the working electrode was evaluated. Figure 17 As shown, a blood glucose sensor with an interfering-reactant layer for reacting with ascorbic acid is fabricated. A blood glucose active region sensing layer 1702 is coated onto a carbon working electrode 1702, which is disposed on a substrate (not shown). The active region sensing layer 1702 has six discontinuous sensing points containing blood glucose oxidase sensing chemicals. The total area of ​​the active region is ~0.1 mm². 2 The working electrode containing the sensing point is immersed in a diffusion-limiting membrane containing either a control polymer composition or an experimental polymer composition containing MnO2 (catalog #217646, available from SIGMA-ALDRICH, St. Louis, Missouri). Both the control and experimental diffusion-limiting membranes are then allowed to cure.

[0239] The sensor was tested in beakers in 100mM PBS at ~30℃, with four copies including two controls, and tested in 1mg / ml ascorbic acid and 5mM blood glucose, respectively. The sensor current results are shown in Table 5.

[0240] Table 5

[0241]

[0242] As shown, experimental sensors containing interfering-reactant substances within a diffusion-limiting membrane exhibited reduced ascorbic acid interference. Therefore, incorporating metal oxide interfering-reactant substances is a feasible method to eliminate or reduce signals at the working electrode attributable to interfering substances.

[0243] Example 7. In this example, the effectiveness of incorporating a scrubbing electrode into an analyte sensor to eliminate or reduce interfering signals at the working electrode was evaluated. The glucose sensor was fabricated by applying the glucose-sensing active region of a glucose oxidase chemical to the working electrode. The working electrode was approximately 200 μm wide. A thin layer of approximately 50 μm was created by applying an adhesive layer to encapsulate the scrubbing electrode. The scrubbing electrode was approximately 2500 μm wide. No diffusion-limiting membrane was included in the sensor.

[0244] The sensor was tested in a beaker at a temperature of ~30°C in 1mM blood glucose in 100mM PBS. Figure 20 The currents of the working electrode 2002 and the wiping electrode 2004 are shown. As illustrated, the working electrode 2002 maintains a substantially stable glucose response, while the wiping electrode 2004 does not respond to glucose, as expected, because it does not contain glucose-sensing chemicals, and this continues for more than two weeks, even without a diffusion-limiting membrane. Therefore, the wiping electrode can be used to achieve the diffusion-limiting function of the membrane.

[0245] Example 8. In this example, the effectiveness of incorporating a scrubbing electrode into an analyte sensor to eliminate or reduce interfering signals at the working electrode was evaluated. A blood glucose sensor including the scrubbing electrode of Example 7 was tested in the presence of blood glucose and ascorbic acid, with a potential applied or removed from the scrubbing electrode. The potential was +40 mV when applied to either the working or scrubbing electrode. Figure 21A As shown, the sensor was tested in a beaker in 100 mM PBS at a temperature of ~30°C. After approximately 24 hours, 250 μM blood glucose was added to 2102, and the functions of the working electrode 2104 and the wash electrode 2106 were observed. As shown, the working electrode 2104 reached a steady state upon detecting blood glucose, while the wash electrode 2106 remained essentially unaffected. After approximately 25 hours, 2 mg / dL (~115 μm) of ascorbic acid 2108 was added. As shown, the response of the working electrode 2104 remained stable (detecting blood glucose), while the response current of the wash electrode 2106 increased instantaneously (detecting ascorbic acid 2108). Subsequently, the potential of the wash electrode 2106 turned off (labeled 2110) and then turned on again (labeled 2110). The relative increase between these effects on the blood glucose signal can be attributed to ascorbic acid 2102. Figure 21B The sensor was shown after approximately 18 days, demonstrating its stability at least during this period. At 2114, 2 mg / ml ascorbic acid was added, and at 2116, the wiping electrode was turned off again at 2106. Therefore, it is clear that wiping the electrode effectively removes ascorbic acid from the working electrode.

[0246] Example 9. In this example, the effectiveness of incorporating a scrubbing electrode into an analyte sensor to eliminate or reduce interfering signals at the working electrode was evaluated. Two blood glucose sensors, each with a different carbon ink type and a different screen printing location, were fabricated according to Example 7. The scrubbing electrodes were manufactured by Steven Label, Inc. (Santa Fe Springs, CA). Figure 22 The middle part is marked "C1" (thick line) and the inside (in) Figure 22 The carbon ink is prepared using a method marked "C2" (fine line). Commercially available carbon ink compositions vary (e.g., different carbon particles, different binders, and / or different carbon-to-binder ratios). Furthermore, the screen-printed positions differ due to proprietary printing processes, temperatures, curing times, etc. Beaker tests were performed on two different sensors at ~30°C in 2.0 mg / dL ascorbic acid in 100 mM PBS, with the ascorbic acid added at approximately 0.8 hours, corresponding to the second current spike. Figure 22 The sensor current for each scrubbing electrode is shown in the diagram, responding to various applied potentials in mV (labeled +40, +100, +200, +300, and +400, and "off" with no applied potential). Clearly, the material composition, location, and applied potential of the scrubbing electrode affect its scrubbing efficiency. Therefore, the scrubbing electrode can be optimized considering factors such as the concentration of interfering substances and / or their components in the body fluids.

[0247] Example 10. In this example, the effectiveness of incorporating an analyte-permeable scrubbing electrode into an analyte sensor to eliminate or reduce interfering signals at the working electrode was evaluated. Figure 23 The fabrication of a blood glucose sensor 2300 comprising a carbon nanotube analyte-permeable electrode is shown. The working electrode 2304 is screen-printed onto a plastic substrate 2302 having a surrounding well 2306 to allow deposition of a solution of additional components of the sensor to be tested. The well 2306 is shown as... Figure 23The "trap boundary" portions 2306a and b. As described above, this trap configuration and its variations can be used in embodiments of this disclosure. The active region 2308 of the ketone-sensing chemical is automatically liquid-dispensed into the trap 2306 and onto (in contact with) the top of the working electrode 2304. While this example utilizes a ketone-sensing chemical, it should be understood that other analytes, such as blood glucose, lactate, creatinine, ethanol, and various combinations thereof, can be used without departing from the scope of this disclosure. The active region 2308 covers a portion of the working electrode, but excess (exposed) working electrode 2304a remains, appearing to the left and right of the active region 2308. Subsequently, an initial diffusion-limiting membrane 2310a (e.g., 10Q5 or other membrane polymers, such as those described herein) is deposited (e.g., manually or automatically deposited) into the trap on top of the active region 2308 and the excess working electrode portion 2304a. A carbon nanotube-permeable scrubbing electrode 2311 is deposited into a trap atop an initial membrane 2310a, followed by dip-coating (or other deposition method) of the entire sensor in a second diffusion-limiting coating (e.g., a membrane polymer, as described herein). Without departing from the scope of this disclosure, the initial and second membrane coatings 2310a, 2310b may be of the same or different compositions, depending at least on the analyte being detected.

[0248] like Figure 24 As shown, in 100mM PBS, the following... Figure 23 A beaker test was performed on the ketone sensor with a carbon nanotube-permeable scrubbing electrode, as shown and described above. After approximately 1 hour (~1.3 hours), 5 mg / dL ascorbic acid was added, and the working electrode 2404 (thick line) and scrubbing electrode 2406 (thin line) were observed. As shown, the addition of ascorbic acid resulted in an interference signal from the working electrode. After applying a potential of +40 mV to the scrubbing electrode, the interference signal from the working electrode decreased by approximately 85%. Upon disconnecting the scrubbing electrode, the interference signal on the bottom electrode returned to its previous level. Reconnecting the scrubbing electrode and adjusting the applied potential to +40, +200, and +600 mV, a moderate increase in scrubbing efficiency was observed at the higher potentials. Although not shown, various other analytes of interest (including blood glucose and β-hydroxybutyrate) were observed to readily diffuse through the scrubbing electrode, thereby generating signals on the working electrode below.

[0249] Therefore, this disclosure provides an analyte sensor for monitoring various analytes in vivo. The analyte sensor may be characterized by enhancements to address signals obtained from interfering substances. Some analyte sensors may include a working electrode comprising an active region disposed thereon and an electrode roughening material laser-grated therefrom. Some analyte sensors may include interfering-reactant substances incorporated therein. Some analyte sensors may include an interfering-washing electrode. Combinations of these enhancements may also be employed.

[0250] Unless otherwise stated, all figures indicating quantities, etc., in this specification and related claims should in all cases be understood to be modified by the term "about". Therefore, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximate values ​​that may vary depending on the desired characteristics sought to be obtained from embodiments of this disclosure. At least, no attempt is made to limit the application of the doctrine of equivalents to the scope of the claims; each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying common rounding techniques.

[0251] This document presents one or more illustrative embodiments combining various features. For clarity, not all features of the physical implementation are described or shown in this application. It should be understood that in the development of physical embodiments incorporating embodiments of this disclosure, many implementation-specific decisions must be made to achieve the developer's goals, such as compliance with system-related, business-related, governmental-related, and other constraints that vary with implementation method and time. While the developer's efforts may be time-consuming, such efforts will be a routine task for those skilled in the art who benefit from this disclosure.

[0252] Although the various systems, tools, and methods are described herein by way of “comprising” various components or steps, they may also be described as “consistently composed of various components and steps” or “comprised of various components and steps.”

[0253] As used herein, the phrase “at least one” preceding a series of items, and the terms “and” or “or” used to separate any items, modify the list as a whole, not each member of the list (i.e., each item). The phrase “at least one” allows for the meaning of including at least one of any item and / or at least one combination of items and / or at least one of each item. For example, the phrases “at least one of A, B, and C” or “at least one of A, B, or C” both refer only to A, B, or C; any combination of A, B, and C; and / or at least one of A, B, and C.

[0254] Therefore, the disclosed systems, tools, and methods are well-suited to achieving the stated purposes and advantages, as well as those inherent therein. The specific embodiments disclosed above are merely illustrative, as the teachings of this disclosure can be modified and practiced in different but equivalent ways, which will be apparent to those skilled in the art who benefit from the teachings herein. Furthermore, no limitation is intended to be made on the details of the constructions or designs shown herein, except as described in the following claims. Therefore, it will be apparent 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 this disclosure. The systems, tools, and methods illustratively disclosed herein can be suitably implemented in the absence of any elements not specifically disclosed herein and / or any optional elements disclosed herein. While the systems, tools, and methods are described as “comprising,” “including,” or “containing” various components or steps, they can also be described as “substantially composed of various components and steps” or “composed of various components and steps.” All numbers and ranges disclosed above can vary. Whenever a numerical range with a lower and upper limit is disclosed, any number falling within that range and any included range is specifically disclosed. In particular, 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 describe each numerical value and range contained within a wider numerical range. Furthermore, the terms in the claims have their simple, general meanings unless the patentee explicitly and clearly defines them otherwise. Additionally, the indefinite articles "a" or "an" used in the claims are defined herein as indicating one or more elements introduced therein. If there is any conflict in the use of words or terms in this specification and in one or more patents or other documents, the definitions consistent with this specification shall prevail.

Claims

1. An in vivo analyte sensor, comprising: A working electrode includes an active region disposed on the working electrode, the active region including an analyte-responsive enzyme; as well as The cleaning electrode, wherein the cleaning electrode: Positioned facing the working electrode, and the working electrode and the wiping electrode are separated by a gap, wherein the gap is a thin layer that allows bodily fluid to pass between the working electrode and the wiping electrode, wherein the width of the wiping electrode is set to be larger than the width of the working electrode, such that the wiping electrode is configured to reduce the signal of the interfering substance attributed to the working electrode by pre-reacting the interfering substance before it reaches the working electrode.

2. The in vivo analyte sensor according to claim 1, wherein, The thin layer is in the range of about 1 μm to about 200 μm.

3. The in vivo analyte sensor according to claim 1, wherein, The scrubbing electrode has a width of approximately 200 μm to approximately 8000 μm.

4. The in vivo analyte sensor according to claim 1, wherein, The width of the scrubbing electrode is in the range of approximately 2:1 to approximately 50:1 compared to the width of the working electrode.

5. The in vivo analyte sensor according to claim 1, wherein, The scrubbing electrode has a potential in the range of about -2000mV to about +2000mV.

6. The in vivo analyte sensor according to claim 1, wherein the analyte sensor further comprises an additional electrode, wherein, The additional electrode is at least one of a counter electrode or a reference electrode.

7. The in vivo analyte sensor according to claim 1, wherein, The scrubbing electrode is configured to react with and deactivate an interfering substance, wherein the interfering substance, which is ascorbic acid, does not contribute to the measurement signal at the working electrode.

8. The in vivo analyte sensor according to claim 1, wherein, The gap between the scrubbing electrode and the working electrode is formed by applying an adhesive, separator, or other separation means along the width edges of the scrubbing electrode and the working electrode.

9. The in vivo analyte sensor according to claim 1, wherein, The thin layer is formed by sealing fluid channels along the two opposite edges of the scrubbing electrode, allowing the body fluid to enter the space between the unsealed thin layer in a controlled manner, so that the body fluid reaches the scrubbing electrode before the working electrode.

10. The in vivo analyte sensor according to claim 1, wherein, The cleaning electrode is coated with an interfering-reactant substance.

11. The in vivo analyte sensor according to claim 1, wherein, The analyte of interest is lactate.

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