Lactate Sensors and Related Methods

Abstract

A lactate-responsive analyte sensor is provided that provides high sensitivity and stable responsiveness to lactate. Such an analyte sensor may include a working electrode having an active area disposed thereon and a mass transport limitation membrane covering at least the active area on the working electrode. The active area includes at least a polymer, albumin, and a lactate-responsive enzyme covalently bound to the polymer. The mass transport limitation membrane may include at least a cross-linked polyvinylpyridine homopolymer or copolymer.

Description

[Technical field]

[0001] The present invention relates to lactate sensors and related methods. [Background technology]

[0002] Detection of various analytes in an individual can be essential for monitoring their health. Deviations from normal analyte concentrations often indicate an underlying physiological condition, such as a metabolic state or disease, or exposure to a particular environmental factor or stimuli. For example, glucose levels can be particularly important for detecting and monitoring individuals with diabetes.

[0003] Lactate is another analyte whose in vivo levels may vary in response to a number of environmental or physiological factors, including, for example, diet, stress, exercise, sepsis or septic shock, infection, hypoxia, the presence of cancerous tissue, and the like. In the case of chronic lactate-altering conditions (e.g., disease), lactate levels may change slowly and therefore can be easily quantified using traditional blood sampling and laboratory measurements. Other lactate-altering conditions may be transient, in which case lactate levels may fluctuate very rapidly and erratically. Traditional laboratory measurements may be inadequate to determine lactate levels in such cases; that is, lactate levels may have changed several times between successive measurements, in which case abnormal lactate levels may be missed entirely, thereby potentially leading to erroneous diagnoses. 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 chronic, slowly varying lactate levels. For example, continuous monitoring of lactate can avoid the pain and expense associated with multiple blood draws to measure lactate levels.

[0004] While continuous analyte monitoring using implantable sensors can be advantageous in some cases, there are certain challenges associated with these types of measurements. Intravenous analyte sensors are invasive and can sometimes be painful for an individual to wear, especially over extended periods of time. Subcutaneous, interstitial, or dermal analyte sensors are often less painful for an individual to wear and can often provide sufficient measurement accuracy.

[0005] Non-intravenous in vivo glucose-responsive analyte sensors have been developed by several manufacturers over the past two decades, and some have recently gained 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 to a transition metal complex (electron transfer agent or electron transfer mediator) that helps transport the electrons released during the oxidation of glucose. In vivo glucose-responsive analyte sensors available from other manufacturers also use glucose oxidase as the sensing principle, but vary in the way that the sensing chemistry / protocol is varied.

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

[0007] A functional lactate-responsive analyte sensor may be constructed by replacing glucose oxidase from a glucose-responsive analyte sensor with lactate oxidase. Unlike the glucose-responsive analyte sensors briefly described above, the corresponding lactate-responsive analyte sensors generally perform poorly when analyzing lactate using similar sensing chemistries. That is, direct replacement of glucose oxidase with lactate oxidase may result in a lactate-responsive analyte sensor with insufficient sensitivity and / or a sensor response with insufficient stability. Thus, lactate-responsive analyte sensors, while functional, have not yet reached a level of development that provides stable and sensitive lactate analysis. Thus, despite the wealth of information that can be obtained through lactate monitoring, the diagnostic value of lactate-responsive analyte sensors remains significantly limited.

[0008] As noted above, lactate-responsive analyte sensors can replace glucose oxidase with lactate oxidase to facilitate the detection of lactate. Such lactate-responsive analyte sensors based on modified glucose-responsive sensor chemistry are described in commonly owned U.S. Patent No. 5,399,633, which is incorporated herein by reference in its entirety. As described therein, enhanced sensor sensitivity to lactate and some response stabilization can be achieved by modifying the glucose-responsive sensor chemistry to include catalase in the active region where lactate oxidase is instead present. While the incorporation of catalase helps to some extent, it does not completely stabilize the long-term response of the analyte sensor. Instead, the lactate signal of analyte sensors containing catalase drops to about 10% over 48 hours of monitoring. Catalase is known to be reactive to hydrogen peroxide, so it is believed that the stabilizing effect of catalase in lactate-responsive analyte sensors involves transient scrubbing of hydrogen peroxide, which may affect the activity of lactate oxidase. Although catalase may improve the performance of lactate-responsive analyte sensors, further performance improvements may be required for such analyte sensors to realize their true potential.

[0009] The drawings are included to illustrate certain aspects of the disclosure and should not be construed as exclusive embodiments. The disclosed subject matter is capable of significant modification, alteration, combination, and equivalents in form and function without departing from the scope of the disclosure. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Pat. No. 9,914,952 Summary of the Invention [Problem to be solved by the invention]

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

[0012] As noted above, measurement of lactate levels (concentrations) can be diagnostic of various physiological conditions and / or exposure to certain environmental factors. While laboratory measurement of lactate concentration may be sufficient for monitoring some physiological conditions, lactate levels may fluctuate rapidly in other instances such that laboratory measurement is not feasible. In vivo lactate-responsive analyte sensors using lactate-responsive enzymes can be used to conveniently measure both slowly and rapidly changing lactate concentrations, which provides particular diagnostic advantages for monitoring a variety of conditions in which lactate concentrations fluctuate rapidly and / or unexpectedly.

[0013] High analytical sensitivity and response stability are key factors required to achieve satisfactory performance of analyte sensors, especially those intended for long-term use in vivo. Despite the potential benefits that can be realized by monitoring lactate levels in vivo, lactate-responsive analyte sensors with sufficient sensitivity and response stability for reliable diagnostic analysis have yet to be developed.

[0014] Although high sensitivity and response stability can be achieved with glucose-responsive analyte sensors containing glucose oxidase, these desirable performance features do not currently translate to lactate-responsive analyte sensors by simply replacing glucose oxidase with lactate oxidase. That is, the sensitivity and response stability of lactate-responsive analyte sensors are significantly inferior to comparable glucose-responsive sensors. At least part of this problem is due to the differences between glucose oxidase and lactate oxidase. Although some progress has been made by utilizing stabilizers (e.g., catalase) to improve the function of lactate-responsive analyte sensors, the desirable levels of sensitivity and response stability have not yet been achieved.

[0015] Furthermore, mass transport limitation membranes for lactate-responsive analyte sensors pose an additional challenge: to date, mass transport limitation membranes successfully used in glucose-responsive analyte sensors have poor sensing properties when analyzing lactate using lactate-responsive enzymes: no membrane polymers or combinations thereof that are more compatible with lactate have 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 enhanced response stability of lactate-responsive analyte sensors. Improvements in these factors can provide high quality analyte data over a long wear life of several days or more, ideally a week or more, which can provide significant diagnostic value. That is, as described further herein, the performance of lactate-responsive analyte sensors can be improved by using different stabilizers instead of catalase and by modifying the mass transport limitation membrane located in the active area. As described herein, several different membrane chemistries or configurations may facilitate improved 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 describes how serum albumin, specifically human serum albumin, is incorporated within the active area of ​​a lactate-responsive analyte sensor to facilitate 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. As such, the introduction of this stabilizer within an analyte sensor intended for use in vivo does not pose any obvious biocompatibility issues. The suitability of human serum albumin as an alternative to catalase stabilizers is particularly surprising, given the lack of known hydrogen peroxide scavenging function associated with human serum albumin. Without being bound by theory or mechanism, the results suggest 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 similarly be incorporated into lactate-responsive analyte sensors to realize advantages comparable to those discussed herein.

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

[0019] Incorporation of serum albumin into the active area of ​​a lactate-responsive analyte sensor can improve sensor properties, particularly response sensitivity, but is usually insufficient by itself to provide a suitable level of both response sensitivity and long-term response stability. Beyond the expectation of those skilled in the art, appropriate selection of mass transport limitation membrane in lactate-responsive analyte sensor provides both sufficient lactate sensitivity and long-term response stability, particularly by coating above the active area with serum albumin. By combining serum albumin, particularly human serum albumin, in the active area, the present disclosure describes various alternative chemistries or configurations of mass transport limitation membrane that can further improve lactate response sensitivity and response stability in lactate-responsive analyte sensor to a suitable level.

[0020] Some glucose-responsive analyte sensors use cross-linked polyvinylpyridine-co-styrene polymers in the mass transport limitation 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 analyte sensors may be achieved by functionalization with bis-epoxides such as polyethylene glycol diglycidyl ether (PEGDGE) or glycerol triglycidyl ether (Gly3). Such membrane polymers are not effective by themselves in providing a stable response when analyzing lactate. This specification shows that these membrane polymers can be replaced with polyethylene glycol cross-linked polyvinylpyridine homopolymers or copolymers (different from polyvinylpyridine-co-styrene) to improve lactate sensing performance in terms of long-term response stability, especially when serum is co-present with lactate oxidase in the active region. Alternatively, and surprisingly, polyethylene glycol cross-linked polyvinylpyridine homopolymers or polyvinylpyridine copolymers, suitably combined (as bilayer membranes or homogeneous mixtures) with cross-linked polyvinylpyridine-co-styrene polymers more commonly used in glucose analysis, provide satisfactory lactate analyte sensor performance. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 illustrates an exemplary sensing system that may incorporate an analyte sensor of the present invention. [Figure 2A] FIG. 2 is a diagram of an exemplary two-electrode analyte sensor configuration suitable for use in certain embodiments of the present disclosure. [Figure 2B] FIG. 1 is a diagram of an exemplary three-electrode analyte sensor configuration suitable for use in certain embodiments of the present disclosure. [Figure 2C] FIG. 1 is a diagram of an exemplary three-electrode analyte sensor configuration suitable for use in certain embodiments of the present disclosure. [Diagram 3] Graph showing exemplary plots of sensor performance for Groups 1-4 of Example 1. [Figure 4] 13 is a graph showing an exemplary plot of the response of Group 4 analyte sensors of Example 1 to lactate solutions having various lactate concentrations. [Diagram 5] Graph showing an exemplary plot of comparative sensor performance of mass transport limitation films deposited from Formulations 3 and 3′. [Figure 6] Graph showing an exemplary plot of sensor performance of a mass transport limitation film deposited from Formulation 4'. [Figure 7] Graph showing an exemplary plot of sensor performance of a mass transport limitation film deposited from Formulation 4″. [Figure 8] Graph showing exemplary plots of sensor performance of mass transport limitation membranes deposited in lactate solutions having various lactate concentrations from Formulation 4″. [Figure 9] Graph showing an exemplary plot of sensor performance of a two-layer mass transport limitation membrane. [Figure 10] Graph showing exemplary plots of sensor performance for bilayer mass transport limitation films with various layer thicknesses. [Figure 11] 1 is a graph showing an exemplary plot of sensor performance of a mixed mass transport limitation film. [Figure 12] 13 is a graph showing an exemplary plot of sensor performance of a mixed mass transport limitation membrane to lactate solutions having various lactate concentrations. [Figure 13] 13 is a graph showing exemplary plots of sensor performance of blended mass transport limitation membranes having various ratios of crosslinked polyvinylpyridine to crosslinked polyvinylpyridine-co-styrene. [Figure 14] FIG. 2 is an exemplary schematic diagram of a portion of an analyte sensor having a bilayer membrane disposed on a working electrode suitable for use in some embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 definition 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 isomeric variations of the same structure). In a crosslinked copolymer, either type of monomer unit may carry a crosslinker, and some of either type of monomer units may have crosslinking groups and some may not have crosslinking groups.

[0023] Before describing the analyte sensors of the present disclosure in more detail, a brief overview of suitable in-vivo analyte sensor configurations and sensor systems that use the analyte sensors will first be provided so that embodiments of the present disclosure may be better understood.

[0024] FIG. 1 illustrates an exemplary sensing system that may incorporate an analyte sensor of the present invention. As shown in the figure, the sensing system 100 includes a sensor control device 102 and a reader device 120 that are configured to communicate with each other via a local communication path or link, which may be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted, wireless or wired. According to some embodiments, the reader device 120 may configure an output medium that allows for one or more user inputs, viewing the analyte concentration, such as lactate concentration, and alerts or notifications determined by the sensor 104 or its associated processor. The reader device 120 may be a general-purpose smartphone or a dedicated electronic reader device. Although only one reader device 120 is shown, in some cases, there may be multiple reader devices 120. The reader device 120 may also communicate with a remote terminal 170 and / or a trusted computer system 180 via communication paths / links 141 and / or 142, which may be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted, respectively. The reader device 120 may additionally or alternatively communicate with a network 150 (e.g., a cellular network, the Internet, or a cloud server) via communication path / link 151. The network 150 may further be communicatively coupled to a remote terminal 170 via communication path / link 152 and / or a trusted computer system 180 via communication path / link 153. Alternatively, the sensor 104 may communicate directly with the remote terminal 170 and / or the trusted computer system 180 without the presence of an intervening reader device 120. For example, the sensor 104 may communicate with the remote terminal 170 and / or the trusted computer system 180 via a direct communication link to the network 150 according to some embodiments, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is incorporated herein by reference in its entirety.Any suitable electronic communication protocol may be used for each of the communication paths or links, such as Near Field Communication (NFC), Radio Frequency Identification (RFID), BLUETOOTH or BLUETOOTH low energy protocols, WiFi, etc. According to some embodiments, the remote terminal 170 and / or the trusted computer system 180 may be accessible to individuals other than the primary user who have an interest in the user's analyte levels. The reader device 120 may include a display 122 and optional input elements 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 house circuitry and a power source for operating the sensor 104. Optionally, the power source and / or active circuitry may be omitted. A processor (not shown) may be communicatively coupled to the sensor 104, the processor being physically located within the sensor housing 103 or the reader device 120. According to some embodiments, the sensor 104 protrudes from an underside 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] The sensor 104 is adapted to be at least partially inserted into a tissue of interest, such as within the dermal or subcutaneous layers of the skin. The sensor 104 may include a sensor tail of sufficient length to insert to a desired depth within a given tissue. The sensor tail may comprise a working electrode and one or more active areas (sensing areas / spots or sensing layers) disposed on the working electrode and active for sensing an analyte of interest according to the present disclosure, in particular lactate. According to one or more embodiments of the present disclosure, each active area may include a lactate-responsive enzyme, suitable examples of which may include lactate oxidase or lactate dehydrogenase. According to some embodiments, the active area may include a polymeric material to which the enzyme is covalently attached. In various embodiments of the present disclosure, lactate may be monitored in any biological fluid of interest, such as dermal fluid, interstitial fluid, plasma, blood, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, etc. In certain embodiments, the analyte sensor of the present disclosure may be adapted to assay dermal fluid or interstitial fluid.

[0027] In some embodiments, the sensor 104 can automatically transfer data to the reader device 120. For example, the analyte concentration data is automatically and periodically transferred when the data is acquired or at a particular frequency, such as after a period of time has passed, with the data stored in memory until transmitted (e.g., every minute, five minutes, or other predetermined period of time). In other embodiments, the sensor 104 can communicate with the reader device 120 in a non-automatic manner rather than according to a set schedule. For example, RFID technology can be used to communicate data from the sensor 104 when the sensor electronics are brought within communication range of the reader device 120. The data can remain stored in the sensor 104's memory until communicated to the reader device 120. Thus, the patient does not need to be in close proximity to the reader device 120 at all times, but can instead upload data at their convenience. In still other embodiments, a combination of automatic and non-automatic data transfer can be performed. For example, data transfer can continue automatically until the reader device 120 is no longer within communication range of the sensor 104.

[0028] The introducer may be present temporarily to facilitate the introduction of the sensor 104 into the tissue. In an exemplary embodiment, the introducer may include a needle or similar sharp. It should be appreciated that in alternative embodiments, other types of introducers, such as a sheath or blade, may be present. More specifically, the needle or other introducer may be present temporarily in proximity to the sensor 104 prior to tissue insertion and then withdrawn. While present, the needle or other introducer may facilitate the insertion of the sensor 104 into the tissue by opening an access path for the sensor 104 to follow. For example, according to one or more embodiments, the needle may facilitate the penetration of the epidermis as an access path to the dermis to allow the implantation of the sensor 104 to occur. After the access path is opened, the needle or other introducer may be withdrawn to avoid posing a sharps hazard. In an exemplary embodiment, a suitable needle may be solid or hollow, beveled or non-beveled, and / or circular or non-circular in cross section. In more particular embodiments, suitable needles may be comparable in cross-sectional diameter and / or tip design to acupuncture needles, which may have a cross-sectional diameter of about 250 microns. However, it should be recognized that suitable needles may have larger or smaller cross-sectional diameters as required for a particular application.

[0029] In some embodiments, the tip of the needle (when present) can be angled over the end of the sensor 104 so that the needle penetrates the tissue first, opening an access path for the sensor 104. In other exemplary embodiments, the sensor 104 can reside within a lumen or channel of the needle, which similarly opens an access path for the sensor 104. In either case, after facilitating insertion of the sensor, the needle is removed.

[0030] Suitable configurations of analyte sensors of the present disclosure may employ two-electrode or three-electrode detection motifs, as further described below with reference to Figures 2A-2C. A three-electrode detection motif may have a working electrode, a counter electrode, and a reference electrode. A related two-electrode detection motif may have a working electrode and a second electrode, with the second electrode functioning as both the counter electrode and the reference electrode (i.e., as a counter / reference electrode). In both two-electrode and three-electrode detection motifs, the active area of ​​the analyte sensor may be in contact with the working electrode. According to embodiments of the invention, the active area may include a lactate-responsive enzyme and a stabilizer, particularly serum albumin. In some embodiments, the various electrodes may be at least partially stacked (layered) on top of each other, as described in more detail below. In some or other embodiments, the various electrodes may be laterally spaced apart from each other on the sensor tail. In either case, the various electrodes may be electrically insulated from each other by a dielectric material or similar insulator.

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

[0032] 2A, according to some embodiments, the membrane 220 covers at least the active area 218 and may optionally cover some or all of the working electrode 214 and / or counter / reference electrode 216, or the entire analyte sensor 200. One or both sides of the analyte sensor 200 may be covered with the membrane 220. The membrane 220 may include one or more polymeric membrane materials capable of limiting the influx of analyte into the active area 218 (i.e., the membrane 220 is a mass transport limiting membrane). The composition of the membrane 220 may be varied to facilitate the desired flux of lactate into the active area 218, thereby providing the desired signal strength and stability, as further described herein. The analyte sensor 200 may be operable to assay lactate by any of the following electrochemical detection techniques: coulometric, amperometric, voltammetric, or potentiometric. Particular compositions of the membrane 220 that may be suitable when assaying for lactate are described in more detail herein below.

[0033] 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 the analyte sensors 201 and 202 (FIGS. 2B and 2C) include an additional electrode 217. With the additional electrode 217, the counter / reference electrode 216 can then function as either a counter electrode or a reference electrode, and the additional electrode 217 performs the other electrode function, unless otherwise specified. The working electrode 214 continues to perform its original function. The additional electrode 217 can be placed on either the working electrode 214 or the electrode 216, with a separating layer of dielectric material between them. For example, as shown in FIG. 2B, dielectric layers 219a, 219, and 219c separate the electrodes 214, 216, and 217 from each other, providing electrical isolation. Alternatively, as shown in FIG. 2C, at least one of the electrodes 214, 216, and 217 may be disposed on opposing sides of the substrate 212. Thus, in some embodiments, the electrode 214 (working electrode) and the electrode 216 (counter electrode) may be disposed on opposing sides 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 location 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 area 218 of the analyte sensors 201 and 202 may include multiple spots or a single spot configured for detection of lactate. Also, the analyte sensors 201 and 202 may be operable to assay lactate by any of the following electrochemical detection techniques: coulometric, amperometric, voltammetric, or potentiometric.

[0034] Similar to analyte sensor 200, membrane 220 also covers active area 218 as well as other sensor components in analyte sensors 201 and 202, thereby functioning as a mass transport limitation membrane. In some embodiments, additional electrode 217 may be covered with membrane 220. Although FIGS. 2B and 2C show all electrodes 214, 216 and 217 as being covered with membrane 220, it should be appreciated that in some embodiments, only working electrode 214 may be covered. Furthermore, the thickness of membrane 220 on each of 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 analyte sensors 201 and 202 may be covered with membrane 220 in the sensor configuration of FIGS. 2B and 2C, or the entire analyte sensors 201 and 202 may be covered. Thus, the three-electrode sensor configuration shown in Figures 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 area of ​​any of the analyte sensors or analyte sensor configurations disclosed herein. A suitable electron transfer agent may facilitate the transport of electrons to or from the working electrode when an analyte (enzyme substrate) such as lactate undergoes an oxidation-reduction reaction. Certain embodiments of the analyte sensors disclosed herein may feature an active area that includes lactate oxidase and serum albumin, particularly human serum albumin, in combination with a mass transport limitation 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) that have redox potentials several hundred millivolts higher or lower than the redox potential of a standard calomel electrode (SCE). According to some embodiments, suitable electron transfer agents include low potential osmium complexes such as those described in U.S. Pat. Nos. 6,134,461 and 6,605,200, which are incorporated herein by reference in their entireties. 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 incorporated herein by reference in their entireties. Other suitable electron transfer agents may 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 polymer-bound electron transfer mediators include those described in U.S. Patent Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are incorporated herein by reference. Suitable ligands for metal complexes may also include, for example, bidentate or higher density ligands, such as bipyridine, biimidazole, phenanthroline, or pyridyl (imidazole). Other suitable bidentate ligands may include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher density ligands may be present in the metal complex to achieve a complete coordination sphere.

[0037] According to various embodiments of the present disclosure, a polymer may be present in the active area of ​​each of the analyte sensors or analyte sensor configurations disclosed herein. Polymers suitable for inclusion in the active area 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 area include those that include monomer units such as, for example, styrene, acrylamide, methacrylamide, or acrylonitrile. In an exemplary embodiment, the polymer in the active area of ​​the analyte sensor disclosed herein may be poly(4-vinylpyridine), some of the monomer units may be functionalized with alkyl carboxylate side chains, some of the monomer units may be attached to an electron transfer agent having an amide spacer group (e.g., see formula 1 below), or some of the monomer units may not be 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 performed by polymerizing a monomer unit having a covalently bonded electron transfer agent, or the electron transfer agent can be reacted with the polymer independently after the polymer has already been synthesized. According to some embodiments, a bifunctional spacer can covalently bond the electron transfer agent to the polymer in the active region, with a first functional group reactive with the polymer (e.g., a functional group capable of quaternizing the pyridine nitrogen atom or the imidazole nitrogen) and a second functional group reacting with the electron transfer agent (e.g., a functional group reacting with the ligand that coordinates the metal ion).

[0039] Similarly, according to some or various other embodiments of the present disclosure, the enzyme in the active region may be covalently linked to the polymer. According to more specific embodiments, the covalent linkage of the enzyme to the polymer may occur via a cross-linking agent when a suitable cross-linking agent is introduced. Cross-linking 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, imidoesters, epichlorohydrin, or derivatized variants thereof. Cross-linking agents suitable for reaction with free carboxylic acid groups in the enzyme may include, for example, carbodiimides. Cross-linking is generally intermolecular, but in some embodiments may be intramolecular.

[0040] The electron transfer agent and / or enzyme can be associated with the polymer of the active region by means other than covalent bonds. 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 yet other embodiments, the electron transfer agent and / or enzyme can be physically associated within the polymer without being bound to it.

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

[0042] Polyvinylpyridine copolymers suitable for inclusion in the mass transport limitation membrane can contain up to about 25% comonomer (based on the total amount of monomers in the copolymer), 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. Suitable comonomers are not particularly limited, provided that the mass transport limitation membrane provides sufficient lactate permeability and provides an analyte sensitivity of about 1 nA / mM or greater when exposed to lactate. The polyvinylpyridine copolymer can be different from the polyvinylpyridine-co-styrene copolymer according to various embodiments.

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

[0044] Alternatively, the analyte sensor of the present invention may include a working electrode having an active area disposed thereon and a mass transport limitation membrane covering at least the active area on the working electrode, the active area comprising a polymer, albumin, and a lactate-responsive enzyme covalently bound to the polymer, the mass transport limitation membrane comprising a membrane polymer that is permeable to lactate. More specifically, the mass transport limitation membrane may 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 analyte sensitivity of the analyte sensor when exposed to lactate is about 1 nA / mM or greater. The membrane polymer may comprise a polyethylene glycol cross-linked polyvinylpyridine homopolymer or copolymer in more specific embodiments, either as a single component membrane or as a multi-component membrane, such as a bilayer of a homogeneous mixture.

[0045] Yet another analyte sensor of the present disclosure may alternatively include a working electrode having an active area disposed thereon and a mass transport limitation membrane covering at least the active area on the working electrode, the active area comprising a polymer, catalase, and a lactate responsive enzyme covalently bound to the polymer, the mass transport limitation membrane comprising at least a cross-linked polyvinylpyridine homopolymer or copolymer.

[0046] According to various embodiments, the lactate responsive enzyme in the active area 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 assays according to 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. In terms of weight percent of the active region, lactate oxidase may be present in an amount ranging from about 10% to about 90% by weight of the active region, or from about 25% to about 75% by weight of the active region, or from about 30% to about 60% by weight of the active region.

[0048] According to various embodiments, the albumin in the active region may include human serum albumin, or alternatively, non-human albumin such as bovine serum albumin may be used without issue. According to the disclosure herein, albumin can be incorporated into the active region in an amount sufficient to stabilize the lactate-responsive enzyme, 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. In terms of weight percentage of the active region, albumin can be present in an amount ranging from about 25% to about 75% by weight of the active region, or from about 30% to about 60% by weight of the active region. In certain embodiments, the weight ratio of lactate oxidase to albumin can be in the range of about 10:1 to about 1:10 (w / w), about 5:1 to about 1:5, or about 1:1 to about 1:5, or about 1:1 to about 1:2.

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

[0050] The specific configuration of the analyte sensors disclosed herein, including the amount of albumin and lactate oxidase incorporated into the active area, will depend on the tissue through which the sensor tail penetrates, the expected concentration of lactate to be analyzed, and the conditions under which the analyte sensor will be operated during the analysis of lactate. In a more specific embodiment, the tissue through which the analyte sensor penetrates may be skin, such that the sensor tail is disposed within the dermal, interstitial, or subcutaneous layer below 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 area of ​​the analyte sensor may include an electron transfer agent covalently bonded to a polymer therein. The manner of 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 may be configured to achieve a steady-state current as soon as the analyte sensor of the present invention is operated at a given potential. Rapid achievement of a steady-state current may be facilitated by selecting an electron transfer agent that rapidly changes its oxidation state when exposed to a potential equal to or greater than its redox potential. Making the active region as thin as possible may also facilitate rapid achievement of a steady-state current. For example, a suitable thickness of the active region may range from about 0.1 microns to about 10 microns. In some or other embodiments, combining conductive materials, such as, for example, carbon nanotubes, graphene, or metal nanoparticles, within the active region may facilitate rapid achievement of a steady-state current. A suitable amount of conductive particles may range from about 0.1% to about 50% by weight, or from about 1% to about 50% by weight, or from about 0.1% to about 10% by weight, or from about 1% to about 10% by weight of the active region.

[0053] The active area on the working electrode of the analyte sensors disclosed herein may include at least one spot or layer disposed on the working electrode. Each spot or layer may range in size from about 0.01 mm2 to about 1 mm2, although larger or smaller active area spots or layers are also contemplated herein. The total size of the active area (total area of ​​all spots or layers) may be up to 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 area may range from about 0.05 mm2 to about 0.1 mm2. In some embodiments, the active area may include one spot or layer, although more typical embodiments of the analyte sensors of the present disclosure feature active areas having multiple spots disposed on the working electrode. The number of spots is not believed to be particularly limited, but may range from 2 to about 10, or from about 3 to about 8, or from about 4 to about 6, according to some embodiments.

[0054] It should further be appreciated that the sensitivity (output current) of the analyte sensor to lactate may be varied by modifying the coverage (area or size) of the active area, the nature and thickness of the mass transport limitation membrane covering the active area, and any combination thereof. Variations of these parameters to achieve the desired sensitivity may be readily implemented by one of ordinary skill in the art given the benefit of the disclosure herein.

[0055] The analyte sensors disclosed herein, particularly those having an active region comprising albumin and covalently bound lactate oxidase, and a mass transport limitation membrane comprising a polyvinylpyridine homopolymer or copolymer, particularly a polyvinylpyridine homopolymer, are characterized by a response stability (changing output current) function that varies by about 10% or less over 190 hours of measurement, or by about 5% or less over 190 hours of measurement, or by about 1% or less over 190 hours of measurement. This degree of variation over a time frame is not expected to be clinically significant (i.e., only marginally impacting Clark Error Grid analysis and MARD or MAD analysis). Thus, the catalase-stabilized lactate-responsive analyte sensors described in the above patents are expected to exhibit even lower response currents over 190 hours of measurement.

[0056] To achieve sufficient response stability, the analyte sensor of the present invention uses a mass transport limitation membrane selected for its compatibility with lactate. More specifically, an advantageous mass transport limitation membrane suitable for use with lactate-responsive enzymes, particularly lactate oxidase, may comprise a crosslinked polyvinylpyridine homopolymer or copolymer. The mass transport limitation 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 multicomponent and 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 specifically, the multicomponent membrane may comprise a first polymer comprising a crosslinked polyvinylpyridine homopolymer and a second polymer comprising a crosslinked polyvinylpyridine copolymer, or 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 more detailed embodiments, the second crosslinked polymer may comprise a crosslinked polyvinylpyridine copolymer, in particular a crosslinked polyvinylpyridine-co-styrene polymer, in which a portion of the pyridine nitrogen atoms are functionalized with non-crosslinked poly(ethylene glycol) tails and a portion of the pyridine nitrogen atoms are functionalized with alkylsulfonic acid groups.

[0057] Multicomponent membranes suitable for use in combination with lactate-responsive enzymes, particularly lactate oxidase, in the analyte sensors of the present invention may include, according to various embodiments, a two-layer membrane. A suitable two-layer membrane may include a first layer comprising a cross-linked polyvinylpyridine homopolymer or copolymer and a second layer comprising a second cross-linked polymer, particularly a cross-linked polyvinylpyridine copolymer. The second layer may include 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 area and the second layer may be disposed on the first layer. In alternative embodiments, the second layer may be disposed directly on the active area and the first layer may be disposed on the second layer. The thickness and order of the first and second layers may be varied to provide the desired sensitivity. Such a two-layer configuration may be prepared in some embodiments by coating a first layer onto the active area (e.g., by spray coating, painting, inkjet printing, roller coating, dip coating, etc.) and then coating the second layer onto the first layer by the same or a different coating technique (e.g., spray coating, painting, inkjet printing, roller coating, dip coating, etc.) In other embodiments, the two-layer membrane is constructed with the first and second layers reversed (thereby reversing the position of the crosslinked polyvinylpyridine homopolymer or copolymer) and each layer coated as described above.

[0058] Bilayer membranes are by their nature heterogeneous, since two different membrane polymers are layered on top of each other. Other multi-component membranes suitable for use in combination with lactate-responsive enzymes, particularly lactate oxidase, in the analyte sensors of the present invention may be homogeneous in composition. More specifically, in some embodiments, suitable multi-component membranes may 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 cross-linked polymers in the homogeneous mixture may be varied over a significant range, and the ratio may be adjusted to provide the desired sensitivity of the analyte sensors disclosed herein.

[0059] Figure 14 is an exemplary schematic diagram of a portion of an analyte sensor having a two-layer membrane disposed on a working electrode suitable for use in some embodiments of the present disclosure. 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 area 618 is disposed on the working electrode 614 and includes a lactate-responsive enzyme in accordance with the present disclosure.

[0060] 14, the active area 618 is covered with a two-layer membrane 621, which includes a membrane layer 621a in direct contact with the active area 618 and a membrane layer 621b covering membrane layer 621a. In accordance with the disclosure herein, membrane layers 621a and 621b may comprise different membrane polymers, at least one of which is a polyvinylpyridine homopolymer or copolymer.

[0061] In another embodiment, a homogenous membrane layer comprising a mixture of different membrane polymers may replace the bi-layer membrane 621 of FIG. 14 in accordance with the disclosure herein. The analyte sensors described above can be utilized in a variety of ways to measure lactate and therefrom determine the concentration of lactate, which can be further correlated to a physiological condition (e.g., resulting from disease or exposure to an environmental factor) according to various embodiments, as described in more detail below.

[0062] According to various embodiments, the method of the invention may comprise exposing an analyte sensor to a fluid, the analyte sensor comprising a working electrode having an active area disposed thereon and a mass transport limitation membrane covering at least the active area on the working electrode. The active area comprises a polymer, albumin, and a lactate responsive enzyme, particularly lactate oxidase, covalently bound to the polymer. The mass transport limitation membrane may in certain embodiments comprise at least a cross-linked polyvinylpyridine homopolymer or copolymer. The method may further comprise obtaining a signal at or above the redox potential of the active area, 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 a more specific embodiment, the fluid is a biological fluid and the analyte sensor is exposed to the biological fluid in vivo in a subject in whom it is desired to measure the concentration of lactate. According to more specific embodiments of the invention, the subject may be a human. Biological fluids suitable for analysis with the analyte sensors of the invention may include any of the biological fluids discussed in more detail above.

[0064] The signal associated with the active area upon exposure to lactate can be correlated to a corresponding lactate concentration by consulting a lookup table or calibration curve. The lookup table or calibration curve can exist in physical form (i.e., written form) or electronic form (i.e., database or computer algorithm). A lookup table of various concentrations of lactate can be created by analyzing multiple samples with known lactate concentrations and recording the response of the analyte sensor at each concentration. Similarly, a calibration curve for lactate can be determined by plotting the analyte sensor response 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 nearly linear with respect to the concentration of lactate. In some embodiments, a 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 described further below, the lookup table or calibration curve can be consulted manually or electronically to determine the concentration of lactate in the fluid.

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

[0066] Similarly, the processor can input the sensor response values ​​of samples with unknown lactate concentrations into the corresponding calibration curve, and the processor then reports the lactate concentration accordingly.

[0067] As discussed above, lactate concentrations determined using the analyte sensors of the present invention may be further correlated to one or more physiological conditions. The one or more physiological conditions may be mediated by lactate in vivo and / or may result in elevated or decreased lactate levels in vivo. More specifically, one or more physiological conditions that may be monitored by the analyte sensors of the present invention include, for example, sepsis, infection, organ function, or any combination thereof. Other conditions in which lactate monitoring may be beneficial include, for example, physiological stress and exercise, both of which may increase lactate levels. Monitoring any of these conditions may include, for example, tracking trends in lactate levels over time and / or determining instantaneous lactate levels at a particular time. Optionally, an alert is issued if the lactate level exceeds a threshold concentration defined for a given condition or if the lactate level trends toward a defined threshold as monitored over time. Thus, the analyte sensors disclosed herein may be configured to provide an alert or otherwise provide an indication that sepsis, infection, organ failure, or any combination thereof may be present. Depending on the physiological condition being monitored, the threshold concentration may vary.

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

[0069] Lactate levels are useful in the diagnosis, monitoring, and / or evaluation of various forms of sepsis and / or associated infections. Thus, determining lactate concentrations according to the present invention may enable sepsis and / or infections to be more effectively monitored, evaluated, and / or managed. Alternatively, the analyte sensors of the present invention may be utilized to monitor subjects (e.g., patients in a hospital) who are at risk for sepsis and / or infection but who are not currently showing signs of either condition. Lactate levels obtained according to the present invention may be combined with other analyte levels and / or physiological markers (e.g., body temperature, heart rate, respiratory rate, blood pressure, decreased urine output, sudden changes in mental status, decreased platelet count) and other markers such as C-reactive protein (CRP), procalcitonin, pancreatic stone protein (PSP), circulating complement (C3 and C4), ferritin, cholesterol, albumin, cortisol, and neutrophil gelatinase-associated lipocalin) in a diagnosis to monitor the progression of sepsis and associated infections in a subject. In certain embodiments, the concentration of lactate can be measured in cerebrospinal fluid to monitor, evaluate, and / or diagnose meningitis and / or septic meningitis. Additional markers of 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 of bacterial sepsis or bacterial infection can include, for example, glutamic acid, malic acid, pseudouridine, acetylcarnitine, glycerophosphocholine, hydroxyphenyllactic acid, acetylneuraminic acid, pseudouridine, and tyrosine. Additional markers of viral sepsis, including septic meningitis, or viral infection can include, for example, hypoxanthine, inosine, and hexanoylcarnitine.

[0070] In some embodiments, the analyte sensors of the present invention may be used to monitor a subject's exposure to an infectious agent. For example, the analyte sensors may monitor a subject's condition following 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 sensors may also be used to monitor a subject's response to anti-infective agents and / or treatments administered to the subject to treat sepsis or infection or symptoms thereof.

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

[0072] The analyte sensors disclosed herein can monitor organ function, particularly liver function, by assessing the activity of lactate dehydrogenase or another suitable lactate-responsive enzyme (e.g., lactate oxidase). The analyte sensors can be used to monitor, diagnose, and / or detect analyte levels in subjects experiencing or at risk of organ dysfunction, potentially identifying organ disease before life-threatening symptoms appear. Lactate concentrations or rates of change in lactate concentrations determined by the analyte sensors disclosed herein can facilitate diagnosis or analysis of organ failure or dysregulation or potential organ failure or dysregulation. The analyte sensors provide an alarm when lactate concentrations exceed a threshold amount or tend to exceed a threshold amount that is characteristic of organ damage or dysfunction.

[0073] Lactate levels obtained according to the present invention can be diagnostically combined with other analyte levels and / or physiological markers to determine organ function and / or failure, particularly of the liver. 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). Lactate levels monitored according to the present invention can further be utilized to shorten the time before initiating a course of treatment to combat organ failure and / or to monitor the progress of a course of treatment or treatment.

[0074] Embodiments disclosed herein include the following: A. Analyte Sensor The analyte sensor comprises a working electrode having an active area disposed thereon, the active area comprising a polymer, albumin, and a lactate-responsive enzyme covalently bound to the polymer, and a mass transport limitation membrane covering at least the active area on the working electrode.

[0075] B. A method for measuring lactate, the method comprising exposing an analyte sensor to the fluid, the analyte sensor comprising a working electrode having an active area disposed thereon, the active area comprising a polymer, albumin, and a lactate responsive enzyme covalently bound to the polymer, and a mass transport limitation membrane covering at least the active area on the working electrode, acquiring a signal above the redox potential of the active area, the signal being proportional to the lactate concentration in the fluid, and correlating the signal to the lactate concentration in the fluid.

[0076] C. Lactate Sensor Responsive to Sepsis, Infection, or Organ Function The lactate sensor includes an analyte sensor comprising a working electrode having an active area disposed thereon, the active area comprising a polymer, albumin, and a lactate-responsive enzyme covalently bound to the polymer, and a mass transport limitation membrane covering at least the active area on the working electrode, wherein 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 a measured lactate concentration.

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

[0078] Element 2: Lactate-responsive enzymes include lactate oxidase. Element 3: The active area includes multiple sensing spots disposed on the working electrode. Element 4: The mass transport limitation membrane comprises a multi-component membrane, the multi-component membrane comprising a cross-linked polyvinylpyridine homopolymer or copolymer and at least a second cross-linked polymer.

[0079] Element 5: 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.

[0080] Element 6: Wherein the multi-component membrane comprises a bi-layer membrane, the bi-layer membrane comprising a first layer comprising a polyvinylpyridine homopolymer or copolymer and a second layer comprising a second cross-linked polymer. Element 7: A first layer is disposed directly on the active area and a second layer is disposed on the first layer.

[0081] Element 8: The mass transport limitation membrane 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 that is configured for insertion into tissue.

[0082] Element 10: The active region further comprises an electron transfer agent covalently attached 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: Wherein the method further comprises determining the presence of one or more conditions in the subject based on the concentration of lactate in the fluid, the one or more conditions being selected from sepsis, infection, organ function, and any combination thereof.

[0083] Element 13: The analyte sensor of claim 1, wherein the mass transport limitation membrane comprises at least a cross-linked polyvinylpyridine homopolymer or copolymer. By way of non-limiting example, exemplary combinations applicable to A through CB include the following:

[0084] The analyte sensor of A or C or the method of B in combination with elements 1 and 2. In the present invention, the present invention relates to a method for manufacturing a semiconductor device comprising: those having elements 1 and 2, those having elements 1, 2, and 13, those having elements 1 and 3, those having elements 1 to 3, those having elements 1 to 3, 13, those having elements 1 and 4, those having elements 1, 4, and 13, those having elements 1 and 13, those having elements 2 and 4, those having elements 2 and 4, 13, those having elements 2 and 13, those having elements 3 and 4, those having elements 3 and 4, 14, those having elements 1 and 13, those having elements 1 to 4, those having elements 1 to 4, 13, those having elements 2 to 4, those having elements 2 to 4, 13, those having elements 1 and 4, 5, those having elements 1 and 4, 5, 13, those having elements 2 and 4, 5, those having elements 2 and 4, 5, 13, those having elements 3 to 5, those having elements 3 to 5, 13, those having elements 4, 5, those having elements 4, 5, 13, those having elements 1 and 4 to 6, and those having elements 4 to 6, 13 , those with 2,4,5,6, those with 2,4,5,6,13, those with 3-6, those with 3-6,13, those with 4-7, those with 4-7,13, those with 4,8, those with 4,8,13, those with 4,5,8, those with 4,5,8,13, those with 1,9, those with 1,9,13, those with 2,9, those with 2,9,13, those with 3,9, those with 3,9,13, those with 4,9, those with 4,9,13, those with 1,10, those with 1,10,13, those with 2,10, those with 2,10,13, those with 3,10, those with 3,10,13, those with 4,10, those with 4,10,13, those with 9,10, those with 9,10,13.

[0085] For method B, the following combinations were used: combination of elements 1 and 11, combination of elements 1, 11, and 13, combination of elements 1 and 13, combination of elements 2, 11, combination of elements 2, 13, combination of elements 3, 11, combination of elements 3, 13, combination of elements 4, 11, combination of elements 4, 13, combination of elements 4, 5, and 11, combination of elements 4, 5, and 11, combination of elements 4 to 6, 11, combination of elements 4, 6, 11, and 13, combination of elements 4 to 7, 11, combination of elements 4 to 7, 11, and 13, combination of elements 4, 8, 11 combination of , combination of 4, 8, 11, 13, combination of 9, 11, combination of 9, 11, 1310, 11, combination of 10, 11, 13, combination of 11, 12, combination of 11 to 13, combination of 1, 12, combination of 1, 12, 13, combination of 2, 12, combination of 2, 12, 13, combination of 3, 12, combination of 3, 12, 13, combination of 4, 12, combination of 4, 12, 13, combination of 4, 5, 12 combination of 4,5,12,13, combination of 4-6,12, combination of 4-6,12,13, combination of 4-7,12, combination of 4-7,12,13, combination of 4-8,12, combination of 4,8,12,13, combination of 9,12, combination of 9,12,13, combination of 10,12, combination of 10,12,13, combination of 1,11,12, combination of 1,11-13, combination of 2,11,12, combination of 2,11- Combinations of 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, 9,A combination of 11 to 13, a combination of 10 to 12, and a combination of 10 to 13.

[0086] To facilitate a better understanding of the embodiments described herein, the following examples of various representative embodiments are given. The following examples should not be read to limit or define the scope of the 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 mass transport limitation membrane fabrication were prepared to characterize various permutations of these formulations and to investigate the performance of lactate-responsive sensors. Details of the formulations and processes used to prepare the analyte sensors are described further below. In general, the analyte sensors were fabricated similarly to 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] Active Area Volume Formulations: 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 polymers are provided in commonly owned U.S. Patent No. 6,605,200, which is incorporated by reference in its entirety. The subscripts for each monomer represent exemplary atomic ratios.

[0089] [ka]

[0090] [Table 1]

[0091] [Table 2]

[0092] To deposit each active area, approximately 20 nL of each solution was deposited onto the carbon working electrode, each with a thickness of approximately 0.01 mm. 2 Formulation 1 was dispensed 4 times and Formulation 2 was dispensed 6 times to form the spots. After deposition, the working electrode was cured overnight at 25° C. Formulation 1 corresponds to that used to deposit the active area of ​​the glucose responsive analyte sensor, except that lactate oxidase was used instead of glucose oxidase.

[0093] Mass Transport Limitation Membrane Fabrication Formulations: Membrane polymer formulations were prepared in aqueous solution formulations as specified in Tables 3 and 4 below.

[0094] [Table 3]

[0095] [Table 4]

[0096] Dip coating was used to deposit the mass transport limitation film on each active area prepared as above. Formulation 3 was deposited using 4 dips and Formulation 4 was deposited using 4 dips. A waiting time of about 10 minutes was used between each dip. After completion of dip coating, the film was cured in a dry vial at 25° C. for 24 hours followed by 56° C. for 48 hours. Alternately, spray coating, screen printing, or similar processes can be used to deposit the mass transport limitation film. Formulation 3 corresponds to that used to deposit the mass transport limitation film in the glucose responsive analyte sensor.

[0097] Lactate-responsive analyte sensors were prepared using the deposition conditions specified above. All possible combinations of active areas and mass transport limitation films 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 buffered saline (PBS) at 37°C for 190 hours, with the operating potential held at +40 mV vs. Ag / AgCl. The combinations of active areas and mass transport limitation films tested are listed in Table 5. Test results are shown in Figure 3.

[0098] [Table 5]

[0099] As shown in Figure 3, lactate-responsive analyte sensors with active areas and mass transport limitation membranes formulated similarly to those successfully used in glucose-responsive analyte sensors (Group 1) performed poorly when exposed to lactate. As shown, signal strength was less than 0.5 nA for all samples tested, which is undesirably low for a viable lactate-responsive sensor. When polyvinylpyridine-co-styrene and a Gly3 crosslinker were used instead of polyvinylpyridine and a different crosslinker in Formulation 3 (Group 2), signal strength was further reduced.

[0100] As further shown in Figure 3, the incorporation of human serum albumin significantly improved the sensor performance. For example, sample group 3 exhibited significantly higher signal intensity than was realized with either group 1 or group 2 samples. However, there was significant variability in the initial signal intensity between this group of samples (>4 nA variance). Additionally, there was a steady decrease in signal intensity from the maximum signal intensity initially observed. Similarly, due to the variability in response and poor signal stability over time, this sample group combination may not be suitable for a viable lactate-responsive analyte sensor.

[0101] Surprisingly, the combination of a human serum albumin-containing active region and a mass transport limitation membrane containing cross-linked polyvinylpyridine homopolymer (group 4) produced a combination of high signal intensity and acceptable signal stability over time. As shown in Figure 3, all replicate sensors in group 4 had initial signal intensities between 4 nA and 5 nA, clustered within 1 nA of each other. This level of signal intensity and variability is within the range in which a commercially viable lactate-responsive analyte sensor may be developed. Furthermore, the signal intensity varied by less than 1 / 10 nA over 190 hours of signal observation, also within a range suitable for the development of a commercially viable sensor.

[0102] As shown in Figure 4 , the current observed for the group 4 sensor responded rapidly and achieved a stable value when the amount of lactate was increased by 1 mM, initially in lactate-free PBS solution.

[0103] Example 2: Alternative Mass Transport Limitation Membranes In these experiments, active regions containing lactate oxidase were deposited from solutions formulated as specified above in Table 2. Deposition and curing of the active regions onto carbon electrodes was carried out as described in Example 1. Mass transport limitation membranes of various samples were prepared as shown below.

[0104] Unless otherwise noted below, deposition of the mass transport limitation membrane onto the active area was accomplished by dip coating (1-5 dippings of the electrode with approximately 10 minutes wait time between dips). The mass transport limitation membrane formulation is further specified below. After completion of dip coating, the membrane was cured in a dry vial at 25°C for 24 hours followed by 56°C for 48 hours.

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

[0106] Formulation 3 / Formulation 3' Alternative Crosslinkers to Formulation 3. Formulation 3 was modified with the alternative crosslinkers specified in Table 6 below. The modified formulation is referred to herein as Formulation 3'.

[0107] [Table 6]

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

[0109] FIG. 5 shows an exemplary plot of the properties of comparative lactate sensors comprising an active region containing human serum albumin covered with mass transport limitation membranes deposited from either formulation 3 or formulation 3'. As shown in FIG. 5, mass transport limitation membranes deposited from formulation 3 had a reduced stability of response when covered with an active region containing lactate oxidase and human serum albumin (corresponding to sensor group 3 above, see FIG. 3). The PEG-DGE crosslinked modification of the mass transport limitation membrane (deposited using formulation 3') also produced a sensor response that changed over time, although less than that deposited using formulation 3. Furthermore, the mass transport limitation membrane deposited from formulation 3' produced a higher response sensitivity than that obtained from formulation 3. In contrast, both of these mass transport limitation membranes provided a stable sensor response in the presence of glucose as an analyte (data not shown).

[0110] Alternative Crosslinkers for Formulation 4 / Formulation 4'. Formulation 4 was modified with alternative crosslinkers as specified in Table 7 below. The modified formulation is referred to herein as Formulation 4'.

[0111] [Table 7]

[0112] Formulation 4′ was prepared by combining 4.3 mL of 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] FIG. 6 is a graph showing an exemplary plot of the sensor performance of the mass transport limitation film deposited from Formulation 4'. Increasing the molecular weight of the crosslinker decreased the sensor performance. As shown in FIG. 6, the mass transport limitation film obtained from Formulation 4' did not provide a stable current response over time. There was a significant decrease in response in the first 48 hours, followed by a relatively stable performance thereafter. Furthermore, the sensitivity was far below the target value of about 1 nA / mM.

[0114] Formulation 4' with Non-crosslinked PEG Side Chains (Formulation 4''). Formulation 4' was modified to incorporate 3-4 wt% non-crosslinked PEG side chains on the PVP polymer backbone and crosslinked with PEGDGE1000 as in Formulation 4' (now designated as Formulation 4''). The composition of Formulation 4'' is set forth below in Table 8.

[0115] [Table 8]

[0116] Formulation 4″ was prepared by mixing 4 mL of 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] FIG. 7 is a graph showing an exemplary plot of sensor performance for a mass transport limitation membrane deposited from Formulation 4″. As shown, the sensor responded quickly and achieved a stable current at a value significantly greater than that provided by either Formulation 4 or Formulation 4′. Furthermore, the sensor responded quickly and achieved a stable current as lactate was added in 1 mM increments (see FIG. 8). Thus, the sensor performance achieved with Formulation 4″ indicates that crosslinkers other than PEGDGE400 can be successfully used as the crosslinker for the membrane polymer.

[0118] Bilayer Membrane Modifications: Bilayer mass transport limitation membranes were formed by depositing Formulation 4' onto the electrode surface, followed by depositing the membrane polymer from Formulation 3' thereon. As noted above, none of these membrane polymers by themselves provided acceptable performance when covering the active area containing human serum albumin and lactate oxidase.

[0119] To prepare the sensor, the polymer from formulation 4' was coated on the electrode surface by repeated dip-coating operations. Then, the polymer from formulation 3' was coated on the deposited cross-linked PVP layer by repeated dip-coating operations. There was a waiting time of 10 minutes between successive 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 sensor performance of a bilayer mass transport limitation membrane. As shown in FIG. 9, surprisingly, the bilayer structure provided a stable current response over time with an acceptable level of sensitivity, even though neither polymer provided acceptable performance. The response data in FIG. 9 is for an electrode dipped twice with Formulation 4' (crosslinked polyvinylpyridine) followed by four dips with Formulation 3' (crosslinked polyvinylpyridine-co-styrene).

[0121] The amount (thickness) of each membrane polymer in the bilayer mass transport limitation membrane can change the sensor performance, as shown in FIG. 10 below. FIG. 10 is a graph showing an exemplary plot of sensor performance for bilayer mass transport limitation membranes with various layer thicknesses. Formulation 4' was used to deposit the bottom layer of FIG. 10, and Formulation 3 (Gly3 crosslinked variant of polyvinylpyridine-co-styrene) was used to deposit the top layer. Thus, different crosslinkers are acceptable for crosslinking the polyvinylpyridine-co-styrene polymer, even those that do not result in acceptable performance when this polymer is used alone in the mass transport limitation membrane. As shown in FIG. 10, dip coating the electrode twice with Formulation 4' and four times with Formulation 3 provided a good balance of sensitivity and stable current response. Varying the number of dip coating operations changed the thickness of each component of the bilayer membrane as well as the mass ratio of the membrane polymers to each other. If the PVP layer is too thin (0 or 1 dip of Formulation 4'), the sensitivity will be high but the response will be less stable, and if it is too thick (more than 3 dips), the electrode may have a low sensitivity and a low response stability, as shown in Figure 10. Spray coating, screen printing, or similar processes can be used alternately to deposit the mass transport limitation membrane.

[0122] Mixed Membrane Modifications A composite membrane formulation (Formulation 5) was prepared by mixing 1.5 mL of polyvinylpyridine in 80:20 ethanol:HEPES buffer (100 mg / mL), 2.5 mL of polyvinylpyridine-co-styrene in 80:20 ethanol:HEPES buffer (140 mg / mL), 0.175 mL of PEGDGE400 in 80:20 ethanol:HEPES buffer (100 mg / mL), and 0.0132 mL of PDMS (100 mg / mL) in ethanol. The composition of Formulation 7 is shown in the table below.

[0123] [Table 9]

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

[0125] The mixed mass transport limitation film described above 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. FIG. 13 is a graph showing an exemplary plot of sensor performance for mixed mass transport limitation films having various ratios of crosslinked polyvinylpyridine to crosslinked polyvinylpyridine-co-styrene. As shown in FIG. 13, acceptable sensitivity and stability of response were obtained even with higher amounts of polyvinylpyridine. However, when the ratio of polyvinylpyridine to polyvinylpyridine-co-styrene was decreased from a 2:2 volume ratio to a 1:3 volume ratio, the overall sensitivity was increased but the stability of response was decreased.

[0126] Unless otherwise indicated, all numbers expressing quantities and the like in the specification and the associated claims should be understood in all instances to be modified by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by embodiments of the invention. At the very least, and without any attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0127] One or more exemplary embodiments incorporating various features are presented herein. For clarity, not all features of a physical implementation are described or shown in this application. It is understood that in developing a physical embodiment incorporating an embodiment of the invention, numerous implementation-specific decisions must be made to achieve the developer's goals, such as system-related, business-related, government-related, and other compliance. Constraints vary from implementation to implementation and from time to time. While the developer's efforts may be time-consuming, such efforts are nevertheless routine for those of ordinary skill in the art having the benefit of this disclosure.

[0128] Although various systems, tools, and methods are described herein in terms of "comprising" various components or steps, the systems, tools, and methods may also be said to "consist essentially of" or "consist of" the various components and steps.

[0129] As used herein, the phrase "at least one" preceding a list of items, together with the terms "and" or "or" separating any of the items, modifies the list as a whole (i.e., each item) and not each member of the list. The phrase "at least one" allows for the meaning to include at least one of any item, and / or at least one of any combination of the items, and / or at least one of each item. By way of example, the phrase "at least one of A, B, and C" or "at least one of A, B, or C" refers to A only, B only, or C only, respectively; any combination of A, B, and C, and / or at least one of each of A, B, and C.

[0130] Thus, the disclosed systems, tools, and methods are well adapted to achieve the objects and advantages mentioned, as well as those inherent therein. The specific embodiments disclosed above are merely exemplary, as the teachings of the present invention may be modified and implemented in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Moreover, no limitations are intended to the details of construction or design shown herein, except as set forth in the appended claims. Thus, it is apparent that the specific exemplary embodiments disclosed above may be altered, combined, or modified, and all such variations are considered to be within the scope of the present invention. The systems, tools, and methods illustratively disclosed herein may be suitably practiced in the absence of elements not specifically disclosed herein and / or any elements disclosed herein. Although the systems, tools, and methods have been described using the terms "comprising," "including," or "including" various components or steps, the systems, tools, and methods may also be said to "consist essentially of" or "consist of" the various components or steps. All numbers and ranges disclosed above may vary to some extent. Whenever a numerical range with a lower limit and an upper limit is disclosed, any number falling within the range and any included range is specifically disclosed. In particular, all value ranges disclosed herein (in the form of "from about a to about b" or, equivalently, "from about a to b" or, equivalently, "from about a to a") refer to all numbers and ranges falling within the broader range of values. Moreover, the terms in the claims have their plain and ordinary meaning unless expressly and unambiguously defined by the patent owner. Moreover, the indefinite article "a" or "an" as used in the claims is defined herein to mean one or more of the element it introduces. In the event of a discrepancy in the use of a term or term in this specification and one or more patents or other documents that may be incorporated herein by reference, the definition consistent with this specification shall be controlled.

Claims

1. An electrochemical analyte sensor for detecting lactate in vivo, comprising: a working electrode having an active region disposed thereon, the active region comprising a polymer, albumin, an electron transfer agent, and a lactate-responsive enzyme, the electron transfer agent and the lactate-responsive enzyme both covalently bonded to the polymer; a mass transport limitation membrane covering at least the active area on the working electrode; the mass transport limitation membrane comprises at least a cross-linked polyvinylpyridine homopolymer or copolymer, and the albumin stabilizes the lactate-responsive enzyme to provide a sensor signal that varies by no more than 10% over a period of at least 190 consecutive hours at a given lactate concentration; the lactate responsive enzyme and the albumin are present in a weight ratio of the lactate responsive enzyme to the albumin ranging from 5:1 to 1:1; The analyte sensor is configured to be partially inserted into the skin of a subject and exposed to a biological fluid of the subject, thereby detecting lactate in the biological fluid in vivo.

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

3. The analyte sensor of claim 1 , wherein the lactate responsive enzyme is 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. exposing the analyte sensor of claim 1 to a biological fluid; acquiring a signal equal to or greater than the redox potential of the active region, the signal being proportional to a lactate concentration in the biological fluid; and correlating the signal to a lactate concentration in the biological fluid.

6. The method of claim 5, wherein the sample sensor is exposed to the biological fluid in vivo.

7. The method of claim 5, wherein the albumin is human serum albumin.

8. 6. The method of claim 5, wherein the lactate responsive enzyme is lactate oxidase.

9. 6. The method of claim 5, The method further comprises determining the presence of one or more conditions in the subject based on the lactate concentration in the biological fluid, wherein the one or more conditions are selected from the group consisting of sepsis, infection, organ function, and any combination thereof.

10. The method of claim 9, wherein the sample sensor is exposed to the biological fluid in vivo.

11. 2. The analyte sensor of claim 1, wherein the crosslinked polyvinylpyridine homopolymer or copolymer is a PEGDGE (polyethylene glycol diglycidyl ether) crosslinked polyvinylpyridine homopolymer.

12. The analyte sensor of claim 1, wherein the analyte sensor generates a signal that is linearly proportional to lactate concentrations from 0 mM to 5 mM.

13. The analyte sensor of claim 1, wherein the sensor signal has a sensitivity of at least 1 nA / mM in vivo.

14. The analyte sensor of claim 1, wherein the sensor signal varies by less than 5% over at least 190 consecutive hours at a given lactate concentration.

15. The analyte sensor of claim 1, wherein the sensor signal varies by less than 1% over at least 190 consecutive hours at a given lactate concentration.

16. The specimen sensor of claim 1, wherein the biological fluid is dermal fluid or interstitial fluid.

17. A method for manufacturing a computer-implemented computer-implemented system comprising: (ii) the analyte sensor of claim 1, acquiring a signal indicative of a lactate concentration in the biological fluid and communicating the signal indicative of the lactate concentration to the processor; a sensor control device including:

18. The sensor control device of claim 17, wherein the processor is configured to correlate the signal indicating the lactate concentration with the lactate concentration in the biological fluid.

19. The sensor control device of claim 17, wherein the processor is configured to determine the presence of one or more conditions in the subject based on the lactate concentration, the one or more conditions selected from the group consisting of sepsis, infection, organ function, and any combination thereof.

20. The sensor control device of claim 17, wherein the processor is configured to issue an alert when the lactate concentration exceeds a predetermined threshold concentration or tends toward a predetermined threshold concentration.

21. (i) an analyte sensor according to claim 1, and (ii) a processor configured to: (a) correlate a signal indicative of lactate concentration acquired by the analyte sensor with a lactate concentration in the biological fluid; and (b) communicate the lactate concentration to a reader device for display. A lactate sensing system comprising:

22. The lactate detection system of claim 21, wherein the processor is configured to determine the presence of one or more conditions in the subject based on the lactate concentration, the one or more conditions selected from the group consisting of sepsis, infection, organ function, and any combination thereof.

23. The lactate detection system of claim 21, wherein the processor is configured to issue an alert when the lactate concentration exceeds a predetermined threshold concentration or tends toward a predetermined threshold concentration.

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