A sulfonated mxene sensing layer, a preparation method thereof and a biosensor

Abstract

The application discloses a sulfonated MXene sensing layer, a preparation method thereof and a biosensor, and belongs to the technical field of electrochemical sensors. The sensing layer contains a sulfonated MXene material, and the sulfonated MXene material is obtained by covalently grafting sulfonic acid groups on the MXene material. The sulfonic acid groups are covalently grafted on the surface of the MXene through plasma treatment, so that the sulfonic acid groups have high specific surface area, excellent electron conductivity, high enzyme immobilization capacity and H2O2 catalytic decomposition activity. The biosensor based on the sensing layer comprises a flexible substrate, a three-electrode system and a diffusion-limiting layer, and the response speed, detection accuracy, stability and anti-interference performance (such as anti-uric acid and anti-ascorbic acid) of the biosensor are significantly improved, and the biosensor is particularly suitable for applications in an implantable continuous glucose monitoring system.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of electrochemical sensors, and in particular to a sulfonated MXene sensing layer, a preparation method thereof and a biosensor. BACKGROUND

[0002] Diabetes is a chronic metabolic disease caused by insufficient insulin secretion or dysfunction, and patients need to monitor blood glucose levels for a long time. Continuous glucose monitoring system (CGMS) can monitor the glucose concentration in the interstitial fluid in real time through the implanted biosensor, which brings revolutionary changes to diabetes management. However, the performance of the core of the existing CGMS, the biosensor, still has obvious bottlenecks, mainly in three aspects:

[0003] First, the response speed and sensitivity are insufficient. The existing sensor mostly adopts the "wired enzyme" technology, which relies on the electron transfer between the enzyme (such as glucose oxidase) and the redox polymer. The process rate is limited, which leads to slow signal response and makes it difficult to achieve real-time monitoring.

[0004] Second, the long-term stability is poor. The enzyme catalytic reaction inevitably produces active byproduct H2O2, which accumulates near the electrode and seriously inhibits the enzyme activity, and can also degrade the sensing layer material, resulting in rapid attenuation of the sensor signal within a few days or weeks, which cannot meet the use requirements of long-term implantation.

[0005] Third, the anti-interference ability is weak. There are many electroactive substances (such as uric acid, ascorbic acid, etc.) in the human tissue fluid environment, which are easy to be non-specifically oxidized on the working electrode, producing interference current that cannot be distinguished from the glucose signal, causing the detection result to be distorted and the accuracy to be reduced.

[0006] MXene, as a new two-dimensional layered material, has been explored for use in the field of electrochemical sensing due to its high specific surface area and excellent electrical conductivity, providing potential solutions to the above problems. However, the functional groups on the surface of the original MXene material (such as -OH, -F) limit its ion transport capacity, and the intrinsic electron transfer and catalytic activity are insufficient, and the ability to immobilize enzyme molecules and selective adsorption is still lacking, making it difficult to directly meet the stringent requirements of high-sensitivity, high-stability CGM sensors.

[0007] Therefore, developing a new type of sensing layer based on material innovation that can simultaneously overcome the three technical difficulties of fast response, long-term stability and precise anti-interference is of great significance for promoting the performance of CGM systems and improving the quality of life of diabetic patients. SUMMARY

[0008] The application aims to solve the above problems, and provides a sulfonated MXene sensing layer, a preparation method thereof, and a biosensor.

[0009] The first aspect of the application provides a sulfonated MXene sensing layer, which adopts the following technical solution:

[0010] The sulfonated MXene sensing layer contains a sulfonated MXene material, and the sulfonated MXene material is obtained by covalently modifying a sulfonic acid group on an MXene material.

[0011] By adopting the above technical solution, the MXene material itself has high specific surface area, high electrical conductivity, and chemical stability. After sulfonation treatment, the enzyme molecules can be better fixed and the enzyme loading capacity can be improved. Since the enzyme molecules are fixed on the surface of the material, the active sites of the fixed enzyme are more easily accessible to the reaction substrate, thereby improving the response speed of the sensing layer. Meanwhile, the sulfonic acid group can serve as an active site for the decomposition of H2O2, improving the decomposition efficiency of H2O2 and further improving the stability of the sensing layer. After introducing the -SO3H group through plasma treatment, the negatively charged interfering substances (such as albumin and uric acid) can be reduced through electrostatic repulsion, reducing non-specific adsorption and improving the anti-interference ability of the sensor.

[0012] Preferably, the MXene material is selected from one or more of Ti3C2T x , Ti2CT x , V2CT x , Nb2CT x , and MoTiC2T x , and is preferably Ti3C2T x .

[0013] Preferably, the preparation material of the sensing layer comprises a redox polymer, a tool enzyme, a sulfonated MXene material, and a crosslinking agent A, wherein the mass ratio of the redox polymer, the tool enzyme, the sulfonated MXene material, and the crosslinking agent A is 30-50:15-30:15-30:1-10, and is preferably 45:25:25:5.

[0014] By adopting the above technical solution, the sulfonated MXene material cooperates with the redox polymer, the tool enzyme, and the crosslinking agent to improve the electron transport characteristics of the sensing layer and improve the sensitivity of potential measurement.

[0015] Preferably, the redox polymer is a redox polymer containing a transition metal, and the transition metal includes ruthenium, rhodium, and osmium, and is preferably a redox polymer containing osmium.

[0016] The tool enzyme is selected from one or more of glucose dehydrogenase, glucose oxidase, uricase, lactate oxidase, cholesterol oxidase, preferably glucose oxidase;

[0017] The crosslinking agent A is selected from one or more of 1,4-butanediol diglycidyl ether, poly(dimethylsiloxane)-diglycidyl ether, glutaraldehyde, polyethylene glycol diglycidyl ether, tetraepoxypropyl-4,4-diaminodiphenylmethane or glycerol triglycidyl ether, preferably polyethylene glycol diglycidyl ether with a molecular weight of between 200 Da and 5000 Da, further preferably polyethylene glycol diglycidyl ether with a molecular weight of 200 Da.

[0018] The second aspect of the application provides a preparation method of the above-mentioned sulfonated MXene sensing layer, which adopts the following technical solution:

[0019] A preparation method of a sulfonated MXene sensing layer, comprising the following steps:

[0020] S1, preparing a sulfonated MXene material;

[0021] S2, using a solvent A to prepare a sensing layer solution containing a redox polymer, a tool enzyme, a sulfonated MXene material and a crosslinking agent A;

[0022] S3, loading the sensing layer solution on the surface of a working electrode, crosslinking and curing at 30-40℃ for 20-28 hours to obtain the sensing layer.

[0023] Preferably, in the technical solution of the above preparation method, S1 comprises: mixing Ti3AlC2 with a 40%-50% hydrofluoric acid solution, reacting at 40-50℃ for 40-60 hours, and obtaining Ti3C2T x powder after centrifugal drying; dispersing the Ti3C2T x powder into a 0.5-2 mg / mL dispersion, drop-coating onto a silicon wafer and drying into a MXene film; placing the MXene film in a plasma reaction chamber, introducing SO3 gas provided by an SO3 cylinder, and treating under the conditions of a gas flow of 100-200 mL / min, a power of 80-120 W, and a gas pressure of 20-60 Pa for 3-10 minutes, and obtaining the sulfonated MXene material after washing and drying.

[0024] Preferably, in the technical solution of the above preparation method, S1 comprises: mixing Ti3AlC2 with a 40% hydrofluoric acid solution, reacting at 50℃ for 48 hours, and obtaining Ti3C2T X powder after centrifugal drying; dispersing the Ti3C2T XThe powder is dispersed into a 1 mg / mL dispersion, drop-coated onto a silicon wafer and dried into a MXene film; the MXene film is placed in a plasma reaction chamber, and SO3 gas provided by an SO3 cylinder is introduced, and the SO3 gas is treated under the conditions of a gas flow of 150 mL / min, a power of 100 W, and a gas pressure of 40 Pa for 5 minutes, and after cleaning and drying, a sulfonated MXene material is obtained.

[0025] Preferably, in S2, the solvent A is HEPES buffer or Tris ethanesulfonic acid buffer with a pH value of 7.5-8.5; in the sensing layer solution, the concentration of the redox polymer is 20-50 mg / mL; the concentration of the tool enzyme is 10-30 mg / mL; the concentration of the sulfonated MXene material is 10-30 mg / mL; and the concentration of the cross-linking agent A is 5-15 mg / mL.

[0026] Preferably, the solvent A is HEPES buffer with a concentration of 20 mmol / L and a pH of 8.0; in the sensing layer solution, the concentration of the redox polymer is 30 mg / mL; the concentration of the tool enzyme is 20 mg / mL; the concentration of the sulfonated MXene material is 20 mg / mL; and the concentration of the cross-linking agent A is 10 mg / mL.

[0027] By adopting the above technical solutions, the steps and parameters of the preferred preparation method improve the performance of the sensing layer.

[0028] The third aspect of the application provides a biosensor for detecting a biomarker in a body fluid, which adopts the following technical solutions:

[0029] A biosensor comprises: a working electrode, the surface of the working electrode being at least partially coated with the above-mentioned sulfonated MXene sensing layer; and the sensing layer being capable of reacting with the biomarker to produce a detectable electrochemical signal.

[0030] Preferably, the biosensor further comprises a counter electrode and a reference electrode, which together with the working electrode form a three-electrode detection system; and / or the working electrode is further coated with a limited diffusion layer that limits the diffusion of the analyte.

[0031] By adopting the above technical solutions, the modified working electrode is combined with the counter electrode and the reference electrode to form a three-electrode system, a biosensor based on the sulfonated MXene material is obtained, the performance of the implantable electrochemical biosensor is improved, the limited diffusion layer is used to widen the monitoring range of the sensor, reduce the interference of non-specific adsorption of proteins, and improve the service life and stability of the sensor.

[0032] Preferably, the diffusion-limiting layer comprises a nitrogen-containing heterocyclic group-containing polymer and a cross-linking agent B, and the mass ratio of the nitrogen-containing heterocyclic group-containing polymer and the cross-linking agent B is 6:1 to 12:1, preferably 9:1;

[0033] The nitrogen-containing heterocyclic group-containing polymer is selected from one or more of polyvinylpyridine, polyvinylpyrrole, and polyvinylpyridine-styrene copolymer, and is preferably polyvinylpyridine-styrene copolymer;

[0034] The cross-linking agent B is selected from one or more of polyethylene glycol diglycidyl ether, triglycidyl-p-aminophenol, and tetra-glycidyl-4,4'-diaminodiphenylmethane, and is preferably polyethylene glycol diglycidyl ether with a molecular weight of 600 Da.

[0035] The fourth aspect of the present application provides the use of the above-mentioned biosensor in a product for detecting biomarkers in a body fluid, the biomarkers including one or more of glucose, lactic acid, uric acid, or cholesterol, and preferably in a product for dynamic glucose monitoring.

[0036] In summary, the sulfonated MXene sensing layer and biosensor provided by the present application have the following remarkable beneficial effects:

[0037] 1. The biosensor provided by the present application exhibits extremely high response speed. This is due to the three-dimensional porous network structure (see Figure 4 a) formed by the sulfonated MXene, which provides a large specific surface area for enzyme immobilization, making it easier for reaction substrates to access the active center of the enzyme. Test results show that the current of the sensor (Example 1) based on the sensing layer reaches a steady state only in about 260 seconds, which is much better than the comparative example 1 (nearly 800 seconds, see Figure 3 ).

[0038] 2. The present application significantly improves the long-term stability of the sensor. The excellent stability is due to the high-efficiency catalytic decomposition ability of the sulfonated MXene material to the reaction byproduct H2O2, thereby reducing its toxic effect on the tool enzyme. In a 15-day test, the current signal attenuation rate of the sensor of Example 1 is only 3%, which is much better than the sensors of comparative examples 1 (27%), 2 (14%), and 4 (10%) (see Figure 5 ).

[0039] 3. The present application greatly enhances the anti-interference ability of the sensor. This feature is derived from the negative charge carried by the material surface after sulfonation treatment, which can effectively reduce the non-specific adsorption of coexisting, negatively charged interference substances (such as uric acid, ascorbic acid) in the body fluid through electrostatic repulsion. In the interference test, the current attenuation amplitude of the sensors of Example 4 and Example 5 is extremely low (2.2% for uric acid and 3.8% for ascorbic acid), which is much lower than that of the sensor of Comparative Example 1 (see Figure 6 , Figure 7 ].

[0040] 4. The present application ultimately achieves extremely high in vivo detection accuracy. The sensor of Example 6 is applied to an implantable continuous glucose monitoring system for human experiments, and the dynamic glucose concentration curve obtained by continuous monitoring is highly consistent with the fingertip blood glucose value (see Figure 8 ), which proves the reliability and accuracy of the sensor of the present application in practical application. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is a structural schematic diagram of the electrode layer in the embodiment of the present application;

[0042] Figure 2 is a structural schematic diagram of the biosensor in the embodiment of the present application;

[0043] Figure 3 is a response speed comparison diagram of the sensors prepared in Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 4 in the present application;

[0044] Figure 4 is a scanning electron microscope image of the sensing layer in the present application, wherein Figure 4 (a) is a scanning electron microscope image of the sensing layer of Example 1, Figure 4 (b) is a scanning electron microscope image of the sensing layer of Comparative Example 1;

[0045] Figure 5 is a stability test result diagram of the sensor of the present application, wherein Figure 5 (a) is a current curve diagram of the sensor without MXene material of Comparative Example 1 for 15 days, Figure 5 (b) is a current curve diagram of the MXene sensor of Comparative Example 2 for 15 days, Figure 5 (c) is a current curve diagram of the sulfonated MWCNTs sensor of Comparative Example 4 for 15 days, Figure 5 (d) is a current curve diagram of the sulfonated MXene sensor of Example 1 for 15 days;

[0046] Figure 6 is a comparison diagram of the anti-interference test (uric acid) results of the sensors prepared in Example 4, Comparative Example 1, Comparative Example 2 and Comparative Example 4 in the present application;

[0047] Figure 7 Figure 5 is a comparison chart of sensor anti-interference test (anti-ascorbic acid) results of the sensor prepared in Example 5, Comparative Example 1, Comparative Example 2 and Comparative Example 4 of the present application;

[0048] Figure 8 Figure 6 is a data chart of in-vivo experiment of the sensor prepared in Comparative Example 1, Comparative Example 3, Comparative Example 4 and Example 6 of the present application applied to an implantable continuous glucose monitoring system. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solutions and advantages of the present application clearer, the present application is further described in detail below in combination with examples and drawings. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application. It should be noted that "crosslinking agent A" described below specifically refers to a crosslinking agent used to construct a sulfonated MXene sensing layer; "crosslinking agent B" specifically refers to a crosslinking agent used to construct a diffusion-limiting layer. The names of the two are only for distinguishing purposes and are not a limitation on the type of crosslinking agent. Those skilled in the art can modify or replace equivalently based on understanding the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application, which should be covered within the protection scope of the present application.

[0050] The reagents, instruments and equipment used in the following examples are not specified by the manufacturer, and are all conventional products that can be purchased on the market. Other specific conditions not specified are carried out according to conventional conditions or manufacturer's recommended conditions.

[0051] Glossary

[0052] In order for those skilled in the art to more accurately understand the present application, some key terms and materials involved in the present application are described as follows:

[0053] 1. MXene: refers to a two-dimensional layered structure material, whose chemical formula is M n+1 X n T x (wherein n is generally 1, 2 or 3), wherein M is an early transition metal (such as Ti, V, Nb, Mo, etc.), X is carbon or nitrogen, T x represents the terminal functional group of the material surface, including but not limited to one or more mixtures of -OH (hydroxyl), -O (oxyl), -F (fluorine group), etc.

[0054] 2. MAX phase: refers to the precursor material of MXene, which is a ternary layered compound, whose chemical formula is M n+1 AX n(where n is typically 1, 2 or 3), wherein M is a early transition metal (such as Ti, V, Nb, Mo, etc.), A is mainly a group IIIA or group IVA element (such as Al, Si, Ga, etc.), and X is carbon or nitrogen.

[0055] 3. Crosslinker A: A crosslinker used to immobilize the redox polymer, the tool enzyme and the sulfonated MXene material in the sensing layer.

[0056] 4. Crosslinker B: A crosslinker used to crosslink the nitrogen-containing heterocyclic polymer in the diffusion-limiting layer.

[0057] 5. Diffusion-limiting layer: Refers to a thin film layer coated outside the sensing layer for controlling the diffusion rate of the analyte (such as glucose) to the sensing layer, which is formed by crosslinking and curing the nitrogen-containing heterocyclic polymer with the crosslinker B.

[0058] 6. Chemical formula is as follows: Ti3AlC2 (titanium aluminum carbide), HF (hydrofluoric acid), HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), MWCNTs (multi-walled carbon nanotubes), and other chemical formulas not specified are based on conventional general knowledge.

[0059] Reference Figure 1 and Figure 2 The present application provides a kind of based on sulfonated MXene material's biosensor, including flexible substrate and electrode layer arranged on flexible substrate, electrode layer includes one counter electrode, one working electrode and one reference electrode, counter electrode, working electrode and reference electrode constitute three electrode system, counter electrode and working electrode are respectively arranged on the two sides of flexible substrate, reference electrode is arranged on the side of working electrode away from flexible substrate, the side of counter electrode away from flexible substrate, between working electrode and reference electrode, the side of reference electrode away from working electrode is equipped with dielectric layer, the surface of working electrode has the bare part of not being equipped with dielectric layer and reference electrode, the surface of bare part is coated with sulfonated MXene sensing layer, for the selection of flexible substrate and each electrode material and the connecting mode between electrode layer and flexible substrate according to the conventional material and connecting mode of existing implantable biosensor, this application is not limited, electrode layer is coated with diffusion-limiting layer outside, this sensor is mainly applied to implantable glucose monitoring system and carries out dynamic glucose level monitoring, it can also be used for monitoring biomarker such as lactic acid, uric acid or cholesterol.

[0060] Sulfonated MXene sensing layer is prepared by the following preparation method:

[0061] S1, preparation of sulfonated MXene material: MXene powder material is obtained after etching MXene precursor material MAX phase with organic solvent, a dispersion solution of the MXene powder material is prepared, and then dropped and coated on a silicon wafer to dry to form a MXene film, the MXene film is treated with SO3 by plasma, and then washed and vacuum dried to obtain the sulfonated MXene material.

[0062] wherein the organic solvent is HF, the MXene precursor material is one or more of Ti3AlC2, Ti2AlC, V2AlC, Nb2AlC, MoTiAlC2, and preferably Ti3AlC2, and the SO3 gas is provided by an SO3 cylinder.

[0063] S2, preparation of sensing layer solution: mixed solutions of redox polymer, tool enzyme, sulfonated MXene material and crosslinking agent A are prepared respectively using solvent A, and then the mixed solutions are mixed in proportion to obtain the sensing layer solution, wherein the sulfonated MXene material is prepared by S1, and in the sensing layer solution, the mass ratio of redox polymer, tool enzyme, sulfonated MXene material and crosslinking agent A is 30-50: 15-30: 15-30: 1-10.

[0064] wherein the solvent A is HEPES buffer or Tris ethanesulfonic acid buffer with pH value of 7.5-8.5, and preferably HEPES buffer with concentration of 20 mmol / L and pH value of 8.0; the redox polymer is a redox polymer containing transition metal, the transition metal includes ruthenium, rhodium, osmium, etc., and preferably a redox polymer containing osmium, the concentration of the redox polymer is 20-50 mg / mL, and preferably 30 mg / mL; the tool enzyme is selected from one or more of glucose dehydrogenase, glucose oxidase, uricase, lactic acid oxidase and cholesterol oxidase, and preferably glucose oxidase, the concentration is 10-30 mg / mL, and preferably 20 mg / mL; the sulfonated MXene material is prepared by S1, and the concentration is 10-30 mg / mL, and preferably 20 mg / mL; the crosslinking agent A is selected from one or more of 1, 4-butanediol diglycidyl ether, poly (dimethylsiloxane) -diglycidyl ether, glutaraldehyde, polyethylene glycol diglycidyl ether, tetraepoxypropyl-4, 4-diaminodiphenyl methane or glycerol triglycidyl ether, and preferably polyethylene glycol diglycidyl ether with molecular weight of 200 Da-5000 Da, and further preferably polyethylene glycol diglycidyl ether with molecular weight of 200 Da, the concentration is 5-15 mg / mL, and preferably 10 mg / mL; when the sensor solution is prepared, the mass ratio of the added amounts of the mixed solutions is preferably 45:25:25:5.

[0065] S3. Preparation of the sensing layer: The sensing layer solution prepared in S2 is drop-coated onto the surface of the working electrode and cross-linked and cured at a temperature of 30-40℃ for 20-28 hours to obtain the sensing layer.

[0066] In the drop-coating operation, 0.1 µL is transferred using a micro-syringe (1 µL) and uniformly drop-coated onto the working electrode surface of the electrode. The cross-linking curing temperature is preferably 35 °C and the time is 24 h.

[0067] After the working electrode with the sensing layer is prepared, a diffusion-limiting layer is dip-coated on the outside of the electrode layer.

[0068] The confined diffusion layer comprises a polymer containing nitrogen-containing heterocyclic groups and a crosslinking agent B, wherein the mass ratio of the polymer containing nitrogen-containing heterocyclic groups to the crosslinking agent B is 6:1 to 12:1, preferably 9:1.

[0069] The polymer containing nitrogen-containing heterocyclic groups is selected from one or more of polyvinylpyridine, polyvinylpyrrole, and polyvinylpyridine-styrene copolymer, preferably polyvinylpyridine-styrene copolymer;

[0070] The crosslinking agent B is selected from one or more of polyethylene glycol diglycidyl ether, triglycidyl p-aminophenol, and tetraglycidyl-4,4'-diaminodiphenylmethane, preferably polyethylene glycol diglycidyl ether with a molecular weight of 600 Da.

[0071] The method for preparing the confined diffusion layer is as follows:

[0072] The first step involves mixing the polymer containing nitrogen-containing heterocyclic groups and crosslinking agent B in a specific ratio to obtain the outer membrane solution.

[0073] The second step involves immersing the electrode layer in the outer film solution obtained in the first step, then removing it and curing it at 40–50°C for 36–48 hours to form a confined diffusion layer.

[0074] The specific operation of the dip coating is as follows: The electrode layer is dipped into the outer film solution obtained in the first step 4 times, with an interval of 20 minutes between each dip coating, until the outer film solution completely covers the outside of the electrode layer. After the dip coating is completed, cross-linking and curing are performed. The preferred temperature for cross-linking and curing is 45℃ and the time is 40h.

[0075] The biosensor based on sulfonated MXene material provided in this application can be applied to products that detect biomarkers in body fluids. The biomarkers include one or more of glucose, lactic acid, uric acid, or cholesterol. For example, when applied to an implantable continuous glucose monitoring system, the existing sensor can be replaced by the biosensor prepared according to this method.

[0076] I. Preparation Example

[0077] Preparation Example 1

[0078] Sulfonated MXene material preparation: Ti3AlC2 was selected as the precursor material, Ti3AlC2 was mixed with 40% hydrofluoric acid solution according to the mass volume ratio of 1 g:20 mL, stirred at 50°C for 48h, centrifuged and washed to remove unreacted substances, dried to obtain Ti3C2T x powder, Ti3C2T x powder was added to deionized water and ultrasonically dispersed to prepare a dispersion liquid with a concentration of 1 mg / mL, Ti3C2T x The dispersion liquid was drop-coated onto a silicon wafer and dried to form a MXene film. The MXene film was placed in a plasma reaction chamber and SO3 gas was introduced. The SO3 gas was provided by an SO3 cylinder. The gas flow was 150 mL / min, the power was 100 W, and the gas pressure was 40 Pa. The treatment was carried out for 5 minutes. After treatment, washing was carried out. After washing, vacuum drying was carried out at 60°C for 30 min to obtain a sulfonated MXene material.

[0079] Preparation Example 2

[0080] Sulfonated MXene material preparation: Ti3AlC2 was selected as the precursor material, Ti3AlC2 was mixed with 40% hydrofluoric acid solution according to the mass volume ratio of 1 g:20 mL, stirred at 50°C for 48h, centrifuged and washed to remove unreacted substances, dried to obtain Ti3C2T x powder, Ti3C2T x powder was added to deionized water and ultrasonically dispersed to prepare a dispersion liquid with a concentration of 1 mg / mL, Ti3C2T x The dispersion liquid was drop-coated onto a silicon wafer and dried to form a MXene film. The MXene film was placed in a plasma reaction chamber and SO3 gas was introduced. The SO3 gas was provided by an SO3 cylinder. The gas flow was 150 mL / min, the power was 100 W, and the gas pressure was 40 Pa. The treatment was carried out for 5 minutes. After treatment, washing was carried out. After washing, vacuum drying was carried out at 60°C for 30 min to obtain a sulfonated MXene material.

[0081] Preparation Example 3

[0082] Sulfonated MXene material preparation: Ti3AlC2 was selected as the precursor material, Ti3AlC2 was mixed with 40% hydrofluoric acid solution according to the mass volume ratio of 1 g:20 mL, stirred at 50°C for 48h, centrifuged and washed to remove unreacted substances, dried to obtain Ti3C2T x powder, Ti3C2T x powder was added to deionized water and ultrasonically dispersed to prepare a dispersion liquid with a concentration of 1 mg / mL, Ti3C2T xThe dispersion was drop-coated onto a silicon wafer and dried to form an MXene film. The MXene film was then placed in a plasma reaction chamber, and SO3 gas was introduced. The SO3 gas was supplied by an SO3 cylinder. The film was treated for 10 minutes at a gas flow rate of 200 mL / min, a power of 120 W, and a pressure of 60 Pa. After treatment, the film was cleaned and then vacuum dried at 60 °C for 30 minutes to obtain the sulfonated MXene material.

[0083] II. Implementation Examples

[0084] Example 1

[0085] A method for preparing a sulfonated MXene sensing layer includes the following steps:

[0086] S1. Preparation of sulfonated MXene material: Sulfonated MXene material was prepared according to Preparation Example 1.

[0087] S2. Preparation of the sensing layer solution: The redox polymer is an osmium-containing redox polymer, the tool enzyme is glucose oxidase, the sulfonated MXene material is the sulfonated MXene material obtained in step S1, and the crosslinking agent A is polyethylene glycol diglycidyl ether with a molecular weight of 200 Da.

[0088] Specifically, using HEPES buffer solution with pH=8.0 and a concentration of 20 mmol / L as a solvent, a redox polymer mixed solution with a concentration of 30 mg / mL, a tool enzyme mixed solution with a concentration of 20 mg / mL, a sulfonated MXene material mixed solution with a concentration of 20 mg / mL, and a crosslinking agent A mixed solution with a concentration of 10 mg / mL were prepared. Then, the mixed solutions were mixed in a mass ratio of 45:25:25:5 to obtain the sensing layer solution.

[0089] S3. Preparation of the sensing layer: Using a microsyringe (1µL), 0.1µL of the sensing layer solution prepared in S2 is transferred and dropped onto the surface of the working electrode. After cross-linking and curing at 35℃ for 24h, a working electrode with a sensing layer is obtained. The sensing layer formed on the surface of the working electrode is the sulfonated MXene sensing layer.

[0090] Reference Figure 1 and Figure 2The application discloses a biosensor based on sulfonated MXene material, which comprises a flexible substrate and an electrode layer arranged on the flexible substrate, and the electrode layer comprises a counter electrode, a working electrode and a reference electrode. The working electrode is prepared by using the working electrode provided with a sensing layer based on the sulfonated MXene material, and the specific operation is as follows: the counter electrode, the working electrode and the reference electrode are sequentially connected to form the electrode layer, the counter electrode and the working electrode are arranged on two sides of the flexible substrate, the reference electrode is arranged on a side, away from the flexible substrate, of the working electrode, a dielectric layer is arranged on a side, away from the flexible substrate, of the counter electrode, between the working electrode and the reference electrode and on a side, away from the working electrode, of the reference electrode, the counter electrode, the working electrode and the reference electrode form a three-electrode system, a bare part without the dielectric layer and the reference electrode is formed on the surface of the working electrode, the surface of the bare part is coated with the sensing layer according to the preparation method of the sensing layer, the selection of the flexible substrate and the electrode materials and the connection mode between the electrode layer and the flexible substrate are selected according to the conventional materials and the connection mode of the existing implantable biosensor, and the application is not limited. After preparation, the diffusion-limiting layer is prepared by dip coating outside the electrode layer.

[0091] The preparation method of the diffusion-limiting layer is as follows:

[0092] In the first step, polyvinylpyrrolidone-styrene copolymer is selected as a nitrogen-containing heterocyclic group polymer, polyethylene glycol diglycidyl ether with a molecular weight of 600 Da is selected as a crosslinking agent B, a mixed solution of ethanol and HEPES buffer solution with a pH value of 8.0 and a concentration of 20 mmol / L is prepared according to a volume ratio of 6:1 as a solvent, a polyvinylpyrrolidone-styrene copolymer solution with a concentration of 50 mg / mL and a polyethylene glycol diglycidyl ether solution with a concentration of 10 mg / mL are respectively prepared, and the polyvinylpyrrolidone-styrene copolymer solution and the polyethylene glycol diglycidyl ether solution are mixed according to a mass percentage of 9:1 to obtain an outer membrane solution.

[0093] In the second step, the electrode layer is placed in the outer membrane solution obtained in the first step and is dip coated for 4 times with an interval of 20 min each time, and then is taken out and is cured at 45 DEG C for 40 h to form a biosensor with a diffusion-limiting layer, and the outer membrane layer formed on the surface of the electrode layer is the diffusion-limiting layer.

[0094] The application of the biosensor based on the sulfonated MXene material is to replace the sensor in the existing implantable continuous glucose monitoring system to realize dynamic glucose level monitoring.

[0095] Example 2

[0096] A preparation method of a sulfonated MXene sensing layer, which is different from that of Example 1 in that S1 adopts the sulfonated MXene material prepared in Preparation Example 2, in S2, the amounts of the mixed solutions for preparing the sensing layer solution are different, and the sensing layer solution is obtained by mixing 20 mg / mL of a redox polymer mixed solution, 10 mg / mL of a tool enzyme mixed solution, 10 mg / mL of a sulfonated MXene material mixed solution, and 5 mg / mL of a crosslinking agent A mixed solution at a mass ratio of 36:30:30:4; and in S3, the crosslinking and curing temperature is 30℃, and the time is 28h.

[0097] A biosensor based on a sulfonated MXene material, which is different from that of Example 1 in that the sensing layer solution dropped on the working electrode adopts the sensing layer solution prepared in the present example, and the preparation of the diffusion limiting layer is different, in the first step, 50 mg / mL of a polyvinylpyridine-styrene copolymer solution and 10 mg / mL of a polyethylene glycol diglycidyl ether solution are mixed at a mass percentage of 6:1 to obtain an outer membrane solution; and in the second step, the crosslinking and curing temperature is 40℃, and the time is 48h.

[0098] Application of a biosensor based on a sulfonated MXene material, which can achieve dynamic glucose level monitoring by replacing the sensor in the existing implantable continuous glucose monitoring system with the biosensor based on a sulfonated MXene material prepared in the present example.

[0099] Example 3

[0100] A preparation method of a sulfonated MXene sensing layer, which is different from that of Example 1 in that S1 adopts the sulfonated MXene material prepared in Preparation Example 3, in S2, the amounts of the mixed solutions for preparing the sensing layer solution are different, and the sensing layer solution is obtained by mixing 50 mg / mL of a redox polymer mixed solution, 30 mg / mL of a tool enzyme mixed solution, 30 mg / mL of a sulfonated MXene material mixed solution, and 15 mg / mL of a crosslinking agent A mixed solution at a mass ratio of 50:15:25:10; and in S3, the crosslinking and curing temperature is 40℃, and the time is 20h.

[0101] A biosensor based on a sulfonated MXene material, which is different from that of Example 1 in that the sensing layer solution dropped on the working electrode adopts the sensing layer solution prepared in the present example, and the preparation of the diffusion limiting layer is different, in the first step, 50 mg / mL of a polyvinylpyridine-styrene copolymer solution and 10 mg / mL of a polyethylene glycol diglycidyl ether solution are mixed at a mass percentage of 12:1 to obtain an outer membrane solution; and in the second step, the crosslinking and curing temperature is 50℃, and the time is 36h.

[0102] Application of a biosensor based on sulfonated MXene material, the biosensor based on sulfonated MXene material prepared in this embodiment can be used to replace the sensor in the existing implantable continuous glucose monitoring system to realize dynamic glucose level monitoring.

[0103] Example 4

[0104] A preparation method of a sulfonated MXene sensing layer, which is different from that of Example 1 in that in S2, the amounts of the mixed solutions for preparing the sensing layer solution are different, and the sensing layer solution is obtained by mixing 30mg / mL redox polymer mixed solution, 20mg / mL tool enzyme mixed solution, 20mg / mL sulfonated MXene material mixed solution and 10mg / mL crosslinking agent A mixed solution in a mass ratio of 30:30:30:10.

[0105] A biosensor based on sulfonated MXene material, the sensing layer solution dropped and coated on the working electrode is the sensing layer solution prepared in this embodiment, and the rest is the same as that of Example 1.

[0106] Application of a biosensor based on sulfonated MXene material, the biosensor based on sulfonated MXene material prepared in this embodiment can be used to replace the sensor in the existing implantable continuous glucose monitoring system to realize dynamic glucose level monitoring.

[0107] Example 5

[0108] A preparation method of a sulfonated MXene sensing layer, which is different from that of Example 1 in that in S2, the amounts of the mixed solutions for preparing the sensing layer solution are different, and the sensing layer solution is obtained by mixing 30mg / mL redox polymer mixed solution, 20mg / mL tool enzyme mixed solution, 20mg / mL sulfonated MXene material mixed solution and 10mg / mL crosslinking agent A mixed solution in a mass ratio of 50:15:30:5.

[0109] A biosensor based on sulfonated MXene material, the sensing layer solution dropped and coated on the working electrode is the sensing layer solution prepared in this embodiment, and the rest is the same as that of Example 1.

[0110] Application of a biosensor based on sulfonated MXene material, the biosensor based on sulfonated MXene material prepared in this embodiment can be used to replace the sensor in the existing implantable continuous glucose monitoring system to realize dynamic glucose level monitoring.

[0111] Example 6

[0112] A method for preparing a sulfonated MXene sensing layer differs from Example 1 in that, in S2, the amounts of each mixed solution used to prepare the sensing layer solution are different. The sensing layer solution is obtained by mixing a 30 mg / mL redox polymer mixed solution, a 20 mg / mL tool enzyme mixed solution, a 20 mg / mL sulfonated MXene material mixed solution, and a 10 mg / mL crosslinking agent A mixed solution in a mass ratio of 45:30:15:10.

[0113] A biosensor based on sulfonated MXene material is provided. The sensing layer solution drop-coated on the working electrode is the sensing layer solution prepared in this embodiment, and the rest is the same as in Example 1.

[0114] An application of a biosensor based on sulfonated MXene material is described, in which the biosensor based on sulfonated MXene material prepared in this embodiment can replace the sensor in an existing implantable continuous glucose monitoring system to achieve dynamic glucose level monitoring.

[0115] III. Comparative Example

[0116] Comparative Example 1

[0117] A method for preparing a sensing layer differs from Example 1 in that it does not contain sulfonated MXene material. A sensing layer solution is obtained by mixing a 30 mg / mL redox polymer mixed solution, a 20 mg / mL tool enzyme mixed solution, and a 10 mg / mL crosslinking agent A mixed solution in a mass ratio of 45:30:25. 0.1 µL of the sensing layer solution prepared in this comparative example is transferred using a microsyringe (1 µL) and drop-coated onto the surface of the working electrode. After crosslinking and curing at 35 °C for 24 h, a working electrode with a sensing layer is obtained.

[0118] A biosensor, wherein the sensing layer solution drop-coated on the working electrode is the sensing layer solution prepared in this comparative example, and the rest is the same as in Example 1.

[0119] An application of a biosensor is described, in which the sensor prepared in this comparative example replaces the sensor in an existing implantable continuous glucose monitoring system to achieve glucose level monitoring.

[0120] Comparative Example 2

[0121] A method for preparing a sensing layer differs from Example 1 in that MXene material is used instead of sulfonated MXene material. The preparation method of MXene material is as follows: Ti3AlC2 is mixed with 40% hydrofluoric acid solution at a mass-to-volume ratio of 1g:20mL, stirred at 50°C for 48 hours, centrifuged and washed to remove unreacted substances, and dried to obtain Ti3C2T. x powder.

[0122] The sensing layer solution is obtained by mixing a 30 mg / mL redox polymer mixed solution, a 20 mg / mL tool enzyme mixed solution, a 20 mg / mL MXene material mixed solution, and a 10 mg / mL crosslinking agent A mixed solution at a mass ratio of 45:25:25:5.

[0123] A biosensor, the sensing layer solution on the working electrode is prepared by using the sensing layer solution of the present example, and the rest is consistent with example 1.

[0124] The application of a biosensor, the sensor prepared by the present example is used to replace the sensor in the existing implantable continuous glucose monitoring system to realize glucose level monitoring.

[0125] Comparative Example 3

[0126] A method for preparing a sensing layer, which is different from comparative example 2 in that the sensing layer solution is obtained by mixing a 30 mg / mL redox polymer mixed solution, a 20 mg / mL tool enzyme mixed solution, a 20 mg / mL MXene material mixed solution, and a 10 mg / mL crosslinking agent A mixed solution at a mass ratio of 30:30:30:10.

[0127] A biosensor, the sensing layer solution on the working electrode is prepared by using the sensing layer solution of the present example, and the rest is consistent with comparative example 2.

[0128] The application of a biosensor, the sensor prepared by the present example is used to replace the sensor in the existing implantable continuous glucose monitoring system to realize glucose level monitoring.

[0129] Comparative Example 4

[0130] A method for preparing a sensing layer, which is different from example 1 in that sulfonated multi-walled carbon nanotubes (MWCNTs) are used to replace sulfonated MXene material, and the preparation method of sulfonated MWCNTs material is as follows: multi-walled carbon nanotubes (MWCNTs) are vacuum dried at 60°C for 24 h to remove adsorbed water, an appropriate amount of multi-walled carbon nanotubes (MWCNTs) is added in a concentrated sulfuric acid / concentrated nitric acid mixture (volume ratio = 3:1), 120°C oil bath reflux for 30 minutes, cool to room temperature, dilute to neutral with ultrapure water, and dry to obtain sulfonated MWCNTs.

[0131] The sensing layer solution is obtained by mixing a 30 mg / mL redox polymer mixed solution, a 20 mg / mL tool enzyme mixed solution, a 20 mg / mL sulfonated MWCNTs mixed solution, and a 10 mg / mL crosslinking agent A mixed solution in a mass ratio of 45:25:25:5.

[0132] A biosensor, the sensing layer solution of the working electrode is prepared by the sensing layer solution of the present comparative example, and the rest is consistent with example 1.

[0133] The application of a biosensor, the sensor prepared by the present comparative example is used to replace the sensor in the existing implantable continuous glucose monitoring system to realize glucose level monitoring.

[0134] IV. Performance test experiment and results

[0135] 1. Response speed test

[0136] The sensors prepared in examples 1-6 and comparative examples 1-4 are respectively tested for response speed by chronoamperometry (i-t), and glucose solutions with concentrations of 2 mmol / L, 5 mmol / L and 15 mmol / L are respectively prepared, the sensors are placed in the solutions for testing until the current curve is stable, and the results of examples 1, comparative example 1, comparative example 2 and comparative example 4 are compared as shown in Figure 3 .

[0137] From Figure 3 , it can be seen that the sensor without MXene material has the slowest polarization speed, the current change curve is relatively slow, and it takes nearly 800 seconds for the current to reach a stable state; the MXene sensor and the sulfonated MWCNTs sensor successively improve the current response speed, and the current change of the sulfonated MXene sensor after replacing different concentration sugar solutions is very rapid, and it takes only about 260 seconds for the current to reach a stable state; it can be seen that the better electron transfer ability of the sulfonated MXene improves the sensitivity of the sensor.

[0138] 2. Structure characterization

[0139] From the response speed test experiment results, the response speed of the sensor with the sulfonated MXene sensing layer is the fastest, it is speculated that the enzyme molecules are fixed on the nanosheet surface, the active sites of the fixed enzyme are easily accessible to the external environment, thereby improving the response speed of the biosensor, on this basis, the sensing layer in example 1 and comparative example 1 is scanned by scanning electron microscopy, and the results are shown in Figure 4 .

[0140] Figure 4 (a) is a scanning diagram of the sensing layer of example 1, Figure 4 (b) is a scanning diagram of the sensing layer of comparative example 1, from which it can be seen thatFigure 4 (a) It can be seen that the enzyme layer containing sulfonated MXene consists of a three-dimensional interconnected network of densely distributed nanoscale pores, and the surface exhibits a porous structure; in contrast... Figure 4 (b) The enzyme layer surface without MXene material has a two-dimensional planar structure, with a significantly smaller specific surface area. Figure 4 It is known that sulfonated MXene provides a high specific surface area for enzyme immobilization. The enzyme molecules are immobilized on the surface of the nanosheet, and the active sites of the immobilized enzyme are easily accessible to the external environment, thereby improving the response speed of the biosensor.

[0141] 3. Stability Test

[0142] The stability of the sensors prepared in Examples 1-6 and Comparative Examples 1-4 was tested using the chronoamperometry (IT) method. The test solution was a glucose solution with a concentration of 15 mmol / L. The sensors were placed in the solution, and the change in current value over time was measured over 15 days. The results of Comparative Examples 1, 2, and 4 were compared with those of Example 1. Figure 5 As shown.

[0143] Figure 5 (a) is the 15-day current curve of the sensor without MXene material in Comparative Example 1. Figure 5 (b) is the 15-day current curve of the MXene sensor in Comparative Example 2. Figure 5 (c) is the 15-day current curve of the sulfonated MWCNTs sensor in Comparative Example 4. Figure 5 (d) is the current curve of the sulfonated MXene sensor of Example 1 after 15 days.

[0144] Depend on Figure 5 It can be seen that, based on the first day, the current value of the sensor without MXene material decreased by 27% within 15 days. The addition of MXene material and sulfonated MWCNTs significantly improved the stability of the sensor, with a decrease of 14% and 10% respectively. The current value of the sulfonated MXene glucose sensor prepared in Example 1 showed a slow decreasing trend over time, with a current value decrease of only 3% after 15 days. This result indirectly but strongly proves that sulfonated MXene effectively decomposes H2O2, ensuring the long-term activity of the enzyme and further improving the long-term stability of the sensor.

[0145] 4. Anti-interference test

[0146] The sensors prepared in Examples 1-6 and Comparative Examples 1-4 were subjected to anti-interference tests by chronoamperometry (i-t), and the first test solution was a mixture of a glucose solution with a concentration of 10 mmol / L and uric acid with a concentration of 0.3 mmol / L, and the second test solution was a mixture of a glucose solution with a concentration of 10 mmol / L and ascorbic acid with a concentration of 0.15 mmol / L.

[0147] Figure 6 The results of Example 4, Comparative Example 1, Comparative Example 2 and Comparative Example 4 in the first test solution are compared in the graph of Figure 6 It can be seen that, for the interfering substance uric acid, the current value of the sulfonated MXene sensor prepared in Example 4 fluctuates less, and the current decay amplitude is 2.2%, while the current curve of the sensor without MXene material changes greatly, and the decay amplitude reaches 22.6%.

[0148] Figure 7 The results of Example 5, Comparative Example 1, Comparative Example 2 and Comparative Example 4 in the second test solution are compared in the graph of Figure 7 It can be seen that, for the interfering substance ascorbic acid, the current curve of the glucose sensor without MXene material decreases significantly after the addition of the interfering substance, and the decay reaches 26.7%, while the decay of the sulfonated MXene sensor prepared in Example 5 is only 3.8%.

[0149] In addition, neither the MXene material nor the sulfonated MWCNTs has better anti-interference performance than the sulfonated MXene for the metabolites uric acid and ascorbic acid, which proves that the high specific surface area and conductivity of the sulfonated MXene provide an efficient electron transport path, and the surface charge regulation reduces the adsorption of interfering substances. The sulfonation treatment makes the MXene surface negatively charged, which can repel negatively charged metabolic interfering substances (such as uric acid and ascorbic acid).

[0150] 5. Detection accuracy test

[0151] The sensors prepared in Comparative Example 1, Comparative Example 3, Comparative Example 4 and Example 6 were applied to an implantable continuous glucose monitoring system for in vivo experiments, and the results were compared with those of fingertip blood glucose tests, as shown in Figure 8

[0152] As shown in Figure 8 ​It can be known that in the human body test for 14 days continuously, the sensor without MXene material has low consistency with the fingertip blood glucose in vivo, and the fingertip blood glucose value at the same time has a large gap with the monitoring value; the MXene sensor and the sulfonated MWCNTs sensor have the conditions of being higher or lower than the fingertip blood glucose in the in vivo test, and the accuracy is not high; and the dynamic glucose concentration (curve) fluctuation monitored by the sulfonated MXene sensor is obvious, and the randomly selected fingertip blood glucose can basically fall on the curve, and the test results of the two are highly consistent. It is comprehensively indicated that the sensor has high precision and reliability in actual application.

[0153] In conclusion, the sensing layer provided by the present application contains sulfonated MXene material, the surface of which is modified by sulfonic groups through covalent bond, has excellent electron transfer ability and high specific surface area for enzyme fixation, the ability of catalyzing H2O2 is enhanced, and the stability of the sensor is enhanced; the sensor comprises a flexible substrate, a working electrode with a sensing layer, a reference electrode and a counter electrode, the sensor prepared by using the sensing layer provided by the present application has the characteristics of high detection precision, fast response speed, strong anti-interference and high stability, and lays a foundation for the development of implantable glucose monitoring system and body fluid biomarker monitoring.

Claims

1. A method for preparing a sulfonated MXene sensing layer, characterized in that, Includes the following steps: S1. Preparation of sulfonated MXene material: Ti3C2T was obtained by etching the MXene precursor material Ti3AlC2 with an organic solvent. x Powder material, Ti3C2T x After the powder material is prepared into a dispersion, it is dropped onto a silicon wafer and dried to form an MXene film. The MXene film is then subjected to plasma treatment with SO3, rinsed, and vacuum dried to obtain sulfonated MXene material. S2. Preparation of the sensing layer solution: The redox polymer, the tool enzyme, the sulfonated MXene material, and the crosslinking agent A are mixed in a certain proportion to obtain the sensing layer solution. The sulfonated MXene material is the sulfonated MXene material prepared in S1. In the sensing layer solution, the mass ratio of the redox polymer, the tool enzyme, the sulfonated MXene material, and the crosslinking agent is 30-50:15-30:15-30:1-10; the tool enzyme is glucose oxidase. S3. Preparation of the sensing layer: The sensing layer solution prepared in S2 is drop-coated onto the surface of the working electrode and cross-linked and cured at a temperature of 30-40℃ for 20-28 hours to obtain the sensing layer.

2. The preparation method according to claim 1, characterized in that, S1 includes: Ti3AlC2 was mixed with a 40%–50% hydrofluoric acid solution and reacted at 40℃–50℃ for 40–60 hours. After centrifugation and drying, Ti3C2T was obtained. x powder; Ti3C2T x The powder was dispersed into a dispersion of 0.5–2 mg / mL, and then drop-coated onto a silicon wafer and dried to form an MXene film. The MXene film was placed in a plasma reaction chamber and SO3 gas was introduced. The mixture was treated for 3 to 10 minutes at a gas flow rate of 100 to 200 mL / min, a power of 80 to 120 W, and a pressure of 20 to 60 Pa. After cleaning and drying, sulfonated MXene material was obtained.

3. The preparation method according to claim 2, characterized in that, S1 includes: Ti3AlC2 was mixed with 40% hydrofluoric acid solution and reacted at 50°C for 48 hours. After centrifugation and drying, Ti3C2T was obtained. x powder; Ti3C2T x The powder was dispersed into a 1 mg / mL dispersion, then drop-coated onto a silicon wafer and dried to form an MXene film. The MXene film was placed in a plasma reaction chamber and SO3 gas was introduced. The mixture was treated for 5 minutes at a gas flow rate of 150 mL / min, a power of 100 W, and a pressure of 40 Pa. After cleaning and drying, sulfonated MXene material was obtained.

4. The preparation method according to claim 1, characterized in that, In S2: Solvent A is a HEPES buffer or Tris ethanesulfonic acid buffer with a pH of 7.5 to 8.5; In the sensing layer solution, the concentration of the redox polymer is 20-50 mg / mL, the concentration of the tool enzyme is 10-30 mg / mL, the concentration of the sulfonated MXene material is 10-30 mg / mL, and the concentration of crosslinking agent A is 5-15 mg / mL.

5. The preparation method according to claim 4, characterized in that, In S2: Solvent A is a HEPES buffer solution with a concentration of 20 mmol / L and a pH of 8.0; In the sensing layer solution, the concentration of the redox polymer is 30 mg / mL; the concentration of the tool enzyme is 20 mg / mL; the concentration of the sulfonated MXene material is 20 mg / mL; and the concentration of crosslinking agent A is 10 mg / mL.

6. The preparation method according to claim 1, characterized in that, In S2, the mass ratio of the redox polymer, the tool enzyme, the sulfonated MXene material, and the crosslinking agent in the sensing layer solution is 45:25:25:

5.

7. The preparation method according to claim 1, characterized in that, In step S2, the redox polymer is an osmium-containing redox polymer.

8. The preparation method according to claim 1, characterized in that, In step S2, the crosslinking agent A is polyethylene glycol diglycidyl ether with a molecular weight of 200 Da.

9. A sulfonated MXene sensing layer, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. A biosensor for detecting biomarkers in bodily fluids, characterized in that, include: A working electrode, the surface of which is at least partially coated with the sulfonated MXene sensing layer according to claim 9; The sensing layer can react with the biomarker to generate a detectable electrochemical signal.

11. The biosensor according to claim 10, characterized in that: The biosensor also includes a counter electrode and a reference electrode, which together with the working electrode form a three-electrode detection system; and / or the working electrode is further coated with a diffusion-limiting layer that restricts the diffusion of the analyte.

12. The biosensor according to claim 11, characterized in that: The confined diffusion layer comprises a polymer containing nitrogen-containing heterocyclic groups and a crosslinking agent B, wherein the mass ratio of the polymer containing nitrogen-containing heterocyclic groups to the crosslinking agent B is 6:1 to 12:1; The polymer containing nitrogen-containing heterocyclic groups is a polyvinylpyridine-styrene copolymer; The crosslinking agent B is polyethylene glycol diglycidyl ether with a molecular weight of 600 Da.

13. The application of a biosensor as described in any one of claims 10 to 12 in a product for detecting biomarkers in body fluids, characterized in that, The biomarkers include glucose.

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