A lactate continuous monitoring electrochemical sensor and a preparation method and application thereof

Abstract

This invention belongs to the field of electrochemical sensor technology, specifically relating to an electrochemical sensor for continuous monitoring of lactic acid, its preparation method, and its application. The sensor includes a substrate electrode, a sensitive layer, and a diffusion confinement layer. The sensitive layer, coated on the surface of the substrate electrode, contains a lactic acid oxidation catalyst and an electron mediator. The diffusion confinement layer is a multilayer cross-linked polymer film or a multilayer non-cross-linked polymer film. The electrochemical sensor of this invention precisely controls the pore size and affinity of the membrane layer by adjusting the monomer ratio and graft modification of the diffusion confinement layer copolymer, achieving both high sensitivity and a wide linear detection range. It is highly adaptable and can meet the needs of different concentration ranges in medical monitoring, exercise physiological monitoring, etc. Simultaneously, the curing conditions simulate the human body environment, ensuring that the actual performance is consistent with laboratory tests, and its stability is reliable.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical sensor technology, specifically relating to an electrochemical sensor for continuous monitoring of lactic acid, its preparation method, and its application. Background Technology

[0002] Real-time monitoring of lactate concentration is of great significance for monitoring lactate abnormalities caused by severe illnesses such as cellular metabolic disorders and oxygen supply-demand imbalances. It can help intensive care units, operating rooms, and emergency rooms to promptly detect patient abnormalities and respond quickly. Simultaneously, it can also be used for exercise health monitoring, providing real-time feedback on changes in interstitial fluid lactate concentration during exercise. This allows for the analysis of the relationship between individual lactate concentration and exercise status, thereby enabling the development of appropriate exercise training programs.

[0003] Currently, most lactate testing systems on the market are traditional benchtop lab machines or point-of-care testing (POCT) products, primarily using whole blood, plasma, or serum as samples. A single sample only reflects the lactate concentration at that specific point in time, and cannot achieve continuous real-time monitoring or generate accurate lactate fluctuation curves. Furthermore, there are currently no products on the market specifically for continuous lactate monitoring. Most products require sampling and testing of whole blood, plasma, or serum.

[0004] Patent CN112057836A discloses a continuous lactate monitoring system and method, comprising: a lactate sensing electrode implanted in the body of a subject and in contact with the tissue fluid within the subject to generate a continuous current signal; a fixed data acquisition unit attached to the skin of the subject and connected to the lactate sensing electrode to receive the current signal and convert it into a digital signal; a data receiving processor to receive the digital signal and convert it into a lactate concentration value, and to send an alarm signal when the lactate concentration value is higher than a pre-stored lactate reference value; and an alarm device to issue an alarm based on the alarm signal. This patent focuses on the signal transmission of the continuous lactate monitor; however, issues regarding how to continuously detect lactate in the body, as well as the detection line and sensitivity, remain to be addressed. Summary of the Invention

[0005] (a) Technical problems to be solved The purpose of this invention is to provide a continuous lactate monitoring electrochemical sensor, its preparation method, and its application in order to solve at least one of the above-mentioned problems. This addresses the issue that existing technologies cannot continuously monitor lactate and can only detect lactate in blood samples, thereby achieving continuous real-time monitoring and plotting a more accurate lactate fluctuation curve.

[0006] (II) Technical Solution The objective of this invention is achieved through the following technical solution: One of the technical solutions of the present invention is a lactic acid continuous monitoring electrochemical sensor, the sensor comprising a base electrode, a sensitive layer, and a diffusion confinement layer; the sensitive layer is coated on the surface of the base electrode and contains a lactic acid oxidation catalyst and an electron mediator; the diffusion confinement layer is a multilayer cross-linked polymer film or a multilayer non-cross-linked polymer film; the diffusion confinement layer is coated on the surface of the sensitive layer.

[0007] The base electrode is configured as a micro-flexible electrode, including a reference electrode, a working electrode, and a counter electrode.

[0008] Furthermore, the reference electrode is any one of a solid Ag / AgCl electrode or a conductive polymer reference electrode; the working electrode is a carbon electrode, a platinum electrode, or a gold electrode; and the counter electrode is a carbon electrode or a platinum electrode.

[0009] Furthermore, the substrate electrode can be prepared by any of the following methods: screen printing, MEMS processing, or inkjet printing.

[0010] Furthermore, the substrate electrode is prepared by stepwise screen printing on a flexible substrate (such as polyimide PI or polyethylene terephthalate PET); the reference electrode is obtained by screen printing Ag / AgCl ink; the working electrode is obtained by screen printing carbon paste (including graphite, carbon nanotube, or graphene paste), platinum paste, or gold paste; and the counter electrode is obtained by screen printing carbon paste (including graphite, carbon nanotube, or graphene paste), platinum paste, or gold paste.

[0011] Furthermore, the lactate oxidation catalyst is lactate oxidase; the electron mediator is a metal-based complex that generates electron transfer with the cofactor FAD in lactate oxidase.

[0012] Furthermore, the metal-based complex includes any one of potassium ferricyanide, Prussian blue, ruthenium complex, and osmium complex.

[0013] Furthermore, the sensitive layer also contains an adjuvant, which includes either albumin or peroxidase, to enhance the stability of lactate oxidase LOD. The core function of the peroxidase is to remove hydrogen peroxide byproducts generated during the lactate reaction catalyzed by lactate oxidase, thereby preventing the accumulation of hydrogen peroxide from inhibiting or damaging the activity of lactate oxidase.

[0014] Furthermore, the peroxidase includes any one of peroxidase, horseradish peroxidase, and catalase. All of the above peroxidases can efficiently decompose hydrogen peroxide byproducts, ensuring the catalytic efficiency and structural stability of lactate oxidase.

[0015] Furthermore, the preparation method of the sensitive layer includes the following steps: mixing lactate oxidase, electron mediator, crosslinking agent, and excipient to obtain a sensitive layer membrane solution; coating the sensitive layer membrane solution onto the surface of the substrate electrode by dip coating, dot coating, spin coating, etc.; after coating the sensitive layer, placing the electrode at 25℃-40℃ for drying and curing for 0.5-24h, allowing the solvent in the sensitive layer to evaporate, and the enzyme and crosslinking agent to undergo a crosslinking reaction, so that each component is stably attached to the electrode surface and crosslinked to form a sensitive layer with a certain structure and function. In this sensitive layer, the peroxidase can continuously remove hydrogen peroxide produced by the lactate oxidase catalytic reaction, providing a stable catalytic environment for the lactate oxidase.

[0016] Furthermore, when the diffusion-limiting layer is a cross-linked polymer membrane, its membrane liquid component includes any one of vinylpyridine-derived hydrophilic polymers at a concentration of 5%-10%, and an epoxy cross-linking agent at a concentration of 0.5%-3%, and the membrane liquid solvent is a mixture of ethanol and water.

[0017] Furthermore, the membrane solvent is a mixture of 75%-100% ethanol and 0-25% water.

[0018] Furthermore, when the diffusion confinement layer is a multilayer cross-linked polymer membrane, the polymer in its membrane liquid component is a copolymer of 4-vinylpyridine and methacrylate. The copolymer is synthesized by free radical polymerization, specifically including the following steps: mixing 4-vinylpyridine and methacrylate in a certain proportion; using azobisisobutyronitrile (AIBN) or ammonium persulfate (APS) as an initiator to carry out the reaction in an organic solvent (such as DMF) or emulsion system; heating to initiate polymerization at 60℃-80℃ for 3-10 hours; purifying the product by precipitation, filtration and vacuum drying to obtain a copolymer with a molecular weight of approximately 180,000.

[0019] Furthermore, the coating process of the cross-linked polymer film includes: using 80% ethanol as a solvent, preparing polymer and epoxy cross-linking agent solutions at a concentration of 100 mg / mL, uniformly mixing the polymer and epoxy cross-linking agent solutions at a volume ratio of 20:1 and coating them onto the surface of the sensitive layer, and curing and cross-linking for 10-30 hours at 30-50℃ and 20-60% humidity.

[0020] Furthermore, the diffusion-limiting layer (polymer coating) involved in this aspect can precisely control the diffusion rate of lactic acid molecules by subtly modifying the surface of its polymer material (such as adjusting the monomer ratio of 4-vinylpyridine to methacrylate and grafting hydrophilic groups), thereby achieving a linear response in different concentration ranges (such as 0.1-10mM for medical monitoring and 5-30mM for motion monitoring).

[0021] The second technical solution of the present invention is a method for preparing a lactic acid continuous monitoring electrochemical sensor as described above, comprising the following steps: coating a sensitive layer on the surface of a substrate electrode; coating a diffusion confinement layer on the surface of the sensitive layer; and curing the coating by heat treatment at 50-80°C to obtain the electrochemical sensor.

[0022] The third technical solution of the present invention is the application of the lactate continuous monitoring electrochemical sensor as described above in the continuous monitoring of lactate in tissue fluid.

[0023] (III) Beneficial Effects Compared with the prior art, the present invention has the following advantages: (1) Wide linear detection range and controllability: This electrochemical sensor significantly expands the detection linear range to 0.1-30mM while ensuring sensitivity by adjusting the monomer ratio and graft modification of the polymer in the diffusion restriction layer. The diffusion layer is a copolymer of 4-vinylpyridine and methacrylate. By adjusting the monomer ratio and graft modification, the pore size and hydrophilic-hydrophobic balance of the polymer membrane can be precisely controlled. The epoxy crosslinking agent restricts the diffusion rate of lactic acid molecules. Curing at 37℃ and 40% humidity for 24 hours ensures that the crosslinking reaction is fully completed and the membrane structure is uniform and stable. This condition simulates the human body environment, ensuring that the performance of the electrochemical sensor in actual use is consistent with that in laboratory testing. Moreover, by changing the ratio of hydrophilic polyurethane or poly4-vinylpyridine in the diffusion layer, it can be adapted to different scenario requirements, including the high sensitivity requirement in medical monitoring (low concentration, 0.1-10mM) and the wide linear range requirement in exercise physiological monitoring (high concentration, 5-30mM).

[0024] (2) High sensitivity and rapid response: The sensitive layer of the electrochemical sensor of the present invention adopts the synergistic effect of lactate oxidase (LOD) and metal-based electron mediator (such as osmium complex) to significantly improve the electron transfer efficiency; the redox potential of osmium complex is highly matched with the FAD cofactor of LOD, which can quickly capture electrons released by enzyme reaction and reduce intermediate reaction steps; LOD is fixed on the surface of electrochemical sensor by crosslinking agent, avoiding the enzyme detachment problem caused by traditional physical adsorption and ensuring effective exposure of enzyme active sites; the addition of cofactor further protects the conformational stability of LOD and reduces the interference of external environment on enzyme activity, so that the electrochemical sensor maintains high sensitivity and rapid response.

[0025] (3) Good long-term stability: The outer diffusion restriction layer, inner sensitive layer and flexible substrate of the lactic acid electrochemical sensor of the present invention together ensure its long-term stability in vivo or on the body surface; In the outer diffusion restriction layer, the vinylpyridine monomer provides a large number of cross-linking sites, and the acrylate material provides good physical properties and anti-adhesion properties, which can restrict the diffusion of lactic acid, delay the enzymatic reaction and protect the lower sensitive layer; The peroxidase added to the sensitive layer can promptly remove the hydrogen peroxide byproduct generated during the lactic acid reaction catalyzed by lactate oxidase, and avoid the accumulation of hydrogen peroxide leading to the decay of lactate oxidase activity; The substrate adopts a micro flexible electrode, which can withstand repeated deformation during human activities and avoid electrode breakage, making the present invention highly adaptable to use.

[0026] (4) The electrochemical sensor of the present invention has a simple structure and clear preparation steps. The formulations of each functional layer (sensitive layer and diffusion layer) can be independently optimized and combined, which facilitates the rapid adjustment of product parameters for different needs. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the diffusion confinement layer copolymer in the continuous lactic acid monitoring electrochemical sensor of the present invention.

[0029] Figure 2 The graph shows the current response of the electrochemical sensor for continuous lactate monitoring in this invention to the lactate concentration in critically ill patients.

[0030] Figure 3 The graph shows the current response of the electrochemical sensor for continuous lactate monitoring in this invention to the lactate concentration in athletes.

[0031] Figure 4 The image shows the response curve of the continuous lactic acid monitoring electrochemical sensor in Experiment Example 1 of this invention, which continuously monitors lactic acid in a 5.0 mM lactic acid solution for fourteen days. Figure 5 The graph shows the response of the sensor obtained in Comparative Example 1 of this invention to different concentrations of lactic acid in phosphate buffer. Figure 6 The graph shows the response of the sensor obtained in Comparative Example 2 of this invention to different concentrations of lactic acid in phosphate buffer. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0033] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. This application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0034] It should be noted that various aspects of embodiments within the scope of the appended claims are described below. It will be apparent that the aspects described herein can be embodied in a wide variety of forms, and any particular structure and / or function described herein is merely illustrative. Based on this application, those skilled in the art will understand that one aspect described herein can be implemented independently of any other aspect, and two or more of these aspects can be combined in various ways. For example, any number and aspects set forth herein can be used to implement the device and / or practice the method. Additionally, this device and / or method can be implemented using structures and / or functionalities other than one or more of the aspects set forth herein.

[0035] Additionally, specific details are provided in the following description to facilitate a thorough understanding of the examples. However, those skilled in the art will understand that practice can be carried out without these specific details.

[0036] Unless otherwise specified, the raw materials or devices used in the following examples are commercially available raw materials or conventional experimental devices.

[0037] This invention proposes an electrochemical sensor for continuous monitoring of lactic acid. The sensor includes a base electrode, a sensitive layer, and a diffusion confinement layer. The base electrode is configured as a micro-flexible electrode, including a reference electrode, a working electrode, and a counter electrode. The reference electrode is either a solid Ag / AgCl electrode or a conductive polymer reference electrode. The working electrode is a flexible carbon-based electrode. The counter electrode is a carbon electrode. The base electrode can be fabricated by screen printing, MEMS processing, inkjet printing, or other methods.

[0038] The sensitive layer is coated on the surface of the substrate electrode and contains a lactate oxidation catalyst and an electron mediator. The sensitive layer also contains an excipient, which includes any one of albumin and peroxidase. The lactate oxidation catalyst is lactate oxidase. The electron mediator is a metal-based complex that generates electron transfer with the FAD cofactor in lactate oxidase. The metal-based complex includes any one of potassium ferricyanide, Prussian blue, ruthenium complex, and osmium complex. The peroxidase includes any one of horseradish peroxidase (HRP) and catalase. The peroxidase can specifically remove hydrogen peroxide byproducts generated during the process of lactate oxidase catalyzing the conversion of lactate to pyruvate, thereby reducing the damage of byproducts to the sensitive layer.

[0039] The diffusion-limiting layer is a multilayer cross-linked polymer membrane or a multilayer non-cross-linked polymer membrane; when the diffusion-limiting layer is a multilayer cross-linked polymer membrane, its membrane liquid component includes 5%-10% of either hydrophilic polyurethane or poly(4-vinylpyridine) polymer, and 0.5%-3% of epoxy cross-linking agent, and the membrane liquid solvent is a mixed solvent of 75%-100% ethanol and 0-25% water.

[0040] The preparation method of the sensitive layer includes the following steps: mixing lactate oxidase, metal-based complex, excipients and crosslinking agent to obtain a sensitive layer membrane solution; coating the sensitive layer membrane solution onto the surface of the substrate electrode; after coating the sensitive layer, placing the electrode at 25℃-40℃ for crosslinking and curing for 0.5-24h to allow the solvent in the sensitive layer to evaporate, and for each component to stably adhere to and crosslink on the electrode surface to form a sensitive layer with a certain structure and function.

[0041] The diffusion confinement layer is preferably a copolymer of 4-vinylpyridine and methacrylate, which is synthesized by free radical polymerization, specifically including the following steps: mixing 4-vinylpyridine and methacrylate in a certain proportion; using azobisisobutyronitrile (AIBN) or ammonium persulfate (APS) as an initiator, and reacting in an organic solvent (such as DMF) or emulsion system; initiating polymerization by heating at 60℃-80℃ for 3-10 hours; purifying the product by precipitation, filtration, and vacuum drying to obtain a copolymer with a molecular weight of approximately 180,000. The polymer is then mixed with an epoxy crosslinking agent at a specific concentration and ratio and coated onto the surface of the sensitive layer, and cured and crosslinked at 30-50℃ and 20-60% humidity for 10-30 hours.

[0042] A method for preparing a continuous lactic acid monitoring electrochemical sensor includes the following steps: coating a sensitive layer on the surface of a substrate electrode; coating a diffusion confinement layer on the surface of the sensitive layer; and curing and crosslinking the coating at 50-80℃ to obtain the electrochemical sensor.

[0043] The technical solutions provided by the various embodiments of this application are described below with reference to the accompanying drawings. Example

[0044] This embodiment provides an electrochemical sensor for continuous monitoring of lactic acid, the sensor comprising a base electrode, a sensitive layer, and a diffusion confinement layer; The reference electrode is a solid Ag / AgCl electrode obtained by printing Ag / AgCl ink on a flexible substrate (such as polyimide); the working electrode is a flexible carbon-based electrode prepared by printing carbon nanotube (CNT) or graphene ink on a polyethylene terephthalate (PET) substrate; and the counter electrode is a carbon electrode prepared by printing conductive graphite paste on an activated carbon mesh.

[0045] The sensitive layer is coated on the surface of the base electrode and contains lactate oxidase (LOD), osmium complex, horseradish peroxidase (HRP), bovine serum albumin, and epoxy crosslinking agent.

[0046] The diffusion confinement layer is a multilayer cross-linked polymer membrane; its membrane liquid components include a copolymer of 4-vinylpyridine and methacrylate at a concentration of 100 mg / mL, an epoxy cross-linking agent at a concentration of 5 mg / mL, and a membrane liquid solvent of a mixture of 80% ethanol and 20% water.

[0047] The diffusion-limiting layer is coated onto the surface of the sensitive layer by dip coating.

[0048] The preparation method of the continuous lactic acid monitoring electrochemical sensor in this embodiment includes the following steps: (1) Pre-treatment of electrodes: The electrode surface is cleaned by solvent cleaning and plasma treatment to ensure that it is clean and free of impurities; (2) Coating the sensitive layer: Lactate oxidase, metal-based complex, excipients, and crosslinking agent are mixed to obtain a sensitive layer membrane solution. The sensitive layer membrane solution is coated onto the surface of the substrate electrode by spot coating, with 20 nL of membrane solution fixed on each sensor surface. After coating, the membrane solution is cured and crosslinked for 10 h at 50°C and 40% humidity to form a sensitive layer; (3) Coating diffusion restriction layer: A copolymer of 4-vinylpyridine and methacrylate is synthesized by free radical polymerization. The polymer is mixed with an epoxy crosslinking agent and dissolved in a mixed solvent of 80% ethanol and 20% water to obtain a diffusion restriction film liquid. The diffusion restriction film liquid is coated onto the surface of the sensitive layer by dip coating. After coating, it is cured and crosslinked for 24 hours at 37°C and 40% humidity to form a stable diffusion restriction layer.

[0049] In Example 1, the monomer ratio in the copolymer was adjusted and the material was grafted modified. The molecular structure of the copolymer and its structure after graft modification are as follows: Figure 1 As shown, where Figure 1 (a) shows the structure before grafting modification. Figure 1(b) shows the structure after grafting modification.

[0050] The continuous lactate monitoring electrochemical sensor prepared in Example 1 was used to monitor different lactate concentrations in the human body, and the results are as follows: Figures 2-3 As shown, the linear range is 0~10mM ( Figure 2 It has sufficient sensitivity for monitoring lactate concentration in critically ill patients, but for monitoring lactate threshold (…). Figure 3 This invention provides a sufficient linear range for problems in sports science fields such as endurance conditions. It demonstrates that by adjusting the comonomer ratio of the diffusion-limiting layer and hydrophilic modification, linear responses can be achieved across different concentration ranges.

[0051] Comparative Example 1 This comparative example provides an electrochemical sensor for lactate monitoring, comprising a substrate electrode, a sensitive layer, and a diffusion confinement layer; wherein, except for the diffusion confinement layer, the substrate electrode and sensitive layer of this comparative example are identical to those of Example 1. The preparation method of the electrochemical sensor for lactate monitoring in this comparative example is identical to that of Example 1.

[0052] The diffusion confinement layer was a commercially available polymer material, poly(4-vinylpyridine-co-styrene) (CAS: 26222-40-2). The material and epoxy crosslinking agent were dissolved in a mixed solvent of 80% and 20% water to obtain a membrane solution. The final concentrations of the polymer material and the epoxy crosslinking agent were 100 mg / mL and 5 mg / mL, respectively. The membrane solution was dip-coated onto the surface of the sensitive layer using the same method as in Example 1 and then crosslinked and cured to obtain the sensor of this comparative example.

[0053] The response data of the sensor obtained in Comparative Example 1 to different concentrations of lactic acid in phosphate buffer were as follows: 0mM current 0nA, 5mM current 8nA, 10mM current 18nA, 15mM current 19nA (without linear increase), 20mM current 20nA, and 25mM current 21nA. The linear range was only 0.1-10mM, and the response saturated above 10mM, making it impossible to measure accurately.

[0055] like Figure 5 As shown, the diffusion confinement layer was prepared using commercially available polymer materials, but the resulting lactic acid sensor had a small response and a linear range of only 10 mM, making it impossible to accurately measure higher lactic acid concentrations.

[0056] Comparative Example 2 This comparative example provides an electrochemical sensor for lactate monitoring, comprising a substrate electrode, a sensitive layer, and a diffusion confinement layer; wherein, except for the diffusion confinement layer, the substrate electrode and sensitive layer of this comparative example are identical to those of Example 1. The preparation method of the electrochemical sensor for lactate monitoring in this comparative example is identical to that of Example 1.

[0057] The diffusion confinement layer is also a copolymer of 4-vinylpyridine and methacrylate, and further hydrophilically grafted (sulfonate modification), with a sulfonate hydrophilic grafting rate of 20% (compared to 5% in Example 1). Except for the hydrophilic grafting ratio, the material preparation method is consistent with Example 1. The material and epoxy crosslinking agent are dissolved in a mixed solvent of 80% and 20% water to obtain a membrane solution. The final concentrations of the copolymer material and the epoxy crosslinking agent are 100 mg / mL and 5 mg / mL, respectively. The membrane solution is dip-coated onto the surface of the sensitive layer using the same method as in Example 1 and crosslinked and cured to obtain the sensor of this comparative example.

[0058] The sensor obtained in Comparative Example 2 showed the following response data for different concentrations of lactic acid in phosphate buffer: current 0 nA at 0 mM, 12 nA at 5 mM, 23 nA at 10 mM, 25 nA at 15 mM, 26 nA at 20 mM, 27 nA at 25 mM, and 28 nA at 30 mM. The linear range was only 0.1-5 mM, and there was no significant increase in current at 30 mM lactic acid, which could not meet the requirements for detecting high concentrations of lactic acid.

[0060] like Figure 6 As shown, Comparative Example 2 verifies the effect of different hydrophilic grafting ratios on the response of the lactic acid sensor proposed in this invention. Increasing the proportion of hydrophilic groups in the copolymer material provided by this invention narrows the linear range, failing to meet the requirements for high-concentration lactic acid detection. This also demonstrates that the proportion of hydrophilic groups in the copolymer material used as a diffusion confinement layer in this invention should be controlled within a suitable range to meet the needs of different monitoring application scenarios.

[0061] Experimental Example 1: Long-term stability test of an electrochemical sensor for continuous lactic acid monitoring This experimental example focuses on the stability of the lactic acid continuous monitoring electrochemical sensor prepared in Example 1 in a 5.0 mM lactic acid solution for fourteen consecutive days to verify its long-term performance.

[0062] Test steps: Sensor pretreatment: The surface of the lactic acid continuous monitoring electrochemical sensor prepared in Example 1 was cleaned with phosphate buffer (PBS, pH 7.4) to remove residual impurities; Test environment setup: Immerse the working part of the sensor in 5.0mM lactic acid solution (solvent is PBS, pH 7.4) and place it in a constant temperature (37℃) environment; Data acquisition: After the low-power Bluetooth signal acquisition device is connected to the sensor, it starts to continuously monitor and record the sensor's current response value for fourteen consecutive days; Data analysis: Plot the "time-current response" curve (e.g.) Figure 4 As shown in the figure, the response decay rate after fourteen days is calculated.

[0063] Test results: like Figure 4 As shown, the lactic acid sensor of this patent continuously monitored in a 5.0 mM lactic acid solution for fourteen days. The initial current response stabilized at approximately 50 nA, and after fourteen days, the current response decayed to approximately 36.5 nA, with a response decay rate of 27%. This result verifies the long-term stability of the sensor—the cross-linked structure of the outer diffusion-limiting layer effectively protects the enzyme activity of the sensitive layer, the peroxidase in the sensitive layer continuously removes hydrogen peroxide byproducts, avoiding irreversible damage to lactate oxidase byproducts, and the flexible substrate withstands long-term immersion environments, ensuring the reliability of the sensor's performance in continuous monitoring scenarios.

[0064] The same or similar parts between the various embodiments in this specification can be referred to mutually. Each embodiment focuses on describing the differences from other embodiments.

[0065] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. An electrochemical sensor for continuous monitoring of lactic acid, characterized in that, The sensor includes a base electrode, a sensitive layer, and a diffusion confinement layer; The sensitive layer is coated on the surface of the substrate electrode and contains a lactic acid oxidation catalyst and an electron mediator. The diffusion confinement layer is a multilayer cross-linked polymer film or a multilayer non-cross-linked polymer film; The diffusion-limiting layer is coated on the surface of the sensitive layer.

2. The electrochemical sensor for continuous lactic acid monitoring according to claim 1, characterized in that, The lactate oxidation catalyst is lactate oxidase; the electron mediator is a metal-based complex that generates electron transfer with the cofactor FAD in lactate oxidase.

3. The electrochemical sensor for continuous lactic acid monitoring according to claim 2, characterized in that, The metal-based complex includes any one of potassium ferricyanide, Prussian blue, ruthenium complex, and osmium complex.

4. The electrochemical sensor for continuous lactic acid monitoring according to claim 1, characterized in that, When the diffusion-limiting layer is a multilayer cross-linked polymer membrane, its membrane liquid component includes 5%-10% of either hydrophilic polyurethane or poly(4-vinylpyridine) polymer, and 0.5%-3% of epoxy cross-linking agent, and the membrane liquid solvent is a mixture of ethanol and water.

5. The electrochemical sensor for continuous lactic acid monitoring according to claim 4, characterized in that, When the diffusion-limiting layer is a multilayer cross-linked polymer membrane, the polymer in its membrane liquid component is a copolymer of 4-vinylpyridine and methacrylate.

6. The electrochemical sensor for continuous lactic acid monitoring according to claim 4, characterized in that, The coating process of the multilayer cross-linked polymer film includes: mixing the polymer with an epoxy cross-linking agent and coating it on the surface of the sensitive layer, and then curing and cross-linking it for 10-30 hours at 30-50℃ and 20-60% humidity.

7. A method for preparing a continuous lactic acid monitoring electrochemical sensor as described in claim 1, characterized in that, The process includes the following steps: coating a sensitive layer on the surface of a substrate electrode; coating a diffusion confinement layer on the surface of the sensitive layer; and curing the coating by heat treatment at 50-80°C to obtain the electrochemical sensor.

8. An application of the electrochemical sensor for continuous monitoring of lactic acid as described in claim 1, characterized in that, The continuous lactate monitoring electrochemical sensor is used for continuous monitoring of lactate in tissue fluid.

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