JP2023531192A - Analyte sensor featuring reduced area working electrode to reduce interfering signals - Google Patents

Abstract

Analyte sensors are increasingly being used to monitor various analytes in vivo. Analyte sensors may feature enhancements to address signals obtained from interfering species. Some analyte sensors can comprise a working electrode having a sensing portion and an exposed electrode portion, the sensing portion comprising an active area with an analyte-reactive enzyme disposed thereon and the exposed electrode portion comprising no active area. The exposed electrode portion and sensing portion can be present in a ratio of about 1:10 to about 10:1.
[Selection drawing] Fig. 1

Description

[Cross reference to related application]
This application claims priority to and benefit from US Provisional Patent Application No. 63/039,743, filed June 16, 2020, which is incorporated herein by reference in its entirety.

Detection of different analytes within an individual is sometimes essential to monitor their health status. Deviations from normal analyte levels can often indicate several physiological conditions. For example, glucose levels can be particularly important to detect and monitor in diabetic patients. By monitoring glucose levels with sufficient regularity, diabetics could take corrective action (e.g., by injecting insulin to lower glucose levels or eating to raise glucose levels) before significant physiological disturbances occur. Monitoring of other analytes for various other physiological conditions may be desirable. Monitoring of multiple analytes may also be desirable in some cases, particularly for concomitant conditions that result in simultaneous dysregulation of two or more analytes in combination with each other.

U.S. Patent Application Publication No. 2011/0213225 U.S. Pat. No. 6,605,200 U.S. Pat. No. 8,268,143 U.S. Patent Application Publication No. 2020/0237275 U.S. patent application Ser. No. 16/774,835 U.S. Patent Application Publication No. 2020/0241015 US patent application Ser. No. 16/582,583 U.S. Patent Application Publication No. 2019/0320947 U.S. Patent Application Publication No. 2020/0060592 U.S. Pat. No. 6,134,461 U.S. Pat. No. 6,736,957 U.S. Pat. No. 7,501,053 U.S. Pat. No. 7,754,093 U.S. Pat. No. 8,444,834 U.S. Pat. No. 6,605,201 U.S. Pat. No. 8,983,568

Many analytes represent interesting targets for physiological analysis, provided that suitable detection chemistries can be identified. To this end, in vivo analyte sensors configured to assay a variety of physiological analytes have been developed and improved over the years, many of which utilize enzyme-based detection strategies to facilitate detection specificity. Indeed, in vivo analyte sensors that utilize glucose-responsive enzymes to monitor blood glucose levels are now commonly used among diabetics. In-vivo analyte sensors for other analytes, including in-vivo analyte sensors capable of monitoring multiple analytes, are at various stages of development. Lack of sensitivity to low volume analytes can be particularly problematic for some analyte sensors, particularly due to background signals resulting from the interaction of interfering substances with the working electrode or other analyte sensing chemistry components.

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

1 is a diagram of an exemplary sensing system that may incorporate analyte sensors of the present disclosure; FIG. FIG. 2 is a cross-sectional view of an analyte sensor with a single active area; FIG. 2 is a cross-sectional view of an analyte sensor with a single active area; FIG. 2 is a cross-sectional view of an analyte sensor with a single active area; FIG. 2 is a cross-sectional view of an analyte sensor with two active areas; FIG. 2 is a cross-sectional view of an analyte sensor with two active areas; FIG. 2 is a cross-sectional view of an analyte sensor with two active areas; FIG. 2 is a cross-sectional view of an analyte sensor comprising two working electrodes each having an active area thereon; 1 is a top view of a conventional carbon working electrode with an active area thereon; FIG. FIG. 4A is a cross-sectional view of an active area grid configuration of a carbon working electrode suitable for use in the analyte sensor of the present disclosure; FIG. 10 illustrates another active area grid configuration; 1 is a top view of a conventional carbon working electrode with an active area thereon; FIG. FIG. 10 shows an exemplary treatment that can enhance the carbon working electrode and active area thereon to reduce interferent signals. FIG. 10 shows an exemplary treatment that can enhance the carbon working electrode and active area thereon to reduce interferent signals. FIG. 10 shows an exemplary treatment that can enhance the carbon working electrode and active area thereon to reduce interferent signals. FIG. 10 shows an exemplary treatment that can enhance the carbon working electrode and active area thereon to reduce interferent signals. FIG. 10 shows an exemplary treatment that can enhance the carbon working electrode and active area thereon to reduce interferent signals.

The present disclosure generally describes analyte sensors suitable for in vivo use and, more specifically, analyte sensors featuring one or more enhancements to reduce or eliminate signals indicative of interfering species to facilitate improved detection sensitivity, and methods of their production and use.

Such enhancements can include reducing the area of the working electrode on the sensor tail, particularly the carbon working electrode, against which interferents may react and contribute to a signal not associated with the analyte. The area of the carbon working electrode can be reduced by one or more means, such as by 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 towards the distal portion of the sensor tail. As used herein, the term "pitch" and grammatical variations thereof means the distance between adjacent sensing spots within the active area sensing layer, measured from the center of each adjacent sensing spot. Specific details and further benefits of each type of reinforcement are described in more detail herein. Analyte sensors of the present disclosure can be configured to detect a single analyte or to detect multiple analytes simultaneously or near-simultaneously, depending on the particular need.

Analyte sensors using enzyme-based detection are commonly used to assay single analytes such as glucose due to the frequent specificity of enzymes for particular substrates or classes of substrates. Analyte sensors using both single enzymes and enzymatic systems comprising multiple enzymes acting cooperatively can be used for this purpose. As used herein, the term "cooperatively" and grammatical variations thereof means a coupled enzymatic reaction in which the product of a first enzymatic reaction becomes a substrate for a second enzymatic reaction, the second enzymatic reaction or subsequent enzymatic reactions serving as a basis for measuring the concentration of an analyte. Additionally, enzymes and/or combinations of enzyme systems can be used to detect more than one analyte type. Using an analyte sensor that features an enzyme or enzyme system to facilitate detection may be particularly advantageous to avoid frequent draws of bodily fluids that may otherwise be required for analyte monitoring.

In vivo analyte sensors monitor one or more analytes in a biological fluid of interest, such as dermal fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, or amniotic fluid. Such fluids may contain one or more interfering substances that can react either directly on the working electrode of the analyte sensor and the working electrode (e.g., carbon working electrode) itself or with one or more sensing chemistries disposed on the electrodes. As used herein, the term "interfering agent" and grammatical variations thereof means any electroactive species present that is not an analyte of interest (e.g., an in vivo electroactive species that is not an analyte of interest). Examples include, but are not limited to, ascorbic acid (vitamin C, also called ascorbate), glutathione, uric acid, paracetamol (acetaminophen), isoniazid, salicylates, etc., and any combination thereof. The reaction of these interfering substances with the working electrode can generate electrochemical signals that are inseparable or cannot be easily separated from the signal emanating from the analyte of interest, which can complicate the accurate detection of such analytes, especially those in small amounts (e.g., low to sub-millimolar concentrations). Electrochemical signals generated by interferents can be particularly problematic when the magnitude of the signal from the interferent approaches the signal from the target analyte. This may occur, for example, when the concentration of the interfering substance approaches or exceeds the concentration of the analyte of interest. Some interfering substances are ubiquitous in the body and cannot be easily avoided. Therefore, techniques that minimize the effects of these interfering substances during in vivo analysis would be highly desirable.

The present disclosure provides analyte sensor enhancements that, either alone or in combination, can improve detection sensitivity for both single analytes and multiple analytes combined with each other, as described in more detail below. That is, the present disclosure provides analyte sensors with small carbon working electrode areas that can provide reduced background signals resulting from in vivo interferents. While certain aspects of this disclosure relate to strengthening carbon working electrodes, it is to be appreciated that other types of electrodes can be similarly strengthened according to the disclosure herein. Electrode types that can be enhanced through use of the disclosure herein also include gold, platinum, PEDOT, and the like.

Generally and without limitation, embodiments of the present disclosure comprising one or more interferent enhancements, as detailed herein, range from at least greater than about 20%, which can be up to 100% compared to sensors lacking these enhancements, or such as from about 20% to about 70% or more, preferably at least about 40% greater, at least about 45% greater, or at least about 50%, including any values and subsets therebetween. and can allow reduction of interferent signals, such as ascorbic acid interferent signals, with separable upper and lower limits. The amount of interferent reduction may depend on a number of 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 interferents in the body fluid, etc. and any combination thereof.

Before describing the presently disclosed analyte sensors and enhancements thereof in more detail, first a brief overview of suitable in-vivo analyte sensor configurations and sensor systems using these analyte sensors will be provided so that embodiments of the present disclosure may be better understood. FIG. 1 illustrates a diagram of an exemplary sensing system that can incorporate analyte sensors of the present disclosure. As shown, the sensing system 100 includes a sensor control device 102 and a reader device 120 configured to communicate with each other over a local communication path or link 140, which can be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. According to some embodiments, reader device 120 may include an output medium for viewing analyte concentrations and alerts or notifications determined by sensor 104 or a processor associated therewith, and may also allow for one or more user inputs. Reader device 120 can be a multi-purpose smart phone or a dedicated electronic reading meter. Although only one reader device 120 is shown, multiple reader devices 120 may be present in certain cases.

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

Any suitable electronic communication protocol, such as Near Field Communication (NFC) protocol, Radio Frequency Identification (RFID) protocol, BLUETOOTH® protocol or BLUETOOTH® Low Energy protocol, or WiFi, etc., can be used for each of the communication paths or links. According to some embodiments, remote terminal 170 and/or trusted computer system 180 may be accessible by individuals other than the primary user who are interested in the user's analyte level. Reader device 120 may comprise display 122 and optional input component 121 . According to some embodiments, display 122 may comprise a touch screen interface.

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

Sensor 104 is adapted to be inserted at least partially into a tissue of interest, such as within the dermal or subcutaneous layer of skin. Alternatively, sensor 104 may be adapted to penetrate the epidermis. As yet another alternative, sensor 104 may be placed on a surface and not penetrate tissue, such as when assaying one or more analytes in perspiration on the skin. Sensor 104 may include a sensor tail of sufficient length for insertion to a desired depth within a given tissue. The sensor tail can comprise at least one working electrode adapted to assay one or more analytes of interest and an active area comprising an enzyme or enzyme system.

A counter electrode can be present in combination with at least one working electrode, optionally in further combination with a reference electrode. Specific electrode configurations on the sensor tail are described in more detail below with reference to FIGS. 2A-4. According to various embodiments, one or more enzymes within the active area can be covalently attached to the polymer comprising the active area. Alternatively, the enzyme can be non-covalently associated within the active area, such as by encapsulation or physical entrainment. One or more analytes can be monitored in biological fluids 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 analyte sensors of the present disclosure can be adapted to analyze dermal or interstitial fluids to determine analyte concentrations in vivo. However, it is to be appreciated that the entire sensor control device 102 may have one or more of a variety of configurations that allow for complete implantation under tissue and within one or more bodily fluids for assaying one or more analytes of interest without departing from the scope of the present disclosure.

Referring again to FIG. 1, sensor 104 can automatically communicate data to reader device 120 . For example, analyte concentration data can be communicated automatically and periodically, such as by the BLUETOOTH protocol or the BLUETOOTH low energy protocol or the like, at a predetermined frequency (e.g., every minute, every 5 minutes, or other predetermined period of time) when the data is obtained or after a predetermined period of time the data is stored in memory prior to transmission. Data regarding different analytes may be communicated at the same or different frequencies and/or using the same or different communication protocols. In other embodiments, sensor 104 may communicate with reader device 120 in a non-automatic 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 brought within communication range of the reader device 120 . The data can remain stored in the memory of sensor 104 until communicated to reader device 120 . Thus, the user need not maintain close proximity to the reader device 120 all the time, but instead can automatically or non-automatically upload data at a convenient time. In still other embodiments, a combination of automatic and non-automatic data transfer can be implemented. For example, data transmission can continue on an automatic basis until reader device 120 is no longer within communication range of sensor 104 .

A temporary introducer may be present to facilitate introduction of the sensor 104 into the tissue. In an exemplary embodiment, the introducer can include a needle or similar point or combination thereof. It should be appreciated that in alternate embodiments, there may be other types of introducers such as sheaths or blades. More specifically, a needle or other introducer can be temporarily near the sensor 104 prior to tissue insertion and then withdrawn. While present, these needles or other introducers can facilitate insertion of sensor 104 into tissue by opening an access path for sensor 104 to follow. For example, according to one or more embodiments, a needle can facilitate epidermal penetration as an access pathway down to the dermis to allow implantation of the sensor 104 . After opening the access path, the needle or other introducer can be withdrawn so that it does not present a cusp. In exemplary embodiments, suitable needles may be solid or hollow, beveled or non-beveled, and/or circular or non-circular in cross-section. In a more specific embodiment, suitable needles can be comparable in cross-sectional diameter and/or tip design to acupuncture needles, which can have a cross-sectional diameter of about 250 microns. However, it should be appreciated that suitable needles may have larger or smaller cross-sectional diameters as desired for a particular application. For example, needles with cross-sectional diameters ranging from about 300 microns to about 400 microns can be used.

In some embodiments, the tip of the needle can be angled above the end of sensor 104 (while it is present) so that the needle first penetrates the tissue and opens an access path for sensor 104. In other exemplary embodiments, sensor 104 can reside within a lumen or groove of a needle, which also opens an access path for sensor 104 . In either case, the needle can be withdrawn after facilitating sensor insertion.

Sensor configurations featuring a single active area configured for detection of a corresponding single analyte can employ two-electrode or three-electrode detection motifs, as further described herein with reference to FIGS. 2A-2C. Subsequently, sensor configurations featuring two different active areas suitable for the detection of separate analytes, either on separate working electrodes or on the same working electrode, are described separately with reference to FIGS. 3A-4. A sensor configuration with multiple working electrodes can be particularly advantageous for incorporating two different active areas within the same sensor tail, as the signal contribution from each active area can be more easily determined by separate evaluation of each working electrode. Each active area can be protected by a mass transport limiting membrane of the same or different composition.

When there is a single working electrode in the analyte sensor, a three electrode sensor configuration can comprise a working electrode, a counter electrode and a reference electrode. A related two-electrode sensor configuration can comprise a working electrode and a second electrode, where 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 "layered", at least partially stacked on top of each other on the sensor tail, and/or laterally spaced from each other. In any of the sensor configurations disclosed herein, the various electrodes can be electrically isolated from each other by dielectric materials or similar insulators.

Analyte sensors featuring multiple working electrodes can also include at least one additional electrode. When one additional electrode is present, this electrode can serve as a counter/reference electrode for each of the multiple working electrodes. When two additional electrodes are present, one of the additional electrodes can serve as a counter electrode for each of the multiple working electrodes and the other of the additional electrodes can serve as a reference electrode for each of the multiple working electrodes.

Any of the working electrode configurations described below can benefit from further disclosure below regarding reducing the area of the working electrode on the sensor tail.

FIG. 2A illustrates a diagram of an exemplary two-electrode analyte sensor configuration suitable for use in the present disclosure. As shown, analyte sensor 200 includes substrate 212 disposed between working electrode 214 and counter/reference electrode 216 . Alternatively, working electrode 214 and counter/reference electrode 216 can be positioned on the same side of substrate 212 with a dielectric material sandwiched between them (configuration not shown). Active area 218 is disposed as at least one layer over at least a portion of working electrode 214 . The active area 218 can comprise multiple discrete spots or a single continuous spot adapted for detection of analytes, as discussed further herein.

With continued reference to FIG. 2A, according to some embodiments, membrane 220 can protect at least active area 218 and optionally some or all of working electrode 214 and/or counter/reference electrode 216 or the entire analyte sensor 200. One or both sides of analyte sensor 200 can be protected by membrane 220 . Membrane 220 can comprise one or more polymeric membrane materials that function to restrict analyte flux to active area 218 (i.e., membrane 220 is a mass transport restricting membrane that is somewhat permeable to the analyte of interest). The composition and thickness of membrane 220 can be varied to facilitate the desired analyte flux into active area 218, thereby providing the desired strength and stability of the signal. Analyte sensor 200 may be operable to assay an analyte by any of coulometric, amperometric, voltmetric, or potentiometric electrochemical detection techniques.

2B and 2C illustrate diagrams of exemplary three-electrode analyte sensor configurations that are also suitable for use with the present disclosure. The three-electrode analyte sensor configuration can be similar to that shown for analyte sensor 200 in FIG. 2A except for the inclusion of additional electrodes 217 within analyte sensors 201 and 202 (FIGS. 2B and 2C). In this case, by using an additional electrode 217, the counter/reference electrode 216 can function as either a counter electrode or a reference electrode, and the additional electrode 217 fulfills other electrode functions not otherwise addressed. Working electrode 214 fulfills its original function. An additional electrode 217 can be placed over either the working electrode 214 or the electrode 216 with a separate layer of dielectric material therebetween. For example, as shown in FIG. 2B, dielectric material layers 219a, 219b, and 219c separate electrodes 214, 216 and 217 from each other and provide electrical isolation. Alternatively, at least one of electrodes 214, 216 and 217 can be positioned on opposite sides of substrate 212, as shown in FIG. 2C. Thus, in some embodiments, electrode 214 (the working electrode) and electrode 216 (the counter electrode) can be positioned on opposite sides of substrate 212, with electrode 217 (reference electrode) positioned above one of electrodes 214 or 216 and spaced from these electrodes by a dielectric material. A reference material layer 230 (eg, Ag/AgCl) can be present over the electrode 217, and the location of the reference material layer 230 is not limited to that shown in FIGS. 2B and 2C. As with sensor 200 shown in FIG. 2A, active area 218 in analyte sensors 201 and 202 can comprise multiple spots or a single spot. In addition, analyte sensors 201 and 202 may also be operable to assay analytes by any of coulometric, amperometric, voltmetric, or potentiometric electrochemical detection techniques.

Similar to analyte sensor 200, membrane 220 can protect active area 218 in analyte sensors 201 and 202, as well as other sensor components, thereby functioning as a mass transport limiting membrane. In some embodiments, additional electrode 217 may be protected by membrane 220 . Film 220 can again be produced by dip coating or in situ photopolymerization, and can be different or the same in composition at various locations. 2B and 2C show all of electrodes 214, 216 and 217 as being protected by membrane 220, it should be appreciated that in some embodiments only working electrode 214 or active area 218 may be protected. Additionally, the thickness of membrane 220 at each of electrodes 214, 216 and 217 may be the same or different. As with the two-electrode analyte sensor configuration (FIG. 2A), in the sensor configuration of FIGS. 2B and 2C one or both sides of analyte sensors 201 and 202 can be protected by membrane 220, or the entirety of analyte sensors 201 and 202 can be protected. Accordingly, the three-electrode sensor configurations shown in FIGS. 2B and 2C should be understood as not limiting the embodiments disclosed herein, and alternative electrode configurations and/or layer configurations remain within the present disclosure.

FIG. 3A illustrates an exemplary configuration for sensor 203 having a single working electrode with two different active areas disposed thereon. FIG. 3A is similar to FIG. 2A except for the presence of two active areas on the working electrode 214, a first active area 218a and a second active area 218b, responsive to different analytes and laterally spaced from each other on the surface of the working electrode 214. Active areas 218a and 218b may comprise multiple spots or a single spot adapted for detection of each analyte. The composition of membrane 220 can be different or the same in active areas 218a and 218b. As discussed further below, the first active area 218a and the second active area 218b can be configured to detect their corresponding analytes at different working electrode potentials.

3B and 3C illustrate cross-sectional views of an exemplary three-electrode sensor configuration for sensor 204 and an exemplary three-electrode sensor configuration for 205, respectively, each featuring a single working electrode with first active area 218a and second active area 218b disposed thereon. Figures 3B and 3C are otherwise similar to Figures 2B and 2C and can be better understood by reference to those figures. As in FIG. 3A, the composition of membrane 220 can be different or the same in active areas 218a and 218b.

FIG. 4 illustrates a cross-sectional view of an exemplary analyte sensor configuration having two working, reference and counter electrodes suitable for use with the disclosure herein. As shown, analyte sensor 400 includes working electrodes 404 and 406 disposed on opposite sides of substrate 402 . A first active area 410 a is located on the side of working electrode 404 and a second active area 410 b is located on the side of working electrode 406 . Counter electrode 420 is electrically isolated from working electrode 404 by dielectric material layer 422 and reference electrode 431 is electrically isolated from working electrode 406 by dielectric material layer 423 . Outer dielectric material layers 430 and 432 are disposed over reference electrode 431 and counter electrode 420, respectively. According to various embodiments, the membrane 440 can protect at least the active areas 410a and 410b, and optionally other components of the analyte sensor 400 or the entire analyte sensor 400 are protected by the membrane 440 as well. Again, membrane 440 may differ in composition in active areas 410a and 410b as desired to provide permeability values suitable for differentially modulating analyte flux at each location.

Alternative sensor configurations having multiple working electrodes and differing from the configuration shown in FIG. 4 can feature counter/reference electrodes instead of separate counter electrodes 420 and reference electrodes 431, and/or feature layer arrangements and/or membrane arrangements different than those specified. For example, the placement of counter electrode 420 and the placement of reference electrode 431 can be reversed from that shown in FIG. Furthermore, working electrodes 404 and 406 need not necessarily reside on opposite sides of substrate 402 in the manner shown in FIG.

A carbon working electrode can suitably constitute the working electrode in any of the analyte sensors disclosed herein. Carbon working electrodes are very commonly used in electrochemical sensing, but their use in electrochemical sensing is not without difficulties. In particular, a current for the analyte of interest is produced only when the active area interacts with the analyte and transfers electrons to the portion of the carbon working electrode adjacent to the active area. Body fluids containing the analyte of interest interact similarly with the carbon faces of the carbon working electrode that are not protected by the active area, but do not contribute to the analyte signal as there are no enzymes or enzymatic systems at these locations to facilitate electron transfer from the analyte to the working electrode. However, interferents can undergo oxidation in portions of the working electrode lacking active area and contribute background to the overall signal. Thus, a carbon working electrode with external (or "exposed") carbon areas on the electrode surface does not contribute significantly to the analyte signal, and in some cases may result in a background signal contribution. Other electrodes with extra surface areas that do not directly detect the analyte of interest may experience similar background signals and can be enhanced by the modifications disclosed herein.

A variety of interfering substances can interact with the working electrodes of the analyte sensors described herein, but ascorbic acid is one example of an interfering substance commonly present in biological fluids that can generate a background signal at the carbon working electrode. For example, ascorbic acid is oxidized at the working electrode to produce dehydroascorbic acid. Various embodiments of the present disclosure are described herein below with respect to an interferent that is ascorbic acid. However, it should be appreciated that the embodiments and analyte sensor configurations described herein are equally applicable to other interfering substances (electroactive species present in body fluids with analytes of interest).

As provided above, the active area described herein can be a single sensing layer having multiple sensing spots or a sensing layer having multiple sensing spots compressed together and thus substantially corresponding to a single sensing layer. Referring now to FIG. 5, illustrated is a top view of a conventional carbon working electrode 500 having an active area 504 containing a plurality of sensing spots 518 disposed thereon. When an analyte interacts with active area 504, only the portion of carbon working electrode 500 that includes sensing spot 518 contributes to the signal for the analyte of interest. Although the carbon working electrode 500 exhibits six sensing spots 518 within the active area 504, it should be appreciated that fewer or more than six sensing spots 518 can be included on the carbon working electrode 500 without departing from the scope of the present disclosure. The outer carbon area 510 is not directly overlaid by the sensing spot 518, and while this area does not contribute to the signal for the analyte, it may generate background signal for one or more interferents. Therefore, the oxidation of interferents at the carbon working electrode 500 is proportional to the area of the external carbon zone 510 that may be used to interact with the interferents. In fact, the oxidation of ascorbic acid at the carbon working electrode 500 scales approximately linearly with the area of the external carbon zone 510 that may be used.

As shown, the active area 504 is discontinuous and in the form of multiple sensing spots 518 . As defined herein, the term "discontinuous" and grammatical variations thereof means that no single spot (sensing element) shares an edge or boundary with (eg, touches) adjacent spots.

The present disclosure reveals how the external carbon area 510 within the carbon working electrode 500 can be reduced to minimize or eliminate interfering signal, while still retaining the ability to generate a signal for the analyte of interest. In particular, to reduce the area of the outer carbon zone 510, the pitch and diameter of the discrete sensing spots 518 of the conventional carbon working electrode 500 can be reduced, as well as the configuration of the discrete sensing spots 518 relative to each other. As used herein, the term "grid" and grammatical variations thereof means a 2D arrangement of active areas along the length of the working electrode (along the axis of the sensor tail 104 (FIG. 1) extending from the sensor housing 103 into the bodily fluid) relative to the width of the working electrode.

For illustration of various lattice configurations, the active areas of the present disclosure can be in the formation of a 1×n lattice, where n is an integer greater than 1, e.g., in the range of 2 to about 20 or 2 to about 10, including any values and subsets therebetween, with upper and lower bounds in separable ranges. In some embodiments, for example, as shown in FIG. 5, the active area can comprise discrete sensing spots in the form of a 1×6 grid. Embodiments described herein can use other grid configurations of active areas, such as those shown in FIGS. 6A-6B, which can be most clearly understood with reference to FIG. 5, in which similar elements retain similar labeling. For example, in some embodiments, the active area can comprise discrete sensing spots in the form of a 2×n grid, where n is an integer from 2 to about 10 or from 2 to about 5, including any values and subsets therebetween, with separable upper and lower bounds. FIG. 6A depicts a carbon working electrode 600 with a 2×3 grid of sensing spots 518 and an outer carbon area 510. FIG. In still other embodiments, the active area may comprise discrete sensing spots in the form of a 3×n grid, where n is an integer from 2 to about 6 or from 2 to about 3, including any values and subsets therebetween, with separable upper and lower bounds. FIG. 6B depicts a carbon working electrode 610 with a 3×2 grid of sensing spots 518 and an outer carbon area 510 . 5, 6A, and 6B each show a variety of lattice configurations suitable for use in the embodiments described herein, each retaining the same area of outer carbon area 510, as the areas of carbon electrodes 500, 600 and 610, respectively, have not yet been reduced in these figures. As can be seen, the grid configurations of FIGS. 6A and 6B are arranged over a shorter longitudinal distance than the grid configuration of FIG. 5, thereby offering the potential to reduce the sensor area with the exposed working electrode.

Embodiments of the present disclosure utilize grating configuration, pitch distance, active area, and/or sensing spot size reduction, and active area location on the sensor tail to minimize the external carbon area and thereby minimize the signal for interferents, as shown in top views of carbon electrodes with various active area configurations. FIG. 7A represents a control (or conventional) 1×6 active area configuration similar to that shown in FIG. The outer carbon area in FIG. 7A is shaded as the working electrode surface absent active areas. Each of Figures 7B-7F was drawn with reference to Figure 7A and illustrates an embodiment of the present disclosure.

Each of FIGS. 7B-7F utilizes reducing the sensing spot pitch to reduce the external carbon area, and in some embodiments coalesce into each other so that each sensing spot is no longer discontinuous. In addition, FIG. 7B further illustrates a reduction in the pitch between adjacent sensing spots, which allows a reduction in the external carbon area (below the double line) shaded as the working electrode surface devoid of sensing spots. FIG. 7C shows the pitch reduction of FIG. 7B combined with displacement of the active area closer to the tip of the sensor tail, which combination allows for yet another reduction in the outer carbon area (below the double line) shaded as the working electrode surface absent sensing spots. FIG. 7D depicts yet another pitch reduction compared to FIG. 7C that allows for yet another reduction in external carbon area (below the double line) shaded as the working electrode surface absent sensing spots. FIG. 7E depicts the pitch reduction of FIG. 7D and the sensor tail displacement of FIG. 7C combined with a 2×3 active area grid configuration that allows for yet another reduction in external carbon area (below the double line) shaded as the active area absent working electrode surface. FIG. 7F depicts the pitch reduction of FIG. 7D and the sensor tail displacement of FIG. 7C combined with a 3×3 active area grid configuration, which allows for yet another reduction in external carbon area (below the double line) shaded as the working electrode surface absent sensing spots. Figures 7D-7F show that the sensing spots become less distinguishable as the pitch reduction width increases, and in some embodiments can be represented as a single active area lacking discrete sensing spots.

表１Ｂ

For illustrative purposes, Tables 1A and 1B illustrate percentage reductions in exposed (or extrinsic) carbon, including those compared based on FIGS. Interferants are measured in terms of signal strength based on tested analyte concentrations and known interferent concentrations.
Table 1A

Table 1B

As shown in Table 1, the interferent signal also decreases approximately linearly as the external carbon area decreases.

The present disclosure provides reduced area working electrodes (eg, carbon electrodes) with one or more active areas disposed thereon. In some embodiments, multiple discrete active areas are disposed on the working electrode. Generally, the discrete active areas of the present disclosure have widths (diameters) ranging from about 50 μm to about 500 μm, such as from about 50 μm to about 300 μm, including any values and subsets therebetween, with separable upper and lower limits. A non-round discontinuous active area (not shown) can have an area range equivalent to a circular feature having the width (diameter) range described above. The pitch between each discrete active area (the distance between adjacent active areas) can be from about 50 μm to about 800 μm, such as from about 50 μm to about 500 μm, including any values and subsets therebetween, with separable upper and lower limits. Generally, the most distal active area is located at least about 200 μm to 300 μm from the tip of the most distally positioned working electrode (which may be equivalent to the tip of the sensor tail) in the bodily fluid (measured from the center of sensing of the active area), but may range from about 50 μm to about 500 μm, including any values and subsets therebetween, where upper and lower limits may be separable ranges.

In total, a working electrode comprising an active area (whether a single active area or multiple discrete active areas) can have an area ranging from about 0.1 ^mm to about 5 ^mm , including any values and subsets therebetween, with separable upper and lower limits. In total, the external working electrode area (excluding any active area) can range from about 0.01 ^mm to about 3 ^mm , including any values and subsets therebetween, with separable upper and lower limits.

To achieve a small external working electrode area to reduce interferent signals while maintaining sensitivity to the analyte or analytes of interest, the ratio of the area of the external working electrode to the area of the active area can range from about 1:10 to about 10:1, including any values and subsets therebetween, with separable upper and lower limits. This ratio is maintained regardless of the grating configuration or pitch distance of the analyte sensors described herein, i.e., the range of the ratio of the area of the external working electrode to the area of the active area is always within the range described above to achieve the desired benefits described herein.

Accordingly, an analyte sensor of the present disclosure comprises a working electrode having a sensing portion and an exposed electrode portion, the sensing portion comprising an active area having an analyte-reactive enzyme disposed thereon, the exposed electrode portion comprising no active area, and a ratio of exposed electrode portion to sensing portion ranging from about 1:10 to about 10:1. The working electrode can be a carbon electrode. At least the sensing portion can have a mass transport limiting membrane overcoated thereon.

Further, the method of the present disclosure can comprise exposing to a bodily fluid an analyte sensor comprising a working electrode having a sensing portion and an exposed electrode portion, the sensing portion comprising an active area having an analyte-reactive enzyme disposed thereon, the exposed electrode portion comprising no active area, and a ratio of exposed electrode portion to sensing portion ranging from about 1:10 to about 10:1. The working electrode can be a carbon electrode. At least the sensing portion can have a mass transport limiting membrane overcoated thereon.

An active area in any of the analyte sensors disclosed herein can comprise one or more analyte-reactive enzymes, either acting singly or acting cooperatively within an enzyme system. One or more enzymes can be covalently attached to the polymer comprising the active area, and also one or more electron transfer agents can be positioned within the active area.

Examples of suitable polymers within each active segment can include, for example, poly(4-vinylpyridine) and poly(N-vinylimidazole), or copolymers thereof, within which quaternized pyridine and imidazole groups serve as attachment points for electron transfer agents or enzymes. Other suitable polymers that can be present in the active zone are those described in U.S. Pat. No. 6,605,200, which is incorporated herein by reference in its entirety, such as poly(acrylic acid), styrene/maleic anhydride copolymers, methyl vinyl ether/maleic anhydride copolymers (GANTREZ polymers), poly(vinylbenzyl chloride), poly(allylamine), polylysine, poly(4-vinylpyridine) quaternized with carboxypentyl groups, and poly(sodium 4-styrenesulfonate).

Enzymes that are covalently attached to the polymer within the active area and that have the function of facilitating analyte detection are not considered to be specifically limited. Suitable enzymes can include those that have the ability to detect glucose, lactate, ketones, creatinine, or the like. Any of these analytes can be detected in combination with each other in an analyte sensor capable of detecting multiple analytes. Enzymes and enzyme systems suitable for detecting these analytes are described below.

In some embodiments, an analyte sensor can comprise a glucose-reactive active area comprising a glucose-reactive enzyme disposed on the sensor tail. Suitable glucose-responsive enzymes can comprise, for example, glucose oxidase or glucose dehydrogenase (e.g., pyrroloquinoline quinone (PQQ) or cofactor-dependent glucose dehydrogenase, such as 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, whereas glucose dehydrogenase transfers electrons to natural or artificial electron acceptors, e.g., enzyme cofactors. Glucose oxidase or glucose dehydrogenase can be used to facilitate detection. Both glucose oxidase and glucose dehydrogenase can be covalently attached to polymers with glucose-responsive active sites, and both can exchange electrons with electron transfer agents (e.g., osmium (Os) complexes or similar transition metal complexes), which can also be covalently attached to the polymer. Suitable electron transfer agents are described in more detail below. Glucose oxidase can directly exchange electrons with an electron transfer agent, whereas glucose dehydrogenase can utilize a cofactor to facilitate electron exchange with an electron transfer agent. FAD cofactors can directly exchange electrons with electron transfer agents. In contrast, a NAD cofactor can utilize diaphorase to facilitate electron transfer from the cofactor to an electron transfer agent. Details regarding glucose-responsive active regions incorporating glucose oxidase or glucose dehydrogenase, as well as glucose detection using same, can be found, for example, in commonly owned U.S. Pat. No. 8,268,143, which is incorporated herein by reference in its entirety.

In some embodiments, active areas of the present disclosure can be configured suitable for detecting ketones. Additional details regarding enzymatic systems responsive to ketones can be found in "Analyte Sensors and Sensing Methods Featuring Dual Detection of Glucose and Ketones", which is incorporated herein by reference in its entirety, filed Jan. 28, 2020 and published as U.S. Patent Application Publication No. 2020/0237275. No. 16/774,835, owned by the present applicant, entitled "section of Glucose and Ketones". In such systems, β-hydroxybutyrate serves as a surrogate for ketones formed in vivo and undergoes reaction with an enzymatic system containing β-hydroxybutyrate dehydrogenase (HBDH) and diaphorase that facilitates detection of ketones within ketone-reactive active areas disposed on the surface of at least one working electrode, as further described herein. Within the ketone-reactive active zone, β-hydroxybutyrate dehydrogenase can convert β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) to acetoacetate and reduced nicotinamide adenine dinucleotide (NADH), respectively. The term "nicotinamide adenine dinucleotide (NAD)" shall be recognized to include phosphate-linked forms of the enzyme cofactors described above. Thus, the use of the term "NAD" herein means both NAD ⁺ phosphate and NADH phosphate, particularly diphosphates that link two nucleotides, one containing an adenine nucleobase and the other a nicotinamide nucleobase. NAD ⁺ /NADH enzymatic cofactors help facilitate the cooperative enzymatic reactions disclosed herein. Once formed, NADH can undergo diaphorase-mediated oxidation in which electrons are transferred to provide the basis for ketone detection at the working electrode. That is, there is a 1:1 molar correspondence between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted. Electron transfer to the working electrode can occur under the further mediated of electron transfer agents such as osmium (Os) compounds or similar transition metal complexes, as described in more detail below. Albumin may also be present as a stabilizer within the active zone. The dehydrogenase and diaphorase of β-hydroxybutyrate can be covalently attached to polymers with ketone-reactive active sites. NAD ⁺ may or may not be covalently attached to the polymer, but if not covalently attached it can be physically retained within the ketone-reactive active area, e.g., a mass transport limiting membrane that is also permeable to ketones protects the ketone-reactive active area.

Other suitable chemistries for enzymatically detecting ketones can be utilized in accordance with embodiments of the present disclosure. For example, β-hydroxybutyrate dehydrogenase (HBDH) can again convert β-hydroxybutyrate and NAD ⁺ to acetoacetate and NADH, respectively. Instead of completing electron transfer to the working electrode by a diaphorase and a suitable redox mediator, the reduced form of NADH oxidase (NADHOx(Red)) undergoes reaction to form the corresponding oxidized form (NADHOx(Ox)). NADHOx (Red) can then be reformed by reaction with molecular oxygen to produce superoxide, which can undergo subsequent conversion to hydrogen peroxide mediated by superoxide dismutase (SOD). The hydrogen peroxide can then undergo oxidation at the working electrode to provide a signal that can be correlated to the amount of ketones originally present. According to various embodiments, the SOD can be covalently attached to the polymer within the ketone-reactive active area. β-Hydroxybutyrate dehydrogenase and NADH oxidase can be covalently attached to the polymer within the ketone-reactive active area, and NAD ⁺ may or may not be covalently attached to the polymer within the ketone-reactive active area. NAD ⁺ can be physically retained within the ketone-reactive active area when not covalently bound, and membrane polymers improve retention of NAD ⁺ within the ketone-reactive active area. 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.

Another enzymatic detection chemistry for ketones can utilize β-hydroxybutyrate dehydrogenase (HBDH) to convert β-hydroxybutyrate and NAD ⁺ to acetoacetate and NADH, respectively. The electron transfer cycle in this case is completed by oxidation of NADH by 1,10-phenanthroline-5,6-dione reforming NAD ⁺ , which then transfers electrons to the working electrode. The 1,10-phenanthroline-5,6-dione may or may not be covalently attached to the polymer within the ketone-reactive active area. β-hydroxybutyrate dehydrogenase can be covalently attached to the polymer within the ketone-reactive active area, and NAD ⁺ may or may not be covalently attached to the polymer within the ketone-reactive active area. Inclusion of albumin within the active area can provide a surprising improvement in response stability. Appropriate membrane polymers can improve retention of NAD ⁺ within the ketone-reactive active area. 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.

In some embodiments, the analyte sensor can further comprise a creatinine-reactive active zone comprising an enzymatic system working cooperatively to facilitate detection of creatinine. Creatinine can be reversibly and hydrolytically reacted to form creatine in the presence of creatinine amidohydrolase (CNH0029). Additionally, creatine can undergo catalytic hydrolysis to form sarcosine in the presence of creatine amidohydrolase (CRH). None of these reactions produce an electron flow (eg, oxidation or reduction) and provide the basis for electrochemical detection of creatinine. Sarcosine produced by hydrolysis of creatine can undergo oxidation in the presence of the oxidized form of sarcosine oxidase (SOX-ox) to form glycine and formaldehyde, thereby generating the reduced form of sarcosine oxidase (SOX-red) in this process. Hydrogen peroxide can also be generated in the presence of oxygen. The reduced sarcosine oxidase can then undergo reoxidation in the presence of an oxidized electron transfer agent (e.g., an Os(III) complex) to produce the corresponding reduced electron transfer agent (e.g., an Os(II) complex) and deliver an electron stream to the working electrode.

Oxygen may interfere with the cooperative reaction sequence used to detect creatinine according to the disclosure above. Specifically, the reduced form of sarcosine oxidase undergoes reaction with oxygen to reform the corresponding oxidized form of the enzyme, but can be reformed without exchanging electrons with an electron transfer agent. When the reaction with oxygen occurs, the enzyme remains all active, but electrons do not flow to the working electrode. Without being bound by theory or mechanism, it is believed that kinetic effects result in a competing reaction with oxygen. Thus, oxidation of the reduced form of sarcosine oxidase by oxygen is thought to occur faster than oxidation facilitated by electron transfer agents. In the presence of oxygen, hydrogen peroxide is also formed.

A desirable reaction pathway to facilitate detection of creatinine can be facilitated by including oxygen scavengers around the enzymatic system. A variety of oxygen scavengers and their placement with oxidase enzymes such as glucose oxidase may be suitable. Small molecule oxygen scavengers may be suitable, but these oxygen scavengers may be completely consumed before the sensor life is otherwise completely exhausted. Enzymes, in contrast, can undergo reversible oxidation and reduction, thereby providing longer sensor lifetimes. By quenching the oxidation of the reduced form of sarcosine oxidase by oxygen, a slow electron exchange reaction with the electron transfer agent can occur, thereby allowing current generation at the working electrode. The intensity of the current produced is proportional to the amount of creatinine initially reacted.

In any embodiment of the disclosure, the oxygen scavenger used to facilitate the desired reaction can be an oxidase enzyme. Any oxidase enzyme can be used to facilitate deoxygenation around the enzymatic system provided there is also a suitable substrate for the enzyme, thereby providing a reagent to react with oxygen in the presence of the oxidase enzyme. In the present disclosure, oxidase enzymes that may be suitable for deoxygenation include, but are not limited to, glucose oxidase, lactate oxidase, xanthine oxidase, and the like. Glucose oxidase can be a particularly desirable oxidase enzyme for promoting deoxygenation due to the ready availability of glucose in various bodily fluids. Reaction 1 below illustrates an enzymatic reaction facilitated by glucose oxidase to provide oxygen removal.
β-D-glucose + O ₂ --> D-glucono-1,5-lactone + H ₂ O ₂
Reaction 1
The concentration of bioavailable lactate is lower than that of glucose, but still sufficient to facilitate deoxygenation.

An oxidase enzyme, such as glucose oxidase, can be placed in any suitable location to facilitate deoxygenation in the analyte sensors disclosed herein. Glucose oxidase, for example, can be placed on the sensor tail to be functional and/or non-functional to facilitate glucose detection. When not functioning well to facilitate glucose detection, glucose oxidase can be positioned on the sensor tail such that electrons produced during glucose oxidation are unable to reach the working electrode, such as by electrically isolating glucose oxidase from the working electrode.

Additional details regarding enzymatic systems responsive to creatinine can be found in Analyte Sensors and Sensing Methods for Detecting Creatinine, which is incorporated herein by reference in its entirety, filed September 25, 2019 and published as U.S. Patent Application Publication No. 2020/0241015. e)”, commonly owned by the present applicant, US patent application Ser. No. 16/582,583.

In some embodiments, an analyte sensor can comprise a lactate-reactive active area comprising a lactate-reactive enzyme disposed on the sensor tail. A suitable lactate-reactive enzyme can comprise, for example, lactate oxidase. A lactate oxidase or other lactate-reactive enzyme can be covalently attached to a polymer with a lactate-reactive active site and can exchange electrons with an electron transfer agent (e.g., an osmium (Os) complex or similar transition metal complex), which can also be covalently attached to the polymer. Suitable electron transfer agents are described in more detail below. Albumin, such as human serum albumin, can be present in the lactate-reactive active area to stabilize sensor reactivity, as described in more detail in commonly owned U.S. Patent Application Publication No. 2019/0320947, which is incorporated herein by reference in its entirety. Lactate levels can 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.

In some embodiments, an analyte sensor can comprise an active area that is responsive to pH. Suitable analyte sensors adapted to determine pH are described in U.S. Patent Application Publication No. 2020/0060592, owned by the assignee of the present invention and incorporated herein by reference in its entirety. Such an analyte sensor may comprise a sensor tail comprising a first working electrode and a second working electrode, a first active area positioned on the first working electrode comprising a substrate having pH-dependent redox chemistry and a second active area positioned on the second working electrode comprising a substrate having redox chemistry that does not substantially vary with pH. By obtaining the difference between the first signal and the second signal, this difference can be correlated with the pH of the fluid to which the analyte sensor is exposed.

Two different types of active areas can be positioned and spaced apart on a single working electrode, such as the carbon working electrode discussed above. Each active area can have a redox potential, wherein the redox potential of the first active area is sufficiently separated from the redox potential of the second active area to allow independent generation of a signal from one of the active areas. As non-limiting examples, these redox potentials can differ by at least about 100 mV, by at least about 150 mV, or by at least about 200 mV. The upper limit of the separation between these redox potentials depends on the in vivo electrochemical window of action. By having the redox potentials of the two active areas sufficiently separated from each other in intensity, an electrochemical reaction can occur in one of the two active areas (i.e., in the first active area or the second active area) without substantially inducing an electrochemical reaction in the other active area. Thus, a signal from one of the first active area or the second active area can be independently generated at its corresponding redox potential (lower redox potential) or at a potential greater than but less than the redox potential of the other active area. Different signals can allow the signal contribution from each analyte to be resolved.

Some or all embodiments of the analyte sensors disclosed herein can feature one or more active areas positioned on the surface of at least one working electrode to detect the same or different analytes. The membrane can protect at least the active area (comprising the analyte-reactive enzyme) and can protect all of the working electrode or portions thereof lacking the active area (exposed or external portions of the working electrode). The membrane can be a mass transport limiting membrane and can be a single layer of membrane, a bilayer of two different membrane polymers, or a hybrid of two different membrane polymers.

An electron transfer agent can be present in any of the active areas disclosed herein. Suitable electron transfer agents are capable of facilitating the transport of electrons to adjacent working electrodes after one or more analytes undergo enzymatic redox reactions within the corresponding active area, thereby generating an electron current indicative of the presence of a particular analyte. The amount of current generated is proportional to the amount of analyte present. Electron transfer agents responsive to different analytes within the active area may be the same or different depending on the sensor configuration used. For example, when two different active areas are placed on the same working electrode, the electron transfer agents in each active area may be different (e.g., chemically different such that the electron transfer agents exhibit different redox potentials). When multiple working electrodes are present, the electron transfer agent within each active area can be the same or different, as each working electrode can be evaluated separately.

Suitable electron transfer agents can comprise electroreducible and electrooxidizable ions, complexes, or molecules (e.g., quinones) that have redox potentials several hundred millivolts above or below the standard calomel electrode (SCE) redox potential. Suitable electron transfer agents, according to some embodiments, can comprise low potential osmium complexes such as those described in U.S. Pat. Additional examples of suitable electron transfer agents include those described in U.S. Pat. Nos. 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are fully incorporated herein by reference. Other suitable electron transfer agents may comprise, for example, metal compounds or metal complexes of ruthenium, osmium, iron (eg, polyvinylferrocene or hexacyanoferrate), or cobalt, including these metallocene compounds. For example, suitable ligands for metal complexes can comprise bidentate or higher dentate ligands such as, for example, bipyridine, biimidazole, phenanthroline, or pyridyl (imidazole). Other suitable bidentate ligands can comprise, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoareines. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher dentate ligands can be present in the metal complex to achieve the largest coordination sphere.

Active areas suitable for detecting any of the analytes disclosed herein can comprise a polymer to which an electron transfer agent is covalently attached. Any of the electron transfer agents disclosed herein can be provided with suitable functional groups to facilitate covalent bonding to the polymer within the active area. Suitable examples of polymer-bound electron transfer agents can include those described in U.S. Pat. Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are fully incorporated herein by reference. Polymers suitable for inclusion within the active zone may comprise, but are not limited to, polyvinylpyridine (eg, poly(4-vinylpyridine)), polyvinylimidazole (eg, poly(1-vinylimidazole)), or copolymers of any of these. Exemplary copolymers that may be suitable for inclusion within the active area include, for example, those containing monomeric units such as styrene, acrylamide, methacrylamide, or acrylonitrile. When there are two or more different active areas, the polymer within each active area may be the same or different.

Covalent attachment of the electron transfer agent to the polymer within the active area can occur by polymerizing monomeric units bearing the covalently attached electron transfer agent, or the electron transfer agent can be separately reacted with the polymer after the polymer has been presynthesized. The bifunctional spacer can covalently bond the electron transfer agent to the polymer within the active area, where a first functional group is reactive with the polymer (e.g., a functional group capable of quaternizing a pyridine nitrogen atom or an imidazole nitrogen atom) and a second functional group is reactive with the electron transfer agent (e.g., a functional group reactive with a ligand that coordinates a metal ion).

Similarly, one or more of the enzymes within the active area can be covalently attached to the polymer comprising the active area. When there is an enzymatic system with multiple enzymes within a given active area, in some embodiments all of the multiple enzymes can be covalently attached to the polymer, and in other embodiments only a portion of the multiple enzymes can be covalently attached to the polymer. For example, one or more enzymes comprising an enzymatic system can be covalently attached to a polymer, and at least one enzyme can be non-covalently associated with the polymer such that the non-covalent enzyme is physically entrained within the polymer. Covalent attachment of the enzyme to the polymer within a given active area can occur through crosslinkers introduced using a suitable crosslinker. Cross-linkers suitable for reaction with free amino groups in enzymes (e.g. with free side chain amines in lysine) can comprise cross-linkers such as, for example, polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derived variants thereof. Cross-linking agents suitable for reaction with free carboxylic acid groups present in enzymes can include, for example, carbodiimides. Cross-linking of enzymes to polymers is generally intermolecular, but can be intramolecular in some embodiments. In certain embodiments, all of the enzymes within a given active area can be covalently attached to the polymer.

The electron transfer agent and/or enzyme can be associated with the polymer within the active area by means other than covalent bonding. In some embodiments, the electron transfer agent and/or enzyme can be ionically or coordinatively associated with the polymer. For example, a charged polymer can be ionically associated with an oppositely charged electron transfer agent or enzyme. In still other embodiments, the electron transfer agent and/or enzyme can be physically entrained within the polymer without being attached to it. Physically entrained electron transfer agents and/or enzymes can still interact adequately with the fluid to facilitate detection of the analyte without substantially leaching out of the active area.

The polymer within the active area can be selected to limit out-diffusion of NAD ⁺ or another cofactor not covalently attached to it. Limiting cofactor outdiffusion can increase sensor lifetime to some extent (days to weeks) while still allowing sufficient internal analyte diffusion to facilitate detection.

In some embodiments, stabilizers can be incorporated within the active areas of the analytes described herein to improve sensor functionality and achieve desired sensitivity and stability. Such stabilizers can comprise, for example, antioxidants and/or enzymatically stabilizing entraining proteins. Examples of suitable stabilizers can include, but are not limited to, serum albumin (e.g., human or bovine serum albumin, or other compatible albumin), catalase, other enzymatic antioxidants, and the like, and any combination thereof. Stabilizers can be conjugated or non-conjugated.

In certain embodiments of the present disclosure, a mass transport restricting membrane protecting one or more active areas can comprise a homopolymer or copolymer of crosslinked polyvinylpyridine. The composition of the mass transport limiting membrane may be the same or different when the membrane protects different types of active areas. When the membrane composition differs at two different locations, the membrane can comprise a bilayer membrane or a homogenous hybrid of two different membrane polymers, one of which can be a homopolymer or copolymer of crosslinked polyvinylpyridine or crosslinked polyvinylimidazole. Suitable techniques for depositing the mass transport limiting film on the active areas can include, for example, spray coating, painting, inkjet printing, screen printing, imprinting, roller coating, dip coating, the like, and combinations thereof. Dip coating techniques can be particularly desirable for polyvinylpyridine and polyvinylimidazole polymers and copolymers.

In certain embodiments, the mass transport limiting membranes discussed above are membranes composed of crosslinked polymers containing heterocyclic nitrogen groups, such as polymers of polyvinylpyridine and polyvinylimidazole. Embodiments also include membranes made of polyurethane, polyetherurethane, or materials chemically related thereto, or made of silicone, or the like.

In some embodiments, membranes can be formed in buffered solutions (e.g., alcoholic buffered solutions) by in situ crosslinking of polymers, including those discussed above, modified with zwitterionic moieties, non-pyridine copolymer components, and optionally another moiety that is either hydrophilic or hydrophobic and/or has other desirable properties. Modified polymers can be prepared from precursor polymers containing heterocyclic nitrogen groups. For example, the precursor polymer can be polyvinylpyridine or polyvinylimidazole. Optionally, hydrophilic or hydrophobic modifiers can be used to "fine tune" the permeability of the resulting membrane to the analyte of interest. Optional hydrophilic modifiers such as poly(ethylene glycol) modifiers, hydroxyl modifiers, or polyhydroxyl modifiers, and similar modifiers, and any combination thereof, can be used to enhance the biocompatibility of the polymer or resulting membrane.

In some embodiments, the membrane comprises poly(styrene-co-maleic anhydride), dodecylamine, and poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propylene glycol) (2-aminopropyl ether). Compounds including, but not limited to, those crosslinked with pyridine derivatives, polyvinylimidazole, polyvinylimidazole derivatives, and the like, and any combination thereof, can be provided. In some embodiments, the membrane can comprise a polyvinylpyridine-co-styrene polymer in which a portion of the pyridine nitrogen atoms are functionalized with non-crosslinked poly(ethylene glycol) tails and a portion of the pyridine nitrogen atoms are functionalized with alkylsulfonic acid groups. Other membrane compounds may comprise suitable copolymers of 4-vinylpyridine, styrene and amine-free polyether arms, either alone or in combination with any of the membrane compounds described above.

The membrane compounds described herein can be further crosslinked with one or more crosslinkers, including those listed above in relation to the enzymes described herein. For example, suitable crosslinkers can include, but are not limited to, polyethylene glycol diglycidyl ether (PEGDGE), glycerol triglycidyl ether (Gly3), polydimethylsiloxane diglycidyl ether (PDMS-DGE), or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derived variants thereof, and any combination thereof. Branches with similar terminal chemistry are also suitable for this disclosure. For example, in some embodiments, Formula 1 can be crosslinked with triglycidyl glycol ether, PEDGE, and/or polydimethylsiloxane diglycidyl ether (PDMS-DGE).

The membrane can be formed in situ by adding an alcohol buffered solution of the crosslinker and the modifying polymer over the active area and any additional compounds (e.g., electron transfer agents) contained therein and allowing the solution to cure for about 1 to 2 days or other suitable period of time. The crosslinker-polymer solution can be provided over the active area by placing one or more droplets of the membrane solution onto at least the sensor elements of the sensor tail, dipping the sensor tail into the membrane solution, spraying the membrane solution onto the sensor, singulating into layers of any size (such as individually or all-inclusive), heat pressing or welding the membrane either before or after vapor deposition of the membrane, powder coating of the membrane, and the like, and any combination thereof. The addition (eg, singulation) of the sensor electron precursors (eg, electrodes) can be followed by the addition of a membrane material to coat the distal and side edges of the sensor. In some embodiments, the analyte sensor is dip coated to apply one or more films following the application of the electron precursors. Alternatively, the analyte sensor could be grid die coated, with each side of the analyte sensor coated separately. Membranes added in the manner described above can have any of a variety of functions including, but not limited to, mass transport restriction (i.e., reducing or eliminating the flux of one or more analytes and/or compounds reaching the active area), enhanced biocompatibility, reduced interferents, etc., and any combination thereof.

In general, the thickness of the membrane is controlled by the concentration of the membrane solution, the number of droplets of membrane solution added, the number of times the sensor is immersed in the membrane solution, and the volume of membrane solution sprayed onto the sensor, and any combination of these factors. In some embodiments, the membranes described herein can have a thickness ranging from about 0.1 micrometers (μm) to about 1000 μm, including any values and subsets between these values, with separable upper and lower limits. As noted above, the membrane can overlie one or more active areas, and in some embodiments, the active areas can have a thickness from about 0.1 μm to about 50 μm, including any values and subsets therebetween, with separable upper and lower limits. In some embodiments, a series of droplets can be applied on top of each other without substantially increasing their diameter (i.e., maintaining the desired diameter or range thereof) to achieve the desired thickness of the active area and/or membrane. For example, each single droplet can be applied, then allowed to cool or dry, followed by one or more additional droplets. The active area and membrane can be of the same thickness or composition throughout, but need not be.

In some embodiments, membrane compositions suitable for use as mass transport limiting layers of the present disclosure can include polydimethylsiloxanes (PDMS), polydimethylsiloxane diglycidyl ethers (PDMS-DGE), and aminopropyl-terminated polydimethylsiloxanes suitable for use as leveling agents (e.g., to reduce the contact angle of the membrane composition or active area composition), and the like, and any combination thereof. Branches with similar terminal chemistry are also suitable for this disclosure. Certain leveling agents can additionally be included, for example, as found in US Pat. No. 8,983,568, the disclosure of which is fully incorporated herein by reference.

In some cases, the membrane can form one or more bonds with the active area. As used herein, the term "bond" and grammatical variations thereof means any type of interaction between atoms or molecules such as, but not limited to, covalent bonds, ionic bonds, dipole-dipole interactions, hydrogen bonding, London dispersion forces, etc., and any combination thereof that allow compounds to form associations with each other. For example, in situ polymerization of the membrane can form crosslinks between the polymer of the membrane and the polymer within the active area. In some embodiments, bridging the membrane to the active areas facilitates reducing the occurrence of delamination of the membrane from the sensor.

Embodiments disclosed herein include:
A. an analyte sensor comprising a working electrode having a sensing portion and an exposed electrode portion, the sensing portion comprising an active area having an analyte-reactive enzyme disposed thereon, the exposed electrode portion comprising no active area, and wherein the ratio of the exposed electrode portion to the sensing portion is in the range of about 1:10 to about 10:1, including any values and subsets between these values, wherein the upper and lower limits are in a separable range;
B. A method comprising exposing said analyte sensor to a bodily fluid wherein the analyte sensor comprises a working electrode having a sensing portion and an exposed electrode portion, the sensing portion comprising an active area having an analyte-reactive enzyme disposed thereon, the exposed electrode portion comprising no active area, and a ratio of the exposed electrode portion to the sensing portion ranging from about 1:10 to about 10:1, including any values and subsets between these values, wherein the upper and lower limits are in a separable range.

Each of embodiments A and B can have one or more of the following additional elements in any combination.

Element 1: The working electrode is a carbon working electrode.

Element 2: The area of the exposed electrode portion ranges from about 0.1 mm ² to about 5 mm ² , including any values and subsets therebetween, with separable upper and lower limits.

Element 3: The area of the sensing portion ranges from about 0.01 mm ² to about 3 mm ² , including any values and subsets therebetween, with separable upper and lower limits.

Element 4: The active area consists of multiple discontinuous active areas or a single continuous active area.

Element 5: The active area comprises a plurality of discrete active areas, each discrete active area having a diameter ranging from about 50 μm to about 500 μm, including any values and subsets therebetween, with separable upper and lower limits.

Element 6: The active area is composed of a plurality of discrete active areas separated by a pitch having a distance in the range of about 50 μm to about 800 μm, including any values and subsets therebetween, where the upper and lower limits are separable.

Element 7: The sensing portion comprises a plurality of discrete active areas arranged in a 1×n grid configuration, where n is an integer ranging from 2 to about 20, including any values and subsets therebetween, with separable upper and lower bounds.

Element 8: The sensing portion comprises a plurality of discrete active areas arranged in a 2×n grid configuration, where n is an integer ranging from 3 to about 10, including any values and subsets therebetween, with separable upper and lower bounds.

Element 9: The sensing portion comprises a plurality of discrete active areas arranged in a 3×n lattice configuration, where n is an integer ranging from 2 to about 6, including any values and subsets therebetween, with separable upper and lower bounds.

Element 10: A mass transport limiting membrane is placed over at least the sensing portion.

Element 11: Analyte-reactive enzyme is a glucose-reactive enzyme.

Element 12: Analyte Sensors Show Reduced Interferent Signal of Interferants Compared to Analyte Sensors with a Larger Ratio of Exposed Electrode Portion to Sensing Portion.

Element 13: The analyte sensor exhibits a reduction in interferent signal compared to an analyte sensor having a greater ratio of exposed electrode portion to sensing portion, wherein the reduction in interferent signal is greater than about 20%.

Element 14: The analyte sensor exhibits a reduction in interferent signal of the interferent as compared to an analyte sensor having a greater ratio of exposed electrode portion to sensing portion, wherein the reduction in interferent signal ranges from about 20% to about 70%, including any value and subset between these values, with separable upper and lower limits.

Element 15: The analyte sensor exhibits a reduced interferent signal of an interferent compared to an analyte sensor having a greater ratio of exposed electrode portion to sensing portion, wherein the interferent is ascorbic acid.

Exemplary combinations applicable to A and B as non-limiting examples include, but are not limited to, A or B having any one or more of 1-7, 10-15 or all in any combination, A or B having any one or more or all of 1-6, 8, 10-15 or all in any combination, A or B having any one or more or all of 1-6 and 9-15 in any combination.

Accordingly, the present disclosure provides an analyte sensor for in vivo monitoring of different analytes. Analyte sensors can feature enhancements to address signals obtained from interfering species. Some analyte sensors can comprise a working electrode having a sensing portion and an exposed electrode portion, the sensing portion comprising an active area having an analyte-reactive enzyme disposed thereon, and the exposed electrode portion comprising no active area. The exposed electrode portion and the sensing portion can be present in a ratio of about 1:10 to about 10:1.

Unless otherwise indicated, all numbers expressing quantities and the like in this specification and the related claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters referred to in the specification and claims are approximations that may vary depending on the desired properties sought to be obtained by embodiments of the present invention. At a minimum and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be interpreted by applying conventional rounding techniques in light of at least the number of significant digits reported.

One or more exemplary embodiments are presented herein that incorporate various features. Not all features of the physical implementation are described or illustrated herein for purposes of clarity. It is recognized that in developing physical embodiments that incorporate embodiments of the present invention, numerous implementation-specific decisions must be made to achieve developer goals such as compliance with system-related, commercial-related, government-related, and other constraints that vary from implementation to implementation and from time to time. While it is believed that developer effort can be time consuming, such effort is nonetheless considered routine work for those of ordinary skill in the art having the benefit of this disclosure.

Although various systems, tools, and methods are described herein in terms of "comprising" various components or steps, the systems, tools, and methods can also "consist essentially of" or "consist of" various components and steps.

As used herein, the phrase "at least one of" following a series of items, along with the terms "and" or "or" to separate any of those items, qualifies the item as a whole rather than each unit of the item (i.e., each item). The phrase “at least one of” allows meaning to include at least one of any one of the items, at least one of any combination of the items, and/or at least one of each of the items. As an example, each of the phrases "at least one of A, B, and C" or "at least one of A, B, or C" means only A, only B, or only C, any combination of A, B, and C, and/or at least one of each of A, B, and C.

Thus, the systems, tools and methods of the present disclosure are well adapted to attain the ends and advantages mentioned as well as those inherent therein. The specific embodiments disclosed above are exemplary only, as the teachings of this disclosure can be modified and implemented in different but equivalent ways that are apparent to those skilled in the art having the benefit thereof. Furthermore, no limitations are intended to the details of construction or design herein shown, other than as described in the claims below. It is therefore evident that the particular illustrative embodiments disclosed above may be altered, combined or modified and all such variations are considered within this disclosure. The systems, tools, and methods illustratively disclosed herein may suitably be practiced without any elements not specifically disclosed herein and/or any optional elements disclosed herein. Although systems, tools, and methods are described in terms of “comprising,” “containing,” or “including” various components or steps, systems, tools, and methods can “consist essentially of” or “consist of” various components and steps. All numbers and ranges disclosed above may vary by some amount. Whenever we disclose a numerical range with lower and upper limits, we specifically disclose every number within that range and every range that is inclusive. In particular, all value ranges disclosed herein (of the form "from about a to about b" or equivalently "from approximately a to b" or equivalently "from approximately a to b") are to be understood to recite all numbers and ranges subsumed within such broad range of values. Similarly, terms in the claims have their plain and ordinary meanings, unless expressly and clearly defined otherwise by the patentee. Further, as used herein, non-plural nouns used in the claims are defined to mean one or more than one element that the claim introduces. In the event of any conflict between the use of a word or term herein and the use of the word or term in one or more of the patent documents or other documents that may be incorporated herein by reference, the definitions consistent with this specification shall prevail.

Claims (26)

an analyte sensor,
a working electrode having a sensing portion and an exposed electrode portion;
said sensing portion comprises an active area having an analyte-reactive enzyme disposed thereon and said exposed electrode portion comprises no active area;
a ratio of the exposed electrode portion to the sensing portion is in the range of about 1:10 to about 10:1;
An analyte sensor characterized by: exhibiting a reduced interferent signal of an interferent compared to an analyte sensor having a greater ratio of the exposed electrode portion to the sensing portion;
The analyte sensor of claim 1. the reduction of the interferent signal of the interferent is greater than about 20%;
3. The analyte sensor of claim 2. the interfering substance is ascorbic acid;
3. The analyte sensor of claim 2. wherein the working electrode is a carbon working electrode;
The analyte sensor of claim 1. an area of the exposed electrode portion is in the range of about 0.1 mm ² to about 5 mm ² ;
The analyte sensor of claim 1. an area of the sensing portion is in the range of about 0.01 mm ² to about 3 mm ² ;
The analyte sensor of claim 1. wherein the active area is composed of a plurality of discontinuous active areas;
The analyte sensor of claim 1. the active area is composed of a plurality of discontinuous active areas,
each discrete active area has a diameter ranging from about 50 μm to about 500 μm;
The analyte sensor of claim 1. said active area is comprised of a plurality of discrete active areas separated by a pitch having a distance ranging from about 50 μm to about 800 μm;
The analyte sensor of claim 1. wherein said active area consists of a single continuous active area;
The analyte sensor of claim 1. a mass transport limiting membrane is disposed over at least the sensing portion;
The analyte sensor of claim 1. the analyte-reactive enzyme is a glucose-reactive enzyme;
The analyte sensor of claim 1. a method,
exposing the analyte sensor to body fluid;
The analyte sensor is
a working electrode having a sensing portion and an exposed electrode portion;
said sensing portion comprising an active area having an analyte-reactive enzyme disposed thereon, said exposed electrode portion comprising no active area;
a ratio of the exposed electrode portion to the sensing portion is in the range of about 1:10 to about 10:1;
A method characterized by: wherein the analyte sensor exhibits a reduced interferant signal of interferants compared to an analyte sensor having a greater ratio of the exposed electrode portion to the sensing portion;
15. The method of claim 14. the reduction of the interferent signal of the interferent is greater than about 20%;
16. The method of claim 15. the interfering substance is ascorbic acid;
16. The method of claim 15. wherein the working electrode is a carbon working electrode;
15. The method of claim 14. an area of the exposed electrode portion is in the range of about 0.1 mm ² to about 5 mm ² ;
15. The method of claim 14. an area of the sensing portion is in the range of about 0.01 mm ² to about 3 mm ² ;
15. The method of claim 14. wherein the active area is composed of a plurality of discontinuous active areas;
15. The method of claim 14. the active area is composed of a plurality of discontinuous active areas,
each discrete active area has a diameter ranging from about 50 μm to about 500 μm;
15. The method of claim 14. said active area is comprised of a plurality of discrete active areas separated by a pitch having a distance ranging from about 50 μm to about 800 μm;
15. The method of claim 14. wherein said active area consists of a single continuous active area;
15. The method of claim 14. a mass transport limiting membrane is disposed over at least the sensing portion;
15. The method of claim 14. the analyte-reactive enzyme is a glucose-reactive enzyme;
15. The method of claim 14.

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