A continuous lactate monitoring sensor and method of making the same

Abstract

The application discloses a continuous lactic acid monitoring sensor and a preparation method thereof, and belongs to the technical field of electrochemical detection. The sensor comprises a substrate layer, an electrode layer and a reaction layer, the electrode layer is arranged on the substrate layer, the electrode layer comprises a working electrode, a reference electrode and a counter electrode; the reaction layer is arranged on the working electrode, and the reaction layer comprises lactic acid oxidase and catalase, wherein the mass ratio of the lactic acid oxidase to the catalase is 1: (1-3), and the catalase is subjected to a purification treatment. The continuous lactic acid monitoring sensor provided by the application has high detection accuracy and high stability, has almost no sensitivity attenuation after being worn in the body for 10 days, and can meet the use requirement of real-time and effective detection when being worn in the body.

Description

TECHNICAL FIELD

[0001] The application relates to a continuous lactate monitoring sensor and a preparation method thereof, and belongs to the technical field of electrochemical detection. BACKGROUND

[0002] Lactate is a key metabolic indicator of the human body, and continuous lactate monitoring is of great significance in the fields of medicine, sports science, etc. In a continuous lactate monitoring sensor (CLM), the detection function is mainly realized through a sensing layer. In the sensing layer, lactate is oxidized by lactate oxidase, and in this process, electrons are transferred to the lactate oxidase. Subsequently, the electrons are transmitted to the working electrode via an electron mediator, and the surface of the working electrode obtains electrons to generate an electric current. The current value is related to the lactate concentration, thereby realizing the function of lactate detection.

[0003] Hydrogen peroxide is a common by-product in the monitoring process of the continuous lactate monitoring sensor, and lactate oxidase is sensitive to this substance. In the presence of hydrogen peroxide, the stability of lactate oxidase is seriously damaged, and the CLM shows obvious late attenuation problems during the wearing period. When no catalase is added as a protective agent for lactate oxidase, the CLM almost completely loses the detection ability for the target lactate after being worn for 5 consecutive days.

[0004] Currently, in order to protect the in-vivo activity of lactate oxidase, catalase is mainly added to the reaction layer formula. The enzyme can quickly remove hydrogen peroxide at a very high efficiency, thereby playing a role in protecting lactate oxidase. However, the continuous lactate monitoring sensor with added hydrogen peroxide still has obvious late sensitivity attenuation and low detection stability problems. SUMMARY

[0005] In order to solve the above problems, the application provides a continuous lactate monitoring sensor, which has high detection accuracy and high stability, almost no sensitivity attenuation in 10 days of in-vivo wearing, and can meet the use requirements of real-time detection in in-vivo wearing.

[0006] The application provides a continuous lactate monitoring sensor, which comprises:

[0007] a substrate layer;

[0008] an electrode layer, which is arranged on the substrate layer and comprises a working electrode, a reference electrode and a counter electrode;

[0009] a reaction layer, which is arranged on the working electrode; wherein the reaction layer comprises lactate oxidase and catalase, the mass ratio of the lactate oxidase and the catalase is 1: (1-3), and the catalase is subjected to a purification treatment.

[0010] The present application sets the mass of catalase in the reaction layer to be not less than that of lactate oxidase, i.e. the proportion of catalase in the reaction layer is large, and it is found that catalase can not only remove peroxide in time, but also effectively protect the activity of lactate oxidase, thereby improving the detection accuracy and sensitivity of the sensor.

[0011] Optionally, the reaction layer further comprises an electron mediator, a crosslinking agent and albumin, and the preset mass ratio of the lactate oxidase, the electron mediator, the crosslinking agent, the albumin and the catalase is 2: (1-2): (0.5-2): (0.5-1): (2-6).

[0012] Optionally, the reaction layer is arranged in a linear shape on the working electrode.

[0013] The length of the reaction layer is 1.1 mm-1.3 mm, and the width is 150 µm-170 µm.

[0014] The length and width of the line are set to keep the sensitivity within a reasonable range of 1-1.6 nA / mM, and the sensitivity is related to the total area of the reaction layer. In the same system, the larger the area of the reaction layer, the higher the sensitivity. If the length or width of the line is too small, the sensitivity of the sensor will be too low, which will result in low signal-to-noise ratio, and if the length or width of the line is too large, the sensitivity of the sensor will be too high, which is more likely to cause attenuation.

[0015] Optionally, the reaction layer is arranged in a circular dot manner on the working electrode.

[0016] The number of circular dots in the reaction layer is at least 4, the diameter of the circular dots is 160 µm-180 µm, and the center-to-center distance between adjacent circular dots is 280 µm-300 µm.

[0017] Under this setting, the circular diameter within this range can keep the sensitivity within 1-1.6 nA / mM, and the circular spacing is set to ensure that the circular dots are not connected together, so that each enzyme solution remains independent.

[0018] Optionally, the enzyme activity of the catalase is ≥3000 u / mg, and the enzyme activity of the lactate oxidase is ≥20 u / mg.

[0019] Preferably, the enzyme activity of the catalase is 6000 u / mg, and the enzyme activity of the lactate oxidase is 80 u / mg.

[0020] The above enzyme activity can further improve the detection sensitivity and stability of the sensor.

[0021] According to another aspect of the present application, a preparation method of the continuous lactate monitoring sensor of any one of the above is provided, comprising:

[0022] S1: The working electrode, the reference electrode, and the counter electrode are disposed at intervals on the substrate layer;

[0023] S2: Purify the catalase;

[0024] S3: The lactate oxidase, the electron mediator, the cross-linking agent, the albumin, and the purified catalase are mixed according to a preset mass ratio to obtain an enzyme solution;

[0025] S4: The enzyme solution is coated onto the working electrode to form the reaction layer, thereby obtaining the continuous lactate monitoring sensor.

[0026] Optionally, step S2 includes:

[0027] The catalase was placed in an ultrafiltration tube, and the ultrafiltration tube was centrifuged at a preset speed. The catalase in the inner tube of the ultrafiltration tube was collected as purified catalase.

[0028] For example, catalase is placed on the inner wall of the ultrafiltration tube of MWCO 30000 and ultrafiltered by centrifugation at 8000-10000 rpm for 10-20 min.

[0029] Optionally, the viscosity of the enzyme solution is in the range of 2-5 mPa·s.

[0030] When the viscosity of the enzyme solution is within the above range, the consistency of the reaction layer can be improved. If the viscosity of the enzyme solution is too high or too low, the enzyme solution will form poorly, resulting in poor consistency of the prepared reaction layer.

[0031] Optionally, step S4 includes: filtering the enzyme solution through a filter membrane and then dotting it onto the working electrode to form 12 longitudinally arranged dots, each dot corresponding to 4 drops of enzyme solution, each drop of enzyme solution having a volume of 300-360 pL, and the center distance between adjacent dots being 110-130 µm.

[0032] Optionally, step S4 includes: filtering the enzyme solution through a filter membrane and then dotting it onto the working electrode to form 4-6 dots arranged longitudinally, each dot corresponding to 8 drops of enzyme solution, each drop of enzyme solution having a volume of 300-360 pL, and the center distance between adjacent dots being 280-300 µm.

[0033] The beneficial effects of this application include, but are not limited to:

[0034] According to the continuous lactate monitoring sensor provided in this application, the mass ratio of lactate oxidase to catalase in the reaction layer is set to 1:(1-3). This effectively removes peroxides, a byproduct of the detection process, and protects the activity of lactate oxidase, thereby improving the detection accuracy and sensitivity of the sensor. Furthermore, by setting the mass of catalase to be no less than that of lactate oxidase, the sensitivity stability of the continuous lactate monitoring sensor when worn in vivo is greatly improved. There is almost no sensitivity decay within 10 days of in vivo wear, and the sensitivity retention rate is greater than 80% after accelerated aging at 55°C for 56 days, ensuring the validity of real-time detection data during in vivo wear. In addition, this application purifies catalase to remove impurities such as small molecules and ions, improving the purity of catalase. This avoids the salting-out effect caused by excessive ions in catalase in the reaction layer, which can seriously affect the sensitivity consistency during mass production of the sensor. The sensitivity range of the same batch of sensors can vary to 1-3 nA / mM. This application further improves the sensitivity stability of the sensor by purifying catalase. Attached Figure Description

[0035] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0036] Figure 1 This is a schematic diagram of the linear arrangement of the reaction layers involved in Embodiment 1 of this application.

[0037] Figure 2 This is a test image of the sensor used in Embodiment 1 of this application being worn in the body.

[0038] Figure 3 This is a test image of the sensor in Embodiment 1 of this application, which has been aged at 55°C for 56 days.

[0039] Figure 4 This is one of the schematic diagrams of the circular arrangement of enzyme solution involved in Embodiment 5 of this application.

[0040] Figure 5 This is the second schematic diagram of the circular arrangement of enzyme solution involved in Comparative Example 1 of this application.

[0041] Figure 6 This is a test image of the sensor used in Comparative Example 2 of this application, taken during in-vivo wear testing.

[0042] Figure 7 This is a test image of the sensor used in Comparative Example 3 of this application, taken during in-vivo wear testing.

[0043] Figure 8The graphs show the in vitro sensitivity test results of the sensors in Comparative Example 2 and Example 1.

[0044] Figure 9 The images show the test results of sensors from Comparative Example 2 and Example 1 after aging at 55°C for different times. Detailed Implementation

[0045] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0046] Unless otherwise specified, the raw materials used in the embodiments and comparative examples of this application were all purchased commercially.

[0047] Unless otherwise specified, the methods used in the embodiments and comparative examples of this application are conventional methods in the prior art.

[0048] In an embodiment achievable under this application, a continuous lactate monitoring sensor is provided. The continuous lactate monitoring sensor includes a substrate layer, an electrode layer, and a reaction layer. The electrode layer is disposed on the substrate layer and includes a working electrode, a reference electrode, and a counter electrode. The reaction layer is disposed on the working electrode. The reaction layer includes lactate oxidase and catalase, with a mass ratio of lactate oxidase to catalase of 1:(1-3), and the catalase is purified.

[0049] In this embodiment of the application, the substrate layer plays a supporting role, which facilitates the adhesion of the electrode layer. Its material can be the conventional material of the substrate layer of the sensor in the prior art, and there is no specific limitation. For example, it can be one of polyethylene terephthalate, polyvinyl chloride and polypropylene.

[0050] The electrode layer includes a working electrode, a counter electrode, and a reference electrode. The working electrode and the counter electrode may be located on opposite surfaces of the substrate layer, wherein the reference electrode may be located on the same side of the substrate layer as the working electrode and separated from it by a dielectric material.

[0051] Specifically, in this sensor, the lactate oxidase on the reaction layer of the working electrode oxidizes lactic acid to generate electrons, which in turn generate current. Different lactic acid contents result in different current values, thus enabling the sensor to detect lactic acid. The working electrode on the electrode layer can be made of carbon or platinum, and the counter electrode can be made of carbon or gold.

[0052] In other embodiments achievable in this application, the continuous lactate monitoring sensor further includes an outer membrane layer for at least covering the working electrode.

[0053] It should be noted that the outer membrane layer has the ability to limit the flux of analytes. Its main function is to limit the flux of lactic acid entering the reaction layer region within the linear range monitored by the reaction layer, thereby enabling the detection of physiological concentrations of lactic acid.

[0054] In one embodiment, the outer membrane layer comprises a cross-linked polyvinylpyridine homopolymer or copolymer.

[0055] In a preferred embodiment, the outer membrane layer comprises: a crosslinked polyvinylpyridine homopolymer or a polyvinylpyridine copolymer.

[0056] In one specific implementation, the outer membrane layer includes a polyethylene pyridine copolymer.

[0057] In one specific implementation, the outer membrane layer includes polyurethane.

[0058] As one implementation method, the mass ratio of lactate oxidase to catalase can be 1:1.2, 1:1.5, 1:1.8, 1:2.0, 1:2.2, 1:2.5, 1:2.8, 1:3, or any ratio between them, preferably 1:3.

[0059] As one implementation method, the activity of catalase is ≥3000u / mg, and the activity of lactate oxidase is ≥20u / mg.

[0060] Specifically, the activity of catalase can be any of the following values: 3000 u / mg, 3500 u / mg, 4000 u / mg, 4500 u / mg, 5000 u / mg, 5500 u / mg, 6000 u / mg, 6500 u / mg, 7000 u / mg, 7500 u / mg, 8000 u / mg, 8500 u / mg, 9000 u / mg, 9500 u / mg, or any activity between these values.

[0061] Specifically, the enzyme activity of lactate oxidase can be any of the following values: 20u / mg, 25u / mg, 30u / mg, 35u / mg, 40u / mg, 45u / mg, 50u / mg, 55u / mg, 60u / mg, 65u / mg, 70u / mg, 75u / mg, 80u / mg, 85u / mg, 90u / mg, 95u / mg, or any value between these values.

[0062] In a preferred embodiment, the activity of catalase is 6000 u / mg, and the activity of lactate oxidase is 80 u / mg.

[0063] In one implementation, the enzyme solution is arranged in a circular or linear manner on the working electrode.

[0064] Optionally, the reaction layer is arranged in a linear shape on the working electrode;

[0065] In one implementation, the length of the reaction layer is 1.1-1.3 mm and the width is 150-170 µm.

[0066] Specifically, the length of the reaction layer can be 1.1 mm, 1.15 mm, 1.2 mm, 1.25 mm, 1.3 mm, or any value between these values, and the corresponding width of the reaction layer can be 150 µm, 155 µm, 160 µm, 165 µm, 170 µm, or any value between these values.

[0067] In one implementation, the reaction layer is arranged in a dot pattern on the working electrode;

[0068] The reaction layer contains at least four dots, each dot having a diameter of 160 µm-180 µm and a center-to-center distance of 280 µm-300 µm between adjacent dots.

[0069] Specifically, the number of dots can be 4, 6, 8, 10, 12, 14, 16, 18, 20, or any number in between.

[0070] Specifically, the diameter of the circle can be 160µm, 165µm, 170µm, 175µm, 180µm, or any value in between.

[0071] Specifically, the distance between the centers of adjacent circles can be 280µm, 285µm, 292µm, 295µm, 300µm, or any value in between.

[0072] This application also relates to a method for preparing a continuous lactate monitoring sensor according to any of the above claims, comprising the following steps:

[0073] S1: The working electrode, reference electrode, and counter electrode are spaced apart on the substrate layer;

[0074] S2: Purification of catalase;

[0075] S3: Mix lactate oxidase, electron mediator, cross-linking agent, albumin and purified catalase according to the preset mass ratio to obtain enzyme solution;

[0076] S4: Coat the working electrode with enzyme solution to form a reaction layer, thus obtaining a continuous lactate monitoring sensor.

[0077] In one implementation, the mass ratio of lactate oxidase, electron mediator, cross-linking agent, albumin and catalase is 2:(1-2):(0.5-2):(0.5-1):(2-6).

[0078] Specifically, the mass ratio of lactate oxidase, electron mediator, cross-linking agent, albumin, and catalase can be 2:1:0.5:0.5:2, 2:1:1:1:2, 2:1:1:1:2.4, 2:2:2:1:2.4, 2:1:1:1:3, 2:1:1:1:4, 2:1:1:1:5, 2:1:1:1:6, 2:1:2:1:6, 2:1:0.5:1:6, and 2:1:1:0.5:6.

[0079] Specifically, the electron mediator used in this application can be at least one of ruthenium compounds, osmium compounds, metallocene compounds, or other transition metal compounds. When it is a ruthenium compound, it can be ruthenium oxide or a ruthenium complex, wherein the ligand can include, for example, bidentate or higher-octagonal ligands, such as bipyridine, biimidazole, o-phenanthroline or pyridinyl (imidazolium), amino acids, oxalic acid, acetylacetone, diaminoalkane or o-diaminoaromatic hydrocarbons, or polyvinylpyridine. Osmium compounds can also be osmium oxide or osmium complexes, whose ligands can include, for example, bidentate or higher-octagonal ligands, such as bipyridine, biimidazole, o-phenanthroline or pyridinyl (imidazolium), and polyvinylpyridine. The transition metal polymer can be a polymer obtained by coordinating a 1,10-phenanthroline derivative with a transition metal mediator.

[0080] Specifically, the crosslinking agent used in this application may be at least one of glutaraldehyde, 1,4-butanediol diglycidyl ether, poly(dimethylsiloxane)-diglycidyl ether, tetraglycidyl-4,4-diaminodiphenylmethane, polyethylene glycol diglycidyl ether, glycerol triglycidyl ether, or 4-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline.

[0081] Specifically, albumin includes human serum albumin and recombinant human serum albumin.

[0082] Specifically, catalase can be purified by ultrafiltration, desalting column chromatography, or dialysis.

[0083] As one implementation method, the specific steps for ultrafiltration of catalase are as follows: place catalase on the inner wall of the ultrafiltration tube of MWCO 30000 and centrifuge and ultrafilter at a preset speed of 8000-10000 rpm for 10-20 minutes.

[0084] Specifically, the ultrafiltration speed can be 8000 rpm, 8500 rpm, 9000 rpm, 9500 rpm, 10000 rpm, or any speed in between.

[0085] The specific ultrafiltration treatment time can be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, or any time in between.

[0086] In one implementation, the mixing time in step S3 is 10-30 min and the mixing speed is 400 rpm.

[0087] Specifically, the mixing time in step S3 can be any time between 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, and any time in between.

[0088] As one implementation method, the viscosity of the enzyme solution is in the range of 2-5 mPa·s.

[0089] Specifically, the viscosity of the enzyme solution can be 2.5 mPa·s, 3.0 mPa·s, 3.5 mPa·s, 4.0 mPa·s, 4.5 mPa·s, 5.0 mPa·s, or any viscosity in between.

[0090] In one embodiment, in step S4, the enzyme solution is filtered through a filter membrane and then coated onto the working electrode.

[0091] As one implementation method, using a filter membrane to filter the enzyme solution can ensure that the enzyme solution does not clog the enzyme dispensing machine and ensures smooth coating of the enzyme solution. A filter membrane of 0.22-0.8µm can be selected.

[0092] Specifically, the filter membrane can be 0.22µm, 0.3µm, 0.4µm, 0.45µm, 0.5µm, 0.6µm, 0.7µm, or 0.8µm.

[0093] Preferably, the filter membrane is a 0.22µm filter membrane.

[0094] As one implementation method, the specific operation of coating the enzyme solution onto the working electrode in step S4 is as follows:

[0095] After filtering the enzyme solution through a filter membrane, it is dotted onto the working electrode to form 12 vertically arranged dots, each dot corresponding to 4 drops of enzyme solution. The volume of each drop of enzyme solution is 300-360 pL, the center distance between adjacent dots is 110-130 µm, and the total volume of the coated enzyme solution is 14.4-17.3 nL.

[0096] As one implementation method, the specific operation of coating the enzyme solution onto the working electrode in step S4 is as follows:

[0097] First pass: After filtering the enzyme solution through a filter membrane, apply it to the working electrode to form 6 dots arranged in a vertical direction. The center distance between adjacent dots is 220-260µm. Each dot corresponds to 4 drops of enzyme solution, and each drop of enzyme solution has a volume of 300-360pL.

[0098] Second pass: Repeat the first pass, but with the dots in the second pass spaced apart from those in the first pass. The center distance between the first circle in the second pass and the first circle in the first pass should be 110-130µm. The center distance between the six adjacent dots in the second pass should be 220-260µm. Each dot corresponds to four drops of enzyme solution, with each drop containing 300-360pL. The total volume of enzyme solution for both passes should be 14.4-17.3nL.

[0099] As one implementation method, the specific operation of coating the enzyme solution onto the working electrode in step S4 is as follows:

[0100] After filtering the enzyme solution through a filter membrane, it is dotted onto the working electrode to form 4-6 dots arranged longitudinally. Each dot corresponds to 8 drops of enzyme solution, with each drop having a volume of 300-360 pL. The center-to-center distance between adjacent dots is 280-300 µm, and the total enzyme solution volume is 14.4-17.3 nL.

[0101] Preferably, when the enzyme solution is arranged in dots on the working electrode, six dots are applied.

[0102] In one implementation, after coating the working electrode with the enzyme solution in step S4, it is placed at 25°C and 60% humidity for 18-24 hours to form a reaction layer.

[0103] During the aforementioned settling time, the enzyme solution can fully cross-link to form a reaction layer.

[0104] Specifically, the settling time in step S4 can be 18h, 19h, 20h, 21h, 22h, 23h, 24h, or any other time.

[0105] It should be noted that the continuous lactate monitoring sensor provided in this application can determine lactate through any of the following electrochemical detection techniques: coulometric method, amperometric method, voltammetric method, or potentiometric method.

[0106] The structure and preparation method of the continuous lactate monitoring sensor involved in this application are listed in detail below.

[0107] Example 1

[0108] This embodiment relates to a continuous lactate monitoring sensor, comprising a base layer, an electrode layer, and a reaction layer. The electrode layer is disposed on the base layer and includes a working electrode, a reference electrode, and a counter electrode. The reaction layer is disposed on the working electrode. The reaction layer includes lactate oxidase and catalase, and the catalase is purified.

[0109] The sensor is prepared by:

[0110] S1: The working electrode, reference electrode, and counter electrode are spaced apart on the substrate layer;

[0111] S2: Ultrafiltration treatment of catalase: Ultrafiltration of 90 mg / mL catalase (enzyme activity 6000 u / mg) was performed by placing the catalase on the inner wall of the MWCO 10000 ultrafiltration tube and centrifuging the ultrafiltration tube at 10000 rpm for 10 minutes. The solution in the inner tube was collected for subsequent enzyme solution preparation.

[0112] S3: Lactate oxidase (enzyme activity 80u / mg), electron mediator bipyridine osmium complex, cross-linking agent polyethylene glycol diglycidyl ether (PEGDGE), recombinant human serum albumin and ultrafiltration-treated catalase were mixed in a mass ratio of 2:1:0.5:0.5:2 and then stirred at 400 rpm for 20 min to obtain enzyme solution;

[0113] S4: Filter the enzyme solution through a 0.22µm filter membrane, collect the filtrate, and apply the filtered enzyme solution to the working electrode area of ​​the sensor. Apply two coats. The first coat applies six vertically arranged dots, with a center-to-center distance of 240µm between adjacent dots. Each dot contains 4 drops of enzyme solution, with a volume of 305pL per drop. Repeat the first coat, applying six dots per dot, with 4 drops of enzyme solution per dot, each drop containing 305pL. The second coat applies dots spaced apart from the first coat, resulting in 12 vertically arranged dots, with a center-to-center distance of 120µm between each dot. The total enzyme solution volume is 14.6nL. The final linear arrangement of the enzyme solution is as follows: Figure 1 The linear electrode is 1.1 mm long and 150 µm wide. After coating, the coated electrode is placed in a constant temperature and humidity chamber at 25 °C and 60% for 24 hours to form a reaction layer, thus obtaining the sensor.

[0114] Example 2

[0115] The difference between this embodiment and Example 1 is that the mass ratio of lactate oxidase, electron mediator bipyridine osmium complex, crosslinking agent PEGDGE, recombinant human serum albumin, and ultrafiltration-treated catalase is 2:2:2:1:2.4.

[0116] Example 3

[0117] The difference between this embodiment and Example 1 is that the mass ratio of lactate oxidase, electron mediator bipyridine osmium complex, crosslinking agent PEGDGE-500, recombinant human serum albumin, and ultrafiltration-treated catalase is 2:1:0.5:0.5:3.

[0118] Example 4

[0119] The difference between this embodiment and Example 1 is that the mass ratio of lactate oxidase, electron mediator bipyridine osmium complex, crosslinking agent PEGDGE-500, recombinant human serum albumin, and ultrafiltration-treated catalase is 2:1:0.5:0.5:4.

[0120] Example 5

[0121] The difference between this embodiment and Example 1 is that the mass ratio of lactate oxidase, electron mediator bipyridine osmium complex, cross-linking agent PEGDGE-500, recombinant human serum albumin, and ultrafiltration-treated catalase is 2:1:0.5:0.5:5; and in step S4, the enzyme solution is spotted into 6 dots (e.g., Figure 4 The center-to-center distance between adjacent dots is 300µm, each dot corresponds to 8 drops of enzyme solution, the diameter of each dot is 160µm, and the total enzyme solution volume is 14.4nL.

[0122] Example 6

[0123] The difference between this embodiment and Example 1 is that the mass ratio of lactate oxidase, electron mediator bipyridine osmium complex, crosslinking agent PEGDGE-500, recombinant human serum albumin, and ultrafiltration-treated catalase is 2:1:0.5:0.5:6.

[0124] Example 7

[0125] The difference between this embodiment and Embodiment 1 is that the activity of catalase is 3000 u / mg and the activity of lactate oxidase is 20 u / mg.

[0126] Comparative Example 1

[0127] The difference between this comparative example and Example 1 is that step S2 is omitted, i.e., the catalase is not subjected to ultrafiltration, and in step S4, the enzyme solution is coated into 6 dots with a center-to-center distance of 300µm between adjacent dots, each dot corresponds to 8 drops of enzyme solution, each drop of enzyme solution has a volume of 360pL, each circle has a diameter of 180µm, and the total enzyme solution volume is 17.3nL.

[0128] This sensor lacks step S2 and does not perform ultrafiltration on the catalase. When the enzyme solution is coated onto the working electrode of the sensor, significant precipitation of the enzyme solution is observed after coating (e.g., Figure 5(As shown in the image). This phenomenon may be due to the presence of a large number of salt ions, small molecules, and other substances in the catalase raw material, which causes the protease in the enzyme solution to precipitate out.

[0129] Comparative Example 2

[0130] The difference between this comparative example and Example 1 is that catalase is not added to the enzyme solution.

[0131] Comparative Example 3

[0132] The difference between this comparative example and Example 1 is that the mass ratio of lactate oxidase, electron mediator bipyridine osmium complex, crosslinking agent PEGDGE-500, recombinant human serum albumin, and pretreated catalase is 2:1:0.5:0.5:1.

[0133] Test case

[0134] The sensors prepared in the above embodiments and comparative examples were tested for enzyme solution viscosity, initial sensitivity, in vivo wearing sensitivity, and sensitivity after being placed at 55°C for 56 days. The test results are shown in Table 1.

[0135] Table 1

[0136]

[0137] As shown in Table 1, the initial sensitivity of all examples and comparative examples is in the range of 1.0-1.6 nA / mM, and the initial sensitivity of the examples is in the range of 1.0-1.4 nA / mM. The viscosity of all examples and comparative examples is in the range of 2-5 mPa·s.

[0138] All the sensors in the embodiments maintained more than 80% of their sensitivity without decay after 10 days in vivo. The in vivo wear test results of the sensor in Embodiment 1 are as follows: Figure 2 As shown, the sensor maintains stable sensitivity even after being worn in the body for ten days. The sensor from Example 1, after being aged at 55°C for 56 days, showed the following results: Figure 3 As shown, it can still maintain 81% sensitivity.

[0139] Comparative Example 2: Sensor wear-in-body test results are as follows Figure 6 As shown, the sensor lost approximately 80% of its sensitivity after being worn in the body for 5 days. The in-body wearing test results for Comparative Example 3 are as follows... Figure 7 As shown, the sensor lost approximately 50% of its sensitivity after being worn in vivo for 4 days. Therefore, it can be concluded that the in vivo sensitivity of Comparative Examples 2 and 3 was lost by 50%-80% within 5 days. Based on this result, it can be concluded that when the mass ratio of lactate oxidase to catalase is 1:(1-3), the in vivo stability of the continuous lactate monitoring sensor can be significantly improved.

[0140] After aging at 55°C for 56 days, all examples maintained more than 80% of their sensitivity without degradation, while Comparative Examples 2 and 3 lost 23% and 68% of their sensitivity, respectively, after 56 days. Furthermore, the higher the proportion of catalase, the better the performance at 55°C aging. The optimal ratio of lactate oxidase to catalase was 1:3, as catalase also protects the thermal stability of lactate oxidase.

[0141] The aging performance of linearly coated enzyme solutions is also better than that of circularly coated enzyme solutions. This may be because the circumference-to-area ratio of circular enzyme solutions is larger, which means that there is more edge portion of the circular enzyme solution. Since the edge of the enzyme solution is often less controllable, the linear enzyme solution performs better.

[0142] Figure 8 The results show the in vitro sensitivity of the sensors in Comparative Example 2 and Example 1. Figure 9 To compare the sensitivity test results of the sensors in Example 2 and Example 1 after aging at 55°C for different times, based on... Figure 8 and Figure 9 It is evident that the in vitro sensitivity of the sensor in this application did not change significantly after the addition of catalase, and the stability of the 55°C accelerated aging test was also effectively improved. When catalase was not added, the sensor almost lost all sensitivity after four weeks of 55°C accelerated aging test (one day of acceleration at 55°C is equivalent to 8 days of shelf life at room temperature). The sensor in Example 1 still maintained more than 80% of its sensitivity after six weeks of accelerated aging at 55°C.

[0143] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A continuous lactate monitoring sensor, characterized in that, include: basal layer; An electrode layer is disposed on the substrate layer, and the electrode layer includes a working electrode, a reference electrode, and a counter electrode. A reaction layer is disposed on the working electrode; wherein the reaction layer includes lactate oxidase, electron mediator, cross-linking agent, albumin and catalase, and the preset mass ratio of lactate oxidase, electron mediator, cross-linking agent, albumin and catalase is 2:(1-2):(0.5-2):(0.5-1):(2-6), and the catalase is purified, with the enzyme activity of catalase ≥3000u / mg and the enzyme activity of lactate oxidase ≥20u / mg. The catalase is placed in an ultrafiltration tube, and the ultrafiltration tube is centrifuged at a preset speed. The catalase in the inner tube of the ultrafiltration tube is collected as purified catalase.

2. The method for preparing the continuous lactate monitoring sensor according to claim 1, characterized in that, include: S1: The working electrode, the reference electrode, and the counter electrode are disposed at intervals on the substrate layer; S2: Purify the catalase; S3: The lactate oxidase, the electron mediator, the cross-linking agent, the albumin, and the purified catalase are mixed according to a preset mass ratio to obtain an enzyme solution; S4: The enzyme solution is coated onto the working electrode to form the reaction layer, thereby obtaining the continuous lactate monitoring sensor.

3. The method for preparing the continuous lactate monitoring sensor according to claim 2, characterized in that, The viscosity range of the enzyme solution is 2-5 mPa·s.

4. The method for preparing the continuous lactate monitoring sensor according to claim 2, characterized in that, Step S4 includes: The enzyme solution is filtered through a filter membrane and then dotted onto the working electrode to form 12 dots arranged longitudinally. Each dot corresponds to 4 drops of enzyme solution, and the volume of each drop of enzyme solution is 300-360 pL. The center distance between adjacent dots is 110-130 µm.

5. The method for preparing the continuous lactate monitoring sensor according to claim 2, characterized in that, Step S4 includes: The enzyme solution is filtered through a filter membrane and then dotted onto the working electrode to form 4-6 dots arranged longitudinally. Each dot corresponds to 8 drops of enzyme solution, and the volume of each drop of enzyme solution is 300-360 pL. The center distance between adjacent dots is 280-300 µm.

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