A sample sensor featuring a reduced-area working electrode to reduce interference signals.

Abstract

The present invention provides a sample sensor and method characterized by enhancement for dealing with signals obtained from interfering material species. [Solution] Some sample sensors may be equipped with an active electrode having a sensing portion and an exposed electrode portion, wherein the sensing portion has an active area on which a sample-reactive enzyme is placed, and the exposed electrode portion does not have an active area. The exposed electrode portion and the sensing portion can be present in a ratio of about 1:10 to about 10:1.

Description

[Technical Field]

[0001] [Cross-reference to related applications] This application claims priority and benefit of U.S. Provisional Patent Application No. 63 / 039,743, filed June 16, 2020, which is incorporated in its entirety by reference herein. [Background technology]

[0002] The detection of different samples within an individual can sometimes be essential for monitoring their health status. Deviations from normal sample levels often indicate several physiological conditions. For example, glucose levels may be particularly important to detect and monitor in diabetic patients. By monitoring glucose levels with sufficient regularity, diabetic patients can take corrective action before significant physiological impairment occurs (e.g., by injecting insulin to lower glucose levels or by eating to raise glucose levels). Monitoring of other samples may be desirable for various other physiological conditions. Monitoring of multiple samples may also be desirable in some cases, particularly for concomitant conditions that result in simultaneous abnormal regulation of two or more samples in combination with each other. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] U.S. Patent Application Publication No. 2011 / 0213225 [Patent Document 2] U.S. Patent No. 6,605,200 [Patent Document 3] U.S. Patent No. 8,268,143 [Patent Document 4] U.S. Patent Application Publication No. 2020 / 0237275 Specification [Patent Document 5] U.S. Patent Application No. 16 / 774,835 [Patent Document 6] U.S. Patent Application Publication No. 2020 / 0241015 [Patent Document 7] U.S. Patent Application No. 16 / 582,583 [Patent Document 8] U.S. Patent Application Publication No. 2019 / 0320947 Specification [Patent Document 9] U.S. Patent Application Publication No. 2020 / 0060592 [Patent Document 10] U.S. Patent No. 6,134,461 [Patent Document 11] U.S. Patent No. 6,736,957 [Patent Document 12] U.S. Patent No. 7,501,053 [Patent Document 13] U.S. Patent No. 7,754,093 [Patent Document 14] U.S. Patent No. 8,444,834 [Patent Document 15] U.S. Patent No. 6,605,201 [Patent Document 16] U.S. Patent No. 8,983,568 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Many specimens represent interesting targets for physiological analysis, assuming that they can identify appropriate detection chemistry. For this purpose, in vivo specimen sensors configured to assay various physiological specimens have been developed and improved over the years, many of which utilize enzyme-based detection strategies to facilitate detection specificity. In fact, in vivo specimen sensors that utilize glucose-responsive enzymes to monitor blood glucose levels are currently commonly used among diabetic patients. In vivo specimen sensors for other specimens, including those that can monitor multiple specimens, are in various stages of development. Insufficient sensitivity to small specimens can be particularly problematic for some specimen sensors, especially due to background signals resulting from interference substance interactions with the working electrode or other specimen sensing chemistry components.

[0005] The following figures are included to illustrate certain aspects of the present disclosure and should not be viewed as limiting embodiments. The disclosed subject matter is capable of considerable modification, alteration, combination, and equivalents in form and function without departing from the scope of the present disclosure.

Brief Description of the Drawings

[0006] [Figure 1] FIG. is a diagram of an exemplary sensing system into which the specimen sensor of the present disclosure can be incorporated. [Figure 2A] FIG. is a cross-sectional view of a specimen sensor having a single active region. [Figure 2B] FIG. is a cross-sectional view of a specimen sensor having a single active region. [Figure 2C] FIG. is a cross-sectional view of a specimen sensor having a single active region. [Figure 3A] FIG. is a cross-sectional view of a specimen sensor having two active regions. [Figure 3B] FIG. is a cross-sectional view of a specimen sensor having two active regions. [Figure 3C] FIG. is a cross-sectional view of a specimen sensor having two active regions. [Figure 4]This is a cross-sectional view of a sample sensor equipped with two working electrodes, each with an active region located on its surface. [Figure 5] This is a top view of a conventional carbon working electrode having an active region on its surface. [Figure 6A] This is a cross-sectional view of the active region grid configuration of a carbon working electrode suitable for use in the sample sensor of this disclosure. [Figure 6B] This figure shows a different active region grid configuration. [Figure 7A] This is a top view of a conventional carbon working electrode having an active region on its surface. [Figure 7B] This figure shows an exemplary treatment that can enhance the carbon working electrode and the active region thereon to reduce interfering substance signals. [Figure 7C] This figure shows an exemplary treatment that can enhance the carbon working electrode and the active region thereon to reduce interfering substance signals. [Figure 7D] This figure shows an exemplary treatment that can enhance the carbon working electrode and the active region thereon to reduce interfering substance signals. [Figure 7E] This figure shows an exemplary treatment that can enhance the carbon working electrode and the active region thereon to reduce interfering substance signals. [Figure 7F] This figure shows an exemplary treatment that can enhance the carbon working electrode and the active region thereon to reduce interfering substance signals. [Modes for carrying out the invention]

[0007] This disclosure describes a sample sensor generally suitable for in vivo use, more specifically, a sample sensor characterized by one or more enhancements for reducing or eliminating signals indicating interfering substance species in order to facilitate improved detection sensitivity, and methods for producing and using the same.

[0008] Such enhancements may include reducing the area of ​​the working electrode on the sensor tail, particularly the carbon working electrode, against which interfering material may react and contribute to signals that are not associated with the sample. The area of ​​the carbon working electrode can be reduced by one or more means, such as reducing the pitch of the active area sensing layer and / or reducing the area of ​​the active area sensing layer and / or positioning the active area sensing layer toward the distal portion of the sensor tail. As used herein, the term “pitch” and its grammatical variations mean the distance between adjacent sensing spots within the active area sensing layer, measured from the center of each adjacent sensing spot. This specification will describe in more detail the specific details and yet another advantage of each type of enhancement. The sample sensors of this disclosure may be configured to detect one sample or multiple samples simultaneously or substantially simultaneously, depending on the specific needs.

[0009] Sample sensors using enzyme-based detection are commonly used to test single samples, such as glucose, due to the high specificity of the enzyme to a particular substrate or category of substrates. Sample sensors using both a single enzyme and an enzyme system comprising multiple enzymes acting cooperatively can be used for this purpose. As used herein, the term “cooperatively” and its grammatical variations mean a conjugated enzyme reaction in which the product of a first enzymatic reaction becomes a substrate for a second enzymatic reaction, and the second or subsequent enzymatic reaction serves as a base for measuring the concentration of the sample. Furthermore, combinations of enzymes and / or enzyme systems can be used to detect more than one sample type. Using sample sensors featuring enzymes or enzyme systems to facilitate detection can be particularly advantageous in avoiding the frequent extraction of bodily fluids that may otherwise be required for sample monitoring.

[0010] In vivo sample sensors monitor one or more samples in a biological fluid of interest, such as dermal fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, or amniotic fluid. Such fluids may contain one or more interfering substances that can react either directly on the working electrode of the sample sensor and on the working electrode (e.g., a carbon working electrode) itself, or with one or more sensing chemical components placed on the electrode. As used herein, the term “interfering substance” and its grammatical variations mean any present electroactive species that are not the sample of interest (e.g., bio-electroactive species that are not the sample of interest). Examples include, but are not limited to, ascorbic acid (vitamin C, also called ascorbate), glutathione, uric acid, paracetamol (acetaminophen), isoniazid, salicylate, and any combination thereof. The reaction of these interfering substances with the working electrode may generate electrochemical signals that are inseparable from or difficult to separate from the signal emitted from the sample of interest, which can complicate the accurate detection of such samples, especially those in small quantities (e.g., from low concentrations to sub-millimole concentrations). The electrochemical signals generated by interfering substances can be particularly problematic when the magnitude of the signal from the interfering substance approaches that of the target sample. This can occur, for example, when the concentration of the interfering substance approaches or exceeds that of the sample of interest. Some interfering substances are ubiquitous in living organisms and are not easily avoided. Therefore, techniques to minimize the effects of these interfering substances during in vivo analysis are highly desirable.

[0011] This disclosure provides sample sensor enhancements that can improve the detection sensitivity of both single samples and multiple samples combined with each other, either individually or in combination, as described in more detail below. Specifically, this disclosure provides a sample sensor having a small carbon working electrode area that can provide a reduction in background signals resulting from bio-interfering substances. Certain aspects of this disclosure relate to the enhancement of carbon working electrodes, but it is acknowledged that other types of electrodes can also be enhanced according to the disclosure herein. Types of electrodes that can be enhanced using the disclosure herein include gold, platinum, and PEDOT, among others.

[0012] Generally and non-limited embodiments of the present disclosure comprising one or more interfering substance enhancements, as detailed herein, can enable reduction of interfering substance signals, such as ascorbic acid interfering substance signals, in a range greater than about 20%, which can be up to 100% compared to sensors lacking these enhancements, or, for example, in a range of about 20% to about 70% or greater, preferably at least about 40% greater, at least about 45% greater, or at least about 50%, encompassing any value and subset between these values, and within a range where the upper and lower limits are separable. The amount of interfering substance reduction may depend on several factors, including but not limited to the particular configuration of the sensor (e.g., which one or more enhancements are selected) and the concentration of the interfering substance in the bodily fluids, and any combination thereof.

[0013] Before describing the sample sensors and their enhancements in more detail, we first provide a brief overview of suitable in vivo sample sensor configurations and sensor systems using these sample sensors to better understand the embodiments of this disclosure. Figure 1 illustrates an exemplary sensing system into which the sample sensors of this disclosure may be incorporated. As shown, the sensing system 100 includes a sensor control device 102 and a reader device 120 configured to communicate with each other through a local communication path or link 140, which can be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. In some embodiments, the reader device 120 includes an output medium for viewing the sample concentration and warnings or notifications determined by the sensor 104 or its associated processor, and can further allow one or more user inputs. The reader device 120 can be a multipurpose smart phone or a dedicated electronic reading instrument. Although only one reader device 120 is shown, in certain cases, multiple reader devices 120 may be present.

[0014] The reader device 120 can also communicate with the remote terminal 170 and / or the highly reliable computer system 180 through communication paths / links 141 and / or 142, which can be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. The reader device 120 can also communicate with the network 150 (e.g., a mobile phone network, the internet, or a cloud server) through communication path / link 151, or alternatively. The network 150 can further communicate with the remote terminal 170 through communication path / link 152 and / or the highly reliable computer system 180 through communication path / link 153. Alternatively, the sensor 104 can communicate directly with the remote terminal 170 and / or the highly reliable computer system 180 without the presence of the intervening reader device 120. For example, in some embodiments, the sensor 104 can communicate with a remote terminal 170 and / or a highly reliable computer system 180 via a direct communication link to a network 150, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is incorporated in whole by reference herein.

[0015] Any suitable electronic communication protocol, such as Near Field Communication (NFC), Radio Frequency Identification (RFID), Bluetooth®, or Bluetooth® Low Energy, or Wi-Fi, can be used for each communication path or link. In some embodiments, the remote terminal 170 and / or the highly reliable computer system 180 can be made accessible to individuals other than the primary user who are interested in the user's sample level. The reader device 120 may comprise a display 122 and an optional input component 121. In some embodiments, the display 122 may comprise a touchscreen interface.

[0016] The sensor control device 102 includes a sensor housing 103 that can house the circuitry and power supply for operating the sensor 104. Optionally, the power supply and / or active circuitry may be omitted. A processor (not shown) can be communicatively coupled to the sensor 104, and the processor is physically located within the sensor housing 103 or the reader device 120. In some embodiments, the sensor 104 protrudes from the underside of the sensor housing 103 and extends through an adhesive layer 105 configured to adhere the sensor housing 103 to a skin-like tissue surface.

[0017] The sensor 104 is designed to be at least partially inserted into the tissue of interest, such as within the dermis or subcutaneous layer of the skin. Alternatively, the sensor 104 can be adapted to penetrate the epidermis. Yet another alternative is that the sensor 104 may be positioned on a surface and not penetrate the tissue, such as when testing one or more samples in sweat on the skin. The sensor 104 may include a sensor tail of sufficient length to be inserted to a desired depth within a given tissue. The sensor tail may comprise an active region comprising at least one working electrode and an enzyme or enzyme system configured to be suitable for testing one or more samples of interest.

[0018] The counter electrode may be present in combination with at least one working electrode, and optionally in yet another combination with a reference electrode. Specific electrode configurations on the sensor tail will be described in more detail below with reference to Figures 2A to 4. In various embodiments, one or more enzymes within the active region may be covalently bonded to a polymer containing the active region. Alternatively, the enzymes may be non-covalently associated within the active region by encapsulation or physical encompassation. One or more specimens can be monitored in a biological fluid of interest, such as dermal fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, or amniotic fluid. In certain embodiments, the specimen sensor of this disclosure may be adapted to analyze dermal or interstitial fluid to determine the concentration of a specimen in vivo. However, without departing from the scope of this disclosure, it will be acknowledged that the entire sensor control device 102 may have one or more different configurations that allow for complete embedding under tissue and in one or more bodily fluids to test one or more specimens of interest.

[0019] Referring again to Figure 1, the sensor 104 can automatically transmit data to the reader device 120. For example, sample concentration data can be communicated automatically and periodically, for example, when data is obtained or at a predetermined frequency (e.g., every minute, every five minutes, or at other predetermined intervals) after a predetermined period has elapsed since the data was stored in memory until transmission, using a BLUETOOTH® protocol or BLUETOOTH® Low Energy protocol, etc. Data for different samples can be transmitted at the same or different frequencies and / or using the same or different communication protocols. In other embodiments, the sensor 104 can communicate with the reader device 120 in an unautomated manner and without following a set schedule. For example, data can be communicated from the sensor 104 using RFID technology when the sensor electronics are within communication range of the reader device 120. The data can remain stored in the sensor 104's memory until it is communicated to the reader device 120. Therefore, the user does not need to maintain close proximity to the reader device 120 at all times, but can instead upload data automatically or non-automatically at a convenient time. In further embodiments, a combination of automatic and non-automatic data transmission can be implemented. For example, data transmission can continue automatically until the reader device 120 is no longer within communication range of the sensor 104.

[0020] A temporary introducer may be present to facilitate the introduction of the sensor 104 into the tissue. In exemplary embodiments, the introducer may include a needle or a similar pointed body or a combination thereof. In alternative embodiments, it is recognized that other types of introducers, such as a sheath or blade, may be present. More specifically, the needle or other introducer may be temporarily present near the sensor 104 before tissue insertion and then withdrawn. While present, these needles or other introducers can facilitate the insertion of the sensor 104 into the tissue by opening an access path for the sensor 104 to follow. For example, in one or more embodiments, the needle may facilitate penetration of the epidermis as an access path to the dermis to enable the implantation of the sensor 104. After opening the access path, the needle or other introducer can be withdrawn so as not to cause injury. In exemplary embodiments, suitable needles may be solid or hollow, beveled or unbeveled, and / or have a circular or non-circular cross-section. In more specific embodiments, a suitable needle may have a cross-sectional diameter and / or tip design equivalent to an acupuncture needle, which may have a cross-sectional diameter of approximately 250 microns. However, it should be recognized that a suitable needle may have a larger or smaller cross-sectional diameter as needed to suit a particular application. For example, needles with cross-sectional diameters ranging from approximately 300 microns to approximately 400 microns can be used.

[0021] In some embodiments, the tip of the needle can be inclined over the end of the sensor 104 (while it is present) so that the needle first penetrates the tissue and opens an access path for the sensor 104. In other exemplary embodiments, the sensor 104 may be located in the lumen or groove of the needle, and the needle also opens an access path for the sensor 104. In either case, the needle can be withdrawn after facilitating the insertion of the sensor.

[0022] Sensor configurations featuring a single active region configured for the detection of a corresponding single sample can utilize two-electrode or three-electrode detection motifs, as will be further described herein with reference to Figures 2A to 2C. Subsequently, sensor configurations featuring two different active regions suitable for the detection of separate samples on either separate working electrodes or the same working electrode will be described separately with reference to Figures 3A to 4. Sensor configurations with multiple working electrodes can be particularly advantageous because the signal contribution from each active region can be more easily determined by separate evaluation of each working electrode, allowing for the incorporation of two different active regions within the same sensor tail. Each active region can be protected by a mass transport restriction membrane of the same or different composition.

[0023] When a single working electrode is present within the sample sensor, a three-electrode sensor configuration may comprise a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration may comprise a working electrode and a second electrode, in which case the second electrode can function as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). The various electrodes can be stacked vertically, at least partially, on the sensor tail, and / or spaced apart from one another laterally. In any of the sensor configurations disclosed herein, the various electrodes can be electrically isolated from one another by a dielectric material or similar insulator.

[0024] A sample sensor characterized by multiple working electrodes may also have at least one additional electrode. When one additional electrode is present, this electrode can function as a pair / reference electrode for each of the multiple working electrodes. When two additional electrodes are present, one of the additional electrodes can function as a pair electrode for each of the multiple working electrodes, and the other additional electrode can function as a reference electrode for each of the multiple working electrodes.

[0025] Any of the working electrode configurations described below can benefit from further disclosures below relating to reducing the area of ​​the working electrode on the sensor tail.

[0026] Figure 2A illustrates an exemplary two-electrode sample sensor configuration suitable for use in the disclosure herein. As shown, the sample sensor 200 includes a substrate 212 positioned between a working electrode 214 and a pair / reference electrode 216. Alternatively, the working electrode 214 and the pair / reference electrode 216 may be positioned on the same plane of the substrate 212, with a dielectric material sandwiched between these electrodes (configuration not shown). The active area 218 is positioned as at least one layer on at least a portion of the working electrode 214. The active area 218 may comprise a plurality of discontinuous spots or a single continuous spot configured to be suitable for sample detection, as will be further discussed herein.

[0027] Continuing to refer to Figure 2A, in some embodiments, the membrane 220 can protect at least the active area 218 and optionally some or all of the working electrode 214 and / or the pair / reference electrode 216 or the entire sample sensor 200. One or both sides of the sample sensor 200 can be protected by the membrane 220. The membrane 220 may comprise one or more polymer membrane materials having the function of restricting the sample flux to the active area 218 (i.e., the membrane 220 is a mass transport restriction membrane with some permeability to the sample of interest). The composition and thickness of the membrane 220 can be modified to facilitate a desired sample flux to the active area 218, thereby providing a desired signal intensity and stability. The sample sensor 200 may be operable to validate the sample by any of the electrochemical detection techniques of coulometry, amperometry, voltometry, or potentiometry.

[0028] Figures 2B and 2C illustrate exemplary three-electrode sample sensor configurations that are also suitable for use in the disclosure herein. The three-electrode sample sensor configuration can be the same as that shown with respect to the sample sensor 200 described in Figure 2A, except for the inclusion of an additional electrode 217 within sample sensors 201 and 202 (Figures 2B and 2C). In this case, by using the additional electrode 217, the pair / reference electrode 216 can function as either the pair electrode or the reference electrode, and the additional electrode 217 satisfies other electrode functions not otherwise addressed. The working electrode 214 satisfies its original function. The additional electrode 217 can be placed on either the working electrode 214 or electrode 216 with a dielectric material isolation layer between them. For example, as shown in Figure 2B, dielectric material layers 219a, 219b, and 219c separate electrodes 214, 216, and 217 from each other and provide electrical isolation. Alternatively, as shown in Figure 2C, at least one of electrodes 214, 216, and 217 can be positioned on opposing surfaces of the substrate 212. Thus, in some embodiments, electrode 217 (reference electrode) can be positioned on one of electrodes 214 or 216, and electrode 214 (working electrode) and electrode 216 (counter electrode) can be positioned on opposing surfaces of the substrate 212, separated from these electrodes 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 those shown in Figures 2B and 2C. Similar to the sensor 200 shown in Figure 2A, the active area 218 in sample sensors 201 and 202 can comprise multiple spots or a single spot. Furthermore, sample sensors 201 and 202 can also be activated to validate a sample by any of the electrochemical detection techniques of coulometry, current measurement, voltage measurement, or potentiometry.

[0029] Similar to the sample sensor 200, the membrane 220 can protect the active areas 218 within the sample sensors 201 and 202, as well as other sensor components, thereby functioning as a mass transport limiting membrane. In some embodiments, an additional electrode 217 can be protected by the membrane 220. The membrane 220 can also be produced by immersion coating or in-situ photopolymerization, and its composition can vary or be the same at different locations. Figures 2B and 2C show all electrodes 214, 216, and 217 protected by the membrane 220, although it should be noted that in some embodiments, only the working electrode 214 or the active area 218 can be protected. Furthermore, the thickness of the membrane 220 at each of electrodes 214, 216, and 217 may be the same or different. Similar to the two-electrode sample sensor configuration (Figure 2A), in the sensor configurations of Figures 2B and 2C, one or both sides of the sample sensors 201 and 202 can be protected by the membrane 220, or the entire sample sensors 201 and 202 can be protected. Accordingly, the three-electrode sensor configuration shown in Figures 2B and 2C should be understood as not limiting the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of this disclosure.

[0030] Figure 3A illustrates an exemplary configuration of a sensor 203 having a single working electrode on which two different active regions are arranged. Figure 3A is similar to Figure 2A except that there are two active regions on the working electrode 214, namely a first active region 218a and a second active region 218b that respond to different samples and are spaced laterally apart from each other on the plane of the working electrode 214. The active regions 218a and 218b may comprise multiple spots or a single spot configured to be suitable for detecting each sample. The composition of the membrane 220 may be different or the same in the active regions 218a and 218b. As will be further discussed below, the first active region 218a and the second active region 218b may be configured to detect their corresponding samples at different working electrode potentials.

[0031] Figures 3B and 3C illustrate cross-sectional views of exemplary three-electrode sensor configurations for sensor 204 and 205, respectively, each featuring a single working electrode with a first active region 218a and a second active region 218b located thereon. Figures 3B and 3C are otherwise similar to Figures 2B and 2C and can be better understood by referencing these figures. As in the case of Figure 3A, the composition of the film 220 may be different or the same in the active regions 218a and 218b.

[0032] Figure 4 illustrates a cross-sectional view of an exemplary sample sensor configuration having two working electrodes, a reference electrode, and a counter electrode, suitable for use in the disclosure herein. As shown, the sample sensor 400 includes working electrodes 404 and 406 positioned on opposing surfaces of a substrate 402. A first active area 410a is positioned on the surface of the working electrode 404, and a second active area 410b is positioned on the surface of the working electrode 406. The counter electrode 420 is electrically isolated from the working electrode 404 by a dielectric material layer 422, and the reference electrode 431 is electrically isolated from the working electrode 406 by a dielectric material layer 423. Outer dielectric material layers 430 and 432 are positioned above the reference electrode 431 and the counter electrode 420, respectively. In various embodiments, the film 440 can protect at least the active areas 410a and 410b, and optionally other components of the sample sensor 400 or the entire sample sensor 400 is also protected by the film 440. Here again, the membrane 440 may have different compositions in the active regions 410a and 410b as needed to provide transmittance values ​​suitable for differentially adjusting the sample flux at each location.

[0033] Alternative sensor configurations having multiple working electrodes, different from the configuration shown in Figure 4, may feature pair / reference electrodes instead of separate pair electrodes 420 and reference electrode 431, and / or different layer and / or film arrangements than those specified. For example, the arrangement of pair electrodes 420 and reference electrode 431 can be reversed from that shown in Figure 4. Furthermore, working electrodes 404 and 406 do not necessarily have to be located on opposing surfaces of the substrate 402 in the manner shown in Figure 4.

[0034] Carbon working electrodes can be appropriately configured as working electrodes in any of the sample sensors disclosed herein. While carbon working electrodes are very commonly used in electrochemical detection, their use in electrochemical sensing is not without its challenges. In particular, a current related to the sample of interest is produced only when the active region interacts with the sample and transfers electrons to the portion of the carbon working electrode adjacent to the active region. Body fluids containing the sample of interest interact with the carbon surface of the carbon working electrode as well as the carbon surface of the carbon working electrode that is not protected by the active region, but these do not contribute to the sample signal because there are no enzymes or enzymatic systems in these locations that facilitate electron transfer from the sample to the working electrode. However, interfering substances may be oxidized in the portion of the working electrode lacking the active region and may contribute to the background of the overall signal. Therefore, carbon working electrodes with an external (or "exposed") carbon region on the electrode surface do not contribute significantly to the sample signal and may, in some cases, lead to a background signal contribution. Other electrodes with extra surface regions that do not directly detect the sample of interest may also receive similar background signals, which can be enhanced by modifications of the disclosure herein.

[0035] Various interfering substances may interact with the working electrode of the sample sensor described herein, but ascorbic acid is one example of an interfering substance that is commonly present in biological fluids and may generate background signals at the carbon working electrode. For example, ascorbic acid is oxidized at the working electrode to produce dehydroascorbic acid. Various embodiments of this disclosure are described below with respect to the interfering substance ascorbic acid. However, it is acknowledged that the embodiments and sample sensor configurations described herein can be equally applied to other interfering substances (electroactive species present in the bodily fluids containing the sample of interest).

[0036] As provided above, the active regions described herein may be a single sensing layer having multiple sensing spots or multiple sensing spots compressed together, and thus may be a sensing layer substantially corresponding to a single sensing layer. Next, referring to Figure 5, an example is shown of a top view of a conventional carbon working electrode 500 on which an active region 504 containing multiple sensing spots 518 is disposed. When a sample interacts with the active region 504, only the portion of the carbon working electrode 500 containing the sensing spots 518 contributes to the signal relating to the sample of interest. The carbon working electrode 500 shows six sensing spots 518 within the active region 504, but it is acknowledged that fewer or more than six sensing spots 518 may be included on the carbon working electrode 500 without departing from the scope of this disclosure. No sensing spots 518 are directly superimposed on an external carbon region 510, and this region does not contribute to the signal relating to the sample, but may generate a background signal relating to one or more interfering substances. Therefore, the oxidation of the interfering substance at the carbon working electrode 500 is proportional to the area of ​​the external carbon region 510 that may be used for interaction with the interfering substance. In fact, the oxidation of ascorbic acid at the carbon working electrode 500 is almost linear in strength with respect to the area of ​​the external carbon region 510 that may be used.

[0037] As shown in the figure, the active area 504 is discontinuous and exists in the form of multiple sensing spots 518. As defined herein, the term “discontinuous” and its grammatical variations mean that no single spot (sensing element) shares an edge or boundary with an adjacent spot (e.g., does not come into contact with it).

[0038] This disclosure reveals how the external carbon region 510 within the carbon working electrode 500 can be reduced to minimize or eliminate interfering substance signals while maintaining its function for generating signals related to the sample of interest. In particular, to reduce the area of ​​the external carbon region 510, the pitch and diameter of the discontinuous sensing spots 518 of the conventional carbon working electrode 500 can be reduced, and the configuration of the discontinuous sensing spots 518 relative to each other can be further reduced. As used herein, the term “grid” and its grammatical variations mean a 2D arrangement of active regions along the length of the working electrode (the length along the axis of the sensor tail 104 (Figure 1) extending from the sensor housing 103 into the body fluid) relative to the width of the working electrode.

[0039] Regarding examples of various grid configurations, the active area of ​​this disclosure may be in the form of a 1×n grid, in which case n is an integer greater than 1, for example, in the range of 2 to about 20 or 2 to about 10, encompassing any value and subset between these values, with upper and lower limits being separable. In some embodiments, the active area may comprise discontinuous sensing spots in the form of a 1×6 grid, as shown in Figure 5, for example. Embodiments described herein can be best understood by referring to Figure 5, and other grid configurations of the active area, such as those shown in Figures 6A to 6B, where similar elements retain similar labeling, may be used. For example, in some embodiments, the active area may comprise discontinuous sensing spots in the form of a 2×n grid, in which case n is an integer greater than 1, for example, in the range of 2 to about 10 or 2 to about 5, encompassing any value and subset between these values, with upper and lower limits being separable. Figure 6A depicts a carbon working electrode 600 having a 2×3 grid of sensing spots 518 and an external carbon area 510. In yet another embodiment, the active area may comprise discontinuous sensing spots in the form of a 3×n grid, where n is an integer encompassing any value and subset between 2 and about 6 or 2 and about 3, with upper and lower limits within a separable range. Figure 6B depicts a carbon working electrode 610 having a 3×2 grid of sensing spots 518 and an external carbon area 510. In particular, Figures 5, 6A, and 6B each show various grid configurations suitable for use in the embodiments described herein, but each figure retains the same area of ​​the external carbon area 510, as the areas of the carbon electrodes 500 and 610 respectively are still not reduced in these figures. As can be seen, the grid configurations of Figures 6A and 6B are arranged over shorter longitudinal distances than the grid configuration of Figure 5, thereby giving the possibility of reducing the sensor area with exposed working electrodes.

[0040] Embodiments of the present disclosure utilize grid configuration, pitch distance, reduction of the size of the active area and / or sensing spot, and the location of the active area on the sensor tail to minimize the external carbon area and thereby minimize the signal from interfering material, as shown in top views 7A to 7E of carbon electrodes having various active area configurations. Figure 7A shows a contrasting (or conventional) 1×6 active area configuration similar to that shown in Figure 5. The external carbon area in Figure 7A is shaded as the working electrode surface where the active area is absent. Figures 7B to 7F are each drawn based on Figure 7A and illustrate embodiments of the present disclosure.

[0041] Figures 7B through 7F each utilize a reduction in the sensing spot pitch to reduce the external carbon area, and in some embodiments, the sensing spots merge with each other so that they are no longer discontinuous. Furthermore, Figure 7B further illustrates a reduction in the pitch between adjacent sensing spots, which allows for a reduction in the external carbon area (below the double line) shaded as the working electrode surface where sensing spots are absent. Figure 7C shows the pitch reduction of Figure 7B combined with a displacement of the active area closer to the tip of the sensor tail, which allows for yet another reduction in the external carbon area (below the double line) shaded as the working electrode surface where sensing spots are absent. Figure 7D shows yet another pitch reduction compared to Figure 7C, which allows for yet another reduction in the external carbon area (below the double line) shaded as the working electrode surface where sensing spots are absent. Figure 7E shows the pitch reduction in Figure 7D and the sensor tail displacement in Figure 7C combined with a 2x3 active area grid configuration. This combination allows for further reduction of the external carbon area (below the double line), which is shaded as the working electrode surface where the active area is absent. Figure 7F shows the pitch reduction in Figure 7D and the sensor tail displacement in Figure 7C combined with a 3x3 active area grid configuration. This combination allows for further reduction of the external carbon area (below the double line), which is shaded as the working electrode surface where the sensing spot is absent. Figures 7D to 7F show that as the pitch reduction width increases, it becomes more difficult to distinguish the sensing spot, and in some embodiments, it can be represented as a single active area lacking discontinuous sensing spots.

[0042] For illustrative purposes, Tables 1A and 1B illustrate the percentage reduction of exposed (or external) carbon, including comparisons based on Figures 7A, 7B, and 7D through 7F, for determining the estimated (Est.) reduction of the interfering substance (e.g., ascorbic acid) signal. The interfering substance was measured in terms of signal intensity based on the concentration of the sample tested and known interfering substance concentrations. Table 1A TIFF2026108628000002.tif33155 Table 1B TIFF2026108628000003.tif55155

[0043] As shown in Table 1, the interfering material signal decreases almost linearly as the external carbon area decreases.

[0044] This disclosure provides a reduced-area working electrode (e.g., a carbon electrode) on which one or more active regions are arranged. In some embodiments, a plurality of discontinuous active regions are arranged on the working electrode. Generally, the discontinuous active regions of this disclosure have a width (diameter) in the range of about 50 μm to about 500 μm, for example, about 50 μm to about 300 μm, encompassing any value and subset between these values, with upper and lower limits that are separable. Non-circular discontinuous active regions (not shown) may have an area range equivalent to that of the circular feature portion having the width (diameter) range described above. The pitch (distance between adjacent active regions) between each discontinuous active region may be about 50 μm to about 800 μm, for example, about 50 μm to about 500 μm, encompassing any value and subset between these values, with upper and lower limits that are separable. Generally, the most distal active area is located at least approximately 200 to 300 μm from the tip of the working electrode (which may be equal to the tip of the sensor tail) that is located most distally within the body fluid (measured from the center of sensing in the active area), but may be in the range of approximately 50 to 500 μm, and is situated in a location where any value and subset between these values ​​can be considered to be within a separable range of upper and lower limits.

[0045] In total, the working electrode, which has an active region (whether a single active region or multiple discontinuous active regions), is approximately 0.1 mm in diameter. 2 From approximately 5mm 2 The area may have a range that includes any value and subset between these values, with upper and lower limits that are separable. In total, the external working electrode area (excluding any active areas) is approximately 0.01 mm². 2 From approximately 3mm 2The range can be defined as encompassing any value and subset between these values, with the upper and lower bounds being separable.

[0046] To achieve a small external working electrode area for reducing interfering substance signals while maintaining sensitivity to one or more samples of interest, the ratio of the external working electrode area to the active area area can be in the range of approximately 1:10 to approximately 10:1, encompassing any value and subset between these values, with upper and lower limits being separable. This ratio is maintained regardless of the grid configuration or pitch distance of the sample sensor described herein; that is, the range of the ratio of the external working electrode area to the active area area is always within the above range to achieve the desired advantages described herein.

[0047] Accordingly, the sample sensor of this disclosure comprises a working electrode having a sensing portion and an exposed electrode portion, wherein the sensing portion has an active region on which a sample-reactive enzyme is disposed, and the exposed electrode portion does not have an active region, and the ratio of the exposed electrode portion to the sensing portion is in the range of about 1:10 to about 10:1. The working electrode may be a carbon electrode. At least the sensing portion may have a mass transport limiting film coated thereon.

[0048] Furthermore, the method of the present disclosure may include a step of exposing a sample sensor, which comprises a working electrode having a sensing portion and an exposed electrode portion, to a body fluid, wherein the sensing portion has an active region on which a sample-reactive enzyme is disposed, and the exposed electrode portion does not have an active region, and the ratio of the exposed electrode portion to the sensing portion is in the range of about 1:10 to about 10:1. The working electrode may be a carbon electrode. At least the sensing portion may have a mass transport limiting membrane coated thereon.

[0049] The active region in any of the sample sensors disclosed herein may comprise one or more sample-reactive enzymes that act either individually or cooperatively within an enzyme system. The one or more enzymes may be covalently bonded to a polymer comprising the active region, and likewise, one or more electron transfer agents may be positioned within the active region.

[0050] Examples of suitable polymers within each active region include, for example, poly(4-vinylpyridine) and poly(N-vinylimidazole) or copolymers thereof, in which quaternized pyridine and imidazole groups function as attachment points to electron transport agents or electron transport enzymes. Other suitable polymers that may be present in the active region include, but are not limited to, those described in U.S. Patent No. 6,605,200, which is incorporated herein in whole by reference, 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 by carboxypentyl groups, and poly(sodium 4-styrene sulfonate).

[0051] The enzymes that are covalently bound to the polymer within the active region and have the function of facilitating sample detection are not considered to be specifically limited. Suitable enzymes may include those that have the function of detecting glucose, lactate, ketones, or creatinine. Any of these samples can be detected in combination with each other in a sample sensor that has the function of detecting multiple samples. Suitable enzymes and enzyme systems for detecting these samples are described below.

[0052] In some embodiments, the sample sensor may include a glucose-reactive active region comprising a glucose-reactive enzyme positioned on the sensor tail. Suitable glucose-reactive enzymes may include, for example, glucose oxidase or glucose dehydrogenase (e.g., pyrroloquinoline quinone (PQQ) or cofactor-dependent glucose dehydrogenase, e.g., flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase or nicotinamide adenine dinucleotide (NAD)-dependent glucose dehydrogenase). Glucose oxidase and glucose dehydrogenase are distinguished by their ability to utilize oxygen as an electron acceptor when oxidizing glucose; glucose oxidase can utilize oxygen as an electron acceptor, while glucose dehydrogenase transfers electrons to a natural or artificial electron acceptor, e.g., an enzyme cofactor. Glucose oxidase or glucose dehydrogenase can be used to facilitate detection. Both glucose oxidase and glucose dehydrogenase can be covalently bonded to polymers containing glucose-reactive active regions, and both can exchange electrons with electron transfer agents (e.g., osmium (Os) complexes or similar transition metal complexes) that can also be covalently bonded to polymers. Suitable electron transfer agents are described in more detail below. Glucose oxidase can directly exchange electrons with electron transfer agents, whereas glucose dehydrogenase can facilitate electron exchange with electron transfer agents using cofactors. FAD cofactors can directly exchange electrons with electron transfer agents. In contrast, NAD cofactors can facilitate electron transfer from cofactors to electron transfer agents using diaphorase. Details regarding glucose-reactive active regions incorporating glucose oxidase or glucose dehydrogenase, and glucose detection using them, can be found, for example, in U.S. Patent No. 8,268,143, owned by the applicant of the present invention, which is incorporated in whole by reference herein.

[0053] In some embodiments, the active region of this disclosure may be configured to be suitable for detecting ketones. Additional details relating to enzyme systems reactive to ketones can be found in the applicant-owned U.S. Patent Application No. 16 / 774,835, entitled “Analyte Sensors and Sensing Methods Featuring Dual Detection of Glucose and Ketones,” which was filed on 28 January 2020 and published as U.S. Patent Application Publication No. 2020 / 0237275, and is incorporated in its entirety by reference herein. In such a system, β-hydroxybutyrate acts as a substitute for ketones formed in vivo and is subjected to a reaction with an enzyme system containing β-hydroxybutyrate dehydrogenase (HBDH) and diaphorase to facilitate the detection of ketones in a ketone-reactive active region located on the surface of at least one working electrode, as further described herein. Within the ketone-reactive region, β-hydroxybutyrate dehydrogenase reacts with β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD). + These can be converted to acetacetate and reduced nicotinamide adenine dinucleotide (NADH), respectively. The term "nicotinamide adenine dinucleotide (NAD)" is acknowledged to include the phosphate-bound form of the enzyme cofactor described above. That is, the use of the term "NAD" in this specification is understood to include NAD + Both phosphates and NADH phosphates refer to diphosphates that bind two nucleotides, one containing an adenine nucleic acid base and the other containing a nicotinamide nucleic acid base. + / NADH enzyme cofactor helps to facilitate the collaborative enzyme reactions disclosed herein. In its formed state, NADH can undergo oxidation under the mediation of diaphorase, and during this process, electrons are transmitted to provide a basis for ketone detection at the working electrode. That is, there is a 1:1 molar correspondence between the amount of electrons transmitted to the working electrode and the amount of β-hydroxybutyrate converted. The transmission of electrons to the working electrode can occur under the further mediation of an electron transfer agent such as an osmium (Os) compound or a similar transition metal complex, as will be described in more detail below. Albumin can further be present as a stabilizer within the active region. β-Hydroxybutyrate dehydrogenase and diaphorase can be covalently bound to a polymer with a ketone-reactive active region. NAD + may or may not be covalently bound to the polymer. If not covalently bound, it can be physically retained within the ketone-reactive active region. For example, a mass transport limiting membrane that is also permeable to ketones protects the ketone-reactive active region.

[0054] Other suitable chemical actions for enzymatically detecting ketones can be utilized in accordance with embodiments of the present disclosure. For example, β-hydroxybutyrate dehydrogenase (HBDH) is also here β-hydroxybutyrate and NAD +These can be converted to acetacetate and NADH, respectively. Instead of completing electron transfer to the working electrode by diaphorase and a suitable redox mediator, reduced NADH oxidase (NADHOx(Red)) undergoes the reaction to form the corresponding oxidized form (NADHOx(Ox)). Next, NADHOx(Red) can be reformed by reaction with molecular oxygen to produce superoxide, which can then be converted to hydrogen peroxide under the mediation of superoxide dismutase (SOD). The hydrogen peroxide is then oxidized at the working electrode to supply a signal that can be correlated to the amount of ketone initially present. In various embodiments, SOD can be covalently bonded to the polymer within the ketone-reactive active region. β-hydroxybutyrate dehydrogenase and NADH oxidase can be covalently bonded to the polymer within the ketone-reactive active region, and NAD + This may or may not involve covalent bonding to the polymer within the ketone-reactive active region. NAD + If not covalently bonded, it can be physically retained within the ketone-reactive active region, and the membrane polymer can retain NAD within the ketone-reactive active region. + This improves retention. Here again, there is a 1:1 molar correspondence between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted, thereby providing a basis for ketone detection.

[0055] Another enzymatic detection chemistry for ketones involves the use of β-hydroxybutyrate dehydrogenase (HBDH) to detect β-hydroxybutyrate and NAD. + These can be converted to acetacetate and NADH, respectively. In this case, the electron transport cycle is NAD +The process is completed by the oxidation of NADH by 1,10-phenanthroline-5,6-dione, which then transfers electrons to the working electrode. 1,10-phenanthroline-5,6-dione may or may not covalently bond to the polymer within the ketone-reactive region. β-hydroxybutyrate dehydrogenase can covalently bond to the polymer within the ketone-reactive region, and NAD + The polymer may or may not covalently bond albumin to the ketone-reactive active region. Inclusion of albumin within the active region can provide a remarkable improvement in response stability. Suitable membrane polymers may contain NAD within the ketone-reactive active region. + The retention of the solution can be improved. Here again, there is a 1:1 molar correspondence between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted, which provides the basis for ketone detection.

[0056] In some embodiments, the sample sensor may further comprise a creatinine-reactive region comprising an enzyme system that works cooperatively to facilitate the detection of creatinine. Creatinine can react reversibly and hydrolytically in the presence of creatinine amide hydrolase (CNH0029) to form creatine. Furthermore, creatine can undergo catalytic hydrolysis in the presence of creatine amide hydrolase (CRH) to form sarcosine. Neither of these reactions generates an electron flow (e.g., oxidation or reduction) and provides a basis for the electrochemical detection of creatinine. Sarcosine, produced by the hydrolysis of creatine, can be oxidized in the presence of oxidative sarcosine oxidase (SOX-ox) to form glycine and formaldehyde, thereby generating reduced sarcosine oxidase (SOX-red) in this process. In the presence of oxygen, hydrogen peroxide may also be generated. Next, the reduced sarcosin oxidase can be reoxidized in the presence of an oxidized electron transport agent (e.g., Os(III) complex), thereby generating a corresponding reduced electron transport agent (e.g., Os(II) complex), which delivers an electron flow to the working electrode.

[0057] Oxygen may interfere with the cooperative reaction sequence used to detect creatinine as disclosed above. Specifically, reduced sarcosine oxidase reacts with oxygen to reformate into the corresponding oxidized form of this enzyme, but this reformation can occur without the exchange of electrons with an electron transport agent. When the reaction with oxygen occurs, the enzyme remains fully active, but electrons do not flow to the working electrode. Regardless of theory or mechanism, it is thought that a competitive reaction with oxygen arises from kinetic effects. That is, the oxidation of reduced sarcosine oxidase by oxygen is thought to occur faster than the oxidation facilitated by an electron transport agent. Hydrogen peroxide is also formed in the presence of oxygen.

[0058] A desirable reaction pathway for facilitating creatinine detection can be facilitated by including an oxygen scavenger around the enzyme system. Various oxygen scavengers and their arrangements, each containing an oxidase enzyme such as glucose oxidase, can be appropriate. While low molecular weight oxygen scavengers may be suitable in some cases, these may be completely consumed before the sensor's lifespan is fully exhausted. In contrast, enzymes can undergo reversible oxidation and reduction, thereby providing a longer sensor lifespan. By deactivating the oxidation of the reduced form of sarcosine oxidase by oxygen, a slow electron exchange reaction with an electron transport agent can be generated, thereby enabling current generation at the working electrode. The intensity of the current generated is proportional to the amount of creatinine initially reacted.

[0059] In any embodiment of this disclosure, the deoxygenating agent used to facilitate the desired reaction may be an oxidase enzyme. Suitable substrates for the enzyme also exist, and any oxidase enzyme can be used to facilitate deoxygenation around an enzyme system, provided that a reagent is supplied for reaction with oxygen in the presence of the oxidase enzyme. In this disclosure, oxidase enzymes that may be suitable for deoxygenation include, but are not limited to, glucose oxidase, lactate oxidase, and xanthine oxidase. Glucose oxidase may be a particularly preferred oxidase enzyme for facilitating deoxygenation due to the immediate availability of glucose in various body fluids. Reaction 1 below illustrates an enzymatic reaction facilitated by glucose oxidase to provide oxygen removal. β-D-glucose + O2 → D-glucono-1,5-lactone + H2O2 Reaction 1 The concentration of lactat available in the body is lower than that of glucose, but it is still sufficient to promote deoxygenation.

[0060] Oxidase enzymes, such as glucose oxidase, can be placed at any location suitable for promoting deoxygenation in the sample sensor disclosed herein. Glucose oxidase can be placed on the sensor tail, for example, to function and / or not function in a manner suitable for facilitating glucose detection. When not functioning to facilitate glucose detection, glucose oxidase can be placed on the sensor tail such that electrons generated during glucose oxidation cannot reach the working electrode, for example, by electrically isolating the glucose oxidase from the working electrode.

[0061] Further details relating to an enzyme system reactive to creatinine can be found in the applicant's U.S. Patent Application No. 16 / 582,583, entitled “Analyte Sensors and Sensing Methods for Detecting Creatinine,” which was filed on 25 September 2019 and published as U.S. Patent Application Publication No. 2020 / 0241015 and is incorporated in its entirety by reference herein.

[0062] In some embodiments, the sample sensor may have a lactate-reactive region comprising a lactate-reactive enzyme located on the sensor tail. Suitable lactate-reactive enzymes may include, for example, lactate oxidase. Lactate oxidase or other lactate-reactive enzymes can be covalently bonded to a polymer comprising the lactate-reactive region and can exchange electrons with an electron transfer agent (e.g., an osmium (Os) complex or a similar transition metal complex) which can also be covalently bonded to the polymer. Suitable electron transfer agents are described in more detail below. Albumin, such as human serum albumin, may be present in the lactate-reactive region to stabilize sensor reactivity, as described in detail in U.S. Patent Application Publication No. 2019 / 0320947, owned by the applicant of the present invention, which is incorporated in whole by reference herein. Lactate levels may change in response to a number of environmental or physiological factors, including, for example, feeding, stress, exercise, sepsis or septic shock, infection, hypoxia, or the presence of cancerous tissue.

[0063] In some embodiments, the sample sensor may include an active region that is reactive to pH. A suitable sample sensor configured for determining pH is described in U.S. Patent Application Publication No. 2020 / 0060592, owned by the applicant of the present invention and incorporated in its entirety by reference herein. Such a sample sensor may include a sensor tail comprising a first working electrode and a second working electrode, wherein the first active region located on the first working electrode comprises a substrate having pH-dependent redox activity, and the second active region located on the second working electrode comprises a substrate having redox activity that is substantially invariant with pH. By obtaining the difference between the first and second signals, this difference can be correlated with the pH of the fluid to which the sample sensor is exposed.

[0064] Two different types of active regions can be positioned 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, and the redox potential of the first active region is sufficiently separated from the redox potential of the second active region to allow independent generation of signals from one of the active regions. As a non-limiting example, these redox potentials can differ by at least about 100 mV, at least about 150 mV, or at least about 200 mV. The upper limit of the separation between these redox potentials depends on the bioactive electrochemical window. By having redox potentials of two active regions that are sufficiently separated in intensity from each other, an electrochemical reaction can occur within one of the two active regions (i.e., within the first or second active region) without substantially inducing an electrochemical reaction in the other active region. Therefore, signals from either the first or second active region can be generated independently at their corresponding redox potentials (lower redox potentials), or at potentials greater than or equal to but less than the redox potential of the other active region. These different signals allow for the decomposition of the signal contributions from each sample.

[0065] Some or all embodiments of the sample sensors disclosed herein may feature one or more active regions positioned on the surface of at least one working electrode to detect the same or different samples. The membrane can protect at least the active regions (which comprise the sample-reactive enzyme) and can protect all or part of the working electrode that lacks the active regions (the exposed or external portion of the working electrode). The membrane may be a mass transport restriction membrane and may be a monolayer of the membrane, a bilayer of two different membrane polymers, or a hybrid of two different membrane polymers.

[0066] Electron transfer agents may be present in any of the active regions disclosed herein. A suitable electron transfer agent can facilitate the transport of electrons to adjacent working electrodes after one or more samples have undergone enzymatic redox reactions within the corresponding active region, thereby generating an electron flow indicating the presence of a particular sample. The amount of current generated is proportional to the number of samples present. Electron transfer agents that are reactive to different samples within the active region may be the same or different, depending on the sensor configuration used. For example, when two different active regions are located on the same working electrode, the electron transfer agents in each active region may be different (e.g., chemically different so that these electron transfer agents exhibit different redox potentials). When multiple working electrodes are present, each working electrode can be evaluated separately, so the electron transfer agents in each active region may be the same or different.

[0067] Suitable electron carriers may comprise electroreducible and electrooxidizing ions, complexes, or molecules (e.g., quinones) having redox potentials exceeding or falling below several hundred millivolts of the redox potential of a standard calomel electrode (SCE). In some embodiments, suitable electron carriers may comprise low-potential osmium complexes, such as those described in U.S. Patents No. 6,134,461 and 6,605,200, which are incorporated herein by reference in their entirety. Additional examples of suitable electron carriers include those described in U.S. Patents No. 6,736,957, 7,501,053, and 7,754,093, whose respective disclosures are incorporated herein by reference in their entirety. Other suitable electron carriers may comprise metal compounds or metal complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt, including these metallocene compounds. For example, suitable ligands for metal complexes may include bidentate or more-locate ligands such as bipyridine, biimidazole, phenanthroline, or pyridyl(imidazole). Other suitable bidentate ligands may include amino acids, oxalic acid, acetylacetone, diaminoalkane, or o-diaminoaleine. Any combination of monodentate, bidentate, tridentate, tetradentate, or more-locate ligands can exist within the metal complex to achieve the maximum coordination sphere.

[0068] An active region suitable for detecting any of the specimens disclosed herein may comprise a polymer to which an electron transfer agent is covalently bonded. Any of the electron transfer agents disclosed herein may comprise a functional group suitable for promoting covalent bonding to a polymer within the active region. Suitable examples of polymer-bonded electron transfer agents include those described in U.S. Patent Nos. 8,444,834, 8,268,143, and 6,605,201, whose disclosures are incorporated herein by reference in their entirety. Polymers suitable for inclusion within the active region may comprise, but are not limited to, polyvinylpyridine (e.g., poly(4-vinylpyridine)), polyvinylimidazole (e.g., poly(1-vinylimidazole)), or copolymers of any of these. Exemplary copolymers that may be suitable for inclusion within the active region include, for example, those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile. When there are two or more different active regions, the polymers within each active region may be the same or different.

[0069] The covalent bonding of an electron transfer agent to the polymer within the active region can be achieved by polymerizing monomer units that support the covalently bonded electron transfer agent, or by reacting the electron transfer agent separately with the polymer after the polymer has been synthesized. A bifunctional spacer can covalently bond an electron transfer agent to the polymer within the active region, in which case the first functional group is reactive with the polymer (e.g., a functional group that has the function of quaternizing pyridine nitrogen atoms or imidazole nitrogen atoms), and the second functional group is reactive with the electron transfer agent (e.g., a functional group that is reactive with ligands that coordinate metal ions).

[0070] Similarly, one or more enzymes within an active region can be covalently bonded to the polymer containing the active region. When an enzyme system comprising multiple enzymes within a given active region exists, 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 comprising an enzyme system can be covalently bonded to a polymer, and at least one enzyme can be non-covalently associated with the polymer such that non-covalent enzymes are physically encombined within the polymer. Covalent bonding of enzymes to a polymer within a given active region can occur through a crosslink introduced using a suitable crosslinking agent. Crosslinking agents suitable for reaction with free amino groups in enzymes (e.g., with free side-chain amines in lysine) may include, for example, polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuric acid chloride, N-hydroxysuccinimids, imide esters, epichlorohydrin, or derived variants thereof. Crosslinking agents suitable for reaction with free carboxylic acid groups in enzymes may include, for example, carbodiimides. Enzyme crosslinking to polymers is generally intermolecular, but in some embodiments it can be intramolecular. In certain embodiments, all of the enzyme within a given active region can be covalently bonded to the polymer.

[0071] Electron transfer agents and / or enzymes can be associated with polymers within the active region by means other than covalent bonding. In some embodiments, electron transfer agents and / or enzymes can be ionically or coordinatively associated with polymers. For example, charged polymers can be ionically associated with inversely charged electron transfer agents or enzymes. In yet other embodiments, electron transfer agents and / or enzymes can be physically encombined within the polymer without being bonded to it. Physically encombined electron transfer agents and / or enzymes can still interact effectively with the fluid without substantially leaching out of the active region, facilitating the detection of the sample.

[0072] The polymer within the active region is NAD that is not covalently bonded to it. + Alternatively, another cofactor can be selected to restrict its outward diffusion. Restricting the outward diffusion of the cofactor can increase the sensor lifespan to some extent (several days to several weeks) while still allowing sufficient inward sample diffusion to facilitate detection.

[0073] In some embodiments, stabilizers may be incorporated into the active region of the sample described herein to improve the sensor's function and achieve desired sensitivity and stability. Such stabilizers may comprise, for example, antioxidants and / or enzymatically stabilizing proteins. Examples of suitable stabilizers may include, but are not limited to, serum albumin (e.g., human or bovine serum albumin, or other suitable albumin), catalase, and other enzyme antioxidants, and any combination thereof. Stabilizers may be conjugated or unconjugated.

[0074] In certain embodiments of this disclosure, a mass transport restriction membrane protecting one or more active areas may comprise a homopolymer or copolymer of crosslinked polyvinylpyridine. The composition of the mass transport restriction membrane may be the same or different when the membrane protects different types of active areas. When the membrane composition differs in two different locations, the membrane may comprise a bilayer or homogeneous hybrid of two different membrane polymers, one of which may be a homopolymer or copolymer of crosslinked polyvinylpyridine or crosslinked polyvinylimidazole. Suitable techniques for depositing the mass transport restriction membrane on the active areas may include, for example, spray coating, coating, inkjet printing, screen printing, smearing, roller coating, dipping coating, similar methods, and combinations thereof. Dipping coating techniques may be particularly preferred for polymers and copolymers of polyvinylpyridine and polyvinylimidazole.

[0075] In certain embodiments, the mass transport limiting membrane discussed above is a membrane composed of a crosslinked polymer containing heterocyclic nitrogen groups, such as polyvinylpyridine and polyvinylimidazole polymers. Embodiments also include membranes made of polyurethane, polyether urethane, or chemically related materials, or membranes made of silicone, etc.

[0076] In some embodiments, the membrane can be formed in a buffer solution (e.g., an alcohol buffer solution) by crosslinking a polymer, including those discussed above, modified with a zwitterionic moiety, a non-pyridine copolymer component, and optionally another moiety having either hydrophilic or hydrophobic properties and / or other desirable properties. The modified polymer can be prepared from a precursor polymer containing heterocyclic nitrogen groups. For example, the precursor polymer can be polyvinylpyridine or polyvinylimidazole. Optionally, the permeability of the resulting membrane to the sample of interest can be "fine-tuned" using hydrophilic or hydrophobic modifiers. The biocompatibility of the polymer or the resulting membrane can be enhanced using optional hydrophilic modifiers such as poly(ethylene glycol) modifiers, hydroxyl modifiers, or polyhydroxyl modifiers, and similar modifiers, and any combination thereof.

[0077] In some embodiments, the film may comprise compounds including, but not limited to, crosslinked with poly(styrene-com-maleate anhydride), dodecylamine, and poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol)(2-aminopropyl ether), 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, polyvinylpyridine derivatives, polyvinylimidazole, and polyvinylimidazole derivatives, and any combination thereof. In some embodiments, the film may comprise a polyvinylpyridine-co-styrene polymer in which a portion of the pyridine nitrogen atoms are functionalized by non-crosslinked poly(ethylene glycol) tails and a portion of the pyridine nitrogen atoms are functionalized by alkylsulfonic acid groups. Other membrane compounds may comprise a suitable copolymer of 4-vinylpyridine, styrene, and an amine-free polyether arm, either alone or in combination with any of the above-mentioned membrane compounds.

[0078] The membrane compounds described herein can be further crosslinked with one or more crosslinking agents, including those listed above in relation to the enzymes described herein. For example, suitable crosslinking agents may include, but are not limited to, polyethylene glycol diglycidyl ether (PEGDGE), glycerol triglycidyl ether (Gly3), polydimethylsiloxane diglycidyl ether (PDMS-DGE), or other polyepoxides, cyanuric acid chlorides, N-hydroxysuccinimids, imide esters, epichlorohydrins, or derived variants thereof, and any combination thereof. Branched compounds with similar terminal chemistry are also suitable for this disclosure. For example, in some embodiments, chemical formula 1 can be crosslinked with triglycidyl glycol ether, PEDGE, and / or polydimethylsiloxane diglycidyl ether (PDMS-DGE).

[0079] The membrane can be formed in situ by adding an alcohol-buffered solution of the crosslinker and modified polymer onto the active area and any additional compounds contained therein (e.g., electron transfer agents), and curing the solution for about one to two days or other suitable period. The crosslinker-polymer solution can be provided on the active area by placing one or more droplets of the membrane solution on at least the sensor elements of the sensor tail, immersing the sensor tail in the membrane solution, spraying the membrane solution onto the sensor, hot-pressing or welding the membrane either before or after assembly into layers of any size (such as individually or all-encompassing), membrane deposition, and powder coating of the membrane, and any combination thereof. To coat the distal and lateral edges of the sensor, the membrane material can be added following the addition (e.g., assembly) of the sensor electron precursor (e.g., electrodes). In some embodiments, the sample sensor is immersion coated to add one or more membranes following the addition of the electron precursor. Alternatively, the sample sensor can be lattice-die coated, with each side of the sample sensor being coated separately. The membrane added in the manner described above may have any of the following functions, including, but not limited to, mass transport restriction (i.e., reduction or elimination of the flux of one or more samples and / or compounds reaching the active region), enhanced biocompatibility, and reduction of interfering substances, and any combination thereof.

[0080] Generally, the thickness of the membrane is controlled by the concentration of the membrane solution, the number of droplets of the membrane solution added, the number of times the sensor is immersed in the membrane solution, and the volume of the membrane solution sprayed onto the sensor, and any combination of these factors. In some embodiments, the membranes described herein can have a thickness that spans a range of about 0.1 micrometers (μm) to about 1000 μm, encompassing any value and subset between these values, with upper and lower limits that are separable. As described above, the membrane can overlap one or more active regions, and in some embodiments, the active regions can have a thickness that spans a range of about 0.1 μm to about 50 μm, encompassing any value and subset between these values, with upper and lower limits that are separable. In some embodiments, to achieve an active region and / or membrane of a desired thickness, a series of droplets can be added on top of each other without substantially increasing their diameter (i.e., while maintaining the desired diameter or range). For example, each single droplet can be added, then cooled or dried, and then one or more additional droplets can be added to it. The active region and membrane may, but do not, have the same thickness or composition throughout.

[0081] In some embodiments, suitable membrane compositions for use as mass transport limiting layers of this disclosure may include polydimethylsiloxane (PDMS), polydimethylsiloxane diglycidyl ether (PDMS-DGE), and aminopropyl-terminated polydimethylsiloxanes, and any combination thereof, suitable for use as leveling agents (e.g., for reducing the contact angle of the membrane composition or active area composition). Branched products with similar terminal chemistry are also suitable for this disclosure. For example, certain leveling agents found in U.S. Patent No. 8,983,568, whose disclosure is incorporated entirely by reference herein, may be additionally included.

[0082] 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 mean any type of interaction between atoms or molecules, including but not limited to covalent bonds, ionic bonds, dipolar-dipolar interactions, hydrogen bonds, and London dispersion forces, and any combination thereof, that allows compounds to form associations with one another. For example, in-situ polymerization of a membrane can form crosslinks between the membrane polymer and the polymer within the active region. In some embodiments, crosslinking of the membrane to the active region facilitates the reduction of membrane delamination from the sensor.

[0083] Embodiments disclosed herein include: A. A sample sensor comprising a working electrode having a sensing portion and an exposed electrode portion, wherein the sensing portion has an active region on which a sample-reactive enzyme is arranged, the exposed electrode portion does not have an active region, and the ratio of the exposed electrode portion to the sensing portion is in the range of approximately 1:10 to approximately 10:1, encompassing any value and subset between these values, with upper and lower limits being separable. B. A method comprising the step of exposing the sample sensor to a body fluid, wherein the sample sensor comprises a working electrode having a sensing portion and an exposed electrode portion, the sensing portion having an active region on which a sample-reactive enzyme is disposed, the exposed electrode portion having no active region, and the ratio of the exposed electrode portion to the sensing portion being in the range of about 1:10 to about 10:1 and encompassing any value and subset between these values, with upper and lower limits being separable.

[0084] Each of embodiments A and B may have one or more of the following additional elements in any combination.

[0085] Element 1: The working electrode is a carbon working electrode.

[0086] Element 2: The area of ​​the exposed electrode portion is approximately 0.1 mm². 2 From approximately 5mm 2 It encompasses any value and subset that lies within the range of these values, and its upper and lower bounds are separable.

[0087] Element 3: The area of ​​the sensing part is approximately 0.01 mm². 2 From approximately 3mm 2 It encompasses any value and subset that lies within the range of these values, and its upper and lower bounds are separable.

[0088] Element 4: The active region consists of multiple discontinuous active regions or a single continuous active region.

[0089] Element 5: The active region comprises multiple discontinuous active regions, each discontinuous active region having a diameter that encompasses any value and subset between approximately 50 μm and approximately 500 μm, with upper and lower limits that are separable.

[0090] Element 6: The active region is in the range of approximately 50 μm to approximately 800 μm, encompassing any value and subset between these values, and consists of multiple discontinuous active regions separated by a pitch with a distance where the upper and lower limits are separable.

[0091] Element 7: The sensing portion comprises multiple discontinuous active regions arranged in a 1 × n grid configuration, where n is an integer encompassing any value and subset between 2 and approximately 20, with upper and lower bounds being separable.

[0092] Element 8: The sensing portion comprises multiple discontinuous active regions arranged in a 2 × n grid configuration, where n is an integer encompassing any value and subset between 3 and approximately 10, with upper and lower bounds being separable.

[0093] Element 9: The sensing portion comprises multiple discontinuous active regions arranged in a 3 × n grid configuration, where n is an integer encompassing any value and subset between 2 and approximately 6, with upper and lower bounds being separable.

[0094] Element 10: A mass transport limiting membrane is placed at least above the sensing portion.

[0095] Element 11: The sample-reactive enzyme is a glucose-reactive enzyme.

[0096] Element 12: The sample sensor shows a reduction in the interference signal of interfering substances compared to a sample sensor having a larger ratio of exposed electrode portion to sensing portion.

[0097] Element 13: The sample sensor exhibits a reduction in the interference signal of interfering substances compared to a sample sensor having a larger ratio of exposed electrode portion to sensing portion, and the reduction in the interference signal is greater than approximately 20%.

[0098] Element 14: The sample sensor exhibits a reduction in the interfering substance signal compared to a sample sensor having a larger ratio of the exposed electrode portion to the sensing portion, the reduction in the interfering substance signal being in the range of approximately 20% to approximately 70% and encompassing any value and subset between these values, with the upper and lower limits being separable.

[0099] Element 15: The sample sensor exhibits a reduction in the interference signal of an interfering substance compared to a sample sensor having a larger ratio of the exposed electrode portion to the sensing portion, the interfering substance being ascorbic acid.

[0100] As an example of non-limiting examples, applicable combinations of A and B include, but are not limited to, A or B having one or two or more or all of 1-7 and 10-15 in any combination, A or B having one or two or more or all of 1-6, 8 and 10-15 in any combination, and A or B having one or two or more or all of 1-6 and 9-15 in any combination.

[0101] Accordingly, this disclosure provides a sample sensor for monitoring different samples in vivo. The sample sensor may be characterized by enhancements to address signals obtained from interfering substance species. Some sample sensors may comprise a working electrode having a sensing portion and an exposed electrode portion, wherein the sensing portion has an active region on which a sample-reactive enzyme is located, and the exposed electrode portion does not have an active region. The ratio of the exposed electrode portion to the sensing portion can range from about 1:10 to about 10:1.

[0102] Unless otherwise indicated, all numbers expressing quantities, etc., within this specification and related claims shall be understood in all cases to be modified by the term “approximately.” Accordingly, unless otherwise indicated, numerical parameters referred to within this specification and claims are approximations, which may vary depending on the desired characteristics to be obtained by embodiments of the invention. Each numerical parameter should be interpreted by applying ordinary rounding techniques, taking into account at least the reported significant digits, and not as an attempt to limit the application of the doctrine of equivalents to the claims.

[0103] This specification presents one or more exemplary embodiments incorporating various features. For clarity purposes, this specification does not describe or illustrate all features of physical implementations. It is acknowledged that in developing physical embodiments incorporating embodiments of the present invention, numerous implementation-specific decisions must be made to achieve the developer's objectives, such as compliance with system-related, commercial-related, government-related, and other constraints, which vary from implementation to implementation and from time to time. While the developer's effort may be time-consuming, such effort will nevertheless be considered routine work for those skilled in the art who are interested in this disclosure.

[0104] In this specification, various systems, tools, and methods are described using the term "comprising" various components or stages, but systems, tools, and methods can also be "basically composed of" or "composed of" various components and stages.

[0105] When used herein, the expression “at least one of” following a set of items, along with the terms “and” or “or” to separate any of these items, modifies the entire enumeration rather than each constituent unit of the enumeration (i.e., each item). The expression “at least one of” may mean at least one of any of the items, at least one of any combination of the items, and / or at least one of each of the items. For example, each of the expressions “at least one of A, B, and C” or “at least one of A, B, or C” means A only, B only, or C only, any combination of A, B, and C, and / or at least one of each of A, B, and C.

[0106] In other words, the systems, tools, and methods of this disclosure are adequately adapted to achieve the purposes and benefits mentioned and those inherent in these systems, tools, and methods. The teachings of this disclosure can be modified and implemented in ways that are clearly different but equivalent to those of a person skilled in the art who are interested in them; therefore, the specific embodiments disclosed above are merely illustrative. Furthermore, no limitation is intended to any structural or design details shown herein other than those described in the claims below. Accordingly, it is clear that the specific exemplary embodiments disclosed above can be modified, combined, or altered, and all such variations will be considered within the scope of this disclosure. The systems, tools, and methods disclosed exemplary herein can be adequately implemented without any elements not specifically disclosed herein and / or any optional elements disclosed herein. While the terms “equipment,” “contains,” or “include” various components or stages are used to describe the systems, tools, and methods, the systems, tools, and methods can “basically constitute” or “constitute” various components and stages. All figures and scopes disclosed above may differ by some degree. When disclosing a numerical range with a lower and upper limit, all numbers and ranges that fall within that range are specifically disclosed. In particular, all value ranges disclosed herein (in the form of "approximately a to approximately b" or equivalently "approximately a to b" or equivalently "approximately ab") should be understood as listing all numbers and ranges that are included within that broad value range. Similarly, terms in the claims have their plain, ordinary meanings unless otherwise explicitly and clearly defined by the Patent Holder. Furthermore, in this specification, non-plural nouns used in the claims are defined as meaning one or more elements introduced by the claims. In the event of any inconsistency between the use of a word or term herein and the use of a word or term in one or more patent documents or other documents that may be incorporated herein by reference, the definition consistent with this specification shall prevail.

Claims

1. It is a sample sensor, It comprises an working electrode having a sensing portion and an exposed electrode portion. The sensing portion comprises an active area on which a sample-reactive enzyme is arranged, while the exposed electrode portion does not comprise an active area. The ratio of the exposed electrode portion to the sensing portion is in the range of approximately 1:10 to approximately 10:

1. A sample sensor characterized by the following features.

2. Compared to a sample sensor having a larger ratio of the exposed electrode portion to the sensing portion, it shows a reduction in the interference signal of the interfering substance. The sample sensor according to claim 1.

3. The reduction of the interference material signal by the aforementioned interference material is greater than approximately 20%. The sample sensor according to claim 2.

4. The interfering substance is ascorbic acid. The sample sensor according to claim 2.

5. The aforementioned working electrode is a carbon working electrode. The sample sensor according to claim 1.

6. The area of ​​the exposed electrode portion is approximately 0.1 mm². 2 From approximately 5 mm 2 Within the range, The sample sensor according to claim 1.

7. The area of ​​the sensing portion is approximately 0.01 mm². 2 From approximately 3 mm 2 Within the range, The sample sensor according to claim 1.

8. The active region is composed of a plurality of discontinuous active regions. The sample sensor according to claim 1.

9. The active region is composed of a plurality of discontinuous active regions. Each discontinuous active region has a diameter ranging from approximately 50 μm to approximately 500 μm. The sample sensor according to claim 1.

10. The active region is composed of a plurality of discontinuous active regions separated by a pitch having a distance ranging from approximately 50 μm to approximately 800 μm. The sample sensor according to claim 1.

11. The active region is composed of a single continuous active region. The sample sensor according to claim 1.

12. The mass transport limiting membrane is positioned at least above the sensing portion. The sample sensor according to claim 1.

13. The aforementioned sample-reactive enzyme is a glucose-reactive enzyme. The sample sensor according to claim 1.

14. It is a method, The procedure includes a step of exposing the sample sensor to bodily fluids. The aforementioned sample sensor is It comprises an working electrode having a sensing portion and an exposed electrode portion. The sensing portion comprises an active area on which a sample-reactive enzyme is arranged, while the exposed electrode portion does not comprise an active area. The ratio of the exposed electrode portion to the sensing portion is in the range of approximately 1:10 to approximately 10:

1. A method characterized by the following:

15. The aforementioned sample sensor exhibits a reduction in the interference signal of interfering substances compared to a sample sensor having a larger ratio of the exposed electrode portion to the sensing portion. The method according to claim 14.

16. The reduction of the interference material signal by the aforementioned interference material is greater than approximately 20%. The method according to claim 15.

17. The interfering substance is ascorbic acid. The method according to claim 15.

18. The aforementioned working electrode is a carbon working electrode. The method according to claim 14.

19. The area of ​​the exposed electrode portion is approximately 0.1 mm². 2 From approximately 5 mm 2 Within the range, The method according to claim 14.

20. The area of ​​the sensing portion is approximately 0.01 mm². 2 From approximately 3 mm 2 Within the range, The method according to claim 14.

21. The active region is composed of a plurality of discontinuous active regions. The method according to claim 14.

22. The active region is composed of a plurality of discontinuous active regions. Each discontinuous active region has a diameter ranging from approximately 50 μm to approximately 500 μm. The method according to claim 14.

23. The active region is composed of a plurality of discontinuous active regions separated by a pitch having a distance ranging from approximately 50 μm to approximately 800 μm. The method according to claim 14.

24. The active region is composed of a single continuous active region. The method according to claim 14.

25. The mass transport limiting membrane is positioned at least above the sensing portion. The method according to claim 14.

26. The aforementioned sample-reactive enzyme is a glucose-reactive enzyme. The method according to claim 14.

Images