Lactate sensors and associated methods

Abstract

To provide a lactate-responsive analyte sensor that provides high sensitivity and stable response to lactate.SOLUTION: Such analyte sensors may include: a working electrode having an active area disposed thereon; and a mass transport limiting membrane overcoating at least the active area upon the working electrode. The active area comprises at least a polymer, an albumin, and a lactate-responsive enzyme that is covalently bonded to the polymer. The mass transport limiting membrane may comprise at least a cross-linked polyvinylpyridine homopolymer or copolymer.SELECTED DRAWING: Figure 9

Description

Technical Field

[0001] The present invention relates to a lactate sensor and related methods.

Background Art

[0002] The detection of various specimens in an individual can sometimes be essential for monitoring their health status. Deviations from normal specimen concentrations often indicate underlying physiological conditions such as metabolic states or diseases, or exposure to specific environmental factors or stimuli. For example, glucose levels may be particularly important for detecting and monitoring individuals with diabetes.

[0003] Lactate is another analyte whose in vivo levels can vary in response to a number of environmental or physiological factors, including, for example, diet, stress, exercise, sepsis or septic shock, infections, hypoxia, the presence of cancerous tissue, etc. In the case of chronic lactate changes (e.g., disease), lactate levels may change slowly and thus can be easily quantified using conventional blood sampling and laboratory measurements. Other lactate change conditions may be transient, in which case lactate levels can vary very rapidly and irregularly. Conventional laboratory measurements may be inappropriate for determining lactate levels in such cases. That is, lactate levels may change several times during continuous measurements, and in such cases, abnormal lactate levels may be completely missed, potentially leading to an incorrect diagnosis. In the case of rapidly fluctuating lactate levels, it may be desirable to continuously measure an individual's lactate levels, such as by using an implanted in vivo lactate sensor. Continuous lactate monitoring is also advantageous for individuals with chronically and slowly changing lactate levels. For example, continuous monitoring of lactate can avoid the pain and cost associated with multiple blood samplings to measure lactate levels.

[0004] Continuous analyte monitoring using implanted sensors can be advantageous in some cases, but there are specific challenges associated with these types of measurements. Intravascular analyte sensors are invasive and can sometimes be painful for an individual to wear, especially over long periods. Subcutaneous, interstitial, or dermal analyte sensors are often less painful for an individual to wear and can often provide sufficient measurement accuracy.

[0005] Non-intravascular in vivo glucose-responsive analyte sensors have been developed by several manufacturers over the past 20 years, and some have recently received regulatory approval for monitoring glucose levels in diabetic patients. Such glucose-responsive analyte sensors use glucose oxidase that is covalently bound to a polymer to facilitate glucose detection and is covalently bound to a transition metal complex (electron transfer agent or electron transfer mediator) that aids in the transport of electrons released during the oxidation of glucose. In vivo glucose-responsive analyte sensors available from other manufacturers also use glucose oxidase as the principle of detection, but the chemical reactions / protocols for detection vary widely.

[0006] In vivo analyte sensors for measuring glucose and other analytes can include a membrane disposed over at least the implanted portion of the analyte sensor. In one aspect, the membrane can improve the biocompatibility of the analyte sensor. In another aspect, the membrane is permeable or semi-permeable to the analyte of interest and can limit the overall influx of analyte to the active region of the analyte sensor such that the membrane functions as a mass transport limiting membrane. Using a mass transport limiting membrane to restrict analyte access to the active region of the analyte sensor can avoid sensor overload (saturation) and improve detection performance and accuracy. Such membranes are highly specific for restricting the mass transport of a particular analyte, and other substances permeate the membrane at significantly different rates. Therefore, it can be difficult to identify membrane polymers that are suitable for incorporation into a mass transport limiting membrane for a given analyte substance, such as lactate, that provide high analyte sensitivity and a stable sensor response.

[0007] As a functional lactic acid-responsive analyte sensor, it can be constructed by replacing glucose oxidase from a glucose-responsive analyte sensor with lactate oxidase. Different from the glucose-responsive analyte sensor briefly described above, the corresponding lactic acid-responsive analyte sensor generally has inferior performance when using similar sensing chemicals to analyze lactic acid. That is, directly replacing glucose oxidase with lactate oxidase may result in a lactic acid-responsive analyte sensor with insufficient sensitivity and / or a sensor response with insufficient stability. Therefore, although the lactic acid-responsive analyte sensor is functional, it has not yet reached the development level to provide stable and highly sensitive lactic acid analysis. Therefore, despite the rich information that may be obtained through the monitoring of lactic acid, the diagnostic value of the lactic acid-responsive analyte sensor remains significantly limited.

[0008] As described above, the lactic acid-responsive analyte sensor can replace glucose oxidase with lactate oxidase to facilitate the detection of lactic acid. Such a lactic acid-responsive analyte sensor based on the modified glucose-responsive sensor chemistry is described in co-owned Patent Document 1, which is hereby incorporated by reference in its entirety. As described therein, enhancement of sensor sensitivity to lactic acid and some response stabilization can be achieved by changing the glucose-responsive sensor chemistry to include catalase in the active region when lactate oxidase is present instead. Incorporation of catalase is somewhat helpful but does not completely stabilize the long-term response of the analyte sensor. Instead, the lactic acid signal of the analyte sensor containing catalase decreases by up to about 10% during 48 hours of monitoring. Since catalase is known to be reactive to hydrogen peroxide, the stabilizing effect of catalase in the lactic acid-responsive analyte sensor is thought to involve the transient scavenging of hydrogen peroxide that may affect the activity of lactate oxidase. Catalase may improve the performance of the lactic acid-responsive analyte sensor, but further performance improvement may be required for such an analyte sensor to realize its true potential.

[0009] The drawings are included to illustrate certain aspects of the present disclosure and should not be regarded as exclusive embodiments. The disclosed subject matter is capable of significant modifications, alterations, combinations, and equivalents in form and function without departing from the scope of the present disclosure.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0011] The present invention generally relates to a lactate-responsive analyte sensor and methods of using such analyte sensors, and more particularly, to a lactate-responsive analyte sensor and methods of providing high sensitivity and stable responsiveness to lactate.

[0012] As described above, the measurement of lactate levels (concentrations) can serve as a diagnosis of various physiological states and / or exposure to certain environmental factors. Laboratory measurements of lactate concentration can be sufficient to monitor some physiological states, but lactate levels can vary rapidly in other instances, making laboratory measurements infeasible. In vivo lactate-responsive analyte sensors using lactate-responsive enzymes can be used to conveniently measure lactate concentrations in both slow-changing and rapidly-changing situations, thereby providing specific diagnostic advantages for monitoring various conditions where lactate concentrations change rapidly and / or unexpectedly.

[0013] High analytical sensitivity and response stability are the major factors necessary to achieve satisfactory performance of analyte sensors, particularly sensors intended for long-term use in the body. Despite the potential benefits achievable by monitoring lactate levels in the body, a lactate-responsive analyte sensor with sufficient sensitivity and response stability for reliable diagnostic analysis has not yet been developed.

[0014] High sensitivity and response stability can be achieved with glucose-responsive analyte sensors containing glucose oxidase, but these desirable performance features are not currently convertible to lactate-responsive analyte sensors simply by replacing glucose oxidase with lactate oxidase. That is, the sensitivity and response stability of lactate-responsive analyte sensors are significantly inferior to those of equivalent glucose-responsive sensors. At least part of this problem is due to the differences between glucose oxidase and lactate oxidase. Some progress has been made by using stabilizers (such as catalase) to improve the function of lactate-responsive analyte sensors, but the desired levels of sensitivity and response stability have not yet been achieved.

[0015] Furthermore, the mass transport limiting membranes of lactate-responsive analyte sensors pose an additional challenge. To date, mass transport limiting membranes that have been successfully used with glucose-responsive analyte sensors have poor sensing characteristics when used with lactate oxidase to analyze lactate. Membrane polymers or combinations thereof that are more compatible with lactate have not yet been disclosed.

Means for Solving the Problem

[0016] The present invention relates to an approach that can be utilized to simultaneously improve the high sensitivity and improved response stability of a lactate-responsive analyte sensor. By improving these factors, high-quality analyte data can be provided over a long wear life of several days or more, ideally for more than one week, which can result in important diagnostic value. That is, as further described herein, the performance of a lactate-responsive analyte sensor can be improved by using different stabilizers instead of catalase and by changing the mast transport-limiting membrane disposed in the active region. As described herein, the chemical properties or configurations of several different membranes may promote an improvement in analyte sensor performance for lactate analysis.

[0017] First, the present disclosure uses an alternative benign stabilizer for lactate oxidase that provides significant performance advantages compared to catalase. That is, the present disclosure explains how serum albumin, particularly human serum albumin, is incorporated into the active region of a lactate-responsive analyte sensor and promotes the response sensitivity (i.e., the magnitude of the observed sensor response). The terms "albumin" and "serum albumin" are used synonymously herein. Human serum albumin is the most abundant protein found in plasma. Therefore, no obvious biocompatibility issues arise when this stabilizer is introduced into an analyte sensor intended for in vivo use. The suitability of human serum albumin as an alternative to the catalase stabilizer is particularly surprising because of the lack of the known function of hydrogen peroxide removal associated with human serum albumin. Without being bound by theory or mechanism, this result suggests that catalase may function independently of the normal hydrogen peroxide clearance function of conventional lactate-responsive analyte sensors. For example, other albumin proteins such as bovine serum albumin may be similarly incorporated into a lactate-responsive analyte sensor to achieve advantages comparable to those discussed herein.

[0018] Albumin proteins may exhibit certain advantages over other types of protein stabilizers, such as catalase, in terms of their thermal stability. Some protein stabilizers may undergo denaturation when heated, which can lead to the loss of their stabilizing function. Advantageously, albumin proteins do not readily denature even when exposed to temperatures close to 60 °C, such that, as further discussed herein, albumin proteins can maintain their stabilizing function even after thermal curing of the active sensor region has occurred.

[0019] Incorporating serum albumin into the active region of a lactate-responsive analyte sensor can improve sensor characteristics, particularly response sensitivity, but is usually insufficient by itself to provide suitable levels of both response sensitivity and long-term response stability. Counterintuitively, appropriate selection of a mass transport-limiting membrane in a lactate-responsive analyte sensor provides both sufficient lactate sensitivity and long-term response stability, particularly by coating over the active region containing serum albumin. By combining serum albumin, particularly human serum albumin, in the active region, the present disclosure describes various alternative chemistries or configurations of mass transport-limiting membranes that can further improve lactate response sensitivity and response stability in a lactate-responsive analyte sensor to more suitable levels.

[0020] Some glucose-responsive analyte sensors use cross-linked polyvinylpyridine-co-styrene polymers in the mass transport-limiting membrane, where some of the pyridine nitrogen atoms are functionalized with non-cross-linked poly(ethylene glycol) side chains and the pyridine nitrogen atoms are functionalized with alkylsulfonic acid groups. Cross-linking of these membrane polymers in the analyte sensor may be performed by functionalization with bisepoxides such as polyethylene glycol diglycidyl ether (PEGDGE) or glycerol triglycidyl ether (Gly3). Such membrane polymers, by themselves, are not effective in providing a stable response when analyzing lactate. This specification shows that replacing these membrane polymers with polyethylene glycol-cross-linked polyvinylpyridine homopolymers or polyvinylpyridine copolymers (different from polyvinylpyridine-co-styrene) can improve lactate sensing performance in terms of long-term response stability, especially when serum coexists with lactate oxidase in the active region. Instead, and surprisingly, polyethylene glycol-cross-linked polyvinylpyridine homopolymers or polyvinylpyridine copolymers, when appropriately combined (as a two-layer membrane or homogeneous mixture) with cross-linked polyvinylpyridine-co-styrene polymers more commonly used in the analysis of glucose, give satisfactory performance of the lactate analyte sensor.

Brief Description of the Drawings

[0021]

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DETAILED DESCRIPTION OF THE INVENTION

[0022] As defined herein, the term "homopolymer" is a polymer that contains only a single type of monomer unit that defines the polymer backbone. In a crosslinked homopolymer, some of the monomer units may have crosslinking groups and some may not. In the definitions herein, monomer units with or without crosslinking groups are considered to represent the same monomer unit. As defined herein, a "copolymer" is a polymer that contains two or more different types of monomer units that define the polymer backbone. The two or more different types of monomer units have different structures (including variations of isomers of the same structure). In a crosslinked copolymer, any type of monomer unit may carry a crosslinking agent, some of any type of monomer unit may have crosslinking groups, and some may not have crosslinking groups.

[0023] Before describing the analyte sensor of the present disclosure in more detail, a brief overview of a suitable in-vivo analyte sensor configuration and sensor system using the analyte sensor is first provided so that the embodiments of the present disclosure can be better understood.

[0024] FIG. 1 is a diagram showing an exemplary detection system into which the analyte sensor of the present invention can be incorporated. As shown in the figure, the sensing system 100 is configured to communicate with each other via a local communication path or link that can be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. The sensing system 100 includes a sensor control device 102 and a reader device 120. According to some embodiments, the reader device 120 may constitute an output medium that enables viewing of analyte concentrations such as lactate concentration, and warnings or notifications determined by the sensor 104 or an associated processor, and enables one or more user inputs. The reader device 120 can be a multi-purpose smartphone or a dedicated electronic reader device. Only one reader device 120 is shown, but in some cases, there may be multiple reader devices 120. The reader device 120 can also communicate with the remote terminal 170 and / or the reliable computer system 180 via communication paths / links 141 and / or 142, each of which can be wired or wireless, single or bidirectional, and encrypted or unencrypted. Additionally or alternatively, the reader device 120 can communicate with the network 150 (e.g., a cellular phone network, the Internet, or a cloud server) via the communication path / link 151. The network 150 may be further communicatively coupled to the remote terminal 170 via the communication path / link 152 and / or to the reliable computer system 180 via the communication path / link 153. Alternatively, the sensor 104 may communicate directly with the remote terminal 170 and / or the reliable computer system 180 without the intervening reader device 120. For example, according to some embodiments, the sensor 104 can communicate with the remote terminal 170 and / or the reliable computer system 180 via a direct communication link to the network 150, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is hereby incorporated by reference in its entirety.Any suitable electronic communication protocol such as Near Field Communication (NFC), Radio Frequency Identification (RFID), BLUETOOTH® or BLUETOOTH® Low Energy protocol, WiFi, etc. may be used for each of the communication paths or links. According to some embodiments, the remote terminal 170 and / or the trusted computer system 180 may be accessible by individuals other than the primary user who is interested in the user's analyte level. The reader device 120 may include a display 122 and an optional input element 121. According to some embodiments, the display 122 may comprise a touch screen interface.

[0025] The sensor control device 102 includes a sensor housing 103 that can accommodate a circuit and a power source for operating the sensor 104. Optionally, the power source and / or the active circuit may be omitted. A processor (not shown) may be communicatively coupled to the sensor 104, and the processor is physically disposed within the sensor housing 103 or the reader device 120. According to some embodiments, the sensor 104 protrudes from the lower side of the sensor housing 103 and extends through an adhesive layer 105 adapted to adhere the sensor housing 103 to a tissue surface such as the skin.

[0026] Sensor 104 is adapted to be at least partially inserted into a target tissue, such as within the dermis or subcutaneous layer of the skin. Sensor 104 may include a sensor tail of sufficient length to insert to a desired depth within a given tissue. The sensor tail can comprise a working electrode and one or more active regions (sensing regions / spots or detection layers) disposed on the working electrode and having activity for sensing a specimen of interest according to the present disclosure, particularly lactate. According to one or more embodiments of the present disclosure, each active region may include a lactate-responsive enzyme, suitable examples of which may include lactate oxidase or lactate dehydrogenase. According to some embodiments, the active region may include a polymeric material to which the enzyme is covalently bound. In various embodiments of the present disclosure, lactate can 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 fluid, amniotic fluid. In certain embodiments, the specimen sensor of the present disclosure can be adapted to assay skin fluid or interstitial fluid.

[0027] In some embodiments, sensor 104 can automatically transfer data to a reader device 120. For example, analyte concentration data is stored in memory until it is transmitted automatically and periodically, such as when the data is acquired or after a certain period of time has elapsed (e.g., every minute, 5 minutes, or other predetermined period). In other embodiments, sensor 104 can communicate with reader device 120 in a non-automatic manner, rather than according to a set schedule. For example, when the sensor electronics are brought within the communication range of reader device 120, data can be communicated from sensor 104 using RFID technology. The data can remain stored in the memory of sensor 104 until it is communicated to reader device 120. Thus, the patient does not need to always be in close proximity to reader device 120 and instead can upload data when convenient. In yet other embodiments, a combination of automatic and non-automatic data transfer can be implemented. For example, data transfer can continue automatically until reader device 120 moves out of the communication range of sensor 104.

[0028] The introducer may be present temporarily to facilitate introduction of the sensor 104 into tissue. In an exemplary embodiment, the introducer may include a needle or similar sharp object. It should be recognized that in alternative embodiments, other types of introducers, such as sheaths or blades, may exist. More specifically, the needle or other introducer is present temporarily proximate to the sensor 104 prior to tissue insertion and is then withdrawn. While present, the needle or other introducer can facilitate insertion of the sensor 104 into tissue by opening an access path for the sensor 104 to follow. For example, according to one or more embodiments, the needle can facilitate penetration of the epidermis as an access path to the dermis, enabling the sensor 104 to be implanted. After opening the access path, the needle or other introducer can be withdrawn so as not to pose a risk from the sharp object. In an exemplary embodiment, a suitable needle may have a solid or hollow, beveled or non-beveled, and / or circular or non-circular cross-section. In a more particular embodiment, a suitable needle can be equivalent to an acupuncture needle that may have a cross-sectional diameter of about 250 microns in cross-sectional diameter and / or tip design. However, it should be recognized that a suitable needle may have a larger or smaller cross-sectional diameter if required for a particular application.

[0029] In some embodiments, the tip of the needle (when present) can be angled across the end of the sensor 104 such that the needle first penetrates the tissue and opens an access path for the sensor 104. In other exemplary embodiments, the sensor 104 can be present within the lumen or groove of the needle, and the needle likewise opens an access path for the sensor 104. In either case, after facilitating insertion of the sensor, the needle is removed.

[0030] A suitable configuration of the analyte sensor of the present disclosure can use a two-electrode or three-electrode detection motif, which is further described below with reference to FIGS. 2A - 2C. The three - electrode detection motif can have a working electrode, a counter electrode, and a reference electrode. The related two - electrode detection motif can have a working electrode and a second electrode, where the second electrode functions as both a counter electrode and a reference electrode (i.e., as a counter / reference electrode). In both the two - electrode and three - electrode detection motifs, the active region of the analyte sensor can contact the working electrode. According to embodiments of the present invention, the active region can include a lactate - responsive enzyme and a stabilizer, particularly serum albumin. In some embodiments, the various electrodes can be at least partially laminated (layered) on top of each other, as described in more detail below. In some or other embodiments, the various electrodes can be arranged laterally spaced from each other on the sensor tail. In any case, the various electrodes can be electrically insulated from each other by a dielectric material or a similar insulator.

[0031] Figure 2A is a diagram of an exemplary two - electrode analyte sensor configuration suitable for use in some embodiments of the present disclosure. As shown, the analyte sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter / reference electrode 216. Alternatively, the working electrode 214 and the counter / reference electrode 216 can be disposed on the same side of the substrate 212 with a dielectric material inserted therebetween (the configuration is not shown). The active region 218 is disposed as at least one layer on at least a portion of the working electrode 214. In various embodiments of the present disclosure, the active region 218 can include a plurality of spots or a single spot configured for the detection of lactate.

[0032] Referring further to FIG. 2A, according to some embodiments, the membrane 220 can cover at least the active region 218 and optionally cover part or all of the working electrode 214 and / or the counter / reference electrode 216, or the entire analyte sensor 200. One or both sides of the analyte sensor 200 may be covered by the membrane 220. The membrane 220 can include one or more polymeric membrane materials having the ability to restrict the influx of the analyte into the active region 218 (i.e., the membrane 220 is a mass transport limiting membrane). The composition of the membrane 220 can vary to promote the desired flow of lactate into the active region 218, thereby providing the desired signal intensity and stability as further described herein. The analyte sensor 200 can be operable to assay lactate by any of the electrochemical detection techniques of coulometry, amperometry, voltammetry, or potentiometry. Specific compositions of the membrane 220 that may be appropriate when analyzing for lactate are described in more detail below herein.

[0033] Figures 2B and 2C show diagrams of an exemplary three - electrode analyte sensor configuration that is also compatible for use in some embodiments of the disclosure herein. The three - electrode analyte sensor configuration can be similar to that shown for the analyte sensor 200 of FIG. 2A, except that it includes an additional electrode 217 in analyte sensors 201 and 202 (FIGS. 2B and 2C). Next, using the additional electrode 217, the counter / reference electrode 216 can function as either a counter electrode or a reference electrode, and the additional electrode 217, unless otherwise specified, serves the other electrode function. The working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed either on the working electrode 214 or the electrode 216 with a separating layer of dielectric material in between. For example, as shown in FIG. 2B, the dielectric layers 219a, 219, 219c separate the electrodes 214, 216, 217 from each other and provide electrical isolation. Alternatively, as shown in FIG. 2C, at least one of the electrodes 214, 216, and 217 may be disposed on opposite surfaces of the substrate 212. Thus, in some embodiments, the electrode 214 (working electrode) and the electrode 216 (counter electrode) can be disposed on opposite surfaces of the substrate 212, and the electrode 217 (reference electrode) is disposed on one of the electrodes 214 or 216 and separated therefrom by a dielectric. A reference material layer 230 (e.g., Ag / AgCl) may be present on the electrode 217, and the position of the reference material layer 230 is not limited to that shown in FIGS. 2B and 2C. Similar to the sensor 200 shown in FIG. 2A, the active regions 218 of the analyte sensors 201 and 202 can include a plurality of spots or a single spot configured for the detection of lactate. Also, the analyte sensors 201 and 202 can be operable to assay lactate by any of the electrochemical detection techniques of charge, current measurement, voltammetry, or potentiometry.

[0034] Similar to the analyte sensor 200, the membrane 220 also covers the active region 218, as well as other sensor components within the analyte sensors 201 and 202, thereby functioning as a mass transport limiting membrane. In some embodiments, the additional electrode 217 may be covered by the membrane 220. FIGS. 2B and 2C are shown with all the electrodes 214, 216, and 217 covered by the membrane 220, but it should be recognized that in some embodiments, only the working electrode 214 may be covered. Further, the thickness of the membrane 220 at each of the electrodes 214, 216, and 217 may be the same or different. As in the two-electrode analyte sensor configuration (FIG. 2A), one or both sides of the analyte sensors 201 and 202 may be covered by the membrane 220 in the sensor configurations of FIGS. 2B and 2C, or the entire analyte sensors 201 and 202 may be covered. Thus, the three-electrode sensor configuration shown in FIGS. 2B and 2C should be understood to be non-limiting of the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of the present disclosure.

[0035] According to various embodiments of the present disclosure, an electron transfer agent may be present in the active region of any of the analyte sensors or analyte sensor configurations disclosed herein. A suitable electron transfer agent can facilitate the transport of electrons to or from the working electrode when an analyte (enzyme substrate), such as lactate, undergoes a redox reaction. Certain embodiments of the analyte sensors disclosed herein may be characterized by an active region that includes lactate oxidase and serum albumin, particularly human serum albumin, in combination with a mass transport limiting membrane that is compatible with lactate, as further described below.

[0036] Suitable electron transfer agents include electroreducible and electrooxidizable ions, complexes, or molecules (e.g., quinones) having a redox potential several hundred millivolts higher or lower than the redox potential of the standard calomel electrode (SCE). According to some embodiments, suitable electron transfer agents include low-potential osmium complexes as described in U.S. Pat. Nos. 6,134,461 and 6,605,200, which are hereby incorporated by reference in their entirety. Further examples 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 hereby incorporated by reference in their entirety. Other suitable electron transfer agents can include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt, including, for example, metallocene compounds. Suitable examples of electron transfer mediators and electron transfer mediators bound to polymers include those described in U.S. Pat. Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are hereby incorporated by reference. Ligands suitable for metal complexes can also include, for example, bidentate or higher density ligands, such as 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 density ligands can be present in the metal complex to achieve a complete coordination sphere.

[0037] According to various embodiments of the present disclosure, the polymer may be present in each active region of the analyte sensors or analyte sensor configurations disclosed herein. Polymers suitable for inclusion in the active region include, but are not limited to, polyvinylpyridine (e.g., poly(4-vinylpyridine)), polyvinylimidazole (e.g., poly(1-vinylimidazole)), any mixture thereof, or any copolymer thereof. Exemplary copolymers suitable for inclusion in the active region include those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile. In an exemplary embodiment, the polymer within the active region of the analyte sensor disclosed herein can be poly(4-vinylpyridine), which may have some monomer units functionalized with alkyl carboxylate side chains, some monomer units attached to an electron transfer agent having an amide spacer group (e.g., see Formula 1 below), or some monomer units that are not functionalized.

[0038] According to various embodiments of the present disclosure, the electron transfer agent can be covalently bonded to the polymer in the active region. The manner of covalent bonding is not particularly limited. The covalent bonding of the electron transfer agent to the polymer can be achieved by polymerizing monomer units having a covalently bonded electron transfer agent, or after the polymer has already been synthesized, the electron transfer agent can be reacted independently with the polymer. According to some embodiments, a bifunctional spacer can covalently bond the electron transfer agent to the polymer within the active region, where the first functional group is reactive with the polymer (e.g., a functional group capable of quaternizing a pyridine nitrogen atom or an imidazole nitrogen), and the second functional group reacts with the electron transfer agent (e.g., a functional group that reacts with a ligand coordinating a metal ion).

[0039] Similarly, according to some or various other embodiments of the present disclosure, the enzyme in the active region can be covalently bonded to the polymer. According to more specific embodiments, the covalent bonding of the enzyme to the polymer can occur via a crosslinking agent when an appropriate crosslinking agent is introduced. Crosslinking agents suitable for reaction with free amino groups in the enzyme (e.g., free amines in lysine) include, for example, polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides (e.g., Gly3), cyanuric chloride, N-hydroxysuccinimide, imide esters, epichlorohydrin, or derivatized variants thereof. Crosslinking agents suitable for reaction with free carboxylic acid groups in the enzyme can include, for example, carbodiimide. Crosslinking is generally intermolecular, but in some embodiments it can be intramolecular.

[0040] The electron transfer agent and / or enzyme can also be bound to the polymer in the active region by means other than covalent bonding. In some embodiments, the electron transfer agent and / or enzyme can be ionically or coordinatively associated with the polymer. For example, a charged polymer can be ionically associated with an oppositely charged electron transfer agent or enzyme. In still other embodiments, the electron transfer agent and / or enzyme can be physically entrapped within the polymer without binding thereto.

[0041] Various configurations of the lactate-responsive analyte sensor of the present invention are described in more detail below. According to various embodiments, the analyte sensor of the present invention can include a working electrode having an active region disposed thereon and a mass transport limiting membrane covering at least the active region on the working electrode. The active region includes a polymer, albumin, and a lactate-responsive enzyme covalently bonded to the polymer. In more specific embodiments, the mass transport limiting membrane can include at least a crosslinked polyvinylpyridine homopolymer or copolymer. The mass transport limiting membrane can consist of a single part or multiple parts. According to more specific embodiments of the present invention, the multi-component membrane embodiment can include a bilayer or homogeneous mixture of crosslinked polyvinylpyridine and another polymer.

[0042] Polyvinyl pyridine copolymers suitable for inclusion in the mass transport limiting membrane can contain up to about 25% comonomer, such as about 0.1% to about 5% comonomer, or about 5% to about 15%, or about 15% to about 25% comonomer, or about 1% to about 10% comonomer (based on the total amount of monomers in the copolymer). If the mass transport limiting membrane provides sufficient lactate permeability and provides a detection sensitivity of at least about 1 nA / mM when exposed to lactate, the appropriate comonomer is not particularly limited. The polyvinyl pyridine copolymer can be different from the polyvinyl pyridine-co-styrene copolymer according to various embodiments.

[0043] The above-described analyte sensor can further include a counter electrode and a reference electrode, or a counter / reference electrode. Suitable configurations for the analyte sensor are described in more detail above with reference to FIGS. 2, 3A, and 3B. Other configurations of the various electrodes and active regions are also within the spirit and scope of the present invention, and the specifically described sensor configurations should not be considered as limiting the scope of the present invention.

[0044] Alternatively, the analyte sensor of the present invention can include a working electrode having an active region disposed thereon, and a mass transport limiting membrane covering at least the active region on the working electrode, wherein the active region includes a polymer, albumin, and a lactate-responsive enzyme covalently bonded to the polymer. The mass transport limiting membrane includes a membrane polymer that is permeable to lactate. More specifically, the mass transport limiting membrane can have a lactate permeability such that the sensitivity of the analyte sensor is at least about 5 nA at a lactate concentration of about 5 mM. That is, the detection sensitivity of the analyte sensor when exposed to lactate is at least about 1 nA / mM. The membrane polymer can include a polyethylene glycol cross-linked polyvinyl pyridine homopolymer or copolymer, either as a single-component membrane or as a multi-component membrane such as a bilayer of a homogeneous mixture, in more specific embodiments.

[0045] Yet other analyte sensors of the present disclosure alternatively include a working electrode having an active region disposed thereon, and a mass transport limiting membrane covering at least the active region on the working electrode, wherein the active region includes a polymer, catalase, and a lactate-responsive enzyme covalently bonded to the polymer, and the mass transport limiting membrane may include at least a crosslinked polyvinylpyridine homopolymer or copolymer.

[0046] According to various embodiments, the lactate-responsive enzyme in the active region may include lactate oxidase or lactate dehydrogenase. In more specific embodiments, the combination of lactate oxidase and albumin, particularly human serum albumin, may be advantageous for performing lactate analysis in accordance with the present invention.

[0047] According to more specific embodiments, lactate oxidase may be present in the active region in an amount ranging from about 0.05 μg to about 5 μg, or from about 0.1 μg to about 4 μg, or from about 0.2 μg to about 3 μg, or from about 0.5 μg to about 2 μg. With respect to the weight percentage of the active region, lactate oxidase may be present in an amount ranging from about 10 wt% to about 90 wt% of the active region, or from about 25 wt% to about 75 wt% of the active region, or from about 30 wt% to about 60 wt% of the active region.

[0048] According to various embodiments, the albumin in the active region may include human serum albumin. Alternatively, non-human albumins such as bovine serum albumin can also be used without problems. According to the disclosure of this specification, albumin can be incorporated into the active region in an amount sufficient to stabilize lactic acid-responsive enzymes, particularly lactate oxidase. In more specific embodiments, albumin can be present in the active region in an amount ranging from about 0.05 μg to about 5 μg, or from about 0.1 μg to about 2 μg, or from about 0.2 μg to about 1.5 μg, or from about 0.3 μg to about 0.8 μg. With respect to the weight percentage of the active region, albumin can be present in an amount ranging from about 25 wt% to about 75 wt% of the active region, or from about 30 wt% to about 60 wt% of the active region. In certain embodiments, the weight ratio of lactate oxidase to albumin can range from about 10:1 to about 1:10 (w / w), from about 5:1 to about 1:5, or from about 1:1 to about 1:5, or from about 1:1 to about 1:2.

[0049] In a more specific embodiment, the analyte sensor of the present invention can include a sensor tail configured for insertion into tissue. The working electrode is disposed on the sensor tail and can be inserted into the tissue to facilitate the analysis of lactate therein. Suitable tissues are not considered to be particularly limited, and specific examples are described in more detail above. Similarly, considerations for deploying the sensor tail to a specific location or depth within the tissue are also described above.

[0050] The specific configuration of the analyte sensor disclosed herein, including the amounts of albumin and lactate oxidase incorporated into the active region, depends on the tissue penetrated by the sensor tail, the expected concentration of lactate to be analyzed, and the conditions under which the analyte sensor operates during the analysis of lactate. In a more specific embodiment, the tissue penetrated by the analyte sensor can be the skin, and as a result, the sensor tail is disposed within the dermal layer, stromal layer, or subcutaneous layer beneath the surface of the skin. The sensor may further be contained within a sensor housing configured to adhere to the skin.

[0051] As described above, the active region of the analyte sensor can include an electron transfer agent covalently bonded to the polymer therein. The mode of the covalent bond between the polymer and the electron transfer agent is not particularly limited. Suitable types of covalent bonds between the polymer and the electron transfer agent are described in more detail above.

[0052] Ideally, the active region can be configured to achieve a steady-state current as soon as the analyte sensor of the present invention is operated at a given potential. The rapid achievement of the steady-state current can be facilitated by selecting an electron transfer agent that rapidly changes its oxidation state when exposed to a potential above its redox potential. Making the active region as thin as possible can also facilitate the rapid achievement of the steady-state current. For example, the appropriate thickness of the active region can range from about 0.1 micron to about 10 microns. In some or other embodiments, for example, combining conductive materials such as carbon nanotubes, graphene, or metal nanoparticles within the active region can facilitate the rapid achievement of a steady current. The appropriate amount of conductive particles can range from about 0.1 wt% to about 50 wt%, or about 1 wt% to about 50 wt%, or about 0.1 wt% to about 10 wt%, or about 1 wt% to about 10% of the active region.

[0053] The active region on the working electrode of the analyte sensor disclosed herein can include at least one spot or layer disposed on the working electrode. Although spots or layers of larger or smaller active regions are contemplated herein, each spot or layer can range in size from about 0.01 mm2 to about 1 mm2. The total size of the active region (the total area of all spots or layers) is at most about 100 mm2, particularly about 25 mm2 or less, or about 10 mm2 or less, or about 5 mm2 or less, or about 1 mm2 or less, or about 0.1 mm2 or less. In more specific embodiments, the total size of the active region can range from about 0.05 mm2 to about 0.1 mm2. In some embodiments, the active region can include one spot or layer, but more typical embodiments of the analyte sensor of the present disclosure feature an active region having a plurality of spots disposed on the working electrode. The number of spots is not considered to be particularly limited, but according to some embodiments, it can range from 2 to about 10, or about 3 to about 8, or about 4 to about 6.

[0054] Furthermore, it should also be recognized that the sensitivity (output current) of the analyte sensor to lactate may vary by changing the coverage (area or size) of the active region, the properties and thickness of the mass transport limiting membrane covering the active region, and any combination thereof. Changes in these parameters to achieve the desired sensitivity can be readily implemented by those skilled in the art if the advantages of the disclosure herein are recognized.

[0055] The analyte sensors disclosed herein, particularly those having an active region containing albumin and covalently bound lactate oxidase, and having a mass transport limiting membrane containing a polyvinylpyridine homopolymer or copolymer, particularly a polyvinylpyridine homopolymer, are characterized by the function of response stability (changing output current), varying by about 10% or less in a 190-hour measurement, or about 5% or less in a 190-hour measurement, or about 1% or less in a 190-hour measurement. Such a degree of difference occurring within the time frame is not expected to be clinically significant (i.e., having only a minor impact on Clark error grid analysis and MARD or MAD analysis). Therefore, the catalase-stabilized lactate-responsive analyte sensor described in the above patent is expected to exhibit an even lower response current in a 190-hour measurement.

[0056] To achieve sufficient response stability, the analyte sensor of the present invention uses a matrix transport limiting membrane selected for its compatibility with lactate. More specifically, advantageous matrix transport limiting membranes suitable for use with lactate-responsive enzymes, particularly lactate oxidase, may include crosslinked polyvinylpyridine homopolymers or copolymers. The matrix transport limiting membrane in the analyte sensor of the present invention may comprise only a single polymer (i.e., a crosslinked polyvinylpyridine homopolymer or copolymer), or the polymer may be multi-component and may comprise two or more polymers (i.e., a crosslinked polyvinylpyridine homopolymer or copolymer and at least a second crosslinked polymer) in a bilayer or homogeneous mixed configuration. More particularly, the multi-component membrane may comprise a first polymer comprising a crosslinked polyvinylpyridine homopolymer and a second polymer comprising a crosslinked polyvinylpyridine copolymer, or may comprise a first polymer comprising a crosslinked polyvinylpyridine copolymer and a second polymer comprising a crosslinked polyvinylpyridine copolymer different from the first crosslinked polyvinylpyridine copolymer. In a more detailed embodiment, the second crosslinked polymer may comprise a crosslinked polyvinylpyridine copolymer, particularly a crosslinked polyvinylpyridine-co-styrene polymer, in which some of the pyridine nitrogen atoms are functionalized with non-crosslinked poly(ethylene glycol) tails and some of the pyridine nitrogen atoms are functionalized with alkylsulfonic acid groups.

[0057] In the analyte sensor of the present invention, a multi-component film suitable for use in combination with a lactate-responsive enzyme, particularly lactate oxidase, may comprise a two-layer film according to various embodiments. A suitable two-layer film may comprise a first layer comprising a crosslinked polyvinylpyridine homopolymer or copolymer and a second layer comprising a second crosslinked polymer, particularly a crosslinked polyvinylpyridine copolymer. The second layer may comprise a polyvinylpyridine copolymer different from the polyvinylpyridine copolymer of the first layer. In more specific embodiments, the first layer may be disposed directly on the active region and the second layer may be disposed on the first layer. In alternative embodiments, the second layer may be disposed directly on the active region and the first layer may be disposed on the second layer. The thicknesses and order of the first and second layers can be varied to provide the desired sensitivity. Such a two-layer configuration can be prepared in some embodiments by coating the first layer on the active region (e.g., by spray coating, painting, inkjet printing, roller coating, dip coating, etc.) and then coating the second layer on the first layer by the same or different coating techniques (e.g., spray coating, painting, inkjet printing, roller coating, dip coating, etc.). In other embodiments, the two-layer film is configured with the first and second layers reversed (thereby reversing the position of the crosslinked polyvinylpyridine homopolymer or copolymer) and each layer is coated as described above.

[0058] Due to its nature, the two-layer film is heterogeneous because two different film polymers are layered on top of each other. In the analyte sensor of the present invention, other multi-component films suitable for use in combination with a lactate-responsive enzyme, particularly lactate oxidase, can have a uniform composition. More specifically, in some embodiments, a suitable multi-component film can include a homogeneous mixture of a cross-linked polyvinylpyridine homopolymer or copolymer and a cross-linked second polymer, particularly a cross-linked polyvinylpyridine copolymer. The two cross-linked polyvinylpyridine copolymers may be different from each other. The ratio of the two cross-linked polymers in the homogeneous mixture can be varied over a significant range, and the ratio can be adjusted to provide the desired sensitivity of the analyte sensor disclosed herein.

[0059] Figure 14 is an exemplary schematic diagram of a portion of an analyte sensor having a two-layer film disposed on a working electrode, suitable for use in some embodiments of the disclosure herein. As shown in Figure 14, the analyte sensor features a sensor tail 600 having a working electrode 614 disposed on a substrate 612. An active region 618 is disposed on the working electrode 614 and contains a lactate-responsive enzyme, in accordance with the disclosure herein.

[0060] As further shown in Figure 14, the active region 618 is covered by a two-layer film 621, which includes a film layer 621a that is in direct contact with the active region 618 and a film layer 621b that covers the film layer 621a. According to the disclosure herein, the film layers 621a and 621b may include different film polymers, and at least one of them is a polyvinylpyridine homopolymer or copolymer.

[0061] In another embodiment, the two-layer film 621 of Figure 14 may be replaced by a homogeneous film layer containing a mixture of different film polymers, in accordance with the disclosure herein. The analyte sensor described above can be utilized in various ways to measure lactate and determine the concentration of lactate therefrom. The concentration of lactate can be further correlated to a physiological state (e.g., resulting from a disease or exposure to environmental factors) in accordance with various embodiments, as described in more detail below.

[0062] According to various embodiments, the method of the present invention can comprise the step of exposing a sample sensor to a fluid, the sample sensor including a working electrode having an active region disposed thereon and a mass transport limiting membrane covering at least the active region on the working electrode. The active region includes a polymer, albumin, and a lactate-responsive enzyme covalently bonded to the polymer, particularly lactate oxidase. The mass transport limiting membrane can, in certain embodiments, include at least a crosslinked polyvinylpyridine homopolymer or copolymer. The method further comprises the steps of obtaining a signal above the redox potential of the active region, the signal being proportional to the concentration of lactate in the fluid, and correlating the signal to the concentration of lactate in the fluid.

[0063] In more specific embodiments, the fluid is a biological fluid and the sample sensor is exposed to the biological fluid in vivo of a subject in whom measurement of the concentration of lactate is desired. According to even more specific embodiments of the present invention, the subject can be a human. Biological fluids suitable for analysis with the sample sensor of the present invention can include any of the biological fluids discussed in more detail above.

[0064] The signals related to the active region upon exposure to lactate can be correlated to the corresponding lactate concentration by examining a look-up table or a calibration curve. The look-up table or calibration curve can exist in a physical form (i.e., in writing) or an electronic form (i.e., a database or a computer algorithm). A look-up table for various concentrations of lactate can be created by analyzing a plurality of samples with known lactate concentrations and recording the responses of the analyte sensor at each concentration. Similarly, a calibration curve for lactate can be determined by plotting the analyte sensor responses of each lactate sample as a function of concentration. According to some embodiments, the calibration curve of the analyte sensor of the present invention can be linear or substantially linear with respect to the concentration of lactate. In some embodiments, the background response can be subtracted from the analyte sensor response before the analyte sensor response is recorded or plotted. The background response can be determined by assaying a liquid that does not contain lactate (i.e., a blank). As further described below, the look-up table or calibration curve can be examined manually or electronically to determine the concentration of lactate in a fluid.

[0065] The processor determines which sensor response value in the look-up table is closest to the measured value of a sample (fluid) with an unknown lactate concentration and reports the lactate concentration accordingly. In some or other embodiments, if the sensor response value of a sample with an unknown lactate concentration is between the recorded values in the look-up table, the processor can interpolate between two look-up table values to estimate the lactate concentration. In interpolation, a linear concentration variation between two values reported in the look-up table can be assumed. Interpolation can be used, for example, when the sensor response of the sample differs from a particular value in the look-up table by a sufficient amount (e.g., a variation of about 10% or more). Interpolation can be performed similarly when manually referring to the look-up table.

[0066] Similarly, the processor can input the sensor response value of a sample having an unknown lactate concentration into the corresponding calibration curve. The processor then reports the concentration of lactate accordingly.

[0067] As described above, the concentration of lactate determined using the analyte sensor of the present invention can be further correlated with one or more physiological states. The one or more physiological conditions are mediated by lactate in vivo and / or may result in an increase or decrease in lactate levels in vivo. More specifically, the one or more physiological states that can be monitored by the analyte sensor of the present invention include, for example, sepsis, infection, organ function, or any combination thereof. Other states in which monitoring of lactate may be beneficial include, for example, physiological stress and exercise, both of which can increase lactate levels. Monitoring any of these states can include, for example, tracking the trend of lactate levels over time and / or determining the instantaneous lactate level at a particular time. Optionally, a warning is issued if the lactate level exceeds a defined threshold concentration for a given condition, or if the lactate level tends towards a defined threshold when monitored over time. Thus, the analyte sensor disclosed herein can be configured to provide a warning that sepsis, an infectious disease, organ failure, or any combination thereof may be present, or otherwise provide an indication. The threshold concentration may vary depending on the physiological state being monitored.

[0068] Sepsis can be considered a three - stage syndrome, starting with sepsis, progressing from severe sepsis to septic shock. The goal is to treat sepsis in its early stages before it becomes more dangerous. Currently, severe sepsis is often diagnosed by a decrease in body temperature, heart rate, respiratory rate, urine output, a sudden change in mental status, and a decrease in platelet count. Septic shock is often diagnosed by the same markers in combination with very low blood pressure.

[0069] Lactic acid levels are useful for the diagnosis, monitoring, and / or evaluation of various forms of sepsis and / or related infectious diseases. Thus, by determining the concentration of lactate according to the present invention, it may be possible to more effectively monitor, evaluate, and / or manage sepsis and / or infection. Alternatively, the sample sensor of the present invention can be used to monitor a subject (e.g., a hospital patient) who is at risk of sepsis and / or infection but currently shows no signs of any condition. The lactic acid levels obtained according to the present invention can be combined with other sample levels and / or physiological markers (e.g., body temperature, heart rate, respiratory rate, blood pressure, decreased urine output, sudden change in mental state, decreased platelet count) and other markers (such as C-reactive protein (CRP), procalcitonin, pancreatitis protein (PSP), circulating complement (C3 and C4), ferritin, cholesterol, albumin, cortisol, and neutrophil gelatinase-associated lipocalin) for the diagnosis of monitoring the progression of infectious diseases associated with sepsis in a subject. In certain embodiments, the concentration of lactic acid can be measured in cerebrospinal fluid to monitor, evaluate, and / or diagnose meningitis and / or septic meningitis. Additional markers for septic meningitis can include, for example, glucose, sTREM-1, procalcitonin, CRP, TNF-α, IL-1β, IL-6, IL-8, and lipopolysaccharide-binding protein. Additional markers for bacterial sepsis or bacterial infectious diseases can include, for example, glutamic acid, malic acid, pseudouridine, acetylcarnitine, glycerophosphocholine, hydroxyphenyl lactic acid, N-acetylneuraminic acid, pseudouridine, and tyrosine. Additional markers for viral sepsis, including septic meningitis, or viral infectious diseases can include, for example, hypoxanthine, inosine, and hexanoyl carnitine.

[0070] In some embodiments, the analyte sensor of the present invention can be used to monitor a subject's exposure to an infectious agent. For example, the analyte sensor can monitor the subject's condition after acute exposure to an infectious agent to monitor for signs of sepsis or infection and / or to monitor the onset and progression of sepsis or infection. The analyte sensor can further be used to monitor an anti-infective agent administered to the subject to treat sepsis or infection or its symptoms and / or the subject's response to the treatment.

[0071] Enzyme activity can be used for the diagnosis of organ function, and the enzyme activity can be either decreased or increased, depending on the specific organ and the given physiological state the subject is experiencing. Within the area of lactate-responsive enzymes, liver function and the physiological state of liver function (i.e., disease) can be characterized with respect to the activity of lactate dehydrogenase. Other enzymes that may be desirable for monitoring organ function (including organs and organ functions different from the liver and liver function) can be used alone or in combination with lactate dehydrogenase, and include, for example, creatine kinase, aspartate transaminase, aspartate aminotransferase, alkaline phosphatase, and 5'-nucleotidase. In addition to the liver, alternative organs whose function can be monitored include, for example, the kidney, heart, brain, lung, pancreas, spleen, stomach, bladder, bone, gallbladder, intestine (small and large), colon, lymph node, thyroid, and the like.

[0072] The analyte sensor disclosed herein can monitor organ function, particularly liver function, by evaluating the activity of lactate dehydrogenase or another suitable lactate-responsive enzyme (e.g., lactate oxidase). The analyte sensor can be used to monitor, diagnose, and / or detect analyte levels in subjects who are experiencing or at risk of organ dysfunction, and may be able to identify organ diseases before life-threatening symptoms appear. The lactate concentration or rate of change of lactate concentration determined by the analyte sensor disclosed herein can facilitate the diagnosis or analysis of organ failure or dysregulation or the likelihood of organ failure or dysregulation. The analyte sensor issues a warning when the lactate concentration exceeds a threshold amount or tends to exceed a threshold amount that is characteristic of organ damage or dysfunction.

[0073] The lactate levels obtained in accordance with the present invention can be diagnostically combined with other analyte levels and / or physiological markers, particularly to determine liver organ function and / or failure. Additional markers that can be measured in combination with lactate to measure organ function include, for example, body temperature, heart rate, respiratory rate, blood pressure, decreased urine output, sudden changes in mental status, decreased platelet count, and other markers (e.g., C-reactive protein (CRP), procalcitonin, pancreatic stone protein (PSP), circulating complement (C3 and C4), ferritin, cholesterol, albumin, cortisol, and neutrophil gelatinase-associated lipocalin). The lactate levels monitored in accordance with the present invention can be further utilized to shorten the time before initiating a treatment course to combat organ failure and / or to monitor the progress of a series of treatments or a treatment.

[0074] The embodiments disclosed herein include the following. A. An analyte sensor. The analyte sensor comprises a working electrode having an active region disposed thereon, the active region comprising a polymer, albumin, and a lactate-responsive enzyme covalently bonded to the polymer, and a mast transport-limitation membrane that covers at least the active region on the working electrode.

[0075] B. Method for measuring lactate. This method comprises the steps of exposing a sample sensor to a fluid, wherein the sample sensor includes a working electrode having an active region disposed thereon, the active region includes a polymer, albumin, and a lactate-responsive enzyme covalently bonded to the polymer, and a mass transport-limiting membrane covers at least the active region on the working electrode; obtaining a signal above the redox potential of the active region, the signal being proportional to the lactate concentration in the fluid; and correlating the signal with the lactate concentration in the fluid.

[0076] C. Lactate sensor responsive to sepsis, infection, or organ function. The lactate sensor includes the following. The sample sensor includes a working electrode having an active region disposed thereon, the active region including a polymer, albumin, and a lactate-responsive enzyme covalently bonded to the polymer, and a mass transport-limiting membrane covering at least the active region on the working electrode. Here, the sample sensor is configured to respond to sepsis, infection, organ function, or any combination thereof and provide a warning or other indication that sepsis, infection, organ failure, or any combination thereof may be present relative to the measured lactate concentration.

[0077] Each of embodiments A - C can have one or more of the following additional elements in any combination. Element 1: Albumin includes human serum albumin.

[0078] Element 2: The lactate-responsive enzyme includes lactate oxidase. Element 3: The active region includes a plurality of detection spots disposed on the working electrode. Element 4: The mass transport-limiting membrane includes a multi-component membrane, and the multi-component membrane includes a cross-linked polyvinylpyridine homopolymer or copolymer and at least a second cross-linked polymer.

[0079] Element 5: The multi-component film comprises a first polymer comprising a crosslinked polyvinylpyridine homopolymer and a second polymer comprising a crosslinked polyvinylpyridine copolymer, or a first polymer comprising a first crosslinked polyvinylpyridine copolymer and a second polymer comprising a second crosslinked polyvinylpyridine copolymer.

[0080] Element 6: Here, the multi-component film comprises a two-layer film, and the two-layer film comprises a first layer comprising a polyvinylpyridine homopolymer or copolymer and a second layer comprising a second crosslinked polymer. Element 7: The first layer is disposed directly on the active region, and the second layer is disposed on the first layer.

[0081] Element 8: The mast transport-limiting film comprises a homogeneous mixture of a crosslinked polyvinylpyridine homopolymer or copolymer and a second crosslinked polymer. Element 9: Here, the working electrode is disposed on a sensor tail configured for insertion into tissue.

[0082] Element 10: The active region further comprises an electron transfer agent covalently bonded to the polymer. Element 11: The fluid is a biological fluid, and the analyte sensor is exposed to the biological fluid in vivo. Element 12: Here, the method further comprises determining the presence of one or more conditions in the subject based on the concentration of lactate in the fluid, and the one or more conditions are selected from sepsis, infection, organ function, and any combination thereof.

[0083] Element 13: The analyte sensor according to claim 1, wherein the mast transport-limiting film comprises at least a crosslinked polyvinylpyridine homopolymer or copolymer. As a non-limiting example, exemplary combinations applicable to A~CB include the following.

[0084] A or C analyte sensor or B method in combination with elements 1 and 2. Among them, those with elements 1 and 2, those with elements 1, 2, and 13, those with elements 1 and 3, those with elements 1, 3, and 13, those with elements 1 to 3, those with elements 1 to 3 and 13, those with elements 1 and 4, those with elements 1, 4, and 13, those with elements 1 and 13, those with elements 2 and 4, those with elements 2, 4, and 13, those with elements 2 and 13, those with elements 3 and 4, those with elements 3, 4, and 14, those with elements 1 and 13, those with elements 1 to 4, those with elements 1 to 4 and 13, those with elements 2 to 4, those with elements 2 to 4 and 13, those with elements 1, 4, and 5, those with elements 1, 4, 5, and 13, those with elements 2, 4, and 5, those with elements 2, 4, 5, and 13, those with elements 3 to 5, those with elements 3 to 5 and 13, those with elements 4 and 5, those with elements 4, 5, and 13, those with elements 1, 4 to 6, those with elements 4 to 6 and 13, those with elements 2, 4, 5, and 6, those with elements 2, 4, 5, 6, and 13, those with elements 3 to 6, those with elements 3 to 6 and 13, those with elements 4 to 7, those with elements 4 to 7 and 13, those with elements 4 and 8, those with elements 4, 8, and 13, those with elements 4, 5, and 8, those with elements 4, 5, 8, and 13, those with elements 1 and 9, those with elements 1, 9, and 13, those with elements 2 and 9, those with elements 2, 9, and 13, those with elements 3 and 9, those with elements 3, 9, and 13, those with elements 4 and 9, those with elements 4, 9, and 13, those with elements 1 and 10, those with elements 1, 10, and 13, those with elements 2 and 10, those with elements 2, 10, and 13, those with elements 3 and 10, those with elements 3, 10, and 13, those with elements 4 and 10, those with elements 4, 10, and 13, those with elements 9 and 10, those with elements 9, 10, and 13.

[0085] For the method of B, combinations of element 1, 11; combinations of 1, 11, 13; combinations of 1, 13; combinations of 2, 11; combinations of 2, 13; combinations of 3, 11; combinations of 3, 13; combinations of 4, 11; combinations of 4, 13; combinations of 4, 5, 11; combinations of 4, 5, 11, 13; combinations of 4 - 6, 11; combinations of 4, 6, 11, 13; combinations of 4 - 7, 11; combinations of 4 - 7, 11, 13; combinations of 4, 8, 11; combinations of 4, 8, 11, 13; combinations of 9, 11; combinations of 9, 11, 13; combinations of 10, 11; combinations of 10, 11, 13; combinations of 11, 12; combinations of 11 - 13; combinations of 1, 12; combinations of 1, 12, 13; combinations of 2, 12; combinations of 2, 12, 13; combinations of 3, 12; combinations of 3, 12, 13; combinations of 4, 12; combinations of 4, 12, 13; combinations of 4, 5, 12; combinations of 4, 5, 12, 13; combinations of 4 - 6, 12; combinations of 4 - 6, 12, 13; combinations of 4 - 7, 12; combinations of 4 - 7, 12, 13; combinations of 4 - 8, 12; combinations of 4, 8, 12, 13; combinations of 9, 12; combinations of 9, 12, 13; combinations of 10, 12; combinations of 10, 12, 13; combinations of 1, 11, 12; combinations of 1, 11 - 13; combinations of 2, 11, 12; combinations of 2, 11 - 13; combinations of 3, 11, 12; combinations of 3, 11 - 13; combinations of 4, 11, 12; combinations of 4, 11 - 13; combinations of 5, 11, 12; combinations of 4, 5, 11 - 13; combinations of 4, 6, 11, 12; combinations of 4 - 6, 11 - 13; combinations of 4 - 7, 11, 12; combinations of 4 - 7, 11 - 13; combinations of 4, 8, 11, 12; combinations of 4, 8, 11 - 13; combinations of 9, 11, 12; combinations of 9,Those combining 11 to 13, those combining 10 to 12, and those combining 10 to 13.,

[0086] For the sake of facilitating a better understanding of the embodiments described in this specification, the following examples of various representative embodiments are provided. The following examples should not be read to limit or define the scope of the present invention.

[0087] (Example) Example 1: Comparison of Lactate Sensor Responses Two different lactate oxidase / polymer formulations for active region fabrication and two different membrane polymer formulations for mast transport limiting membrane fabrication were prepared to examine the performance of lactate-responsive sensors that characterize various permutations of these formulations. Details of the formulations and processes used to prepare the analyte sensors are further described below. Generally, the analyte sensors were fabricated in the same manner as the corresponding glucose-responsive analyte sensors, except that lactate oxidase (with or without albumin) was used instead of glucose oxidase, and in some cases, different membrane polymers were used.

[0088] Formulation of active region volume: Lactate oxidase was combined with the polymer of Formula 1 in an aqueous solution formulation as specified in Tables 1 and 2 below. Further details regarding the polymer are provided in co-owned U.S. Patent No. 6,605,200, which is hereby incorporated by reference in its entirety. The subscripts of each monomer represent exemplary atomic ratios.

[0089] [Chemical formula]

[0090] [Table 1]

[0091] [Table 2]

[0092] To form each active region, approximately 20 nL of each solution was deposited on the carbon working electrode to form six individual spots each having an area of approximately 0.01 mm 2 . Formulation 1 was dispensed four times and Formulation 2 was dispensed six times to form the spots. After film formation, the working electrode was cured overnight at 25 °C. Formulation 1 corresponds to that used to form the active region of a glucose-responsive analyte sensor, except that lactate oxidase was used instead of glucose oxidase.

[0093] Formulation for making the mast transport-limiting membrane: The membrane polymer formulation was prepared with the aqueous formulations specified in Tables 3 and 4 below.

[0094]

Table 3

[0095]

Table 4

[0096] To form a mast transport-limiting membrane on each active region prepared as described above, dip coating was used. Formulation 3 was deposited using four dips and Formulation 4 was deposited using four dips. A waiting time of approximately 10 minutes was used as the time between each dip. After completion of the dip coating, the membrane was cured in a dried vial at 25 °C for 24 hours, followed by 48 hours at 56 °C. Spray coating, screen printing, or a similar process can be used interchangeably to form the mast transport-limiting membrane. Formulation 3 corresponds to that used to form the mast transport-limiting membrane within a glucose-responsive analyte sensor.

[0097] The lactate-responsive analyte sensor was prepared using the film-forming conditions specified above. All possible combinations of the active region and the mass transport-limiting membrane were prepared, and eight sensors were fabricated for each possible combination. After fabrication, each sensor was exposed to a 5 mM lactate solution in 100 mM phosphate buffer saline (PBS) at 37 °C for 190 hours, and the operating potential was maintained at +40 mV versus Ag / AgCl. The combinations of the active regions and the mass transport-limiting membranes tested are shown in Table 5. The test results are shown in Figure 3.

[0098] [[Table 5]]

[0099] As shown in Figure 3, the analyte sensor for lactate with an active region and a mass transport-limiting membrane formulated in the same way as those that are well used in the analyte sensor (Group 1) that reacts to glucose showed a performance degradation when exposed to lactate. As shown, the signal intensity was less than 0.5 nA in all samples tested, which is unacceptably low for a viable lactate-responsive sensor. When polyvinylpyridine-co-styrene and Gly3 crosslinking agents were used instead of different crosslinking agents for formulation 3 (Group 2), the signal intensity further decreased.

[0100] As further shown in Figure 3, incorporating human serum albumin significantly improved the performance of the sensor. For example, sample group 3 showed a significantly higher signal intensity than that achieved in either sample of group 1 or group 2. However, there was a significant variation in the initial signal intensity among the samples of this group (>4 nA variance). Furthermore, there was a gradual decrease in the signal intensity from the initially observed maximum signal intensity. Similarly, due to the variability of the response and the low signal stability over time, this combination of sample groups may not be suitable for a viable lactate-responsive analyte sensor.

[0101] Surprisingly, the combination of the active region containing human serum albumin and the mast transport limitation membrane containing a crosslinked polyvinylpyridine homopolymer (Group 4) produced a combination of high signal intensity and an acceptable range of long-term signal stability. As shown in Figure 3, all replicated sensors in Group 4 had an initial signal intensity clustered within 1 nA of each other between 4 nA and 5 nA. This level of signal intensity and variability is within the range where a commercially viable lactate-responsive analyte sensor could potentially be developed. Furthermore, the signal intensity had a variation of less than 1 / 10 nA over 190 hours of signal observation, which is also within the range suitable for the development of a commercially realizable sensor.

[0102] As shown in Figure 4, the current observed in the Group 4 sensors responded rapidly and initially reached a stable value when the amount of lactate was increased by 1 mM at a time in a PBS solution without lactate.

[0103] Example 2: Alternative Mast Transport Limitation Membrane In these experiments, the active region containing lactate oxidase was formed into a film from a solution prepared as specified in Table 2 above. The deposition and curing of the active region onto the carbon electrode were performed as described in Example 1. The mast transport limitation membranes for various samples were prepared as follows.

[0104] Unless otherwise specifically indicated below, the formation of the mast transport limitation membrane on the active region was performed by dip coating (1 to 5 dips of the electrode with an approximate waiting time of 10 minutes between dips). The formulation of the mast transport limitation membrane is further specified below. After completion of the dip coating, the membrane was cured in a dried vial at 25°C for 24 hours and then at 56°C for 48 hours.

[0105] The sensor response was measured by placing the active region of the electrode in a beaker containing PBS at 37°C with 100 mM and pH = 7.5. The potential was raised to +40 mV with respect to Ag / AgCl, and then the current was continuously monitored.

[0106] Formulation 3 / Alternative cross-linking agent for Formulation 3'. Formulation 3 was changed to the alternative cross-linking agent specified in Table 6 below. The modified formulation is designated as Formulation 3' herein.

[0107] [Table 6]

[0108] Formulation 3' was prepared by mixing 4 mL of membrane polymer in 80:20 ethanol:HEPES buffer (140 mg / mL), 0.2 mL of PEGDGE400 in 80:20 ethanol:HEPES buffer (100 mg / mL), and 0.0132 mL of aminopropyl-terminated polydimethylsiloxane (PDMS) in ethanol (100 mg / mL).

[0109] Figure 5 shows an exemplary plot of the characteristics of a comparative example of a lactate sensor comprising an active region containing human serum albumin covered with a mass transport-limiting membrane formed from either Formulation 3 or Formulation 3'. As shown in Figure 5, when the mass transport-limiting membrane formed from Formulation 3 covered the active region containing lactate oxidase and human serum albumin (corresponding to Sensor Group 3 above, see Figure 3), the stability of the response decreased. The PEGDGE cross-linking modification example of the mass transport-limiting membrane (formed using Formulation 3') also produced a sensor response that changed over time, although less than that formed using Formulation 3. Furthermore, the mass transport-limiting membrane formed from Formulation 3' provided a higher response sensitivity than that obtained from Formulation 3. In contrast, both of these mass transport-limiting membranes provided a stable sensor response in the presence of glucose as an analyte (data not shown).

[0110] Alternative cross-linking agent for Formulation 4 / Formulation 4'. Formulation 4 was changed to the alternative cross-linking agent specified in Table 7 below. The modified formulation is designated as Formulation 4' herein.

[0111]

Table 7

[0112] The formulation 4' was prepared by combining 4.3 mL of the membrane polymer in 80:20 ethanol HEPES buffer (100 mg / mL), 0.25 mL of PEGDGE1000 in 80:20 ethanol:HEPES buffer (200 mg / mL), and 0.0132 mL of aminopropyl-terminated polydimethylsiloxane (PDMS) in ethanol (100 mg / mL).

[0113] Figure 6 is a graph showing an exemplary plot of the sensor performance of the mass transport-limiting membrane formed from formulation 4'. Increasing the molecular weight of the crosslinker decreased the performance of the sensor. As shown in Figure 6, the mass transport-limiting membrane obtained from formulation 4' did not provide a stable current response over a long period. The reaction decreased significantly in the first 48 hours, followed by relatively stable performance. Furthermore, the sensitivity was far below the target value of about 1 nA / mM.

[0114] Formulation 4' containing non-crosslinked PEG side chains (formulation 4''). Formulation 4' was modified to incorporate 3 - 4 wt% of non-crosslinked PEG side chains on the PVP polymer backbone and was crosslinked with PEGDGE1000 as in the case of formulation 4' (here designated as formulation 4''). The composition of formulation 4'' is specified in Table 8 below.

[0115]

Table 8

[0116] Complex 4’’ was prepared by mixing 4 mL of the membrane polymer in 80:20 ethanol:HEPES buffer (100 mg / mL), 0.025 mL of PEGDGE1000 in 80:20 ethanol HEPES buffer (200 mg / mL), and 0.0132 mL of aminopropyl-terminated polydimethylsiloxane (PDMS) in ethanol (100 mg / mL).

[0117] Figure 7 is a graph showing an exemplary plot of the sensor performance of a mast transport-limiting membrane formed from Complex 4’’. As shown, the sensor responded rapidly and achieved a stable current at a value significantly greater than that provided by either Complex 4 or Complex 4’. Further, as lactate was added in 1 mM increments, the sensor responded rapidly and achieved a stable current (see Figure 8). Thus, the sensor performance achieved with Complex 4’’ indicates that crosslinking agents other than PEGDGE400 can be used well as crosslinking agents for the membrane polymer.

[0118] Alternative of the two-layer membrane: The two-layer mast transport-limiting membrane was formed by depositing a polymer from Complex 4’ on the electrode surface and subsequently depositing the membrane polymer from Complex 3’ thereon. As described above, none of these membrane polymers provided acceptable performance on their own when covering the active region containing human serum albumin and lactate oxidase.

[0119] To prepare the sensor, the polymer from Complex 4’ was coated on the electrode surface by repeated dip-coating operations. Subsequently, the polymer from Complex 3’ was coated on the formed crosslinked PVP layer by repeated dipping coating operations. There was a 10-minute waiting time between consecutive dips. After all dip operations were completed, the sensor was cured in a dry vial at 25 °C for 24 hours and then at 56 °C for 48 hours.

[0120] FIG. 9 is a graph showing an exemplary plot of the sensor performance of a two-layer mast transport limitation membrane. As shown in FIG. 9, surprisingly, this two-layer structure provided a current response that was stable over time with an acceptable level of sensitivity, even though neither polymer provided acceptable performance. The response data in FIG. 9 relate to an electrode that was dipped twice in formulation 4' (crosslinked polyvinylpyridine) and then four times in formulation 3' (crosslinked polyvinylpyridine-co-styrene).

[0121] The amount (thickness) of each membrane polymer in the two-layer mast transport limitation membrane can potentially change the performance of the sensor, as shown in FIG. 10 below. FIG. 10 is a graph showing an exemplary plot of the sensor performance of two-layer mast transport limitation membranes of various layer thicknesses. The lower layer of FIG. 10 was formed using formulation 4', and the upper layer was formed using formulation 3 (Gly3 crosslinked variant of polyvinylpyridine-co-styrene). Thus, different crosslinking agents are tolerated, even if this polymer does not provide acceptable performance when used alone in the mast transport limitation membrane, in order to crosslink the polyvinylpyridine-co-styrene polymer. As shown in FIG. 10, dipping and coating the electrode twice with formulation 4' and four times with formulation 3 resulted in a good balance of sensitivity and stable current response. Changing the number of dipping and coating operations changed not only the thickness of each component of the two-layer membrane, but also the mass ratio of the membrane polymers to each other. As shown in FIG. 10, when the PVP layer is too thin (0 or 1 dip of formulation 4'), the sensitivity is high but the stability of the response is low, and when it is too thick (more than 3 dips), the electrode may have low sensitivity and low response stability. Spray coating, screen printing, or similar processes can be used alternately to form the mast transport limitation membrane.

[0122] Example of Mixed Membrane: 1.5 mL of polyvinylpyridine was added to 80:20 ethanol:HEPES buffer (100 mg / mL), 2.5 mL of polyvinylpyridine-co-styrene was added to 80:20 ethanol:HEPES buffer (140 mg / mL), 0.175 mL of PEGDGE400 was added to 80:20 ethanol:HEPES buffer (100 mg / mL), and 0.0132 mL of PDMS (100 mg / mL) was added to ethanol to prepare a composite membrane formulation (Formulation 5). The composition of Formulation 7 is shown in the following table.

[0123]

Table 9

[0124] Therefore, after crosslinking, Formulation 5 contained each polymer crosslinked with PEGDGE400. Figure 11 is a graph showing an exemplary plot of the sensor performance of a mixed mass transport-limitation membrane. As shown here, the mixed membrane provided a stable current response over time and an acceptable level of sensitivity. Furthermore, as the amount of lactate was increased by 1 mM each time, the current responded rapidly and reached a stable value (Figure 12).

[0125] The above-mentioned mixed mass transport-limitation membrane contained polyvinylpyridine and polyvinylpyridine-co-styrene polymers in a volume ratio of 3:5. Alternative ratios of these two polymers may also produce acceptable performance. Figure 13 is a graph showing an exemplary plot of the sensor performance of mixed mass transport-limitation membranes having various ratios of crosslinked polyvinylpyridine to crosslinked polyvinylpyridine-co-styrene. As shown in Figure 13, even when the amount of polyvinylpyridine was higher, acceptable sensitivity and response stability were obtained. However, when the ratio of polyvinylpyridine to polyvinylpyridine-co-styrene was decreased from a volume ratio of 2:2 to a volume ratio of 1:3, the overall sensitivity increased, but the response stability decreased.

[0126] Unless otherwise specified, all numbers representing amounts and the like in this specification and the related patent claims should be understood to be modified in all cases by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended patent claims are approximations that may vary depending on the desired characteristics sought to be obtained by embodiments of the present invention. At a minimum, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the reported significant digits and by applying ordinary rounding techniques.

[0127] In this specification, one or more exemplary embodiments incorporating various features are presented. For clarity, not all functions of a physical implementation are described or shown in this application. It is understood that in developing a physical implementation incorporating embodiments of the present invention, numerous implementation-specific decisions must be made to achieve the developer's goals, such as system-related, business-related, government-related compliance, etc. Constraints vary by implementation and sometimes over time. Although the developer's efforts may be time-consuming, such efforts are routine work for those skilled in the art and are benefited by this disclosure.

[0128] Various systems, tools, and methods are described herein using the term "comprising" various components or steps, but the systems, tools, and methods can also be said to "consist essentially of" or "consist of" various components and steps.

[0129] As used herein, the phrase "at least one" preceding a series of items, together with the term "and" or "or" that separates any of the items, modifies the entire list rather than each member of the list (i.e., each item). The phrase "at least one of" enables the meaning of including at least one of any of the items, and / or at least one of any combination of the items, and / or at least one of each item. By way of example, the phrases "at least one of A, B, and C" or "at least one of A, B, or C" each refer to only A, only B, or only C, respectively, to any combination of A, B, and C, and / or to at least one of each of A, B, and C.

[0130] Accordingly, the disclosed systems, tools, and methods are well adapted to attain the recited objects and advantages, as well as those inherent therein. The teachings of the present invention may be modified and practiced in different but equivalent manners that will be apparent to those skilled in the art having the benefit of the teachings herein, so the specific embodiments disclosed above are merely illustrative. Further, no limitations are intended as to the details of structure or design shown herein, other than as described in the appended claims. Accordingly, it is manifest that the specific exemplary embodiments disclosed above may be varied, combined, or modified, and all such variations are considered to be within the scope of the present invention. The systems, tools, and methods exemplified herein may be suitably practiced without the elements specifically disclosed herein and / or in the absence of any of the elements disclosed herein. The systems, tools, and methods have been described using the terms "comprising," "including," or "containing" various components or steps, but the systems, tools, and methods may also "consist essentially of" or "consist of" various components or steps. All of the numbers and ranges disclosed above may vary to some degree. When numerical ranges with lower and upper limits are disclosed, any number and any included range within that range are specifically disclosed. In particular, all ranges of values disclosed herein (in the form of "about a to about b" or equivalently "approximately a to b" or equivalently "from approximately a - b") represent all numbers and ranges included within the broader range of values. Also, the terms of the claims have their plain and ordinary meaning unless explicitly and clearly defined by the patentee. Further, the indefinite articles "a" or "an" used in the claims are defined herein to mean one or more of the elements introduced by them. In the event of any conflict in the usage of terms or terminology in this specification and one or more patents or other documents incorporated herein by reference, the definitions consistent with this specification should be adopted.

Claims

1. 1. An analyte sensor comprising: a working electrode having an active region disposed thereon, the active region comprising a polymer, albumin, and a lactate-responsive enzyme covalently bound to the polymer; a mass transport limitation membrane covering at least the active area on the working electrode.

2. The analyte sensor of claim 1 , wherein the albumin comprises human serum albumin.

3. The analyte sensor of claim 1 , wherein the lactate responsive enzyme comprises lactate oxidase.

4. The analyte sensor of claim 1 , wherein the active area comprises a plurality of sensing spots disposed on the working electrode.

5. 10. The analyte sensor of claim 1, wherein the mass transport limitation membrane comprises at least a cross-linked polyvinylpyridine homopolymer or copolymer.

6. 6. The analyte sensor of claim 5, wherein the mass transport limitation membrane comprises a multi-component membrane, said multi-component membrane comprising a cross-linked polyvinylpyridine homopolymer or copolymer and at least one second cross-linked polymer.

7. 7. The analyte sensor of claim 6, wherein the multi-component membrane comprises a first polymer comprising a cross-linked polyvinylpyridine homopolymer and a second polymer comprising a cross-linked polyvinylpyridine copolymer, or a first polymer comprising a first cross-linked polyvinylpyridine copolymer and a second polymer comprising a second cross-linked polyvinylpyridine copolymer.

8. 7. The analyte sensor of claim 6, wherein the multi-component membrane comprises a bilayer membrane, the bilayer membrane comprising a first layer comprising a polyvinylpyridine homopolymer or copolymer and a second layer comprising a second crosslinked polymer.

9. The analyte sensor of claim 8 , wherein the first layer is disposed directly on the active area and the second layer is disposed on the first layer.

10. 7. The analyte sensor of claim 6, wherein the mass transport limitation membrane comprises a homogeneous mixture of a cross-linked polyvinylpyridine homopolymer or copolymer and a second cross-linked polymer.

11. The analyte sensor of claim 1 , wherein the working electrode is disposed on a sensor tail configured for insertion into tissue.

12. The analyte sensor of claim 1 , wherein the active region further comprises an electron transfer agent covalently attached to the polymer.

13. exposing the analyte sensor to a fluid, The analyte sensor comprises: a working electrode having an active area disposed thereon, the active area including a polymer, albumin, and a lactate-responsive enzyme covalently bound to the polymer; exposing an analyte sensor, the analyte sensor comprising a mass transport limitation membrane covering at least the active area on the working electrode, to a fluid; acquiring a signal equal to or greater than the redox potential of the active area, the signal being proportional to a lactate concentration in the fluid; and correlating the signal to a lactate concentration in the fluid.

14. The method of claim 13 , wherein the fluid is a biological fluid and the analyte sensor is exposed to the biological fluid in vivo.

15. 14. The method of claim 13, wherein the albumin comprises human serum albumin.

16. 14. The method of claim 13, wherein the lactate responsive enzyme comprises lactate oxidase.

17. The method of claim 13 , wherein the mass transport limitation membrane comprises at least a cross-linked polyvinylpyridine homopolymer or copolymer.

18. 20. The method of claim 17, wherein the mass transport limitation membrane comprises a multi-component membrane, the multi-component membrane comprising a crosslinked polyvinylpyridine homopolymer or copolymer and at least a second crosslinked polymer.

19. 20. The method of claim 18, wherein the multi-component membrane comprises a first polymer comprising a cross-linked polyvinylpyridine homopolymer and a second polymer comprising a cross-linked polyvinylpyridine copolymer, or a first polymer comprising a first cross-linked polyvinylpyridine copolymer and a second polymer comprising a second cross-linked polyvinylpyridine copolymer.

20. 20. The method of claim 18, wherein the multi-component membrane comprises a bilayer membrane, the bilayer membrane comprising a first layer comprising a polyvinylpyridine homopolymer or copolymer and a second layer comprising a second crosslinked polymer.

21. 21. The method of claim 20, wherein the first layer is disposed directly on the active region and the second layer is disposed on the first layer.

22. 20. The method of claim 17, wherein the mass transport limitation membrane comprises a homogeneous mixture of a crosslinked polyvinylpyridine homopolymer or copolymer and a second crosslinked polymer.

23. The method of claim 13 , wherein the active region further comprises an electron transfer agent covalently attached to the polymer.

24. 14. The method of claim 13, The method further comprises determining the presence of one or more conditions in the subject based on the concentration of lactate in the bodily fluid, wherein the one or more conditions are selected from the group consisting of sepsis, infection, organ function, and any combination thereof.

25. 25. The method of claim 24, wherein the fluid is a biological fluid and the analyte sensor is exposed to the biological fluid in vivo.

26. a working electrode having an active region disposed thereon, the active region comprising a polymer, albumin, and a lactate-responsive enzyme covalently bound to the polymer; a mass transport limitation membrane covering at least the active area on the working electrode, The analyte sensor is configured to respond to sepsis, infection, organ function, or any combination thereof, and to provide a warning or other indication that sepsis, infection, organ failure, or any combination thereof may be present relative to the measured lactate concentration.

27. 27. The analyte sensor of claim 26, wherein the mass transport limitation membrane comprises at least a cross-linked polyvinylpyridine homopolymer or copolymer.

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