Lactic acid sensors and related methods

Abstract

A lactic acid responsive enzyme may constitute a basis for lactic acid detection and quantification using an electrochemical analyte sensor. Various features may be incorporated within an analyte sensor comprising a lactic acid responsive enzyme, particularly a lactic acid oxidase, to improve the sensitivity and response stability of the analyte sensor. Such an analyte sensor may include: a working electrode having an active region disposed thereon; and a mass transfer confinement membrane coating at least the active region above the working electrode. The active region includes at least a polymer, albumin, and a lactic acid responsive enzyme covalently bonded to the polymer. The mass transfer limiting membrane may comprise at least a cross-linked polyvinyl pyridine homopolymer or copolymer. The analyte sensor may determine lactic acid concentrations in a biological fluid, especially in vivo, which may be associated with various physiological conditions.

Description

[0001] This application is a divisional application of Chinese patent application No. 201980026577.0 (PCT / US2019 / 017608), filed on February 12, 2019, and named “Lactic Acid Sensor and Related Methods”.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] not applicable. background

[0005] Detection of various analytes in an individual is sometimes essential for monitoring their health. Deviations from normal analyte levels can often indicate underlying physiological conditions, such as metabolic conditions or diseases, or exposure to specific environmental factors or stimuli. For example, glucose levels are particularly important for detecting and monitoring diabetic individuals.

[0006] Lactic acid is another analyte, and its level in the body can change in response to many environmental or physiological factors, including, for example, diet, stress, exercise, sepsis or septic shock, infection, hypoxia, the presence of cancerous tissue, etc. In the case of chronic lactic acid changes (such as diseases), lactic acid levels may change slowly, so that they can be easily quantified using conventional blood draws and laboratory measurements. Other situations that change lactic acid may be sporadic in nature, in which the lactic acid level may fluctuate very quickly and irregularly. In this case, conventional laboratory measurements may not be suitable for determining lactic acid levels. That is, the lactic acid level may have changed several times between consecutive measurements, and in this case, abnormal lactic acid levels may be completely missed, resulting in potential misdiagnosis. In the case of rapid fluctuations in lactic acid levels, it may be desirable to continuously measure the lactic acid level of an individual, such as by using an implanted in vivo lactic acid sensor. Continuous lactic acid monitoring is also beneficial for individuals with chronic, slowly changing lactic acid levels. For example, continuous lactic acid monitoring can avoid the pain and expense associated with implementing multiple blood draws to determine lactic acid levels.

[0007] In some cases, continuous analyte monitoring using implantable sensors may be advantageous, but there are certain challenges associated with these types of measurements. Intravenous analyte sensors are invasive and sometimes painful for the individual, especially when worn for extended periods of time. Subcutaneous, interstitial, or dermal analyte sensors are generally less painful for the individual to wear and can provide adequate measurement accuracy in many cases.

[0008] Over the past two decades, several manufacturers have developed non-intravenous in vivo glucose-responsive analyte sensors, and some have recently received regulatory approval for use in monitoring glucose levels in diabetic patients. Such glucose-responsive analyte sensors employ glucose oxidase, which is covalently bound to a polymer to facilitate glucose detection, and to a transition metal complex (electron transfer agent or electron transfer mediator) to help transport the electrons released during glucose oxidation. In vivo glucose-responsive analyte sensors available from other manufacturers also employ glucose oxidase as the basis for sensing, but alter the sensing chemistry / scheme in various ways.

[0009] In vivo analyte sensors for analyzing glucose and other analytes may include a film disposed above at least the implanted portion of the analyte sensor. On the one hand, the film can improve the biocompatibility of the analyte sensor. On the other hand, the film may be permeable or semi-permeable for the analyte of interest, and all analyte fluxes are limited to the active area of ​​the analyte sensor so that the film is used as a mass transfer limiting membrane. Limiting the entry of analytes into the active area of ​​the analyte sensor with a mass transfer limiting membrane can help avoid sensor overload (saturation), thereby improving detection performance and accuracy. Such a film may have a high degree of specificity for limiting the mass transfer of a specific analyte, while other substances penetrate the film at significantly different rates. Therefore, it can challenge the identification of a membrane polymer suitable for adding a mass transfer limiting membrane for a given analyte (e.g., lactic acid), which provides high analyte sensitivity and a stable sensor response.

[0010] A functional lactate responsive analyte sensor can be constructed by replacing the glucose oxidase from the glucose responsive analyte sensor with lactate oxidase. Unlike the glucose responsive analyte sensor briefly discussed above, the corresponding lactate responsive analyte sensor generally shows poor performance when analyzing lactate using similar sensing chemistry methods. That is, directly replacing glucose oxidase with lactate oxidase will provide a lactate responsive analyte sensor with low sensitivity and / or insufficient sensor response stability. Therefore, although the lactate responsive analyte sensor is powerful, it has not yet reached the level of development that provides stable, highly sensitive lactate analysis. In this way, although a large amount of possible information can be obtained by monitoring lactate, the diagnostic value of the lactate responsive analyte sensor is still quite limited.

[0011] As previously mentioned, the lactate responsive analyte sensor can replace glucose oxidase with lactate oxidase to facilitate the detection of lactic acid. This lactate responsive analyte sensor based on improved glucose responsive sensor chemistry is described in co-owned U.S. Patent No. 9,914,952, which is incorporated herein by reference in its entirety. As described therein, when lactate oxidase is present instead, the glucose responsive sensor chemistry can be changed to include catalase in the active region to achieve enhanced analytical sensitivity and some response stabilization to lactic acid. Although the addition of catalase helps to a certain extent, it cannot completely stabilize the long-term response of the analyte sensor. On the contrary, during the monitoring of 48 hours, the lactic acid signal in the analyte sensor containing catalase dropped to as high as about 10%. Since catalase is known to be reactive to hydrogen peroxide, the stabilizing effect of catalase in the lactate responsive analyte sensor is believed to involve purifying transient hydrogen peroxide, which may additionally affect the activity of lactate oxidase. Although catalase can improve the performance of lactate-responsive analyte sensors, further performance improvements may be required for such analyte sensors to realize their true potential.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The following drawings are included to illustrate certain aspects of the disclosure and should not be considered to be the exclusive embodiments. The disclosed subject matter is capable of numerous modifications, alterations, combinations, and equivalents in form and function without departing from the scope of the disclosure.

[0014] Figure 1 Diagram showing an exemplary sensing system into which the analyte sensors disclosed herein may be incorporated.

[0015] Figure 2A A diagram showing an exemplary two-electrode analyte sensor configuration that is compatible for use with some embodiments disclosed herein. Figure 2B and 2C A diagram showing an exemplary three-electrode analyte sensor configuration that is compatible for use with some embodiments disclosed herein.

[0016] Figure 3 Exemplary line graph showing sensor performance for Groups 1-4 in Example 1.

[0017] Figure 4 Exemplary line graphs showing the response of the Group 4 analyte sensors of Example 1 to lactic acid solutions having varying lactic acid concentrations.

[0018] Figure 5 Exemplary line graphs showing comparative sensor performance for mass transfer limiting membranes deposited from Formulations 3 and 3'.

[0019] Figure 6 Exemplary line graph showing sensor performance for a mass transfer limiting membrane deposited from Recipe 4'.

[0020] Figure 7 Exemplary line graph showing sensor performance for a mass transfer limiting membrane deposited from Recipe 4".

[0021] Figure 8 Exemplary line graphs showing sensor performance of a mass transfer limiting membrane deposited from Recipe 4" for lactic acid solutions with varying lactic acid concentrations.

[0022] Fig. 9 Exemplary line graph showing sensor performance of a double-layer mass transfer limiting membrane.

[0023] Fig.10 Exemplary line graph showing sensor performance of a double-layer mass transfer limiting membrane with variable layer thickness.

[0024] Fig.11 Exemplary line graphs showing sensor performance for a hybrid mass transfer limiting membrane.

[0025] Fig.12 Exemplary line graphs showing sensor performance of a hybrid mass transfer limiting membrane for lactic acid solutions with varying lactic acid concentrations.

[0026] Fig.13 Exemplary line graphs showing sensor performance for mixed mass transfer limiting membranes having various ratios of cross-linked polyvinylpyridine to cross-linked polyvinylpyridine-co-styrene.

[0027] Fig.14 An exemplary schematic diagram showing a portion of an analyte sensor having a bilayer membrane disposed on a working electrode, the sensor being compatible for use with some embodiments disclosed herein. Detailed description

[0029] The present disclosure generally describes lactate-responsive analyte sensors and methods using such analyte sensors, and more particularly, provides lactate-responsive analyte sensors and methods having high sensitivity and stable responsiveness to lactate.

[0030] As described above, measurement of lactate levels (concentrations) can be used to diagnose various physiological conditions and / or exposure to certain environmental factors. Although laboratory-determined lactate concentrations may be sufficient to monitor certain physiological conditions, in other cases, fluctuations in lactate levels may be too rapid to be feasible for laboratory determination. In vivo lactate-responsive analyte sensors employing lactate-responsive enzymes can be used to conveniently measure both slowly and rapidly fluctuating lactate concentrations, which can provide particular diagnostic advantages to monitor various situations in which lactate concentrations fluctuate rapidly and / or unexpectedly.

[0031] High analytical sensitivity and response stability are major factors that are desirable properties required of analyte sensors, especially those intended for long-term use in vivo. Despite the potential benefits that can be achieved by monitoring lactate levels in vivo, a lactate-responsive analyte sensor with analytical sensitivity and response stability for reliable diagnosis has not yet been developed.

[0032] Although high sensitivity and response stability can be achieved in glucose-responsive analyte sensors comprising glucose oxidase, these desirable performance characteristics cannot currently be converted into lactic acid-responsive analyte sensors when glucose oxidase is simply substituted with lactate oxidase. That is, the sensitivity and response stability of lactic acid-responsive analyte sensors are much worse than similar glucose-responsive sensors. At least some of these difficulties are due to the differences between glucose oxidase and lactate oxidase. By utilizing stabilizers (e.g., catalase) to improve the function of lactic acid-responsive analyte sensors, some progress has been made, but the desired sensitivity and response stability levels have not yet been achieved.

[0033] Additionally, the mass transfer limiting membranes of lactate responsive analyte sensors are a further source of difficulty. To date, the mass transfer limiting membranes successfully used in glucose responsive analyte sensors provide poor sensing properties for lactate analysis using lactate responsive enzymes. Membrane polymers or combinations thereof that are more compatible with lactate have not yet been described.

[0034] The disclosure describes a parallel method that can be used to improve the sensitivity of lactic acid responsive analyte sensors and the response stability of extension. The improvement of these factors can provide high-quality analyte data in the extended service life of up to several days or longer (ideally one week or longer), which may have important diagnostic value. That is, as further discussed herein, the performance of lactic acid responsive analyte sensors can be improved by replacing catalase with different stabilizers and by changing the mass transfer limiting membrane arranged on the active area. As discussed herein, several different membrane chemistries or structures can promote the improved analyte sensor performance for lactic acid analysis.

[0035] First, the present invention replaces a good stabilizer for lactate oxidase, which provides a significant performance advantage over catalase. That is, the present disclosure describes how serum albumin, particularly human serum albumin, is incorporated into the active region of a lactate-responsive analyte sensor to promote response sensitivity (i.e., the magnitude of the observed sensor response). The terms "albumin" and "serum albumin" are used synonymously herein. Human serum albumin is the most abundant protein found in plasma. Therefore, there is no obvious biocompatibility problem in introducing this stabilizer into an analyte sensor intended for in vivo use. The applicability of human serum albumin as a substitute for catalase stabilizers is particularly surprising because there is no known hydrogen peroxide scavenging function associated with human serum albumin. Without being bound by theory or mechanism, the results indicate that catalase may work in a manner unrelated to its usual hydrogen peroxide scavenging function in previous lactate-responsive analyte sensors. Other albumin proteins, such as bovine serum albumin, can be similarly incorporated into lactate-responsive analyte sensors to achieve advantages comparable to those discussed herein.

[0036] In terms of its thermal stability, albumin may have particular advantages over other types of protein stabilizers, such as catalase. Some protein stabilizers may denature when heated, resulting in loss of their stabilizing function. Advantageously, albumin protein is not easily denatured when exposed to temperatures approaching 60°C, allowing the albumin protein to retain its stabilizing function after thermal curing of the active sensor region occurs, as discussed further herein.

[0037] Although incorporation of serum albumin in the active region of a lactate-responsive analyte sensor can improve sensor performance, particularly response sensitivity, it alone is generally insufficient to provide desired levels of response sensitivity and extended response stability. Surprisingly, appropriate selection of a mass transfer limiting membrane in a lactate-responsive analyte sensor can provide adequate lactate responsiveness and extended response stability, particularly when coating the active region containing serum albumin. In conjunction with serum albumin, particularly human serum albumin, in the active region, the present disclosure describes various alternative chemistries or configurations of mass transfer limiting membranes in lactate-responsive analyte sensors that can improve lactate response sensitivity and response stability to more desirable levels.

[0038] Some glucose-sensitive analyte sensors can use cross-linked polyvinylpyridine-co-styrene polymers as mass transfer limiting membranes, wherein a portion of the pyridine nitrogen atoms are functionalized with uncross-linked poly(ethylene glycol) side chains and a portion of the pyridine nitrogen atoms are functionalized with alkyl sulfonic acid groups. The cross-linking of these membrane polymers in the analyte sensor can be performed by functionalization with a diepoxide (e.g., polyethylene glycol diglycidyl ether (PEGDGE) or glycerol triglycidyl ether (Gly3)). Such membrane polymers themselves are not effective in providing a stable response when analyzing lactic acid. The present disclosure shows that these membrane polymers can be replaced with polyvinylpyridine homopolymers or polyvinylpyridine copolymers (different from polyvinylpyridine-co-styrene) cross-linked with polyethylene glycol to provide improved lactic acid sensing performance, particularly when serum albumin and lactate oxidase are co-present in the active region, in the form of extended response stability. Alternatively and surprisingly, polyethylene glycol cross-linked polyvinylpyridine homopolymers or polyvinylpyridine copolymers can be appropriately combined (either in a bilayer membrane or as a homogeneous mixture) with the cross-linked polyvinylpyridine-co-styrene polymers more commonly used for glucose analysis to provide satisfactory sensor performance when analyzing lactate.

[0039] As defined herein, a "homopolymer" is a polymer that contains only a single type of monomeric unit that defines the polymer backbone. In a cross-linked homopolymer, some monomeric units may carry a cross-linking group, while some monomeric units may not carry a cross-linking group. As defined herein, monomeric units with or without cross-linking groups are considered to represent the same monomeric unit. As defined herein, a "copolymer" is a polymer that contains two or more different types of monomeric units that define the polymer backbone. Two or more different types of monomeric units have different structures (including isomeric variants of the same structure). In a cross-linked copolymer, any monomeric unit may carry a cross-linking agent, and some of any type of monomeric units may carry a cross-linking group, while some do not.

[0040] Before describing the analyte sensors disclosed herein in greater detail, a brief overview of suitable in vivo analyte sensor configurations and sensor systems employing analyte sensors will first be provided so that the embodiments disclosed herein may be better understood.

[0041] Figure 1Figures representing exemplary sensing systems of analyte sensors disclosed herein can be incorporated. As shown, sensing system 100 includes sensor control device 102 and reader device 120, which are configured to communicate with each other through local communication paths or links, which can be wired or wireless, unidirectional or bidirectional, and encrypted or non-encrypted. According to some embodiments, reader device 120 can constitute an output medium for viewing analyte concentrations, such as lactate concentrations, and alarms or notifications determined by sensor 104 or a processor associated therewith, and allowing one or more user inputs. Reader device 120 can be a multi-function smart phone or a dedicated electronic reader instrument. Although only one reader device 120 is shown, multiple reader devices 120 can exist in some cases. Reader device 120 can also communicate with remote terminal 170 and / or trusted computer system 180 via communication paths / links 141 and / or 142, respectively, and communication paths / links 141 and / or 142 can also be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. The reader device 120 may also or alternatively communicate with a network 150 (e.g., a mobile phone network, the Internet, or a cloud server) via a communication path / link 151. The network 150 may be further communicatively coupled to a remote terminal 170 via a communication path / link 152 and / or to a trusted computer system 180 via a communication path / link 153. Alternatively, the sensor 104 may communicate directly with the remote terminal 170 and / or the trusted computer system 180 without an intervening reader device 120. For example, according to some embodiments, as described in U.S. Patent Application Publication 2011 / 0213225, the entire contents of which are incorporated herein by reference, 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. Any suitable electronic communication protocol may be used for each communication path or link, such as near field communication (NFC), radio frequency identification (RFID), or Low power protocol or WiFi, etc. According to some embodiments, the remote terminal 170 and / or the trusted computer system 180 can be accessed by individuals other than the primary user who is interested in the user's analyte level. The reader device 120 can include a display 122 and an optional input component 121. According to some embodiments, the display 122 can include a touch screen interface.

[0042] The sensor control device 102 includes a sensor housing 103, which can house circuitry and a power source for operating the sensor 104. Optionally, the power source and / or active circuitry can be omitted. A processor (not shown) can be communicatively coupled to the sensor 104, wherein the processor is physically located within the sensor housing 103 or the reader device 120. According to some embodiments, the sensor 104 protrudes from the bottom side of the sensor housing 103 and extends through an adhesive layer 105, which is suitable for adhering the sensor housing 103 to a tissue surface such as skin.

[0043] The sensor 104 is suitable for at least partially inserting into the tissue of interest, such as in the dermis or subcutaneous layer of the skin. The sensor 104 may include a sensor tail having a sufficient length to be inserted into the desired depth in a given tissue. The sensor tail may include a working electrode and one or more active areas (sensing areas / spots or sensing layers) located on the working electrode, and the active area is active for sensing an analyte of interest, particularly lactic acid according to the present invention. According to one or more embodiments disclosed herein, each active area may include a lactate responsive enzyme, and suitable examples thereof may include lactate oxidase or lactate dehydrogenase. According to some embodiments, the active area may include a polymer material to which the enzyme is covalently bonded. In various embodiments disclosed herein, lactic acid in any biological fluid of interest can be monitored, and the biological fluid is, for example, skin fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, etc. In a specific embodiment, the analyte sensor disclosed herein may be suitable for analyzing skin fluid or interstitial fluid.

[0044] In some embodiments, the sensor 104 can automatically forward data to the reader device 120. For example, the analyte concentration data can be automatically sent regularly, such as at a certain frequency when the data is obtained or after a certain time, and the data is stored in the memory until the transmission (for example, every minute, five minutes, or other predetermined time period). In other embodiments, the sensor 104 can communicate with the reader device 120 in a non-automatic manner and not according to a set schedule. For example, when the sensor electronics are brought into the communication range of the reader device 120, RFID technology can be used to transmit data from the sensor 104. Before being communicated to the reader device 120, the data can remain stored in the memory of the sensor 104. Therefore, the patient does not have to keep close to the reader device 120 all the time, but can upload data at a convenient time. In other embodiments, a combination of automatic and non-automatic data transmission can be achieved. For example, data transmission can be continued on an automatic basis until the reader device 120 is no longer in the communication range of the sensor 104.

[0045] An introducer may be present momentarily to facilitate the introduction of sensor 104 into tissue. In an exemplary embodiment, the introducer may include a needle or similar tip. It should be appreciated that other types of introducers, such as a sheath or a blade, may be present in alternative embodiments. More specifically, a needle or other introducer may be temporarily placed near sensor 104 before tissue insertion and then removed afterwards. When present, a needle or other introducer may facilitate the insertion of sensor 104 into tissue by opening an entry path for sensor 104 to follow. For example, according to one or more embodiments, a needle may facilitate the penetration of the epidermis as a pathway to the dermis to allow the implantation of sensor 104 to occur. After the entry path is opened, the needle or other introducer may be removed to avoid sharp hazards. In an exemplary embodiment, the cross-section of a suitable needle may be solid or hollow, oblique or non-oblique and / or circular or non-circular. In a more specific embodiment, the cross-sectional diameter and / or tip design of a suitable needle may be comparable to an acupuncture needle, which may have a cross-sectional diameter of about 250 microns. However, it will be appreciated that a suitable needle may have a larger or smaller cross-sectional diameter if desired for a particular application.

[0046] In some embodiments, the tip of the needle (when present) may be angled on the end of the sensor 104 so that the needle first penetrates the tissue and opens an access path for the sensor 104. In other exemplary embodiments, the sensor 104 may reside within a lumen or groove of the needle, where the needle similarly opens an access path for the sensor 104. In either case, the needle is subsequently withdrawn after facilitating sensor insertion.

[0047] Suitable configurations of the analyte sensors disclosed herein may employ a two-electrode or a three-electrode detection pattern, which will be referred to hereinafter. Figures 2A-2C Further description.

[0048] The three-electrode detection pattern may include a working electrode, a counter electrode and a reference electrode. The related two-electrode detection pattern may include a working electrode and a second electrode, wherein the second electrode is used as both a counter electrode and a reference electrode (e.g., a counter / reference electrode). In two-electrode and three-electrode detection patterns, the active area of ​​the analyte sensor may be in contact with the working electrode. According to the embodiments disclosed herein, 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 (laminated) on each other, as described in further detail below. In some or other embodiments, the various electrodes may be spaced laterally from each other on the sensor tail. In either case, the various electrodes may be electrically isolated from each other by dielectric materials or similar insulators.

[0049] Figure 2AFigure representing an exemplary two-electrode analyte sensor configuration, which is applicable to some embodiments disclosed herein. As shown, the analyte sensor 200 includes a substrate 212 disposed between a working electrode 214 and a pair / reference electrode 216. Alternatively, the working electrode 214 and the pair / reference electrode 216 can be located on the same side of the substrate 212, and a dielectric material is inserted between them (not shown in the configuration). The active area 218 is disposed as at least one layer on at least a portion of the working electrode 214. In various embodiments disclosed herein, the active area 218 may include a plurality of spots or a single spot configured to detect lactic acid.

[0050] Still reference Figure 2A According to some embodiments, the membrane 220 coats at least the active area 218, and may optionally coat a portion or all of the working electrode 214 and / or the reference electrode 216, or the entirety of the analyte sensor 200. One or both faces of the analyte sensor 200 may be coated with the membrane 220. The membrane 220 may include one or more polymer membrane materials having the ability to limit the analyte flux to the active area 218 (i.e., the membrane 220 is a mass transfer limiting membrane). The composition of the membrane 220 may vary to facilitate the desired lactate flux to the active area 218, thereby providing the desired signal strength and stability, as further described herein. The analyte sensor 200 may be used for measuring lactic acid by any of the coulometric, amperometric, voltammetric or potentiometric electrochemical detection techniques. The specific composition of the membrane 220 that may be applicable when analyzing lactic acid is further discussed herein.

[0051] Figure 2B and 2C Figure 2 shows an exemplary three-electrode analyte sensor configuration, which is also suitable for use in some embodiments disclosed herein. The three-electrode analyte sensor configuration can be used with Figure 2A 200 in FIG. 201 . In addition to including an additional electrode 217 in analyte sensors 201 and 202 ( Figure 2B and 2C ). When the additional electrode 217 is present, the counter electrode / reference electrode 216 can be used as a counter electrode or a reference electrode, and there is no need to explain that the additional electrode 217 performs another electrode function. The working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed on the working electrode 214 or the electrode 216 with a dielectric material separating layer therebetween. For example, Figure 2B As shown, dielectric layers 219a, 219b and 219c separate electrodes 214, 216 and 217 from each other and provide electrical isolation. Alternatively, at least one of electrodes 214, 216 and 217 may be located on an opposite side of substrate 212, such as Figure 2CThus, in some embodiments, electrode 214 (working electrode) and electrode 216 (counter electrode) may be located on opposite sides of substrate 212, wherein electrode 217 (reference electrode) is located on one of electrodes 214 or 216 and separated therefrom by a dielectric material. Reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, and the location of reference material layer 230 is not limited to Figure 2B and 2C As depicted in Figure 2A As shown in sensor 200, active area 218 in analyte sensors 201 and 202 can include multiple spots or a single spot configured to detect lactate. In addition, analyte sensors 201 and 202 can be used to measure lactate by any of coulometric, amperometric, voltammetric or potentiometric electrochemical detection techniques.

[0052] Like analyte sensor 200, membrane 220 may also coat active area 218 and other sensor components in analyte sensors 201 and 202, thereby acting as a mass transfer limiting membrane. In some embodiments, additional electrode 217 may be coated with membrane 220. Figure 2B and 2C All electrodes 214, 216, and 217 have been depicted as being coated with membrane 220, but it should be appreciated that in some embodiments, only working electrode 214 may be coated. Furthermore, the thickness of membrane 220 at each electrode 214, 216, and 217 may be the same or different. As with the dual electrode analyte sensor configuration ( Figure 2A ),exist Figure 2B and 2C In the sensor configuration of FIG. 2 , one or both faces of the analyte sensors 201 and 202 may be coated with the membrane 220 , or the entire analyte sensors 201 and 202 may be coated. Figure 2B and Figure 2C The three-electrode sensor configuration shown in should be understood as a non-limiting embodiment of the disclosure herein, and alternative electrode and / or layer configurations are still within the scope of the disclosure herein.

[0053] According to various embodiments disclosed herein, an electron transfer agent may be present in the active region of any analyte sensor or analyte sensor configuration disclosed herein. When an analyte (enzyme substrate), such as lactic acid, undergoes a redox reaction, a suitable electron transfer agent may facilitate the transfer of electrons to or from a working electrode. As further described below, a specific embodiment of an analyte sensor disclosed herein is characterized in that an active region comprises lactate oxidase and serum albumin, particularly human serum albumin, and a combination of a mass transfer limiting membrane compatible with lactic acid.

[0054] Suitable electron transfer agents may include electroreducible and electrooxidizable ions, complexes or molecules (e.g., quinones) having a redox potential that is several hundred millivolts higher or lower than the redox potential of a standard calomel electrode (SCE). According to some embodiments, suitable electron transfer agents may include low potential osmium complexes, such as those described in U.S. Patents 6,134,461 and 6,605,200, which are incorporated herein by reference in their entirety. Additional examples include those described in U.S. Patents 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are incorporated herein by reference in their entirety. Other suitable electron transfer agents may include, for example, metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate) or cobalt, including metallocene compounds thereof. Suitable examples of electron transfer mediators and polymer-bound electron transfer mediators can include those described in U.S. Patents 8,444,834, 8,268,143 and 6,605,201, the disclosure of which is incorporated herein by reference in its entirety. Suitable ligands for metal complexes can also include, for example, bidentate or higher denticity ligands, such as bipyridine, biimidazole, o-phenanthroline or pyridyl (imidazole). Other suitable bidentate ligands can include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkane or o-diaminoarene. Monodentate, bidentate, tridentate, tetradentate or any combination of higher denticity ligands can be present in metal complexes to achieve a complete coordination range.

[0055] According to various embodiments disclosed herein, polymers may be present in each active region of an analyte sensor or analyte sensor construct disclosed herein. Suitable polymers included in the active region may 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 that may be suitable for inclusion in the active region include, for example, copolymers comprising monomer units, such as styrene, acrylamide, methacrylamide, or acrylonitrile. In exemplary embodiments, the polymer in the active region of an analyte sensor disclosed herein may be poly(4-vinylpyridine), wherein a portion of the monomer units are functionalized by alkyl carboxylate side chains, a portion of the monomer units are attached to an electron transfer agent having an amide spacer (e.g., see Formula 1 below), and a portion of the monomer units are not functionalized.

[0056] According to various embodiments disclosed herein, the electron transfer agent can be covalently bonded to the polymer in the active region. It is believed that the mode of covalent bonding is not particularly limited. The covalent bonding of the electron transfer agent to the polymer can be carried out by polymerizing the monomer units of the covalently bonded electron transfer agent, or after the polymer has been synthesized, the electron transfer agent can react with the polymer alone. According to some embodiments, the bifunctional spacer can covalently bond the electron transfer agent to the polymer in the active region, wherein the first functional group reacts with the polymer (e.g., a functional group capable of quaternizing a pyridine nitrogen atom or an imidazole nitrogen atom) and the second functional group reacts with the electron transfer agent (e.g., a functional group reacting with a ligand of a coordinated metal ion).

[0057] Similarly, according to some or other various embodiments disclosed herein, the enzyme in the active region can be covalently bonded to a polymer. According to a more specific embodiment, the covalent bonding of the enzyme to the polymer can be carried out by adopting a cross-linking agent introduced by a suitable cross-linking agent. Suitable cross-linking agents for reacting with the free amino groups in the enzyme (for example, with the free amines in lysine) may include cross-linking agents, for example, polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides (for example, Gly3), cyanuric chloride, N-hydroxysuccinimide, iminoesters, epichlorohydrin or its derivative variants. Suitable cross-linking agents for reacting with the free carboxylic acid groups in the enzyme may include, for example, carbodiimide. Cross-linking is usually intermolecular, but can be intramolecular in some embodiments.

[0058] The electron transfer agent and / or enzyme may also be associated with the polymer in the active region by methods other than covalent bonding. In some embodiments, the electron transfer agent and / or enzyme may be associated with the polymer ionically or by coordination. For example, a charged polymer may be associated with an electron transfer agent or enzyme ion with an opposite charge. In other embodiments, the electron transfer agent and / or enzyme may be physically entrained within the polymer without being bonded thereto.

[0059] Various configurations of the lactate-responsive analyte sensors disclosed herein will now be described in more detail. According to various embodiments, the analyte sensors disclosed herein may include: a working electrode having an active region disposed thereon; and a mass transfer limiting membrane coating at least the active region above the working electrode. The active region comprises a polymer, an albumin, and a lactate-responsive enzyme covalently bonded to the polymer. In a more specific embodiment, the mass transfer limiting membrane may comprise at least a cross-linked polyvinylpyridine homopolymer or copolymer. The mass transfer limiting membrane may be monocomponent or multicomponent. According to a more specific embodiment disclosed herein, an embodiment of a multicomponent membrane may comprise a double layer or a homogeneous mixture of cross-linked polyvinylpyridine and another polymer.

[0060] Suitable polyvinylpyridine copolymers included in the mass transfer limiting membrane may 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% comonomer, or about 15% to about 25% comonomer, or about 1% to about 10% comonomer. Suitable comonomers are not particularly limited, provided that the mass transfer limiting membrane provides sufficient lactic acid permeability to provide an analyte sensitivity of about 1 nA / mM or more when exposed to lactic acid. According to various embodiments, the polyvinylpyridine copolymer may be different from a polyvinylpyridine-co-styrene copolymer.

[0061] The aforementioned analyte sensor may further include a counter electrode and a reference electrode, or a counter / reference electrode. Figure 2A , 2B Suitable configurations for analyte sensors are described in more detail in 2C and 2D. Other configurations of various electrodes and active areas are also within the spirit and scope of the disclosure herein, and the depicted sensor configurations should not be viewed as limiting the scope of the disclosure.

[0062] The analyte sensor disclosed herein may alternatively include: a working electrode having an active area disposed thereon; and a mass transfer limiting membrane coating at least the active area above the working electrode, wherein the active area comprises a polymer, an albumin, and a lactate responsive enzyme covalently bonded to the polymer, and the mass transfer limiting membrane comprises a membrane polymer permeable to lactic acid. More specifically, the mass transfer limiting membrane may have lactic acid permeability such that the sensitivity of the analyte sensor is at least about 5nA at a lactic acid concentration of about 5mM. That is, when exposed to lactic acid, the analyte sensitivity of the analyte sensor is about 1nA / mM or more. In a more specific embodiment, the membrane polymer may comprise a polyvinylpyridine homopolymer or copolymer cross-linked with polyethylene glycol, as a single component membrane or a multi-component membrane, such as a double layer homogeneous mixture.

[0063] Other analyte sensors of the present invention may alternatively include: a working electrode having an active area disposed thereon; and a mass transfer limiting membrane coating at least the active area on the working electrode, wherein the active area comprises a polymer, catalase, and a lactate responsive enzyme covalently bonded to the polymer, and the mass transfer limiting membrane comprises at least a cross-linked polyvinylpyridine homopolymer or copolymer.

[0064] According to various embodiments, the lactate responsive enzyme within the active region may comprise lactate oxidase or lactate dehydrogenase. In more specific embodiments, a combination of lactate oxidase and albumin, in particular human serum albumin, may be advantageous for performing lactate analysis according to the present invention.

[0065] According to more specific embodiments, lactate oxidase may be present in the active region in an amount of about 0.05 μg to about 5 μg, or about 0.1 μg to about 4 μg, or about 0.2 μg to about 3 μg, or about 0.5 μg to about 2 μg. In terms of weight percentage of the active region, lactate oxidase may be present in an amount of about 10% to about 90% by weight of the active region, or about 25% to about 75% by weight of the active region, or about 30% to about 60% by weight of the active region.

[0066] According to various embodiments, the albumin within the active region may comprise human serum albumin. Alternatively, non-human albumins, such as bovine serum albumin, may be satisfactorily used.

[0067] 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 a more specific embodiment, albumin can be present in the active region in an amount of about 0.05 μg to about 5 μg, or about 0.1 μg to about 2 μg, or about 0.2 μg to about 1.5 μg, or 0.3 μg to about 0.8 μg. In terms of the weight percentage of the active region, the amount of albumin present can be about 25% to about 75% of the weight of the active region, or about 30% to about 60% of the weight of the active region. In certain embodiments, the weight ratio of lactate oxidase to albumin can range from about 10: 1 to about 1: 10 (w / w), or from about or about 5: 1 to about 1: 5, or about 5: 1 to about 1: 1, or about 2: 1 to about 1: 1, or about 1: 1 to about 1: 5, or about 1: 1 to about 1: 2.

[0068] In a more specific embodiment, the analyte sensor disclosed herein may include a sensor tail configured for insertion into tissue. The working electrode is arranged on the sensor tail and can be inserted into the tissue to facilitate lactate analysis therein. It is believed that suitable tissues are not particularly limited, and specific examples are discussed in more detail above. Similarly, considerations for arranging the sensor tail at a specific location or depth within the tissue are discussed above.

[0069] The specific configuration of the analyte sensors disclosed herein, including the amount of albumin and lactate oxidase to be bound to the active region, may depend on the tissue penetrated by the sensor tail, the expected concentration of lactate to be analyzed, and the specific conditions under which the analyte sensor is intended to operate when analyzing lactate. In more specific embodiments, the tissue penetrated by the analyte sensor can be skin, such that the sensor tail is located within the dermis, interstitial layer, or subcutaneous layer below the surface of the skin. The sensor can be further contained within a sensor housing configured to adhere to the skin.

[0070] As described above, the active region of the analyte sensor may include an electron transfer agent that is covalently bonded to a polymer therein. It is believed that the covalent bonding 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 further described in detail above.

[0071] Ideally, the active region can be configured to rapidly obtain steady-state current when operating the analyte sensor disclosed herein at a given potential. Rapid steady-state current can be facilitated by selecting an electron transfer agent that rapidly changes its oxidation state when exposed to a potential equal to or higher than its redox potential. Making the active region as thin as possible can also facilitate rapid acquisition of steady-state current. For example, a suitable thickness for the active region can be in the range of about 0.1 micron to about 10 microns. In some or other embodiments, in the active region, conductive materials such as, for example, carbon nanotubes, graphene or metal nanoparticles can be combined to facilitate rapid acquisition of steady-state current. The appropriate amount of conductive particles can be about 0.1% by weight to about 50% by weight of the active region, or about 1% by weight to about 50% by weight, or about 0.1% by weight to about 10% by weight, or about 1% by weight to about 10% by weight.

[0072] The active area on the working electrode of the analyte sensor disclosed herein may include at least one spot or layer disposed on the working electrode. The size of each spot or layer may range from about 0.01 mm to about 0.1 mm. 2 To about 1mm 2 , although larger or smaller spots or layers of active area are also contemplated herein. The total size of the active area (total area of ​​all spots or layers) may be up to about 100 mm 2 , especially about 25mm 2 or smaller, or about 10mm 2 or smaller, or about 5mm 2 or smaller, or about 1mm 2 or smaller, or about 0.1mm 2 In more specific embodiments, the total size of the active area can be about 0.05 mm 2 To about 0.1mm 2 Although in some embodiments the active area may include one spot or one layer, more typical embodiments of the analyte sensors disclosed herein are characterized by having an active area with a plurality of spots arranged on the working electrode. According to some embodiments, the number of spots is not considered to be particularly limited, but may be in the range of 2 to about 10, or about 3 to about 8, or about 4 to about 6.

[0073] It should be further understood that the sensitivity (output current) of the analyte sensor to lactic acid can be varied by changing the coating extent (area or size) of the active region, the properties and thickness of the mass transfer limiting membrane coating the active region, and any combination thereof. Once having the benefit of the disclosure herein, one of ordinary skill in the art can readily make changes to these parameters to obtain the desired sensitivity.

[0074] The analyte sensors disclosed herein, particularly those having an active region comprising albumin and covalently bound lactate oxidase and a mass transfer limiting membrane comprising a polyvinylpyridine homopolymer or copolymer, particularly a polyvinylpyridine homopolymer, can be functionally characterized as having a response stability (change in output current) of about 10% or less over 190 hours of measurement, or about 5% or less over 190 hours of measurement, or about 1% or less over 190 hours of measurement. Any changes occurring within this time frame are expected to be clinically insignificant (i.e., only marginally affecting Clark error grid analysis and / or MARD or MAD analysis). Therefore, it is expected that the catalase-stabilized lactate-responsive analyte sensors described in the above patents will still show a lower response current at 190 hours of measurement.

[0075] In order to achieve sufficient response stability, the analyte sensor disclosed herein adopts a mass transfer limiting membrane, which is selected to match lactic acid. More particularly, a favorable mass transfer limiting membrane suitable for use in combination with a lactic acid responsive enzyme, particularly lactate oxidase, can include a cross-linked polyvinyl pyridine homopolymer or copolymer. The mass transfer limiting membrane in the analyte sensor of the present disclosure can include only a single polymer (i.e., a cross-linked polyvinyl pyridine homopolymer or copolymer), or it can be multi-component and include two or more polymers (i.e., a cross-linked polyvinyl pyridine homopolymer or copolymer and at least a second cross-linked double layer or homogeneous mixture configuration polymer). More specifically, the multi-component membrane can include: a first polymer containing a cross-linked polyvinyl pyridine homopolymer, a second polymer containing a cross-linked polyvinyl pyridine copolymer, or a first polymer containing a first cross-linked polyvinyl pyridine copolymer and a second polymer containing a second cross-linked polyvinyl pyridine copolymer different from the first cross-linked polyvinyl pyridine copolymer. In a more specific embodiment, the second crosslinked polymer may comprise a crosslinked polyvinylpyridine copolymer, particularly a polyvinylpyridine-co-styrene polymer, wherein a portion of the pyridinic nitrogen atoms are functionalized with uncrosslinked poly(ethylene glycol) tails and a portion of the pyridinic nitrogen atoms are functionalized with alkyl sulfonic acid groups.

[0076] According to various embodiments, in the analyte sensor disclosed herein, the multi-component membrane suitable for use in combination with lactate response enzyme, particularly lactate oxidase, may include a double-layer membrane. Suitable double-layer membranes may include: a first layer comprising a cross-linked polyvinyl pyridine homopolymer or copolymer and a second layer comprising a second cross-linked polymer, particularly a cross-linked polyvinyl pyridine copolymer. The polyvinyl pyridine copolymer included in the second layer may be different from the polyvinyl pyridine copolymer in the first layer. In a more specific embodiment, the first layer may be directly disposed on the active area, and the second layer may be disposed on the first layer. In an alternative embodiment, the second layer may be directly disposed on the active area, and the first layer may be disposed on the second layer. The thickness and order of the first layer and the second layer may vary so as to provide the desired sensitivity. In some embodiments, the first layer may be coated on the active area (e.g., by spraying, smearing, inkjet printing, roller coating, dip coating, etc.), and then the second layer may be coated on the first layer by the same or different coating techniques (e.g., by spraying, smearing, inkjet printing, roller coating, dip coating, etc.), to prepare such a double-layer structure. In other embodiments, a two-layer membrane may be configured with the first and second layers reversed (thereby reversing the position of the cross-linked polyvinylpyridine homopolymer or copolymer), with each layer being coated as described above.

[0077] Bilayer membrane is heterogeneous in nature, because two different membrane polymers are stacked on each other. In analyte sensor disclosed herein, other multi-component membranes suitable for use in combination with lactate response enzyme, particularly lactate oxidase, can be homogeneous in composition. More particularly, in some embodiments, suitable multi-component membrane can include a homogeneous mixture of a cross-linked polyvinylpyridine homopolymer or copolymer and a cross-linked second polymer (especially a cross-linked polyvinylpyridine copolymer). Two cross-linked polyvinylpyridine copolymers can be different from each other. The ratio of two cross-linked cross-linked polymers can change in a considerable range in the homogeneous mixture, and the ratio can be adjusted to provide the desired sensitivity of the analyte sensor disclosed herein.

[0078] Fig.14 Schematic diagram showing a portion of an analyte sensor having a bilayer membrane disposed on a working electrode, which sensor is compatible for use with some embodiments disclosed herein. Fig.14 As shown, the analyte sensor features a sensor tail 600 having a working electrode 614 disposed on a substrate 612. An active region 618 is disposed on the working electrode 614 and includes a lactate responsive enzyme in accordance with the present disclosure.

[0079] like Fig.14As further shown, active area 618 is coated by a double layer membrane 621, which includes a membrane layer 621a directly contacting active area 618 and a membrane layer 621b coating membrane layer 621a. According to the disclosure herein, membrane layers 621a and 621b can include different membrane polymers, at least one of which is a polyvinylpyridine homopolymer or copolymer.

[0080] In alternative embodiments, a homogenous membrane layer comprising a mixture of different membrane polymers may replace the membrane layer according to the present disclosure. Fig.14 Double-layer membrane 621.

[0081] The analyte sensor described above can be used in various methods for analyzing lactate and determining lactate concentration. According to various embodiments, the concentration of lactate can be further related to physiological conditions (e.g., caused by disease or exposure to environmental factors), as described in further detail below.

[0082] According to various embodiments, the method disclosed herein may include exposing an analyte sensor to a fluid, wherein the analyte sensor includes a working electrode, an active region is disposed thereon, and a mass transfer limiting membrane, which at least coats the active region above the working electrode. The active region comprises a polymer, albumin, and a lactate responsive enzyme covalently bonded to the polymer, particularly lactate oxidase. In a specific embodiment, the mass transfer limiting membrane may at least comprise a cross-linked polyvinylpyridine homopolymer or copolymer. The method also includes obtaining a signal of a redox potential equal to or higher than the active region, wherein the signal is proportional to the concentration of lactic acid in the fluid, and the signal is associated with the concentration of lactic acid in the fluid.

[0083] In a more specific embodiment, the fluid is a biological fluid, and the analyte sensor is exposed to the biological fluid in a subject in which lactate concentration needs to be measured. According to still more specific embodiments disclosed herein, the subject can be a human. Suitable biological fluids for analyzing using the analyte sensor disclosed herein can include any of the biological fluids discussed in more detail above.

[0084] By consulting a lookup table or calibration curve, the signal associated with the active area exposed to lactic acid can be associated with the corresponding lactic acid concentration. The lookup table or calibration curve can exist in physical form (i.e., written form) or electronic form (i.e., database or computer algorithm). The lookup table of various lactic acid concentrations can be filled by analyzing multiple samples with known lactic acid concentrations and recording the analyte sensor response of each concentration. Similarly, the calibration curve of lactic acid can be determined by drawing a graph of the analyte sensor response of each lactic acid sample as a function of concentration. According to some embodiments, the calibration curve of the analyte sensor disclosed herein can be linear or nearly linear relative to the concentration of lactic acid. In some embodiments, before recording or drawing the analyte sensor response graph, the background response can be subtracted from the analyte sensor response. The background response can be determined by analyzing a fluid (i.e., blank) that does not contain lactic acid. The lookup table or calibration curve can be manually or electronically consulted to determine the lactic acid concentration in the fluid, as further described below.

[0085] The processor can determine which sensor response value in the lookup table is closest to the measured value of the sample (fluid) with unknown lactic acid concentration, and then report the lactic acid concentration accordingly. In some or other embodiments, if the sensor response value of the sample with unknown lactic acid concentration is between the recorded values ​​in the lookup table, the processor can interpolate between the two lookup table values ​​to estimate the lactic acid concentration. The interpolation can assume that there is a linear concentration change between the two values ​​reported in the lookup table. For example, when the sensor response of the sample differs sufficiently from a given value in the lookup table (e.g., a change of about 10% or more), interpolation can be used. When manually consulting the lookup table, interpolation can be performed similarly.

[0086] Likewise, the processor can input the sensor response value of a sample with an unknown lactate concentration into a corresponding calibration curve. The processor can then report the concentration of lactate accordingly.

[0087] The lactate concentration determined using the analyte sensor disclosed herein, as described above, can be further associated with one or more physiological conditions. One or more physiological conditions can be mediated by lactic acid in the body and / or produce an increased or decreased lactate level in the body. More specifically, one or more physiological conditions that can be monitored by the analyte sensor disclosed herein include, for example, sepsis, infection, organ function, or any combination thereof. Other conditions in which monitoring lactate may be beneficial include, for example, physiological stress and exercise, both of which may produce elevated lactate levels. Monitoring any of these conditions can include, for example, tracking the trend of lactate levels over time and / or determining the instantaneous lactate level at a specific time. Optionally, if the lactate level exceeds a defined threshold concentration under given conditions, or if the lactate level tends to a defined threshold when monitored over time, an alarm can be provided. Thus, the analyte sensor disclosed herein can be configured to provide an alarm or otherwise provide an indication that sepsis, infection, organ failure, or any combination thereof may be present. Depending on the physiological condition monitored, the threshold concentration can vary.

[0088] Sepsis can be viewed as a three-stage syndrome, starting with sepsis and progressing through severe sepsis to septic shock. The goal is to treat sepsis in its early stages, before it becomes more dangerous. Currently, severe sepsis is usually diagnosed by decreased body temperature, heart rate, respiratory rate, urine output, sudden changes in mental status, and decreased platelet count. Septic shock is usually diagnosed by the same markers in combination with very low blood pressure.

[0089] Lactic acid levels can be used to diagnose, monitor and / or evaluate various forms of sepsis and / or related infections. Therefore, determining lactic acid concentration according to the present disclosure can allow more effective monitoring, evaluation and / or control of sepsis and / or infection. Alternatively, the analyte sensor disclosed herein can be used to monitor subjects (e.g., patients in hospitals) who are in sepsis and / or infection risk but do not currently show any signs of illness. The lactic acid level obtained according to the present disclosure can be combined diagnostically with other analyte levels and / or physiological markers (e.g., body temperature, heart rate, respiratory rate, blood pressure, urine output reduction, sudden changes in mental state, platelet count reduction 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 lipoprotein)) to diagnose and / or monitor the progress of sepsis and related infections in subjects. In certain embodiments, the concentration of lactic acid in the cerebrospinal fluid can be measured to monitor, evaluate and / or diagnose meningitis and / or septic meningitis. Other 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. Other markers for bacterial sepsis or bacterial infection can include, for example, glutamate, malic acid, pseudouridine, acetylcarnitine, glycerophosphocholine, hydroxyphenyllactic acid, acetylneuraminic acid, pseudouridine and tyrosine. Other markers for viral sepsis (including septic meningitis) or viral infection can include, for example, hypoxanthine, inosine and caproylcarnitine.

[0090] In some embodiments, the analyte sensors disclosed herein can be used to monitor a subject's exposure to an infectious agent. For example, the analyte sensor can monitor a subject's condition after acute exposure to an infectious agent to monitor for signs of sepsis or infection and / or to monitor the development and progression of sepsis or infection. The analyte sensor can also be used to monitor a subject's response to an anti-infective agent and / or treatment administered to a subject to treat sepsis or infection or a symptom thereof.

[0091] The activity of enzyme can also be the diagnosis of organ function, wherein the activity of enzyme can be too low or too high, depending on the specific organ of experimenter and the existing physiological condition that is just experienced.In the field of lactic acid response enzyme, the physiological condition (that is, disease) of liver function and liver function can be characterized according to the activity of lactate dehydrogenase.Other enzymes that may be needed for carrying out organ function monitoring (including organs and organ functions different from liver and liver function) separately or in conjunction with lactate dehydrogenase include, for example, creatine kinase, aspartate aminotransferase, aspartate aminotransferase, alkaline phosphatase, and 5' nucleotidase.Except liver, the optional organ that can monitor its organ function includes, for example, kidney, heart, brain, lung, pancreas, spleen, stomach, bladder, skeleton, gallbladder, intestine (large and small), colon, lymph node and thyroid.

[0092] Analyte sensors disclosed herein can monitor organ function, particularly liver function, by assessing the activity of lactate dehydrogenase or another suitable lactate response enzyme (e.g., lactate oxidase). Analyte sensors can be used to monitor, diagnose and / or detect analyte levels in subjects experiencing organ dysfunction or at risk of organ dysfunction, possibly allowing organ diseases to be identified before life-threatening symptoms develop. The lactate concentration or the rate of change of lactate concentration determined by the analyte sensor disclosed herein can facilitate diagnosis or analysis of organ failure or disorder or the possibility of organ failure or disorder. When the lactate concentration exceeds a threshold amount or tends to exceed a threshold amount that may be a feature of organ damage or failure, the analyte sensor can provide an alarm.

[0093] Lactate levels obtained according to the present disclosure can be combined diagnostically with other analyte levels and / or physiological markers to determine organ function and / or failure, particularly liver function and / or failure. Other markers that can be measured in combination with lactate to determine organ function include, for example, 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 lipoprotein. Lactate levels monitored according to the disclosure herein can be further used to shorten the time before starting a treatment course to combat organ failure and / or monitor the progress of a treatment or treatment process.

[0094] Embodiments disclosed herein include: A. An analyte sensor. The analyte sensor includes: a working electrode having an active region disposed thereon, the active region comprising a polymer, albumin, and a lactate responsive enzyme covalently bonded to the polymer; and a mass transfer limiting membrane coating at least the active region on the working electrode.

[0095] B. A method for determining lactate. The method comprises: exposing an analyte sensor to a fluid; wherein the analyte sensor comprises: a working electrode having an active region disposed thereon, the active region comprising a polymer, an albumin, and a lactate-responsive enzyme covalently bonded to the polymer; and a mass transfer limiting membrane coating at least the active region on the working electrode; obtaining a signal equal to or higher than the redox potential of the active region, the signal being proportional to the concentration of lactate in the fluid; and correlating the signal with the concentration of lactate in the fluid.

[0096] C. A lactate sensor responsive to sepsis, infection, or organ function. The lactate sensor comprises: an analyte sensor including: a working electrode having an active region disposed thereon, the active region comprising a polymer, an albumin, and a lactate responsive enzyme covalently bonded to the polymer; and a mass transfer limiting membrane coating at least the active region on the working electrode; wherein the analyte sensor is responsive to sepsis, infection, organ failure, or any combination thereof, and is configured to provide an alarm or other indication that sepsis, infection, organ failure, or any combination thereof may be present for a measured lactate concentration.

[0097] Each of embodiments AC may have one or more of the following additional elements in any combination:

[0098] Element 1: wherein the albumin comprises human serum albumin.

[0099] Element 2: wherein the lactate responsive enzyme comprises lactate oxidase.

[0100] Element 3: wherein the active area comprises a plurality of sensing sites disposed on the working electrode.

[0101] Element 4: wherein the mass transfer limiting membrane comprises a multi-component membrane comprising a cross-linked polyvinylpyridine homopolymer or copolymer and at least a second cross-linked polymer.

[0102] Element 5: wherein the multi-component membrane comprises: a first polymer comprising a cross-linked polyvinyl pyridine homopolymer and a second polymer comprising a cross-linked polyvinyl pyridine copolymer, or a first polymer comprising a first cross-linked polyvinyl pyridine copolymer and a second polymer comprising a second cross-linked polyvinyl pyridine copolymer.

[0103] Element 6: wherein the multi-component film comprises a two-layer film, the two-layer film comprising a first layer comprising a polyvinylpyridine homopolymer or copolymer and a second layer comprising a second cross-linked polymer.

[0104] Element 7: wherein the first layer is disposed directly on the active area, and the second layer is disposed on the first layer.

[0105] Element 8: wherein the mass transfer limiting membrane comprises a homogeneous mixture of a cross-linked polyvinylpyridine homopolymer or copolymer and a second cross-linked polymer.

[0106] Element 9: wherein the working electrode is disposed on a sensor tail configured for insertion into tissue.

[0107] Element 10: wherein the active region further comprises an electron transfer agent covalently bonded to the polymer.

[0108] Element 11: wherein the fluid is a biological fluid, and the analyte sensor is exposed to the biological fluid in vivo.

[0109] Element 12: wherein the method further comprises: determining the presence of one or more conditions in the subject based on the lactate concentration in the fluid, the one or more conditions being selected from sepsis, infection, organ function, and any combination thereof.

[0110] Element 13: The analyte sensor of claim 1, wherein the mass transfer limiting membrane comprises at least a cross-linked polyvinylpyridine homopolymer or copolymer.

[0111] As non-limiting examples, exemplary combinations suitable for use with A-CB include:

[0112] The analyte sensor of A or C or the method of B in combination with elements 1 and 2; 1, 2 and 13; 1 and 3; 1, 3 and 13; 1-3; 1-3 and 13; 1 and 4; 1, 4 and 13; 1 and 13; 2 and 4; 2, 4 and 13; 2 and 13; 3 and 4; 3, 4 and 13; 3 and 13; 1-4; 1-4 and 13; 2-4; 2-4 and 13; 1, 4 and 5; 1, 4, 5 and 13; 2, 4 and 5; 2, 4, 5 and 13; 3-5; 3-5 and 13; 4 and 5; 4, 5 and 13; 1 and 4-6; 1, 4-6 and 13; 2, 4, 5 and 6; 2, 4, 5, 6 and 13; 3-6; 3-6 and 13; 4-7; 4-7 and 13; 4 and 8; 4, 8 and 13; 4, 5 and 8; 4, 5, 8 and 13; 1 and 9; 1, 9 and 13; 2 and 9; 2 , 9 and 13; 3 and 9; 3, 9 and 13; 4 and 9; 4, 9 and 13; 1 and 10; 1, 10 and 13; 2 and 10; 2, 10 and 13; 3 and 10; 3, 10 and 13; 4 and 10; 4, 10 and 13; 9 and 10; 9, 10 and 13. The method of B and any combination of the following elements: 1 and 11; 1, 11 and 13; 1 and 13; 2 and 11; 2 and 13; 3 and 11; 3 and 13; 4 and 11; 4 and 13; 4, 5 and 11; 4, 5, 11 and 13; 4-6 and 11; 4, 6, 11 and 13; 4-7 and 11; 4-7, 11 and 13; 4, 8 and 11; 4, 8, 11 and 13; 9 and 11; 9, 11 and 13; 10 and 11; 10, 11 and 13; 11 and 12; 11-13; 1 and 12; 1, 12 and 13; 2 and 12; 2, 12 and 13; 3 and 12; 3, 12 and 13; 4 and 12; 4, 12 and 13; 4, 5 and 12; 4, 5, 12 and 13; 4-6 and 12; 4-6, 12 and 13; 4-7 and 12; 4-7, 12 and 13; 4, 8 and 12; 4, 8, 12 and 13; 9 and 12; 9, 12 and 13; 10 and 1 2; 10, 12 and 13; 1, 11 and 12; 1 and 11-13; 2, 11 and 12; 2 and 11-13; 3, 11 and 12; 3 and 11-13; 4, 11 and 12; 4 and 11-13; 4, 5, 11 and 12; 4, 5 and 11-13; 4-6, 11 and 12; 4-6 and 11-13; 4-7, 11 and 12; 4-7 and 11-13; 4, 8, 11 and 12; 4, 8 and 11-13; 9, 11 and 12; 9 and 11-13; 10-12; and 10-13.

[0113] In order to facilitate a better understanding of the embodiments described herein, the following examples of various representative embodiments are given.The following examples should in no way be construed as limiting or defining the scope of the invention. Example

[0114] Example 1: Comparison of Lactate Sensor Response. Two different lactate oxidase / polymer formulations for active area deposition and two different membrane polymer formulations for mass transfer limiting membrane deposition were prepared to determine the performance of lactate responsive sensors having various arrangements of these formulations. The formulation details and methods used to prepare the analyte sensors are further discussed below. In general, the preparation of the analyte sensors was similar to the corresponding glucose responsive analyte sensors, except that lactate oxidase was used instead of glucose oxidase (with or without albumin) and in some cases different membrane polymers were used.

[0115] Recipes for active area deposition : Lactate oxidase is mixed with the polymer of Formula 1 in an aqueous solution formulation as described in Tables 1 and 2 below. Further details regarding the polymer are provided in commonly owned US Patent 6,605,200, which is incorporated by reference in its entirety. The subscripts for each monomer represent exemplary atomic ratios.

[0116]

[0117] Table 1

[0118]

[0119] Table 2

[0120]

[0121] To deposit each active area, ~20 nL of each solution was deposited on the carbon working electrode to form 6 discrete spots, each with a diameter of approximately 0.01 mm. 2 The area of ​​​​the electrode was formed. Formulation 1 was dispensed 4 times and formulation 2 was dispensed 6 times to form the dots. After deposition, the working electrode was cured overnight at 25°C. Formulation 1 corresponds to the formulation used to deposit the active area of ​​the glucose-responsive analyte sensor, except that lactate oxidase was used instead of glucose oxidase.

[0122] Recipe for mass transfer limited film deposition: The membrane polymer formulations described in Tables 3 and 4 below were prepared as aqueous solution formulations.

[0123] Table 3

[0124]

[0125] Table 4

[0126]

[0127] The mass transfer limiting membrane was deposited on each active area prepared as described above using dip coating. Formulation 3 was deposited using 4 dip coatings, and Formulation 4 was deposited using 4 dip coatings. A waiting time of about 10 minutes was used between two dip coatings. After the dip coating was completed, the membrane was cured at 25°C for 24 hours and then at 56°C for 48 hours in a dry vial. Spray coating, screen printing or similar processes can be used alternately to deposit the mass transfer limiting membrane. Formulation 3 corresponds to the formulation for depositing the mass transfer limiting membrane within the glucose responsive analyte sensor.

[0128] Lactic acid responsive analytes were prepared using the precipitation conditions described above. All possible combinations of active area and mass transfer limiting membrane were prepared, and 8 sensors were prepared for each possible combination. After fabrication, each sensor was exposed to a 5 mM lactic acid solution in 100 mM phosphate buffered saline (PBS) at 37°C for 190 hours, with the working potential maintained at +40 mV vs. Ag / AgCl. The combinations of active area and mass transfer limiting membrane tested are listed in Table 5. The test results are shown in Figure 3 middle.

[0129] Table 5

[0130]

[0131] like Figure 3 As shown, lactate responsive analyte sensors (Group 1) with active areas and mass transfer limiting membranes similar to those successfully used in glucose responsive analyte sensors provide poor performance when exposed to lactate. As shown, the signal intensities of all tested samples were below 0.5 nA, which is an undesirably low value for a viable lactate responsive sensor. When polyvinylpyridine and a different cross-linker were substituted for the polyvinylpyridine-co-styrene and Gly3 cross-linker in Formulation 3, the signal intensity was even worse (Group 2).

[0132] like Figure 3 As further shown, incorporation of human serum albumin significantly improved sensor performance. For example, sample set 3 showed significantly higher signal intensities than achieved by either of the samples in set 1 or set 2. However, there was considerable variability in the initial signal intensities between this set of samples (>4 nA variation). In addition, the signal intensity steadily decreased from the maximum signal intensity initially observed. The response variability and poor signal stability over time also make this sample set combination unlikely to be suitable for a viable lactate-responsive analyte sensor.

[0133] Surprisingly, the combination of an active region containing human serum albumin and a mass transfer limiting membrane comprising a cross-linked polyvinylpyridine homopolymer (Group 4) yielded an acceptable combination of high signal intensity and prolonged signal stability over time. Figure 3As shown, all replicate sensors of Group 4 had initial signal intensities that were clustered within 1 nA of each other between 4 nA and 5 nA. This level of signal intensity and variability is within the range that can develop a commercially viable lactate-responsive analyte sensor. In addition, over 190 hours of signal observation, the signal intensity varied by only a few tenths of a nA or less, which is also within the range that can be suitable for developing a commercially viable sensor.

[0134] like Figure 4 As shown, when increasing amounts of lactate were added in 1 mM increments to a PBS solution initially free of lactate, the current of the sensors of Group 4 was observed to respond rapidly and reach a stable value.

[0135] Example 2: Alternative Mass Transfer Limiting Membranes For these experiments, active regions containing lactate oxidase were deposited from solutions formulated as described above in Table 2. Active region deposition and curing on carbon electrodes were performed as described in Example 1. Mass transfer limiting membranes for various samples were formulated as described below.

[0136] Unless otherwise specified below, the deposition of the mass transfer limiting membrane on the active area was performed by dip coating (1-5 dips of the electrode with a waiting time of about 10 minutes between dips). The formulation of the mass transfer limiting membrane is further described below. After the dip coating was completed, the membrane was cured at 25°C for 24 hours and then at 56°C for 48 hours in a dry vial.

[0137] The sensor response was measured by placing the active area of ​​the electrode in a beaker containing 100 mM PBS pH=7.5 at 37° C. The potential was raised to +40 mV vs. Ag / AgCl, after which the current was continuously monitored.

[0138] Alternative cross-linking agents in Formulation 3 (Formulation 3'). Formulation 3 was modified with alternative cross-linkers as described below in Table 6. The modified formulation is designated herein as Formulation 3'.

[0139] Table 6

[0140]

[0141] Formulation 3' was prepared by mixing 4 mL of membrane polymer (140 mg / mL) in 80:20 ethanol:HEPES buffer, 0.2 mL of PEGDGE400 (100 mg / mL) in 80:20 ethanol:HEPES buffer, and 0.0132 mL of aminopropyl terminated polydimethylsiloxane (PDMS) (100 mg / mL) in ethanol.

[0142] Figure 5An exemplary line graph showing the comparative performance of a lactate sensor comprising an active area containing human serum albumin coated with mass transfer limiting membranes deposited from Formulations 3 and 3'. Figure 5 As shown, the mass transfer limiting film deposited from Formulation 3 resulted in poor response stability when coated on an active area containing lactate oxidase and human serum albumin (corresponding to Sensor Set 3 above - see Figure 3 ). The PEGDGE-crosslinked variant of the mass transfer limiting membrane (deposited using Formulation 3') also produced a variable sensor response over time, but with less variation than that deposited using Formulation 3. In addition, the response sensitivity obtained from the mass transfer limiting membrane deposited from Formulation 3' was higher than that obtained from Formulation 3. In contrast, both mass transfer limiting membranes provided stable sensor responses in the presence of glucose as the analyte (data not shown).

[0143] Alternative crosslinking agents in Formulation 4 (Formulation 4'). Formulation 4 was modified with alternative cross-linkers as described below in Table 7. The modified formulation is designated herein as Formulation 4'.

[0144] Table 7

[0145]

[0146] Formulation 4' was prepared by mixing 4.3 mL of membrane polymer (100 mg / mL) in 80:20 ethanol:HEPES buffer, 0.25 mL of PEGDGE1000 (200 mg / mL) in 80:20 ethanol:HEPES buffer, and 0.0132 mL of aminopropyl terminated polydimethylsiloxane (PDMS) (100 mg / mL) in ethanol.

[0147] Figure 6 An exemplary line graph showing sensor performance for a mass transfer limiting membrane deposited from Formulation 4'. Increasing the molecular weight of the cross-linking agent resulted in worse sensor performance. Figure 6 As shown, the mass transfer limiting membrane produced by formulation 4' did not provide a stable current response over time. There was a large drop in response within the first 48 hours, after which the performance was relatively stable. In addition, the sensitivity was well below the target value of about 1 nA / mM.

[0148] Formulation containing non-cross-linked PEG side chains (Formulation 4") Formulation 4' was modified to introduce 3-4 wt% non-crosslinked PEG side chains on the PVP polymer backbone and then crosslinked with PEGDGE1000 as for Formulation 4' (referred to herein as Formulation 4"). The composition of Formulation 4" is described in Table 8 below.

[0149] Table 8

[0150]

[0151] Formulation 4" was prepared by mixing 4 mL of membrane polymer (100 mg / mL) in 80:20 ethanol:HEPES buffer, 0.025 mL of PEGDGE1000 (200 mg / mL) in 80:20 ethanol:HEPES buffer, and 0.0132 mL of aminopropyl terminated polydimethylsiloxane (PDMS) (100 mg / mL) in ethanol.

[0152] Figure 7 An exemplary line graph showing sensor performance for a mass transfer limiting membrane deposited from Recipe 4″. As shown, the sensor responds quickly and obtains a stable current at a value significantly greater than that provided by Recipe 4 or Recipe 4′. In addition, the sensor responds quickly and obtains a stable current as increasing amounts of lactic acid are added in increments of 1 mM (see Figure 8 ), therefore, the sensor performance obtained with formulation 4″ confirms that crosslinkers other than PEGDGE400 can be successfully used as crosslinkers for membrane polymers.

[0153] Double membrane variant : A double layer mass transfer limiting membrane was formed by depositing Formulation 4' onto the electrode surface and then depositing the membrane polymer from Formulation 3' thereon. As shown above, none of these membrane polymers by themselves provided acceptable performance when coated onto the active area containing human serum albumin and lactate oxidase.

[0154] To prepare the sensor, the polymer from formulation 4' was applied to the electrode surface by repeated dip-coating operations. Then, the polymer from formulation 3' was applied to the deposited cross-linked PVP layer by repeated dip-coating operations. There was a 10-minute waiting time between consecutive dip-coatings. After all dip-coating operations were completed, the sensor was cured at 25°C for 24 hours and then at 56°C for 48 hours in a dry vial.

[0155] Fig. 9 Example line graph showing sensor performance for a double mass transfer limiting membrane. Fig. 9 As shown, the bilayer structure surprisingly provides a stable current response over time at an acceptable level of sensitivity, even though neither polymer alone provides acceptable performance. Fig. 9 The response data in are for an electrode dip-coated twice in Formulation 4' (cross-linked polyvinylpyridine) and then four times in Formulation 3' (cross-linked polyvinylpyridine-co-styrene).

[0156] The amount (thickness) of each membrane polymer in the double-layer mass transfer limiting membrane can change the sensor performance as follows Fig.10 as shown in . Fig.10Example line graph showing sensor performance of a dual-layer mass transfer limiting membrane with variable layer thickness. Recipe 4' was used to deposit Fig.10 The lower layer in Formulation 1 was used, while Formulation 3 (Gly3 cross-linked variant of polyvinylpyridine-co-styrene) was used to deposit the upper layer. Thus, different cross-linking agents are tolerable for cross-linking polyvinylpyridine-co-styrene polymers, even those that would not produce acceptable performance when the polymer is used alone in a mass transfer limiting membrane. Fig.10 As shown, dipping the electrode twice in Formulation 4' and four times in Formulation 3 provided a good balance of sensitivity and stable current response. Changing the number of dipping operations changes the thickness of each component of the bilayer membrane, as well as the mass ratio of the membrane polymers to each other. Fig.10 As shown, if the PVP layer is too thin (0 or 1 dip coating of Formulation 4'), the sensitivity is high but the response stability is poor, while if it is too thick (3 or more dip coatings), the electrode shows low sensitivity and poor response stability in some cases. Spray coating, screen printing or similar processes can alternatively be used to deposit the mass transfer limiting membrane.

[0157] Hybrid membrane variants: A hybrid membrane formulation (Formula 5) was prepared by mixing 1.5 mL of polyvinylpyridine (100 mg / mL) in 80:20 ethanol:HEPES buffer, 2.5 mL of polyvinylpyridine-co-styrene (140 mg / mL) in 80:20 ethanol:HEPES buffer, 0.175 mL of PEGDGE400 (100 mg / mL) in 80:20 ethanol:HEPES buffer, and 0.0132 mL of PDMS (100 mg / mL) in ethanol.

[0158] Table 9

[0159]

[0160] Thus, after cross-linking, Formulation 5 contained each polymer cross-linked with PEGDGE400.

[0161] Fig.11 Graph showing exemplary sensor performance for a hybrid mass transfer limiting membrane. As shown, the hybrid membrane provides a stable current response and acceptable levels of sensitivity over time. In addition, when increasing amounts of lactic acid are added in increments of 1 mM, the current response is rapid and stable values ​​are obtained ( Fig.12 ).

[0162] The hybrid mass transfer limiting membrane described above contains polyvinylpyridine and polyvinylpyridine-co-polystyrene in a 3:5 volume:volume ratio.Alternative ratios of these two polymers may also produce acceptable performance. Fig.13An exemplary line graph showing sensor performance for mixed mass transfer limiting membranes having various ratios of cross-linked polyvinylpyridine to cross-linked polyvinylpyridine-co-styrene. Fig.13 As shown, higher amounts of polyvinylpyridine also produced acceptable sensitivity and response stability. However, when the ratio of polyvinylpyridine to polyvinylpyridine-copolystyrene was reduced from 2:2 vol:vol to 1:3 vol:vol, even though the overall sensitivity was higher, the resulting response stability was poorer.

[0163] Unless otherwise indicated, all numerical values ​​indicating quantities and the like in this specification and the related claims should be understood as being modified in all cases by the term "about". Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the attached claims are approximate values, which may vary depending on the desired properties sought to be obtained according to the embodiments of the present invention. At a minimum, and without attempting to limit the application of the doctrine of equivalents to the scope of protection of the claims, each numerical parameter should at least be interpreted in light of the number of reported significant figures and by applying ordinary rounding techniques.

[0164] One or more exemplary embodiments in combination with various features are presented herein. For the sake of clarity, all features of physical implementation are not described or shown in this application. It should be understood that when developing a physical implementation in combination with an embodiment of the present invention, many specific implementation-specific decisions must be made to achieve the goals of the developers, such as complying with system-related, business-related, government-related constraints and other constraints, which are changed and changed from time to time due to specific implementation. Although the efforts of the developers may be time-consuming, these efforts will be routine tasks for those of ordinary skill in the art and benefit from the present disclosure.

[0165] Although various systems, tools, and methods are described herein in terms of "comprising" various components or steps, the systems, tools, and methods may also "consist essentially of" or "consist of" the various components and steps.

[0166] As used herein, the phrase "at least one" preceding a series of terms (with the terms "and" or "or" separating any of the terms) will modify the list as a whole, rather than each member of the list (i.e., each item). The phrase "at least one" is permitted to refer to at least one of any of the items, and / or at least one of any combination of the items, and / or at least one of each of the items, including the terms. For example, the phrases "at least one of A, B, and C" or "at least one of A, B, or C" each refer to only A, only B, or only C; any combination of A, B, and C; and / or at least one of each of A, B, and C.

[0167] Therefore, the disclosed systems, tools and methods are well suited to obtain the mentioned results and advantages and the results and advantages inherent therein. The specific embodiments disclosed above are exemplary only, because the teachings disclosed herein can benefit from the teachings herein and be modified and implemented in different but equivalent ways that are obvious to those skilled in the art. In addition, except as described in the following claims, there is no intended limitation on the details of the construction or design shown herein. Therefore, it is obvious that the specific exemplary embodiments disclosed above can be changed, combined or modified, and all these changes are considered to be within the scope disclosed herein. The systems, tools and methods exemplarily disclosed herein can be implemented appropriately in the absence of any elements not specifically disclosed herein and / or any optional elements disclosed herein. Although systems, tools and methods are described according to "including", "containing", "comprising" various components or steps, the systems, tools and methods can also be "essentially composed of various components and steps" or "composed of various components and steps". All numerical values ​​and ranges disclosed above can vary by a certain amount. Whenever a numerical range with a lower limit and an upper limit is disclosed, any numerical value and any included range falling within the range are specifically disclosed. Specifically, each numerical range disclosed herein (in the form of "about a to about b", or equivalently "about a to b", or equivalently "about ab") should be understood to list each numerical value and range covered within the broader numerical range. Moreover, the terms in the claims have their ordinary general meaning unless otherwise clearly and clearly defined by the patentee. In addition, the indefinite articles "a" or "an" used in the claims are defined herein to refer to one or more elements it introduces rather than one element. If there is any conflict in the use of a word 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 should be adopted.

Claims

1. Analyte sensor, comprising: a working electrode having an active region disposed thereon, the active region comprising a polymer, albumin, and a lactate responsive enzyme covalently bonded to the polymer; and a mass transfer limiting membrane coating at least the active region above the working electrode.

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

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

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

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

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

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

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

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

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

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

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

13. Methods, comprising: exposing the analyte sensor to the fluid; Wherein the analyte sensor comprises: a working electrode having an active region disposed thereon, the active region comprising a polymer, albumin, and a lactate responsive enzyme covalently bonded to the polymer; and a mass transfer limiting membrane coating at least the active region above the working electrode; A signal is obtained that is equal to or greater than the redox potential of the active region, the signal being proportional to the concentration of lactic acid in the fluid; and the signal is correlated to the concentration of lactic acid in the fluid.

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

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

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

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

18. The method of claim 17, wherein the mass transfer limiting membrane comprises a multicomponent membrane comprising a cross-linked polyvinylpyridine homopolymer or copolymer and at least a second cross-linked polymer.

19. The method of claim 18, wherein the multicomponent membrane comprises: a first polymer comprising a cross-linked polyvinyl pyridine homopolymer and a second polymer comprising a cross-linked polyvinyl pyridine copolymer, or a first polymer comprising a first cross-linked polyvinyl pyridine copolymer and a second polymer comprising a second cross-linked polyvinyl pyridine copolymer.

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

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

22. The method of claim 17, wherein said mass transfer limiting membrane comprises a homogeneous mixture of said cross-linked polyvinylpyridine homopolymer or copolymer and said second cross-linked polymer.

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

24. The method of claim 13, further comprising: The presence of one or more conditions in the subject is determined based on the lactate concentration in the fluid, the one or more conditions being selected from sepsis, infection, organ failure, and any combination thereof.

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

26. An analyte sensor comprising: a working electrode having an active region disposed thereon, the active region comprising a polymer, albumin, and a lactate responsive enzyme covalently bonded to the polymer; and a mass transfer limiting membrane coating at least the active area overlying the working electrode; wherein the analyte sensor is responsive to sepsis, infection, organ failure, or any combination thereof, and is configured to provide an alarm or other indication that sepsis, infection, organ failure, or any combination thereof may be present for the measured lactate concentration.

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

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