Analyte sensor for sensing glutamate and methods of use thereof - Patents.com

Abstract

The present disclosure provides an analyte sensor for use in detecting glutamate. In certain embodiments, the glutamate-responsive active site of the analyte sensor of the present disclosure comprises a glutamate oxidase and a redox mediator disposed on the surface of a working electrode. The present disclosure further provides a method for detecting glutamate using the disclosed analyte sensor.

Description

[Technical field]

[0001] The subject matter described herein relates to analyte sensors for sensing glutamate and methods of use thereof. [Background technology]

[0002] Detection of various analytes in an individual can sometimes be crucial for monitoring their health, since deviations from normal analyte levels can indicate physiological conditions. For example, monitoring glucose levels can enable a person suffering from diabetes to take appropriate corrective measures to avoid significant physiological harm from hypoglycemia, hyperglycemia, or ketoacidosis. Other analytes, such as glutamate, may be desirable for monitoring other physiological conditions.

[0003] Glutamate or glutamic acid is one of the excitatory signaling molecules in the central nervous system (CNS) and contributes to neurotransmission in the brain. Under physiological conditions, blood / plasma levels of glutamate remain relatively stable. However, after traumatic brain injury (TBI), such as stroke and intracerebral hemorrhage, plasma glutamate levels can increase significantly and remain elevated for extended periods of time, leading to worse neurological outcomes and other complications, such as acute lung injury (ALI). Therefore, it is important to monitor glutamate levels after traumatic brain injury to achieve better prognosis and neurological outcomes.

[0004] Analyte monitoring in an individual can be performed periodically or continuously over a period of time. Periodic analyte monitoring can be performed by taking samples of bodily fluids, such as blood or urine, at set time intervals and analyzing ex vivo. Periodic ex vivo analyte monitoring can be sufficient to determine the physiological status of many individuals. However, ex vivo analyte monitoring can be inconvenient or painful in some cases. Furthermore, if analyte measurements are not obtained at the appropriate time, there is no way to recover lost data. Continuous analyte monitoring can be performed using one or more sensors that remain at least partially implanted within the individual's tissue (e.g., transdermally, subcutaneously or intravenously) so that analysis can be performed in vivo. The implanted sensors can collect analyte data on demand, on a set schedule, or continuously, depending on the individual's particular health needs and / or previously measured analyte levels. Analyte monitoring using in vivo implanted sensors may be a more desirable approach for individuals with severe analyte dysregulation and / or rapidly fluctuating analyte levels, but may be beneficial for other individuals as well.

[0005] Enzyme-based amperometric sensors configured to continuously assay glucose in vivo have been developed and improved in recent years to aid in monitoring the health of diabetic individuals. However, analyte sensors configured to detect analytes other than glucose in vivo are known but currently less improved. Thus, there is a need in the art for sensors for detecting analytes such as glutamate in vivo. Summary of the Invention

[0006] The objects and advantages of the disclosed subject matter will be set forth in and apparent from the following description, as well as will be learned by practice of the disclosed subject matter. Additional advantages of the disclosed subject matter will be realized and attained by the devices particularly pointed out in the description and claims hereof, as well as the appended drawings.

[0007] To achieve these and other advantages, and in accordance with the objects of the disclosed subject matter, as embodied and broadly described, the disclosed subject matter provides an analyte sensor for detecting glutamate. In certain embodiments, the disclosed analyte sensor includes a sensor tail including at least a first working electrode, a glutamate-responsive active region disposed on a surface of the first working electrode, and a mass transport limiting membrane permeable to glutamate overlying at least a portion of the glutamate-responsive active region. In certain embodiments, the glutamate-responsive active region includes glutamate oxidase and an electron transfer agent.

[0008] The present disclosure further provides a method for detecting glutamate. In certain embodiments, the method may include providing an analyte sensor including: (a) a sensor tail including at least a first working electrode; (b) a glutamate-responsive active region disposed on a surface of the first working electrode, the glutamate-responsive active region including glutamate oxidase and an electron transfer agent; and (c) a mass transport limiting membrane permeable to glutamate overlying at least the glutamate-responsive active region. In certain embodiments, the method further includes applying a potential to the first working electrode, obtaining a first signal equal to or greater than the redox potential of the glutamate-responsive active region, the first signal being proportional to the concentration of glutamate in a fluid in contact with the glutamate-responsive active region, and correlating the first signal to the concentration of glutamate in the fluid. In certain embodiments, the fluid is interstitial fluid.

[0009] In certain embodiments, the glutamate-responsive region further comprises a polymer. For example, but not limited to, the electron transfer agent and / or glutamate oxidase are crosslinked to the polymer in the glutamate-responsive active region. In certain embodiments, the electron transfer agent and / or glutamate oxidase are covalently bound to the polymer in the glutamate-responsive active region. In certain embodiments, the glutamate-responsive active region further comprises a stabilizer, such as albumin.

[0010] In certain embodiments, the mass transport limiting membrane comprises a polyurethane or a copolymer thereof. In certain embodiments, the mass transport limiting membrane further comprises an ion exchange polymer, such as a perfluorosulfonic acid polymer, or a polyvinylpyridine-based polymer.

[0011] In certain embodiments, the analyte sensor of the present disclosure further includes a second working electrode and a second active area disposed on a surface of the second working electrode and responsive to a second analyte different from glutamate. In certain embodiments, the second active area includes at least one enzyme responsive to the second analyte. In certain embodiments, a second portion of the mass transport limiting membrane overlies the second active area, or a second mass transport limiting membrane overlies the second active area.

[0012] In certain embodiments, the analyte is implanted in a subject who is at risk of having a neurological condition or has a neurological condition.In certain embodiments, the neurological condition is brain injury.In certain embodiments, the brain injury is traumatic brain injury.In certain embodiments, the traumatic brain injury is stroke and / or intracerebral hemorrhage.

[0013] In certain embodiments, the analyte sensor is implanted in the subject for about 15 days, hi certain embodiments, the analyte sensor retains at least about 90% sensitivity during its use. The following figures are included to illustrate certain aspects of the present disclosure and should not be considered exclusive embodiments. The disclosed subject matter is capable of considerable modification, permutations, combinations, and equivalents in form and function without departing from the scope of the present disclosure. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a diagram of an exemplary sensing system that can incorporate an analyte sensor of the present disclosure. [Diagram 2] 2A-2C are cross-sectional views of an analyte sensor that includes a single active area. [Diagram 3] 3A-3C are cross-sectional views of an analyte sensor that includes two active areas. [Figure 4] 1 is a cross-sectional view of an analyte sensor including two active areas. [Diagram 5] 5A-5C are perspective views of an analyte sensor including two active areas on separate working electrodes. [Figure 6] FIG. 1 is a diagram of a particular enzyme system that can be used to detect glutamate in accordance with the present disclosure. [Figure 7] 1 is a plot of sensor current (nA) versus time (hr) for an exemplary glutamate sensor of the present disclosure. [Figure 8] FIG. 8 shows exemplary plots of sensor current response (nA) versus glutamate concentration (μM) for the glutamate sensor of FIG. 7 on days 1 and 5. [Figure 9] FIG. 13 shows exemplary plots of sensor current response (nA) versus time (hr) for glutamate sensors including alternative membrane compositions. [Figure 10] FIG. 13 shows exemplary plots of sensor current (nA) versus glutamate concentration (μM) for glutamate sensors including alternative membrane compositions. [Figure 11] FIG. 13 shows the stability of the glutamate sensor on days 1, 6 and 12 as measured by sensor current (nA) versus glutamate concentration (mM). [Figure 12]FIG. 1 shows an exemplary plot of sensor current (nA) versus glutamate concentration (mM) for glutamate sensors coated with HydroMed™ D1 or HydroMed™ D7 membranes. [Figure 13] FIG. 1 shows exemplary plots of sensor current (nA) versus time (hrs) for glutamate sensors coated with membranes composed of HydroMed™ D1 and polyvinylpyridine or HydroMed™ D1 and 10Q5. [Figure 14] FIG. 1 shows exemplary plots of sensor current (nA) versus glutamate concentration (μM) for glutamate sensors coated with membranes composed of HydroMed™ D1 and polyvinylpyridine or HydroMed™ D1 and 10Q5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present disclosure relates to an analyte sensor that uses one or more enzymes for the detection of glutamate.In certain embodiments, the present disclosure further provides an analyte sensor that uses multiple enzymes for the detection of two different analytes, for example, multiple enzymes for the detection of glutamate and a second analyte.Depending on the sensor configuration, the analyte sensor of the present disclosure can be configured to detect one analyte, for example, glutamate, or multiple analytes simultaneously or nearly simultaneously.The present disclosure further provides a method of detecting one or more analytes, for example, glutamate, using the disclosed analyte sensor.

[0016] The present disclosure provides a sensor chemistry and mass transport limiting membrane suitable for detecting glutamate with good response stability and sensitivity over a range of glutamate concentrations.In certain embodiments, the glutamate sensor of the present disclosure is stable for up to about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 20 days after first use, for example after implantation.In certain embodiments, the glutamate sensor of the present disclosure exhibits less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, or less than about 5% sensitivity loss during sensor use. In certain embodiments, the continuous glutamate sensors of the present disclosure exhibit less than about 10% loss in sensitivity during use of the sensor (e.g., over a wear period of about 12 days). In certain embodiments, the continuous glutamate sensors of the present disclosure exhibit less than about 15% (e.g., 13%) loss in sensitivity during use of the sensor (e.g., over a wear period of about 15 days).

[0017] For purposes of clarity, but not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections. I. Definition; II. Analyte sensors; 1. General Structure of Analyte Sensor 2. Enzymes; 3. Redox mediators; 4. Polymer backbone; 5. Mass-transfer limiting membranes; and 6. Manufacturing; III. How to use.

[0018] I. Definition The terms used herein generally have their ordinary meaning in the art, within the context of this disclosure and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.

[0019] As used herein, the use of the words "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0020] The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not exclude additional acts or structures. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0021] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by a person skilled in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, according to the convention in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, for example, within 5-fold or within 2-fold.

[0022] The term "biological fluid" as used herein refers to any bodily fluid or fluid derivative in which an analyte can be measured. Non-limiting examples of biological fluids include dermal fluid, interstitial fluid, plasma, blood, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, sweat, tears, etc. In certain embodiments, the biological fluid is dermal fluid or interstitial fluid.

[0023] As used herein, the term "redox mediator" refers to an electron transfer agent for carrying electrons between an analyte or an enzyme that has reduced or oxidized the analyte and an electrode, either directly or via one or more additional electron transfer agents. In certain embodiments, redox mediators that include a polymer backbone may also be referred to as "redox polymers."

[0024] The term "electrolysis," as used herein, refers to the electro-oxidation or electro-reduction of a compound, either directly at an electrode or via one or more electron transfer agents (e.g., redox mediators or enzymes).

[0025] As used herein, the term "reference electrode" can refer to either a reference electrode or an electrode that functions as both a reference electrode and a counter electrode. Similarly, as used herein, the term "counter electrode" can refer to both a counter electrode and a counter electrode that also functions as a reference electrode.

[0026] As used herein, the term "homogeneous membrane" refers to a membrane that includes a single type of membrane polymer. As used herein, the term "multi-component membrane" refers to a membrane that includes two or more types of membrane polymers.

[0027] As used herein, the term "single-component membrane" refers to a membrane that includes one type of membrane polymer. As used herein, the term "ion exchange polymer" refers to a polymer that is capable of exchanging ions (cations or anions) with ionic components in a solution.

[0028] As used herein, the term "polyvinylpyridine-based polymer" refers to a polymer or copolymer that includes polyvinylpyridine (eg, poly(2-vinylpyridine) or poly(4-vinylpyridine)) or a derivative thereof.

[0029] II. Analyte Sensors 1. General Structure of Analyte Sensor Before describing the analyte sensors of the present disclosure and their components in further detail, a brief overview of suitable in vivo analyte sensor configurations and sensor systems employing analyte sensors is provided so that embodiments of the present disclosure can be better understood. FIG. 1 shows a diagram of an exemplary sensing system that can incorporate the analyte sensors of the present disclosure. As shown, the sensing system 100 includes a sensor control device 102 and a reader device 120 that are configured to communicate with each other via a local communication path or link 140, which can be wired or wireless, one-way or two-way, and encrypted or unencrypted. The reader device 120 can configure an output medium for viewing analyte concentrations and alerts or notifications determined by the sensor 104 or its associated processor, as well as for enabling one or more user inputs, according to certain embodiments. The reader device 120 can be a general-purpose smartphone or a dedicated electronic reader instrument. Although only one reader device 120 is shown, in some cases, there may be multiple reader devices 120. Reader device 120 may also communicate with remote terminal 170 and / or trusted computer system 180 via communication paths / links 141 and / or 142, respectively, which may also be wired or wireless, one-way or two-way, and encrypted or unencrypted. Reader device 120 may also, or alternatively, communicate with network 150 (e.g., a cellular network, the Internet, or a cloud server) via communication path / link 151. Network 150 may be further communicatively coupled to remote terminal 170 via communication path / link 152 and / or to trusted computer system 180 via communication path / link 153. Alternatively, sensor 104 may communicate directly with remote terminal 170 and / or trusted computer system 180 without the presence of an intervening reader device 120.For example, but not by way of limitation, the sensor 104 may communicate with the remote terminal 170 and / or the trusted computer system 180 via a direct communication link to the network 150 according to certain embodiments, 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 may be used for each of the communication paths or links, such as Near Field Communication (NFC), Radio Frequency Identification (RFID), BLUETOOTH® or BLUETOOTH® Low Energy protocols, WiFi, etc. The remote terminal 170 and / or the trusted computer system 180 may be accessible by individuals other than the primary user who are interested in the user's analyte levels according to certain embodiments. The reader device 120 may include a display 122 and an optional input component 121. The display 122 may include a touch screen interface according to certain embodiments.

[0030] The sensor control device 102 includes a sensor housing 103 that can house circuitry and a power source for operating the sensor 104. Optionally, the power source and / or active circuitry can be omitted. A processor (not shown) can be communicatively coupled to the sensor 104, the processor being physically located within the sensor housing 103 or the reader device 120. The sensor 104, according to certain embodiments, protrudes from an underside of the sensor housing 103 and extends through an adhesive layer 105 that is adapted for adhering the sensor housing 103 to a tissue surface such as skin.

[0031] The sensor 104 is adapted to be at least partially inserted into a tissue of interest, such as into the dermis or subcutaneous layer of the skin. The sensor 104 may include a sensor tail of sufficient length to insert to a desired depth within a given tissue. The sensor tail may include at least one working electrode. In certain configurations, the sensor tail may include an active area for detecting an analyte. A counter electrode may be present in combination with the at least one working electrode. Particular electrode configurations on the sensor tail are described in more detail below.

[0032] The active regions can be configured to detect a particular analyte. For example, and without limitation, the disclosed analyte sensors include at least one active region configured to detect glutamate. In certain embodiments, the disclosed sensors include two active regions, each active region configured to detect a different analyte. Alternatively, the two active regions can be configured to detect the same analyte. In certain embodiments, the first active region can be configured to detect glutamate and the second active region can be configured to detect glutamate or a second analyte different from glutamate.

[0033] In certain embodiments of the present disclosure, one or more analytes may be monitored in any biological fluid of interest, such as dermal fluid, interstitial fluid, plasma, blood, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, etc. In certain embodiments, the analyte sensor of the present disclosure may be adapted to assay dermal or interstitial fluid to determine the concentration of one or more analytes in vivo. In certain embodiments, the biological fluid is interstitial fluid.

[0034] With further reference to FIG. 1 , the sensor 104 may automatically transfer data to the reader device 120. For example, but not by way of limitation, analyte concentration data (i.e., glutamate concentration) may be communicated automatically and periodically, such as when data is obtained or at a particular frequency, such as after a particular period of time has passed, and the data is stored in memory until transmission (e.g., every minute, every five minutes, or other predetermined period of time). In certain other embodiments, the sensor 104 may communicate with the reader device 120 non-automatically, rather than according to a set schedule. For example, but not by way of limitation, RFID technology may be used to communicate data from the sensor 104 when the sensor electronics are brought within communication range of the reader device 120. The data may remain stored in the memory of the sensor 104 until communicated to the reader device 120. Thus, a user need not maintain proximity to the reader device 120 at all times, but may instead upload data at their convenience. In certain other embodiments, a combination of automatic and non-automatic data transfer may be implemented. For example, and not by way of limitation, data transfer may continue automatically until the reader device 120 is no longer within communication range of the sensor 104 .

[0035] An introducer may be temporarily present to facilitate the introduction of the sensor 104 into the tissue. In certain exemplary embodiments, the introducer may include a needle or similar sharp. As would be readily recognized by one of ordinary skill in the art, in alternative embodiments, other types of introducers, such as a sheath or blade, may be present. More specifically, the needle or other introducer may be temporarily present in proximity to the sensor 104 prior to tissue insertion and then subsequently removed. While present, the needle or other introducer may facilitate the insertion of the sensor 104 into the tissue by opening an access path for the sensor 104 to follow. For example, and not by way of limitation, according to one or more embodiments, the needle may facilitate the penetration of the epidermis as an access path to the dermis to allow the implantation of the sensor 104 to occur. After opening the access path, the needle or other introducer may be removed so as not to pose a sharps hazard. In certain embodiments, a suitable needle may be solid or hollow, beveled or non-beveled, and / or circular or non-circular in cross section. In more specific embodiments, suitable needles may be comparable in cross-sectional diameter and / or tip design to acupuncture needles, which may have a cross-sectional diameter of about 250 microns. However, suitable needles may have larger or smaller cross-sectional diameters as required for a particular application.

[0036] In certain embodiments, the tip of the needle (while present) can be angled over the end of the sensor 104 such that the needle penetrates the tissue first, opening an access path for the sensor 104. In certain embodiments, the sensor 104 can reside within a lumen or channel of the needle, which likewise opens an access path for the sensor 104. In either case, after facilitating insertion of the sensor, the needle is subsequently removed.

[0037] Sensor configurations featuring a single active region configured for detection of a corresponding single analyte can employ two-electrode or three-electrode detection motifs, as further described herein with reference to Figures 2A-2C. Sensor configurations featuring two different active regions for detection of separate analytes, either on separate working electrodes or on the same working electrode, are described separately below with reference to Figures 3A-5C. Sensor configurations having multiple working electrodes can be particularly advantageous for incorporating two different active regions within the same sensor tail, since the signal contribution from each active region can be more easily determined.

[0038] When a single working electrode is present in the analyte sensor, a three-electrode sensor configuration can include a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration can include a working electrode and a second electrode, which can function as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). The various electrodes can be at least partially stacked (layered) on top of each other and / or laterally spaced apart from each other on the sensor tail. Suitable sensor configurations can be substantially flat, substantially cylindrical, or any other suitable shape. In any of the sensor configurations disclosed herein, the various electrodes can be electrically insulated from each other by a dielectric material or similar insulator.

[0039] Analyte sensors featuring multiple working electrodes can also include at least one additional electrode. If one additional electrode is present, the one additional electrode can function as a counter / reference electrode for each of the multiple working electrodes. If two additional electrodes are present, one of the additional electrodes can function as a counter electrode for each of the multiple working electrodes, and the other of the additional electrodes can function as a reference electrode for each of the multiple working electrodes.

[0040] 2A shows a diagram of an exemplary two-electrode analyte sensor configuration suitable for use in the disclosure herein. As shown, the analyte sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter / reference electrode 216. Alternatively, the working electrode 214 and the counter / reference electrode 216 may be located on the same side of the substrate 212 with a dielectric material interposed therebetween (configuration not shown). An active area 218 is disposed as at least one layer over at least a portion of the working electrode 214. The active area 218 may include multiple spots or a single spot configured for detection of an analyte, as discussed further herein.

[0041] 2A, the membrane 220 overlays at least the active area 218. In certain embodiments, the membrane 220 may also overlay some or all of the working electrode 214 and / or counter / reference electrode 216, or the entire analyte sensor 200. One or both sides of the analyte sensor 200 may be overlayed with the membrane 220. The membrane 220 may include one or more polymeric membrane materials capable of limiting analyte flux to the active area 218 (i.e., the membrane 220 is a mass transport limiting membrane having some permeability to the analyte of interest). In accordance with the disclosure herein and as further described below, the membrane 220 may be crosslinked with a branched crosslinker in certain sensor configurations. For example, without limitation, the membrane 220 is crosslinked with a crosslinker as described herein. The composition and thickness of the membrane 220 may be varied to facilitate the desired analyte flux to the active area 218, thereby providing the desired signal strength and stability. The analyte sensor 200 may be operable to assay the analyte by either coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

[0042] 2B and 2C show diagrams of an exemplary three-electrode analyte sensor configuration that is also suitable for use in the present disclosure. The three-electrode analyte sensor configuration can be similar to that shown for analyte sensor 200 of FIG. 2A, except for the inclusion of an additional electrode 217 in analyte sensors 201 and 202 (FIGS. 2B and 2C). The additional electrode 217 allows counter / reference electrode 216 to function as either a counter electrode or a reference electrode, and additional electrode 217 performs other electrode functions unless otherwise specified. Working electrode 214 continues to perform its original function. Additional electrode 217 can be disposed on either working electrode 214 or electrode 216, with a separating layer of dielectric material therebetween. For example, but not by way of limitation, as shown in FIG. 2B, dielectric layers 219a, 219b, and 219c separate electrodes 214, 216, and 217 from one another and provide electrical insulation. Alternatively, as shown in FIG. 2C, at least one of the electrodes 214, 216, and 217 may be located on both sides of the substrate 212. Thus, in certain embodiments, the electrodes 214 (working electrode) and 216 (counter electrode) may be located on both sides of the substrate 212, and the electrode 217 (reference electrode) is located on one of the electrodes 214 or 216 and spaced therefrom using a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) may be present on the electrode 217, and the location of the reference material layer 230 is not limited to those shown in FIGS. 2B and 2C. Similar to the sensor 200 shown in FIG. 2A, the active area 218 of the analyte sensors 201 and 202 may include multiple spots or a single spot. Additionally, the analyte sensors 201 and 202 may be operable to assay the analyte by any of coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

[0043] As with analyte sensor 200, membrane 220 can also overlay active area 218 and other sensor components in analyte sensors 201 and 202, thereby functioning as a mass transport limiting membrane. In certain embodiments, additional electrode 217 can be overlayed with membrane 220. Although FIGS. 2B and 2C show electrodes 214, 216, and 217 as overlayed with membrane 220, it should be appreciated that in certain embodiments, only working electrode 214 is overlayed. Also, the thickness of membrane 220 on each of electrodes 214, 216, and 217 can be the same or different. As in the two-electrode analyte sensor configuration (FIG. 2A), one or both sides of analyte sensors 201 and 202 can be overlayed with membrane 220 in the sensor configuration of FIGS. 2B and 2C, or the entire analyte sensors 201 and 202 can be overlayed. Thus, the three-electrode sensor configuration shown in Figures 2B and 2C should be understood as non-limiting of the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of the present disclosure.

[0044] FIG. 3A shows an exemplary configuration of a sensor 203 having a single working electrode with two different active regions disposed thereon. FIG. 3A is similar to FIG. 2A, except that there are two active regions on the working electrode 214, a first active region 218a and a second active region 218b, which are responsive to different analytes and are laterally spaced apart from each other on the surface of the working electrode 214. The active regions 218a and 218b can include multiple spots or a single spot configured for detection of each analyte. The composition of the membrane 220 can vary in the active regions 218a and 218b or can be compositionally the same. The first active region 218a and the second active region 218b can be configured to detect their corresponding analytes at different working electrode potentials from each other, as discussed further below.

[0045] Figures 3B and 3C show cross-sectional views of exemplary three-electrode sensor configurations of sensors 204 and 205, respectively, each featuring a single working electrode having a first active area 218a and a second active area 218b disposed thereon. Figures 3B and 3C are otherwise similar to, and may be better understood by reference to, Figures 2B and 2C. As with Figure 3A, the composition of membrane 220 may vary or be compositionally the same in active areas 218a and 218b.

[0046] Exemplary sensor configurations having multiple working electrodes, specifically two working electrodes, are described in further detail with reference to Figures 4-5C. The following description primarily relates to sensor configurations having two working electrodes, but it should be understood that more than two working electrodes can be incorporated through extension of the disclosure herein. Additional working electrodes can be used to impart additional sensing capabilities to the analyte sensor beyond just the first and second analytes, e.g., for detection of a third and / or fourth analyte.

[0047] 4 illustrates a cross-sectional view of an exemplary analyte sensor configuration having two working electrodes, a reference electrode, and a counter electrode suitable for use in the present disclosure. As shown, the analyte sensor 300 includes working electrodes 304 and 306 disposed on opposite sides of a substrate 302. A first active area 310a is disposed on the surface of the working electrode 304, and a second active area 310b is disposed on the surface of the working electrode 306. The counter electrode 320 is electrically insulated from the working electrode 304 by a dielectric layer 322, and the reference electrode 321 is electrically insulated from the working electrode 306 by a dielectric layer 323. Outer dielectric layers 330 and 332 are disposed on the reference electrode 321 and the counter electrode 320, respectively. A membrane 340 can overlay at least the active areas 310a and 310b according to various embodiments, with other components of the analyte sensor 300 or the entire analyte sensor 300 optionally overlayed with the membrane 340. The membrane 340 may be continuous, but compositionally varied on the active area 310a and / or on the active area 310b to provide different permeability values ​​to regulate analyte flux differently at each location. For example, different membrane formulations may be sprayed and / or printed on opposing sides of the analyte sensor 300. In particular, dip-coating techniques may also be suitable to deposit at least a portion of a bilayer membrane on one of the active areas 310a and 310b. In certain embodiments, the membrane 340 may be the same in the active areas 310a and 310b, or may be compositionally varied. For example, but not limited to, the membrane 340 may be a homogeneous membrane including a bilayer overlying the active area 310a and overlying the active area 310b, or the membrane 340 may be a homogeneous membrane including a bilayer overlying the active area 310b and overlying the active area 310a. In certain embodiments, the analyte sensor may include more than one membrane 340, e.g., two or more membranes. For example, without limitation, the analyte sensor may include a membrane overlying one or more active areas, e.g., 310a and 310b, and an additional membrane overlying the entire sensor, as shown in FIG.

[0048] Similar to analyte sensors 200, 201 and 202, analyte sensor 300 may be operable to assay glutamate by either coulometric, amperometric, voltammetric, or potentiometric electrochemical detection techniques.

[0049] Alternative sensor configurations having multiple working electrodes and differing from the configuration shown in Figure 4 may feature counter / reference electrodes instead of separate counter and reference electrodes 320, 321, and / or may feature layer and / or film arrangements different from those explicitly shown. For example, and not by way of limitation, the arrangement of counter electrode 320 and reference electrode 321 may be reversed from that shown in Figure 4. Additionally, working electrodes 304 and 306 need not necessarily be on opposing sides of substrate 302 in the manner shown in Figure 4.

[0050] Although preferred sensor configurations may feature electrodes that are substantially planar in nature, it should be understood that sensor configurations featuring non-planar electrodes may be advantageous and particularly suitable for use in the present disclosure. In particular, substantially cylindrical electrodes arranged concentrically relative to one another may facilitate deposition of a mass transport limiting membrane, as described below. Figures 5A-5C show perspective views of an analyte sensor featuring two working electrodes arranged concentrically relative to one another. It should be understood that sensor configurations having a concentric electrode arrangement, but lacking a second working electrode, are also possible in the present disclosure.

[0051] 5A shows a perspective view of an exemplary sensor configuration in which multiple electrodes are substantially cylindrical and concentrically arranged with respect to one another around a central substrate. As shown, the analyte sensor 400 includes a central substrate 402 around which all of the electrodes and dielectric layers are concentrically arranged with respect to one another. In particular, a working electrode 410 is disposed on a surface of the central substrate 402, and a dielectric layer 412 is disposed on a portion of the working electrode 410 distal to the sensor tip 404. A working electrode 420 is disposed on the dielectric layer 412, and a dielectric layer 422 is disposed on a portion of the working electrode 420 distal to the sensor tip 404. A counter electrode 430 is disposed on the dielectric layer 422, and a dielectric layer 432 is disposed on a portion of the counter electrode 430 distal to the sensor tip 404. A reference electrode 440 is disposed on the dielectric layer 432, and a dielectric layer 442 is disposed on a portion of the reference electrode 440 distal to the sensor tip 404. Thus, the exposed surfaces of working electrode 410 , working electrode 420 , counter electrode 430 , and reference electrode 440 are spaced apart from one another along the longitudinal axis B of analyte sensor 400 .

[0052] 5A, first and second active areas 414a and 414b responsive to different or the same analyte are disposed on exposed surfaces of working electrodes 410 and 420, respectively, thereby allowing contact with fluid for sensing. Although active areas 414a and 414b are shown as three separate spots in FIG. 5A, it should be understood that there may be fewer or more than three spots comprising a continuous layer of active area in alternative sensor configurations.

[0053] In Fig. 5A, the sensor 400 is partially coated with a membrane 450 over the working electrodes 410 and 420 and the active areas 414a and 414b disposed thereon. Fig. 5B shows an alternative sensor configuration in which substantially the entire sensor 401 is overcoated with a membrane 450. The membrane 450 may be the same in the active areas 414a and 414b or may be compositionally varied. For example, the membrane 450 may include a bilayer overcoating the active area 414a and may be a homogenous membrane overcoating the active area 414b.

[0054] It should be further understood that the arrangement of the various electrodes in Figures 5A and 5B may differ from those explicitly shown. For example, the positions of the counter electrode 430 and the reference electrode 440 may be reversed from the configuration shown in Figures 5A and 5B. Similarly, the positions of the working electrodes 410 and 420 are not limited to those explicitly shown in Figures 5A and 5B. Figure 5C shows an alternative sensor configuration to that shown in Figure 5B, where the sensor 405 contains the counter electrode 430 and the reference electrode 440 located more proximally to the sensor tip 404 and the working electrodes 410 and 420 located more distally to the sensor tip 404. A sensor configuration in which the working electrodes 410 and 420 are located more distally to the sensor tip 404 may be advantageous by providing a larger surface area for deposition of the active regions 414a and 414b (five separate sensing spots exemplarily shown in Figure 5C), thereby facilitating increased signal strength in some cases. Similarly, the center substrate 402 can be omitted in any of the concentric sensor configurations disclosed herein, and the innermost electrode can instead support subsequently deposited layers.

[0055] Some parts of the sensor are further described below. 2. Enzymes The active area of ​​the analyte sensor of the present disclosure may be configured to detect one or more analytes, for example, glutamate. In certain embodiments, the analyte sensor of the present disclosure may include more than one active area, each active area configured to detect the same analyte or a different analyte. The analyte sensor of the present disclosure includes one or more active areas configured to detect glutamate. In certain embodiments, the analyte sensor of the present disclosure may further include one or more active areas configured to detect a second analyte other than glutamate.

[0056] In certain embodiments, the analyte sensor of the present disclosure may include one or more glutamate-responsive regions. In certain embodiments, the glutamate-responsive region may include one or more enzymes for detecting glutamate. For example, but not by way of limitation, the glutamate-responsive region may include glutamate oxidase as shown in FIG. 6. In certain embodiments, the glutamate-responsive active region contains glutamate oxidase ("GlutOx" in FIG. 6), which converts L-glutamate to α-ketoglutarate (also referred to as "α-ketoglutaric acid") and reduces glutamate oxidase. The reduced form of glutamate oxidase can then transfer electron(s) to a redox mediator, which can then be oxidized at the anode, i.e., working electrode. The electrons transferred during this reaction provide the basis for glutamate detection at the working electrode. The resulting electrochemical signal can then be correlated with the amount of glutamate originally present in the sample.

[0057] In certain embodiments, an analyte sensor of the present disclosure can include a sensor tail including at least one working electrode and one or more glutamate-responsive active regions disposed on a surface of the working electrode, the glutamate-responsive active region including glutamate oxidase. In certain embodiments, the glutamate oxidase is immobilized to the glutamate-responsive active region, for example, by covalent attachment to a polymer present within the glutamate-responsive active region.

[0058] In certain embodiments, the glutamate-responsive active area is disposed on a portion of the working electrode. For example, without limitation, the glutamate-responsive active area is disposed on a portion of the working electrode in a spotted pattern, such as two or more spots on the working electrode. In certain embodiments, the glutamate-responsive active area is disposed on a portion of the working electrode in a slotted pattern. In certain embodiments, the glutamate-responsive active area is disposed on the entire length of the working electrode or in a continuous pattern on the working electrode. In certain embodiments, the glutamate-responsive active area is disposed on a portion of the working electrode within about 0.01 mm. 2 ~ approx. 2.0 mm 2 , for example, about 0.1 mm 2 ~ approx. 1.0 mm 2 or about 0.2 mm 2 ~about 0.5mm 2 It has an area of.

[0059] In certain embodiments, the glutamate-responsive active region may further comprise a stabilizer, for example, to stabilize the enzyme. For example, but not limited to, the stabilizer may be albumin, for example, serum albumin. Non-limiting examples of serum albumin include bovine serum albumin and human serum albumin. In certain embodiments, the stabilizer is human serum albumin. In certain embodiments, the stabilizer is bovine serum albumin.

[0060] In certain embodiments, the glutamate responsive active region can comprise a ratio of stabilizer to glutamate oxidase of about 40:1 to about 1:40, e.g., about 35:1 to about 1:35, about 30:1 to about 1:30, about 25:1 to about 1:25, about 20:1 to about 1:20, about 15:1 to about 1:15, about 10:1 to about 1:10, about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, or about 1:1. In certain embodiments, the glutamate responsive active region can comprise a ratio of stabilizer to glutamate oxidase of about 1:1 to about 1:10, e.g., about 1:1 to about 1:9, about 1:1 to about 1:8, about 1:1 to about 1:7, about 1:1 to about 1:6, about 1:1 to about 1:5, about 1:2 to about 1:9, about 1:3 to about 1:8, about 1:3 to about 1:7, or about 1:4 to about 1:6.

[0061] In certain embodiments, the analyte sensor may include two working electrodes, e.g., a first active area disposed on the first working electrode and a second active area disposed on the second working electrode. In certain embodiments, the analyte sensor disclosed herein may feature a glutamate-responsive active area and a second active area for detecting an analyte different from glutamate. For example, without limitation, such an analyte sensor may include a sensor tail having at least a first working electrode and a second working electrode, a glutamate-responsive active area disposed on the surface of the first working electrode, and a second active area, e.g., a second enzyme-responsive active area, configured to detect a different analyte disposed on the surface of the second working electrode. In certain embodiments, the additional analyte, e.g., the second analyte, detected by the analyte sensor of the present disclosure may be glucose, lactate, ketone, creatinine, and / or alcohol. In certain embodiments, when a sensor is configured to detect more than one analyte, detection of each analyte may include applying a potential to each working electrode separately such that a separate signal is obtained from each analyte. The signal obtained from each analyte can then be correlated to the analyte concentration through the use of a calibration curve or function or by using a look-up table. In certain embodiments, the correlation of analyte signal to analyte concentration can be performed through the use of a processor.

[0062] In certain embodiments, the second enzyme-responsive active region of the disclosed analyte sensor, e.g., on the second working electrode, may include one or more enzymes that may be used to detect glucose. For example, without limitation, the disclosed analyte sensor may include an active region that includes one or more enzymes for detecting glucose, e.g., disposed on the second working electrode. In certain embodiments, the analyte sensor may include active sites that include glucose oxidase and / or glucose dehydrogenase for detecting glucose.

[0063] In certain embodiments, the second enzyme-responsive active region of the disclosed analyte sensor, e.g., on the second working electrode, can include one or more enzymes that can be used to detect ketones. For example, without limitation, the disclosed analyte sensor can include an active region that includes one or more enzymes, e.g., an enzyme system, for detecting ketones, disposed, e.g., on the second working electrode. In certain embodiments, the analyte sensor can include active sites that include β-hydroxybutyrate dehydrogenase and / or diaphorase for detecting ketones.

[0064] In certain embodiments, the second enzyme-responsive active region of the disclosed analyte sensor, e.g., on the second working electrode, can include one or more enzymes that can be used to detect lactate. For example, without limitation, the disclosed analyte sensor can include an active region that includes one or more enzymes, e.g., an enzyme system, for detecting lactate, disposed, e.g., on the second working electrode. In certain embodiments, the analyte sensor can include an active site that includes lactate dehydrogenase and / or lactate oxidase.

[0065] In certain embodiments, the second enzyme-responsive active region of the analyte sensor of the present disclosure, e.g., on the second working electrode, can include one or more enzymes that can be used to detect alcohol. For example, without limitation, the analyte sensor of the present disclosure can include an active region that includes one or more enzymes, e.g., an enzyme system, for detecting alcohol, e.g., disposed on the second working electrode. In certain embodiments, the analyte sensor can include an active site that includes alcohol dehydrogenase.

[0066] In certain embodiments, the second enzyme-responsive active region of the analyte sensor of the present disclosure, e.g., on the second working electrode, may include one or more enzymes that can be used to detect creatinine. For example, without limitation, the analyte sensor of the present disclosure may include an active region that includes one or more enzymes, e.g., an enzyme system, for detecting creatinine, disposed, e.g., on the second working electrode. In certain embodiments, the analyte sensor may include active sites that include amidohydrolase, creatine amidinohydrolase, and / or sarcosine oxidase.

[0067] In certain other analyte sensor configurations, the first active area and the second active area can be disposed on a single working electrode. A first signal can be obtained from the first active area, for example at a low potential, and a second signal including signal contributions from both active areas can be obtained at a higher potential. The signal contribution resulting from the second analyte can then be determined by subtracting the first signal from the second signal. The signal contribution from each analyte can then be correlated to the analyte concentration in a manner similar to that described for sensor configurations having multiple working electrodes. In certain embodiments, when a glutamate-responsive active area and a second active area configured to detect a different analyte (e.g., a second analyte-responsive active area) are disposed on a single working electrode in this manner, one of the active areas can be configured to be interrogated separately to facilitate detection of each analyte. For example, either the glutamate-responsive active area or the second active area responsive to the second analyte can generate a signal independently of the other active area.

[0068] It should also be understood that the sensitivity (output current) of the analyte sensor to each analyte may be varied by varying the coverage (area or size) of the active regions, the area ratio of the active regions to each other, the identity, thickness, and / or composition of the mass transport limiting membrane overlying the active regions. Modification of these parameters may be readily effected by one of ordinary skill in the art having the benefit of the disclosure herein.

[0069] 3. Redox mediators In certain embodiments, the analyte sensors disclosed herein may include an electron transfer agent, e.g., a redox mediator. In certain embodiments, one or more active regions of the analyte sensors disclosed herein may include an electron transfer agent, e.g., a redox mediator.

[0070] In certain embodiments, the glutamate-responsive active region can include one or more electron transfer agents. For example, but not by way of limitation, an analyte sensor of the present disclosure can include a sensor tail having at least a first working electrode and a glutamate-responsive active region disposed on a surface of the first working electrode, the glutamate-responsive active region including glutamate oxidase and an electron transfer agent.

[0071] In certain embodiments, the analyte sensor of the present disclosure may include two or more active regions, each of which includes an electron transfer agent. For example, without limitation, the analyte sensor of the present disclosure may include a sensor tail having at least a first working electrode and a second working electrode, a glutamate-responsive active region including glutamate oxidase and a first electron transfer agent disposed on the surface of the first working electrode, and a second analyte-responsive active region including at least one enzyme responsive to a second analyte and a second electron transfer agent disposed on the surface of the second working electrode. Alternatively, the analyte sensor of the present disclosure may include two or more active regions, with only one active region including an electron transfer agent. For example, and without limitation, an analyte sensor of the present disclosure can include a sensor tail having at least a first working electrode and a second working electrode, a glutamate-responsive active region comprising glutamate oxidase and an electron transfer agent disposed on a surface of the first working electrode, and a second analyte-responsive active region comprising at least one enzyme responsive to a second analyte disposed on a surface of the second working electrode, where the second analyte-responsive active region does not comprise an electron transfer agent.

[0072] Electron transfer agents suitable for use in the analyte sensors of the present disclosure can facilitate the transfer of electrons to an adjacent working electrode after the analyte undergoes an enzymatic redox reaction within the corresponding active area, thereby generating a current indicative of the presence of that particular analyte, the amount of current generated being proportional to the amount of analyte present.

[0073] In certain embodiments, suitable electron transfer agents can include electroreducible and electrooxidizable ions, complexes or molecules (e.g., quinones) that have redox potentials several hundred millivolts higher or lower than the redox potential of the standard calomel electrode (SCE). In certain embodiments, electron transfer agents can include osmium complexes and other transition metal complexes (e.g., those described in U.S. Pat. Nos. 6,134,461 and 6,605,200, which are incorporated herein by reference in their entirety). Further examples of suitable redox mediators 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 also incorporated herein by reference in their entirety. Other examples of suitable electron transfer agents include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt, including metallocene compounds thereof. Suitable ligands for metal complexes can also include bidentate or higher densities, such as, for example, bipyridine, biimidazole, phenanthroline, or pyridyl (imidazole). Other suitable bidentate ligands can include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher densities ligands can be present in the metal complex to achieve a complete coordination sphere. In certain embodiments, the electron transfer agent is an osmium complex. In certain embodiments, the electron transfer agent is osmium complexed with a bidentate ligand.

[0074] In certain embodiments, the electron transfer agent disclosed herein may include suitable functional groups to facilitate covalent attachment to the polymer (also referred to herein as the polymer backbone) in the active region, as discussed further below. For example, without limitation, the electron transfer agent for use in the present disclosure may include a polymer-bound electron transfer agent, such as a redox polymer. Suitable non-limiting examples of polymer-bound electron transfer agents include those described in U.S. Pat. Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are incorporated herein by reference in their entirety. In certain embodiments, the electron transfer agent is a bidentate osmium complex bound to a polymer (e.g., the polymer backbone described in Section 4 below) described herein. In certain embodiments, the polymer-bound electron transfer agent shown in FIG. 3 of U.S. Pat. No. 8,444,834 (referred to as "X7") may be used in the sensor of the present disclosure.

[0075] In certain embodiments, the glutamate responsive active region can comprise a ratio of glutamate oxidase to redox mediator of about 10:1 to about 1:10, e.g., about 9:1 to about 1:9, about 8:1 to about 1:8, about 7:1 to about 1:7, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, about 1.5:1 to about 1:1.5, or about 1:1.

[0076] 4. Polymer backbone In certain embodiments, one or more active sites for facilitating analyte detection may include a polymer to which an enzyme and / or redox mediator is covalently attached. Any suitable polymer backbone may be present in the active region to facilitate detection of analytes via covalent attachment of enzymes and / or redox mediators to the active region. Non-limiting examples of suitable polymers in the active region include polyvinylpyridine, e.g., poly(4-vinylpyridine), and polyvinylimidazole, e.g., poly(N-vinylimidazole) and poly(1-vinylimidazole), or copolymers thereof, e.g., quaternized pyridine groups serve as attachment points for redox mediators or enzymes. In certain embodiments, the polymer is a poly(4-vinylpyridine) polymer or a derivative thereof. Non-limiting polymers for use in the present disclosure are disclosed in U.S. Pat. No. 8,444,834.

[0077] Exemplary copolymers that may be suitable for inclusion in the active region include those that contain monomer units such as, for example, styrene, acrylamide, methacrylamide, or acrylonitrile. In certain embodiments, the polymer is a copolymer of vinylpyridine and styrene. Further non-limiting examples of polymers that may be present in the active region include those described in U.S. Pat. No. 6,605,200, which is incorporated by reference in its entirety, such as poly(acrylic acid), styrene / maleic anhydride copolymer, methyl vinyl ether / maleic anhydride copolymer (GANTREZ™ polymer), poly(vinylbenzyl chloride), poly(allylamine), polylysine, poly(4-vinylpyridine) quaternized with carboxypentyl groups, and poly(sodium 4-styrenesulfonate). In certain embodiments in which the analyte sensor includes two active sites, the polymers in each active region may be the same or different.

[0078] In certain embodiments, the enzyme of a given active region can be immobilized. In certain embodiments, the enzyme of the active region is covalently bound to a polymer. Alternatively, or in addition, the enzyme of the active region can be non-covalently associated with a polymer such that the non-covalently bound enzyme is physically retained within the polymer. In certain embodiments, the glutamate oxidase present can be covalently bound to a polymer within the glutamate-responsive active region of the disclosed analyte sensors. For example, and without limitation, glutamate oxidase can be covalently bound to a polymer of a redox mediator, i.e., a redox polymer, within the glutamate-responsive active region of the disclosed analyte sensors. In certain embodiments, glutamate oxidase can be non-covalently associated with a polymer.

[0079] In certain embodiments, the covalent attachment of one or more enzymes and / or redox mediators to the polymer in a given active area can occur via crosslinking introduced with a suitable crosslinking agent. Crosslinking agents suitable for reaction with free amino groups in the enzyme (e.g., with free side chain amines in lysine) can include, for example, crosslinkers such as polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuric chloride, glutaraldehyde, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatized variants thereof. In certain embodiments, the crosslinking agent is a polymer having a number average molecular weight (M) of, for example, about 200 to 1,000, e.g., about 400. n ) is PEGDGE. In certain embodiments, the crosslinker is PEGDGE400. In certain embodiments, the crosslinker can be glutaraldehyde. Crosslinkers suitable for reaction with free carboxylic acid groups in the enzyme can include, for example, carbodiimides. In certain embodiments, the crosslinking of the enzyme to the polymer is generally intermolecular. In certain embodiments, the crosslinking of the enzyme to the polymer is generally intramolecular.

[0080] 5. Mass-transfer limiting membrane In certain embodiments, the analyte sensor disclosed herein further comprises a membrane overlying at least one active area of ​​the analyte sensor, e.g., the first active area and / or the second active area. In certain embodiments, the membrane is permeable to the analyte (e.g., glutamate) to be detected in the active area. In certain embodiments, the membrane overlies each of the active areas of the analyte sensor. Alternatively, the first membrane overlies one of the multiple active areas, and the second membrane overlies the second active area. Alternatively, the first membrane overlies one of the multiple active areas, and then the second membrane overlies both the first and second active areas.

[0081] In certain embodiments, the membrane overlying the analyte-responsive active area can function as a mass transport limiting membrane and / or to improve biocompatibility. The mass transport limiting membrane can act as a diffusion limiting barrier to reduce the rate of mass transport of the analyte. For example, but not limited to, using a mass transport limiting membrane to restrict the access of the analyte, such as glutamate, to the analyte-responsive active area can help avoid overloading (saturation) of the sensor, thereby improving detection performance and accuracy.

[0082] In certain embodiments, the mass transport limiting membrane can be homogeneous and single component (e.g., containing a single membrane polymer or a copolymer of two or more polymers). Alternatively, the mass transport limiting membrane can be multicomponent (e.g., containing two or more different membrane polymers, e.g., as a composite). In certain embodiments, the multicomponent membrane can exist as a multilayer membrane, e.g., a bilayer membrane or a trilayer membrane. In certain embodiments, the multicomponent membrane can exist as a homogeneous mixture of two or more membrane polymers. In certain embodiments, the homogeneous mixture can be deposited by combining two or more membrane polymers in a solution and then depositing the solution, e.g., by dip coating, on the working electrode. In certain embodiments, the multilayer membrane can be deposited on the analyte-responsive active region by depositing a first layer, e.g., by dip coating, and depositing a second layer, e.g., by dip coating, on the first layer to produce a bilayer membrane. In certain embodiments, the third layer can be deposited on the second layer, e.g., by dip coating, to produce a trilayer membrane.

[0083] In certain embodiments, the mass transport limiting membrane of the present disclosure can have a neutral charge, hi certain embodiments, a mass transport limiting membrane having a neutral charge allows glutamate to readily diffuse from the interstitial fluid to a glutamate-responsive active area present on the working electrode to generate a concentration-dependent current signal.

[0084] In certain embodiments, the mass transport limiting membrane of the present disclosure can include a hydrophilic polymer. For example, but not limited to, the mass transport limiting membrane can be a single component membrane or a multi-component membrane that includes a hydrophilic polymer.

[0085] In certain embodiments, the polymer can be polyvinylpyridine-co-polystyrene sulfonate or polyvinylimidazole-co-poly(n-isopropylacrylamide), as described herein.

[0086] In certain embodiments, the polymer is a polyurethane. For example, and without limitation, the mass transport limiting membrane can be a single component membrane or a multi-component membrane including polyurethane. In certain embodiments, the polymer (e.g., polyurethane) for use in the present disclosure can absorb about 30% to about 95% (e.g., about 30% to about 70%) of its weight of water. In certain embodiments, the polyurethane can absorb at least about 30% of its weight of water. In certain embodiments, the polyurethane can absorb at least about 40% of its weight of water. In certain embodiments, the polyurethane can absorb at least about 50% of its weight of water. In certain embodiments, the polyurethane can absorb at least about 60% of its weight of water. In certain embodiments, the polyurethane can absorb at least about 70% of its weight of water. In certain embodiments, the polyurethane for use in the present disclosure is a low thermoset. For example, and without limitation, polymers for use in the present disclosure, such as polyurethanes, can be cured at temperatures of about 20° C. to about 90° C., e.g., about 25° C. to about 85° C., about 30° C. to about 80° C., about 35° C. to about 75° C., about 40° C. to about 70° C., about 45° C. to about 65° C., about 20° C. to about 70° C., about 20° C. to about 60° C., about 20° C. to about 50° C., about 30° C. to about 90° C., about 40° C. to about 90° C., about 50° C. to about 90° C., about 60° C. to about 90° C., or about 70° C. to about 90° C. In certain embodiments, polymers for use in the present disclosure (e.g., polyurethanes) have a molecular weight of about 50 to about 500 kDa.

[0087] In certain embodiments, the polyurethane can be a commercially available hydrophilic polyurethane. In certain embodiments, the hydrophilic polyurethane can include the HydroMed™ series of polyurethanes from AdvanSource biomaterials (Wilmington, MA). For example, without limitation, commercially available hydrophilic polyurethanes can include HydroMed™ D1, HydroMed™ D2, HydroMed™ D3, HydroMed™ D4, HydroMed™ D6, HydroMed™ D640, HydroMed™ D7, HydroMed™ Hydroslip C, or combinations thereof. In certain embodiments, the polyurethane can include HydroMed™ D7. In certain embodiments, the polyurethane can include HydroMed™ D1.

[0088] In certain embodiments, the membrane, e.g., a single-component membrane or a multi-component membrane, may include a copolymer of polyurethane. In certain embodiments, the membrane (e.g., a single-component membrane) may include a copolymer of polyurethane and one or more additional polymers (e.g., a second polymer). In certain embodiments, the second polymer is a hydrophilic polymer. In certain embodiments, the polyurethane may be copolymerized with a hydrophilic polymer (e.g., but not limited to, polyethers, polyesters, polyalkenes, polyamines, and polyalkylene oxides). In certain embodiments, the polyurethane is copolymerized with polyethylene oxide, polybutylene oxide, polypropylene oxide, or polytetramethylene oxide.

[0089] In certain embodiments, the mass transport limiting membrane may be a multi-component membrane. For example, but not limited to, the mass transport limiting membrane may be a composite of two or more polymers, for example, three or more, four or more, or five or more polymers. In certain embodiments, the mass transport limiting membrane may be a composite of at least two polymers. In certain embodiments, the mass transport limiting membrane is a composite of a polyurethane polymer or its copolymer and a second polymer. In certain embodiments, the mass transport limiting membrane may be a homogenous mixture of a polyurethane polymer or its copolymer and a second polymer. In certain embodiments, the mass transport limiting membrane may have a multi-layer configuration, each layer containing a different polymer. For example, but not limited to, the mass transport limiting membrane may be composed of at least two layers, each layer containing a different polymer. In certain embodiments, the mass transport limiting membrane may be composed of three layers, at least two of the three layers containing different polymers.

[0090] In certain embodiments, the second polymer is a fluoropolymer. In certain embodiments, the second polymer for use in the mass transport limiting membrane of the present disclosure is an ion exchange polymer. In certain embodiments, the ion exchange polymer is a short side chain ion exchange polymer. In certain embodiments, the ion exchange polymer is a long side chain ion exchange polymer. In certain embodiments, the ion exchange polymer is a perfluorosulfonic acid (PFSA) polymer or a perfluorocarboxylic acid (PFCA) polymer. In certain embodiments, the ion exchange polymer is a PFSA polymer. In certain embodiments, the ion exchange polymer has a softening point of greater than about 75°C, greater than about 80°C, greater than about 85°C, greater than about 90°C, or greater than about 95°C.

[0091] In certain embodiments, the ion exchange polymer can be a commercially available polymer. For example, but not limited to, the ion exchange polymer can be a polymer from the AQUIVION® product line from Solvay and / or a polymer from the NAFION® product line from Sigma Aldrich. In certain embodiments, the short side chain ion exchange polymer is an AQUIVION® product. In certain embodiments, the long side chain ion exchange polymer is a NAFION® product.

[0092] In certain embodiments, the second polymer is a polyvinylpyridine-based polymer. In certain embodiments, the polyvinylpyridine can be poly(2-vinylpyridine) or poly(4-vinylpyridine). In certain embodiments, the second polymer can be a copolymer of a polyvinylpyridine-based polymer or a derivative thereof. For example, without limitation, the second polymer can be a copolymer of a polyvinylpyridine-based polymer or a derivative thereof with styrene. In certain embodiments, the styrene can be derivatized (e.g., sulfonated). In certain embodiments, the second polymer can be a derivatized polyvinylpyridine-co-styrene copolymer. In certain embodiments, the second polymer is polyvinylpyridine-co-polystyrene sulfonate. In certain embodiments, the polyvinylpyridine-co-polystyrene sulfonate polymer comprises about 15% to about 50% polystyrene sulfonate per molar content. Non-limiting examples of polyvinylpyridine-based polymers are disclosed in US Patent Publication No. 2003 / 0042137 (e.g., Formula 2b), the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the second polymer can be the 10Q5 polymer described in US Patent No. 8,761,857, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, the polyvinylpyridine-based polymer has a molecular weight of about 50 to about 300 kDa.

[0093] In certain embodiments, the second polymer is a polyvinylimidazole-based polymer. In certain embodiments, the second polymer can be a copolymer of a polyvinylimidazole-based polymer or a derivative thereof. For example, but not limited to, the second polymer can be a copolymer of a polyvinylimidazole-based polymer or a derivative thereof and isopropylacrylamide. In certain embodiments, the polyvinylimidazole-based polymer comprises about 30% to about 70% polyvinylimidazole per molar content. In certain embodiments, the second polymer is polyvinylimidazole-co-poly(n-isopropylacrylamide). In certain embodiments, the polyvinylimidazole-based polymer has a molecular weight of about 50 to about 300 kDa.

[0094] In certain embodiments, the membrane comprises at least about 10% polyurethane or copolymer thereof. For example, and without limitation, the membrane comprises at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% polyurethane or copolymer thereof. In certain embodiments, the membrane comprises from about 1% to about 100% polyurethane or copolymers thereof, e.g., from about 1% to about 95%, from about 1% to about 90%, from about 1% to about 85%, from about 1% to about 80%, from about 1% to about 75%, from about 1% to about 70%, from about 1% to about 65%, from about 1% to about 60%, from about 1% to about 55%, from about 1% to about 50%, from about 1% to about 45%, from about 1% to about 40%, from about 1% to about 35%, from about 1% to about 30%, from about 1% to about 25%, from about 1% to about 20%, from about 1% to about 15%, from about 1% to about 10%, from about 5% to about 100%, from about 10% to about 100%, from about 15% to about 20%. The membrane may comprise about 100%, about 20% to about 100%, about 25% to about 100%, about 30% to about 100%, about 35% to about 100%, about 40% to about 100%, about 45% to about 100%, about 50% to about 100%, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 95% to about 100%, about 20% to about 80%, about 30% to about 70%, or about 40% to about 60% polyurethane or copolymer thereof. In certain embodiments, the membrane comprises about 1% to about 30% polyurethane or copolymer thereof.

[0095] In certain embodiments, the membrane, e.g., the multi-component membrane, comprises at least about 10% additional polymer. For example, but not limited to, the membrane comprises at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% additional polymer, e.g., a second polymer, e.g., an ion exchange polymer. In certain embodiments, the membrane comprises from about 1% to about 95% polyurethane or copolymer thereof, e.g., from about 1% to about 90%, from about 1% to about 85%, from about 1% to about 80%, from about 1% to about 75%, from about 1% to about 70%, from about 1% to about 65%, from about 1% to about 60%, from about 1% to about 55%, from about 1% to about 50%, from about 1% to about 45%, from about 1% to about 40%, from about 1% to about 35%, from about 1% to about 30%, from about 1% to about 25%, from about 1% to about 20%, from about 1% to about 15%, from about 1% to about 10%, from about 5% to about 95%, from about 10% to about 95%, from about 1 The membrane may comprise 5% to about 95%, about 20% to about 95%, about 25% to about 95%, about 30% to about 95%, about 35% to about 95%, about 40% to about 95%, about 45% to about 95%, about 50% to about 95%, about 55% to about 95%, about 60% to about 95%, about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 95% to about 95%, about 20% to about 80%, about 30% to about 70%, or about 40% to about 60% polyurethane or copolymer thereof. In certain embodiments, the membrane comprises about 1% to about 30% of an additional polymer, e.g., a second polymer. For example, and without limitation, the membrane comprises about 1% to about 30%, e.g., about 1% to about 25% or about 1% to about 20% ion exchange polymer. In certain embodiments, the membrane comprises about 1% to about 20% additional polymer, e.g., a second polymer. In certain embodiments, the membrane comprises about 1% to about 10% additional polymer, e.g., a second polymer.In certain embodiments, the membrane comprises about 1% to about 10%, e.g., about 2% to about 9%, about 3% to about 8%, about 4% to about 8%, or about 5% to about 8% of a polyvinylpyridine-based polymer or copolymer thereof or a derivative of a polyvinylpyridine-based copolymer. In certain embodiments, the second polymer is present in a layer separate from the layer comprising polyurethane.

[0096] In certain other embodiments, the membrane polymer overlying one or more active regions may be crosslinked with a branched crosslinker, which may reduce the amount of extractables that may be obtained from the mass transport limiting membrane. Non-limiting examples of branched crosslinkers include branched glycidyl ether crosslinkers, such as branched glycidyl ether crosslinkers that contain three or more crosslinkable groups, such as polyethylene glycol tetraglycidyl ether.

[0097] In certain embodiments, polydimethylsiloxane (PDMS) can be incorporated into any of the mass transfer limiting membranes disclosed herein.For example, but not limited to, PDMS can be incorporated into the multi-component mass transfer limiting membranes disclosed herein.In certain embodiments, PDMS can be incorporated into the multi-component mass transfer limiting membranes comprising polyvinylpyridine-based polymers or their copolymers or derivatives of polyvinylpyridine copolymers, such as polyvinylpyridine, 10Q5 and / or polyvinylpyridine-co-polystyrenesulfonate.

[0098] In certain embodiments, the composition of a mass transport limiting membrane disposed on an analyte sensor having two active regions may be the same or different when the mass transport limiting membrane overlies each active region. For example, but not by way of limitation, the portion of the mass transport limiting membrane overlying the glutamate-responsive active region may be multicomponent and / or the portion of the mass transport limiting membrane overlying the second analyte-responsive active region may be single component. Alternatively, the portion of the mass transport limiting membrane overlying the glutamate-responsive active region may be single component and / or the portion of the mass transport limiting membrane overlying the second analyte-responsive active region may be multicomponent.

[0099] In certain embodiments of the present disclosure, the glutamate-responsive active area can be overcoated with a single-component membrane comprising polyurethane (or a copolymer thereof) and the second active area responsive to a second analyte can be overcoated with a multi-component membrane comprising polyvinylpyridine and / or polyvinylpyridine-co-styrene copolymer. Alternatively, the glutamate-responsive active area can be overcoated with a multi-component membrane comprising polyurethane (or a copolymer thereof) and a second polymer (e.g., an ion-exchange polymer or a polyvinylpyridine-based polymer), either as a bi- or tri-layer membrane or homogenous mixture, and the active area responsive to the second analyte can be overcoated with a single-component membrane comprising polyvinylpyridine or polyvinylpyridine-co-styrene copolymer.

[0100] In certain embodiments, the portion of the mass transport limiting membrane overlying the glutamate-responsive active area and the portion of the mass transport limiting membrane overlying the second analyte-responsive active area can both be single component but can comprise different polymers. For example, and without limitation, the glutamate-responsive active area can be overlying with a single component membrane comprising polyurethane (or a copolymer thereof) and the second active area responsive to the second analyte can be overlying with a single component membrane comprising polyvinylpyridine or a polyvinylpyridine-co-styrene copolymer.

[0101] In certain embodiments, the portion of the mass transport limiting membrane overlying the glutamate-responsive active area and the portion of the mass transport limiting membrane overlying the second analyte-responsive active area can both be multicomponent, but comprise different polymers. For example, but not limited to, the glutamate-responsive active area can be overlying with a multicomponent membrane comprising polyurethane (or a copolymer thereof) and a second polymer, such as an ion exchange polymer or a polyvinylpyridine-based polymer, and the second active area responsive to the second analyte can be overlying with a multicomponent membrane comprising polyvinylpyridine and / or a polyvinylpyridine-co-styrene copolymer.

[0102] In certain embodiments, when a first active area and a second active area configured to analyze different analytes are disposed on separate working electrodes, the mass transport limiting membrane can have different permeability values ​​for the first analyte and the second analyte. For example, without limitation, the mass transport limiting membrane overlying at least one of the multiple active areas can include a mixture of a first membrane polymer and a second membrane polymer, a bilayer of a first membrane polymer (e.g., the first membrane) and a second membrane polymer (e.g., the second membrane), or a trilayer of a first membrane polymer (e.g., the first membrane), a second membrane polymer (e.g., the second membrane), and a third membrane polymer (e.g., the third membrane). In certain embodiments, the third membrane polymer is the same as the first or second membrane polymer. A homogeneous membrane can overlying an active area that is not overlying with a mixture or bilayer, the homogeneous membrane including only one of the first membrane polymer or the second membrane polymer. Advantageously, the structure of the analyte sensor disclosed herein readily allows for a continuous membrane having a homogeneous membrane portion to be disposed on a first active area of ​​the analyte sensor, and for a multi-component membrane portion to be disposed on a second active area of ​​the analyte sensor, thereby simultaneously equalizing the transmittance values ​​of each analyte to provide improved sensitivity and detection accuracy. The continuous membrane deposition can be performed, in certain embodiments, by sequential dip-coating operations.

[0103] In certain embodiments, the analyte sensors described herein can include a sensor tail including at least a first working electrode, a first active area disposed on a surface of the first working electrode, and a mass transport limiting membrane permeable to the first analyte overlying at least the first active area. In certain embodiments, the first active area includes an enzyme system responsive to a first analyte, e.g., glutamate, including at least one enzyme responsive to the first analyte, e.g., glutamate oxidase. For example, without limitation, the analyte sensors described herein can include a sensor tail including at least a first working electrode, a glutamate-responsive active area including an enzyme system including glutamate oxidase disposed on a surface of the first working electrode, and a mass transport limiting membrane permeable to glutamate overlying the glutamate-responsive active area. In certain embodiments, the mass transport limiting membrane includes polyurethane or a copolymer thereof. In certain embodiments, the mass transport limiting membrane further comprises a second polymer, for example, an ion exchange polymer or a polyvinylpyridine-based polymer.

[0104] In certain embodiments, the analyte sensor of the present disclosure may include a second active region configured to detect the same or a different analyte as the first active region. In certain embodiments, at least a portion of the mass transport limiting membrane overlying the first active region may overlying the second active region. Alternatively, or in addition, a second mass transport limiting membrane may be used to overlying the second active region. In certain embodiments, at least a portion of the second mass transport limiting membrane overlying the second active region may overlying the first active region. In certain embodiments, the mass transport limiting membrane overlying the first active region is of a different composition than the second mass transport limiting membrane.

[0105] In certain embodiments, the mass transport limiting membrane has a thickness of about 5 μm to about 100 μm, for example, about 10 μm to about 90 μm, about 10 μm to about 80 μm, about 10 μm to about 70 μm, about 10 μm to about 60 μm, about 10 μm to about 50 μm, about 10 μm to about 40 μm, about 10 μm to about 30 μm, about 10 μm to about 20 μm, about 10 μm to about 15 μm, about 10 μm to about 100 μm. , about 20 μm to about 100 μm, about 30 μm to about 100 μm, about 40 μm to about 100 μm, about 50 μm to about 100 μm, about 60 μm to about 100 μm, about 70 μm to about 100 μm, about 80 μm to about 100 μm, about 90 μm to about 100 μm, about 20 μm to about 60 μm, or about 30 μm to about 50 μm, e.g., a total thickness, e.g., a dry thickness.

[0106] In certain embodiments where the mass transport limiting membrane comprises a single polymer, e.g., polyurethane, e.g., a single layer of one polymer, the mass transport limiting membrane can have a thickness, e.g., total thickness, e.g., dry thickness, of about 10 μm to about 30 μm. In certain embodiments where the mass transport limiting membrane comprises two or more polymers, e.g., polyurethane and a second polymer (e.g., a single layer comprising two polymers or a multilayer membrane comprising two polymers), the mass transport limiting membrane can have a thickness, e.g., total thickness, e.g., dry thickness, of about 30 μm to about 50 μm. In certain embodiments, the polyurethane layer of the mass transport limiting membrane disclosed herein has a thickness, e.g., dry thickness, of about 10 μm to about 30 μm. In certain embodiments, the layer comprising the second polymer, e.g., an ion exchange polymer and / or a polyvinylpyridine-based polymer, described herein, can have a thickness, e.g., dry thickness, of about 10 μm to about 40 μm, e.g., about 15 μm to about 35 μm.

[0107] In certain embodiments, the mass transport limiting membrane of the present disclosure can include a single layer of polyurethane polymer. In certain embodiments, the mass transport limiting membrane of the present disclosure can include two or more layers of polyurethane polymer, for example, two, three, or four layers of polyurethane polymer. In certain embodiments, the mass transport limiting membrane of the present disclosure can include a single layer of a second polymer, for example, an ion exchange polymer, as described herein. In certain embodiments, the mass transport limiting membrane of the present disclosure can include two or more layers of a second polymer, for example, two, three, or four layers of a second polymer (e.g., an ion exchange polymer). In certain embodiments, the mass transport limiting membrane can have alternating layers of polyurethane polymer and layers of a second polymer.

[0108] 6.Manufacturing The present disclosure further provides a method for manufacturing an analyte sensor of the present disclosure comprising one or more active sites. In certain embodiments, the method includes producing a working electrode, e.g., a carbon electrode, e.g., by carbon printing.

[0109] In certain embodiments, the method may further include adding a composition comprising one or more enzymes onto the surface of the working electrode to generate active sites on the working electrode, e.g., in a pattern as described herein. For example, but not limited to, the composition may include glutamate oxidase. In certain embodiments, the composition may further include a crosslinker, e.g., polyethylene glycol diglycidyl ether, a buffer, e.g., HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), and / or a stabilizer, e.g., serum albumin. In certain embodiments, the composition may further include a redox mediator, e.g., a redox polymer. In certain embodiments, the method may further include curing, e.g., thermally curing, the enzyme composition.

[0110] In certain embodiments, the method can further include adding one or more membrane compositions onto the cured enzyme composition, for example, by dip coating. In certain embodiments, the membrane composition can include a single polymer, for example, polyurethane or its copolymer. In certain embodiments, the membrane composition can include two or more polymers. For example, but not limited to, the membrane composition can include a first polymer, for example, polyurethane or its copolymer, and a second polymer, for example, an ion exchange polymer, for example, as a mixture or multilayer film.

[0111] In certain embodiments, the method can include adding a first layer comprising a polyurethane or copolymer thereof over the cured enzyme composition, for example, by dip coating. In certain embodiments, the method can include adding a layer comprising a second polymer over the first layer comprising a polyurethane or copolymer thereof, for example, by dip coating. In certain embodiments, the method can include adding a second layer comprising a polyurethane or copolymer thereof over the second polymer layer, for example, by dip coating, to produce a three-layer membrane composition.

[0112] In certain embodiments, the method can include curing the film composition. III.How to use The present disclosure further provides a method of using the analyte sensor disclosed herein. In certain embodiments, the present disclosure provides a method for detecting glutamate in a subject in need thereof. In certain embodiments, the subject in need of glutamate monitoring can be a subject at risk of developing or has developed one or more disorders and / or symptoms associated with glutamate dysregulation. For example, but not limited to, the subject in need of glutamate monitoring can be a subject at risk of developing or has developed one or more disorders and / or symptoms associated with elevated glutamate levels as described herein. In certain embodiments, the subject in need of glutamate monitoring can be a subject at risk of developing or has developed one or more disorders and / or symptoms associated with glutamate deficiency as described herein.

[0113] In certain embodiments, the glutamate sensor of the present disclosure can be used to continuously monitor glutamate levels in subjects at risk for or having a neurological disorder or injury, such as Parkinson's disease, multiple sclerosis (MS), Alzheimer's disease, stroke, amyotrophic lateral sclerosis or Lou Gehrig's disease (ALS), and traumatic brain injury. Further examples of diseases and disorders associated with glutamate dysregulation are disclosed in Li et al., Frontiers in Psychiatry 9:767 (2019); Guerriero et al., Curr. Neurol. Neurosci. Rep., 15:27 (2015); and Miladinovic et al., Biomolecules, 5(4):3112-3141 (2015), the contents of which are incorporated herein by reference in their entireties.

[0114] In certain embodiments, a method for detecting glutamate (e.g., in a subject in need thereof) includes the steps of: (i) providing an analyte sensor comprising: (a) a sensor tail comprising at least a first working electrode; (b) a glutamate-responsive active region disposed on a surface of the first working electrode and responsive to glutamate, the glutamate-responsive active region comprising glutamate oxidase and, optionally, a first polymer and / or electron transfer agent, e.g., a redox polymer; and (c) a mass transport limiting membrane permeable to glutamate overlying the glutamate-responsive active region; (ii) applying a potential to the first working electrode; (iii) obtaining a first signal equal to or greater than the redox potential of the glutamate-responsive active region, the first signal being proportional to a concentration of glutamate in a fluid contacting the glutamate-responsive active region; and (iv) correlating the first signal to the concentration of glutamate in the fluid. In certain embodiments, the mass transport limiting membrane can include a polymer, such as polyurethane, or a copolymer thereof. In certain embodiments, the mass transport limiting membrane can include a polymer, such as polyurethane, or a copolymer thereof, and a second polymer, such as an ion exchange polymer.

[0115] In certain embodiments, a method of the present disclosure may include (i) exposing an analyte sensor to a fluid (e.g., a body fluid containing glutamate), the analyte sensor including (a) a sensor tail including at least a first working electrode; (b) a glutamate-responsive active region responsive to glutamate disposed on a surface of the first working electrode, the glutamate-responsive active region including glutamate oxidase and optionally a first polymer and / or electron transfer agent, e.g., a redox polymer; and (c) a mass transport limiting membrane permeable to glutamate overlying the glutamate-responsive active region; (ii) applying a potential to the first working electrode; (iii) obtaining a first signal equal to or greater than a redox potential of the glutamate-responsive active region, the first signal being proportional to a concentration of glutamate in the fluid; and (iv) correlating the first signal to a concentration of glutamate in the fluid. In certain embodiments, the mass transport limiting membrane can include a polymer, such as polyurethane, or a copolymer thereof. In certain embodiments, the mass transport limiting membrane can include a polymer, such as polyurethane, or a copolymer thereof, and a second polymer, such as an ion exchange polymer.

[0116] In certain embodiments, the present disclosure further provides a method for detecting glutamate and a second analyte. For example, but not limited to, the method of the present disclosure may further include providing an analyte sensor comprising a second active area and / or detecting a second analyte by exposing the analyte sensor comprising a second active area to a fluid (e.g., a body fluid comprising glutamate and a second analyte). In certain embodiments, the analyte sensor used in the method for detecting glutamate and a second analyte may further include a second working electrode and a second active area disposed on the surface of the second working electrode and responsive to a second analyte different from the first analyte, the second active area comprising at least one enzyme responsive to the second analyte and optionally a second polymer and / or an electron transfer agent, and a portion of the mass transfer limiting membrane, e.g., the second portion, overlies the second active area. Alternatively, the second active site may be covered by a second mass transport limiting membrane that is separate and / or different from the mass transport limiting membrane overlying the glutamate-responsive active region.

[0117] In certain embodiments, the glutamate sensors of the present disclosure can be used for up to about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 20 days. In certain embodiments, the glutamate sensors of the present disclosure can be used for up to about 15 days.

[0118] The present disclosure is further illustrated by the following embodiments. [1] An analyte sensor comprising: (i) a sensor tail including at least a first working electrode; (ii) a glutamate-responsive active region disposed on a surface of the first working electrode, the glutamate-responsive active region including glutamate oxidase and an electron transfer agent; and (iii) a mass transport limiting membrane permeable to glutamate overlying at least a portion of the glutamate-responsive active region.

[0119] [2] The analyte sensor of [1], wherein the glutamate-responsive active region further comprises a polymer, and the glutamate oxidase and / or the electron transfer agent are crosslinked to the polymer. [3] The analyte sensor of [1] or [2], wherein the glutamate-responsive activity further comprises a stabilizer.

[0120] [4] The analyte sensor of [3], wherein the stabilizer comprises albumin. [5] The analyte sensor according to any one of [1] to [4], wherein the mass transport limiting membrane comprises polyurethane or a copolymer thereof.

[0121] [6] The analyte sensor of [5], wherein the mass transport limiting membrane further comprises an ion exchange polymer or a polyvinylpyridine-based polymer. [7] The analyte sensor according to any one of [1] to [6], comprising: (iv) a second working electrode; and (v) a second active area disposed on a surface of the second working electrode and responsive to a second analyte other than glutamate, the second active area comprising at least one enzyme responsive to the second analyte, wherein a second portion of the mass transport limiting membrane covers the second active area from above.

[0122] [8] A method for detecting glutamate comprising: (i) providing an analyte sensor comprising: (a) a sensor tail including at least a first working electrode; (b) a glutamate-responsive active region disposed on a surface of the first working electrode, the glutamate-responsive active region including glutamate oxidase and an electron transfer agent; and (c) a mass transport limiting membrane permeable to glutamate overlying at least a portion of the glutamate-responsive active region; (ii) applying a potential to the first working electrode; (iii) obtaining a first signal equal to or greater than a redox potential of the glutamate-responsive active region, the first signal being proportional to a concentration of glutamate in a fluid contacting the glutamate-responsive active region; and (iv) correlating the first signal to the concentration of glutamate in the fluid.

[0123] [9] The method according to [8], wherein the glutamate responsive active region further comprises a polymer.

[10] The method of [9], wherein the glutamate oxidase and / or the electron transfer agent are covalently attached to the polymer at a glutamate-responsive active region.

[0124]

[11] Any one of the methods [8] to

[10] , wherein the glutamate responsive active substance further comprises a stabilizer.

[12] The method according to

[11] , wherein the stabilizer is albumin.

[0125]

[13] Any one of the methods [8] to

[12] , wherein the mass transport limiting membrane comprises polyurethane or a copolymer thereof.

[14] The method of

[13] , wherein the mass transport limiting membrane further comprises an ion exchange polymer or a polyvinylpyridine-based polymer.

[0126]

[15] Any one of the methods according to [8] to

[14] , wherein the analyte sensor further comprises: (d) a second working electrode; and (e) a second active area disposed on a surface of the second working electrode and responsive to a second analyte other than glutamate, the second active area comprising at least one enzyme responsive to the second analyte; and wherein a second portion of the mass transport limiting membrane covers the second active area from above.

[0127]

[16] Any one of the methods [8] to

[15] , wherein the fluid is interstitial fluid.

[17] Any one of the methods of [8]-

[16] , wherein the analyte sensor is implanted in a subject at risk of having or having a neurological condition.

[0128]

[18] The method according to

[17] , wherein the neurological condition is brain damage.

[19] The method described in

[18] , wherein the brain injury is traumatic brain injury.

[20] The method according to

[19] , wherein the traumatic brain injury is stroke and / or intracerebral hemorrhage.

[0129]

[21] Any one of the methods [8]-

[20] , wherein an analyte sensor is implanted in a subject for approximately 15 days.

[22] Any one of the methods [8] to

[21] , wherein the analyte sensor retains at least about 90% sensitivity during its use.

[0130]

[23] The analyte sensor of any one of [1] to [7] for use in monitoring a subject at risk of having or having a neurological condition. Working Example The presently disclosed subject matter will be better understood by reference to the following examples, which are provided by way of illustration of the presently disclosed subject matter and not by way of limitation.

[0131] Example 1: Glutamate Sensor This embodiment provides a sensor for detecting glutamate. As shown in Figure 6, the sensor includes glutamate oxidase and a redox mediator to detect L-glutamate in a sample. The chemical composition for the glutamate-responsive active region of the sensor includes glutamate oxidase, a stabilizer, and a crosslinker.

[0132] The glutamate sensor was produced by depositing and thermally curing a composition containing the ingredients provided in Table 1 onto a working electrode. The glutamate sensor was subsequently tested by sequentially adding various glutamate concentrations in the presence of 100 mM phosphate buffered saline (PBS) at physiological pH (7.4) and 33°C. Figure 7, which provides the chronoamperometric response of the glutamate sensor, shows that when a concentration of glutamate was introduced into the supporting electrolyte solution coating the glutamate sensor, electrons generated as a result of the enzymatic process led to a current increase over several minutes until a stable current was achieved. The current recorded for the glutamate sensor was dependent on glutamate concentration, indicating that the glutamate-responsive region, which contains glutamate oxidase and redox mediator, is well suited to detect glutamate levels in samples.

[0133] [Table 1]

[0134] To assess the stability of the sensor, the glutamate sensor was recalibrated at different time points under the same test conditions. Figure 8 shows the current response vs. glutamate concentration plots obtained at two different time points. As shown in Figure 8, the glutamate sensor exhibited stability for 5 days without the presence of a mass transport limiting membrane, with less than 10% sensitivity loss observed over the tested period.

[0135] Example 2: Mass transport limiting membrane The addition of a mass transport limiting membrane can improve the linear concentration range of the sensor for target molecules by controlling the analyte diffusion rate into the glutamate-sensitive layer. This mass transport membrane can also act as a protector for the glutamate-responsive region against mechanical and chemical stressors. The composition of the mass transport limiting membrane can be tailored for different analyte sensitivity targets.

[0136] In this example, polyurethane-based membranes were tested as mass transport limiting membranes. Polyurethane-based membranes can offer several advantages to glutamate sensors over other membrane polymers. For example, polyurethane membranes do not have positively or negatively charged functional groups, such as polyvinylpyridine and 10Q5. This charge neutrality allows glutamate (a molecule with one positively charged group and two negatively charged groups) to easily diffuse into the glutamate-sensitive layer to generate a concentration-dependent current signal. In addition, polyurethane membranes do not require a crosslinker to form a hydrogel, and the density of the hydrogel is primarily dictated by the concentration of the PU solution and its solvent system. Due to these properties, polyurethane membranes can offer a wider working range for glutamate detection compared to polyvinylpyridine-based membranes.

[0137] Membranes containing polyurethane (PU) polymers (e.g., HydroMed™ D1 or D7 polymers) or composite membranes containing PU and ion-exchange polymers were tested by coating the glutamate sensing layer described in Example 1 with the polymers by dip coating. Short and long side chain ion-exchange polymers such as AQUIVION® (Solvay) and NAFION® (Sigma Aldrich), respectively, were used in the composite membranes. The polymer concentrations were 7.5% (w / v%) for HydroMed™ D1, 20% (w / v%) for NAFION®, and 25% (w / v%) for AQUIVION®. The single layer HydroMed™ D1 membrane had a dry thickness of about 11 μm. A HydroMed™ D1 / AQUIVION™ composite membrane with a three-layer configuration was produced by dip coating, with the first HydroMed™ D1 layer having a dry thickness of about 11 μm, the AQUIVION™ layer having a dry thickness of about 22.5 μm, and the second HydroMed™ D1 layer having a dry thickness of about 11 μm. A HydroMed™ D1 / NAFION™ composite membrane with a three-layer configuration was produced by dip coating, with the first HydroMed™ D1 layer having a dry thickness of about 11 μm, the NAFION™ layer having a dry thickness of about 18 μm, and the second HydroMed™ D1 layer having a dry thickness of about 11 μm.

[0138] Figures 9 and 10 show the current response curves of different membrane composites coated on the glutamate sensing layer. The coated glutamate sensors were tested at 33°C over the same glutamate concentration range in 100 mM PBS. As shown in Figures 9 and 10, the highest analyte sensitivity was obtained with a PU membrane (HydroMed™, AdvanSource Biomaterials). In addition, the sensitivity of the sensor was tuned by making composite membranes containing polyurethane and AQUIVION® or NAFION® (Figures 9 and 10). As shown in Figure 11, the sensor coated with HydroMed™ D1 membrane with a dry thickness of about 11 μm showed better stability compared to the sensor without the membrane. Figure 11 shows the calibration plots obtained on days 1, 6 and 12 within the same glutamate concentration range. The sensor nearly retained its sensitivity over the 12 days, with a loss of sensitivity of <10%.

[0139] A comparison of different PU polymers was also performed. As shown in FIG. 12, the PU polymer HydroMed™ D7 was compared to the PU polymer HydroMed™ D1. As shown in FIG. 12, the sensor coated with HydroMed™ D7 showed a decrease in glutamate sensitivity compared to the sensor coated with HydroMed™ D1. HydroMed™ D7 showed a lower water absorption capacity compared to HydroMed™ D1 (30% vs. 70% water absorption capacity, respectively), and without being limited to a particular theory, this change in absorption capacity may be the reason for the decrease in glutamate sensitivity compared to the sensor coated with HydroMed™ D1. However, the 10-day stability data suggested that the sensor with HydroMed™ D7 membrane was more stable (<6% decrease) than the one coated with HydroMed™ D1 (25% decrease).

[0140] Additional PU composite membranes were tested. As mentioned above, 10Q5 and PVP polymers cannot be used as single-component mass-transport limiting membranes because the resulting sensor is not sensitive enough to detect physiologically relevant glutamate concentrations. However, it was tested whether a composite membrane containing PU and a polyvinylpyridine-based polymer, such as polyvinylpyridine (PVP) or 10Q5, could be used to coat a glutamate sensor. Such a glutamate sensor was produced by depositing the glutamate sensing chemistry of Example 1 on a substrate. The sensor was then coated with a 75 mg / ml HydroMed™ D1 solution, followed by a 65 mg / ml PVP or 80 mg / ml 10Q5 solution. Both the PVP and 10Q5 membrane solutions contained 10% PEGDGE crosslinker at 5% v / v. As shown in Figures 13 and 14, both 10Q5 and PVP can be used to obtain functional sensors in the presence of HydroMed™ D1 inner membrane. Although PU-10Q5 and PU-PVP composite membranes reduced glutamate sensitivity (Figures 13 and 14), they improved sensor stability over 14 days of continuous operation. The change in sensitivity of the sensor over 14 days was <20%, <4% and <2% for HydroMed™ D1, HydroMed™ D1-PVP and HydroMed™ D1-10Q5 membrane compositions, respectively.

[0141] The data in this example show that PU-based membranes can improve the sensitivity and stability of glutamate sensors. Although the subject matter of the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the disclosed subject matter. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, and compositions of matter, methods and processes described herein.

[0142] As one skilled in the art will readily appreciate from the disclosed subject matter of the presently disclosed subject matter, any existing or later developed process, machine, manufacture, composition of matter, method, or step that performs substantially the same function or achieves substantially the same result as the corresponding embodiments described herein can be utilized in accordance with the presently disclosed subject matter, and it is therefore intended that the appended claims include within their scope such processes, machines, manufacture, compositions of matter, methods, or steps.

[0143] Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the inventions of which are incorporated herein by reference in their entireties for all purposes.

Claims

1. (i) a sensor tail including at least a first working electrode; (ii) a glutamate-responsive active area disposed on a surface of the first working electrode, the glutamate-responsive active area comprising glutamate oxidase and an electron transfer agent; (iii) a mass transport limiting membrane permeable to glutamate overlying at least a portion of the glutamate-responsive active region; and an analyte sensor comprising:

2. 10. The analyte sensor of claim 1, wherein the glutamate-responsive active region further comprises a polymer, and the glutamate oxidase and / or the electron transfer agent are cross-linked to the polymer.

3. The analyte sensor of claim 1 or 2, wherein the glutamate-responsive active region further comprises a stabilizer.

4. The analyte sensor of claim 3 , wherein the stabilizer comprises albumin.

5. The analyte sensor of claim 1 or 2, wherein the mass transport limiting membrane comprises polyurethane or a copolymer thereof.

6. The analyte sensor of claim 5 , wherein the mass transport limiting membrane further comprises an ion exchange polymer or a polyvinylpyridine-based polymer.

7. (iv) a second working electrode; and (v) a second active area disposed on the surface of the second working electrode and responsive to a second analyte different from glutamate, the second active area comprising at least one enzyme responsive to the second analyte; further comprising The analyte sensor of claim 1 or 2, wherein the second portion of the mass transport limiting membrane overlies the second active area.

8. A method for detecting glutamate, (i) (a) a sensor tail including at least a first working electrode; (b) a glutamate-responsive active region disposed on a surface of the first working electrode, the glutamate-responsive active region comprising glutamate oxidase and an electron transfer agent; (c) a mass transport limiting membrane permeable to glutamate overlying at least a portion of the glutamate-responsive active region; providing an analyte sensor comprising: (ii) applying a potential to the first working electrode; (iii) obtaining a first signal equal to or greater than the redox potential of the glutamate-responsive active region, the first signal being proportional to the concentration of glutamate in a fluid in contact with the glutamate-responsive active region; (iv) correlating the first signal to the concentration of glutamate in the fluid; A method comprising:

9. The method of claim 8 , wherein the glutamate responsive active region further comprises a polymer.

10. 10. The method of claim 9, wherein the glutamate oxidase and / or the electron transfer agent are covalently attached to the polymer at the glutamate-responsive active region.

11. The method of any one of claims 8 to 10, wherein the glutamate-responsive active region further comprises a stabilizer.

12. The method of claim 11 , wherein the stabilizer is albumin.

13. The method of any one of claims 8 to 10, wherein the mass transport limiting membrane comprises polyurethane or a copolymer thereof.

14. 14. The method of claim 13, wherein the mass transport limiting membrane further comprises an ion exchange polymer or a polyvinylpyridine-based polymer.

15. the analyte sensor (d) a second working electrode; (e) a second active area disposed on the surface of the second working electrode and responsive to a second analyte different from glutamate, the second active area including at least one enzyme responsive to the second analyte; further comprising The method of any one of claims 8 to 10, wherein the second portion of the mass transport limiting membrane overlies the second active area.

16. The method according to any one of claims 8 to 10, wherein the fluid is interstitial fluid.

17. The method of any one of claims 8 to 10, wherein the analyte sensor is implanted in a subject at risk of having or having a neurological condition.

18. 18. The method of claim 17, wherein the neurological condition is brain injury.

19. 19. The method of claim 18, wherein the brain injury is a traumatic brain injury.

20. 20. The method of claim 19, wherein the traumatic brain injury is a stroke and / or intracerebral hemorrhage.

21. The method of any one of claims 8 to 10, wherein the analyte sensor is implanted in the subject for about 15 days.

22. The method of any one of claims 8 to 10, wherein the analyte sensor retains at least about 90% sensitivity during its use.

23. 3. The analyte sensor of claim 1 or 2 for use in monitoring a subject at risk of having or having a neurological condition.

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