Sulfonated MXene sensing layer, preparation method thereof and biosensor

Abstract

The invention 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 performing covalent bond modification on a sulfonic acid group on the MXene material. According to the preparation method, sulfonic acid groups are covalently grafted on the surface of MXene through plasma treatment, so that the MXene has high specific surface area, excellent electron conductivity, efficient enzyme immobilization capability and H2O2 catalytic decomposition activity. The biosensor constructed based on the sensing layer comprises a flexible substrate, a three-electrode system and a limited diffusion layer, the response speed, the detection precision, the stability and the anti-interference performance (such as uric acid resistance and ascorbic acid resistance) of the sensor are remarkably improved, and the biosensor is particularly suitable for being applied to 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 by-product 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, and 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, tetraglycidyl-4,4-diaminodiphenylmethane, or glycerol triglycidyl ether, preferably polyethylene glycol diglycidyl ether with a molecular weight between 200 Da and 5000 Da, and more preferably polyethylene glycol diglycidyl ether with a molecular weight of 200 Da.

[0018] A second aspect of the present invention provides a method for preparing the above-mentioned sulfonated MXene sensing layer, which adopts the following technical solution:

[0019] A method for preparing a sulfonated MXene sensing layer includes the following steps:

[0020] S1. Preparation of sulfonated MXene materials;

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

[0022] S3. Load the sensing layer solution onto the surface of the working electrode and crosslink and cure it at 30-40°C for 20-28 hours to obtain the sensing layer.

[0023] Preferably, in the above preparation method, step S1 includes: mixing Ti3AlC2 with a 40%–50% hydrofluoric acid solution, reacting at 40°C–50°C for 40–60 hours, and then centrifuging and drying to obtain Ti3C2T. x Powder; Ti3C2T x The powder is 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 is placed in a plasma reaction chamber, and SO3 gas supplied by an SO3 cylinder is introduced. The mixture is treated for 3–10 minutes at a gas flow rate of 100–200 mL / min, a power of 80–120 W, and a gas pressure of 20–60 Pa. After cleaning and drying, sulfonated MXene material is obtained.

[0024] Preferably, in the above preparation method, step S1 includes: mixing Ti3AlC2 with a 40% hydrofluoric acid solution, reacting at 50°C for 48 hours, and then centrifuging and drying to obtain Ti3C2T. X Powder; Ti3C2T XThe powder was dispersed into a 1 mg / mL dispersion, which was 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 supplied by an SO3 cylinder 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.

[0025] Preferably, in step S2, solvent A is a HEPES buffer or Tris ethanesulfonic acid buffer with a pH 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 crosslinking agent A is 5–15 mg / mL.

[0026] Preferably, 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.

[0027] By adopting the above technical solution and optimizing the steps and parameters of the preparation method, the performance of the sensing layer can be improved.

[0028] A third aspect of the present invention provides a biosensor for detecting biomarkers in body fluids, employing the following technical solution:

[0029] A biosensor includes: a working electrode, the surface of which is at least partially coated with a sulfonated MXene sensing layer according to any one of claims 1 to 5; the sensing layer is capable of reacting with the biomarker to generate a detectable electrochemical signal.

[0030] Preferably, the biosensor further includes a counter electrode and a reference electrode, which together with the working electrode constitute 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.

[0031] By adopting the above technical solution, the modified working electrode is combined with the counter electrode and the reference electrode to form a three-electrode system, thereby obtaining a biosensor based on sulfonated MXene material. This improves the performance of the implantable electrochemical biosensor. In conjunction with the diffusion-limiting layer, the sensor's monitoring range is broadened, interference from non-specific adsorbed proteins is reduced, and the sensor's timeliness and stability are improved.

[0032] Preferably, 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;

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

[0034] 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.

[0035] A fourth aspect of the present invention provides the application of the above-described biosensor in products for detecting biomarkers in body fluids, wherein the biomarkers include one or more of glucose, lactic acid, uric acid or cholesterol, preferably in dynamic glucose monitoring products.

[0036] In summary, the sulfonated MXene sensing layer and biosensor provided by this invention have the following significant advantages:

[0037] 1. The biosensor provided by this invention exhibits extremely high response speed. This is due to the three-dimensional porous network structure formed by sulfonated MXene (see...). Figure 4 a) This provides a large specific surface area for enzyme immobilization, allowing the reaction substrate to more easily access the enzyme's active site. Test results show that the sensor based on this sensing layer (Example 1) reaches a steady-state current in only about 260 seconds, a performance far superior to Comparative Example 1 (nearly 800 seconds, see [example]) which did not use MXene material. Figure 3 ).

[0038] 2. This invention significantly improves the long-term stability of the sensor. The excellent stability stems from the highly efficient catalytic decomposition of the reaction byproduct H2O2 by the sulfonated MXene material, thereby reducing its toxicity to the enzyme. During a 15-day test, the sensor current signal attenuation rate of Example 1 was only 3%, and its stability was far superior to that of Comparative Example 1 (27%), Comparative Example 2 (14%), and Comparative Example 4 (10%) (see...). Figure 5 ).

[0039] 3. This invention significantly enhances the sensor's anti-interference capability. This characteristic stems from the negative charge carried on the material surface after sulfonation treatment, which effectively reduces the non-specific adsorption of negatively charged interfering substances (such as uric acid and ascorbic acid) coexisting in body fluids through electrostatic repulsion. In the interfering substance test, the sensor current attenuation of Examples 4 and 5 was extremely low (2.2% for uric acid and 3.8% for ascorbic acid), far lower than that of the sensor in Comparative Example 1 (see Comparative Example 1). Figure 6 , Figure 7 ).

[0040] 4. This invention ultimately achieves extremely high in vivo detection accuracy. When the sensor from Example 6 was applied to an implantable continuous glucose monitoring system in human trials, the dynamic glucose concentration curve obtained from continuous monitoring showed a high degree of agreement with fingertip blood glucose levels (see...). Figure 8 This demonstrates the reliability and accuracy of the sensor of the present invention in practical applications. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the electrode layer structure in an embodiment of this application;

[0042] Figure 2 This is a schematic diagram of the structure of the biosensor in the embodiments of this application;

[0043] Figure 3 This is a comparison chart of the response speeds of the sensors prepared in Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 4 of this application;

[0044] Figure 4 This is a scanning electron microscope image of the sensing layer in this application, in which... Figure 4 (a) is a scan of the sensing layer in Example 1. Figure 4 (b) is a scan of the sensing layer in Comparative Example 1;

[0045] Figure 5 This is a graph showing the stability test results of the sensor in this application. 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;

[0046] Figure 6 This is a comparison chart 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 of this application;

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

[0048] Figure 8 These are data maps of the sensors prepared in Comparative Examples 1, 3, 4 and 6 of this application, used in an in vivo experiment on an implantable continuous glucose monitoring system. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. It should be noted that "crosslinking agent A" as used below specifically refers to the crosslinking agent used to construct the sulfonated MXene sensing layer; "crosslinking agent B" specifically refers to the crosslinking agent used to construct the confined diffusion layer. These names are only for distinguishing uses and are not intended to limit the type of crosslinking agent. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the technical solutions of this invention, should all be covered within the protection scope of this invention.

[0050] Unless otherwise specified, all reagents, instruments and equipment used in the following examples are commercially available products. Other unspecified conditions shall be performed in accordance with standard conditions or conditions recommended by the manufacturer.

[0051] Glossary

[0052] To enable those skilled in the art to more accurately understand the present invention, some key terms and materials involved in this application are explained below:

[0053] 1. MXene: refers to a two-dimensional layered material with the general chemical formula M. n+1 X n T x (where n is generally 1, 2, or 3), where M is an early transition metal (such as Ti, V, Nb, Mo, etc.), X is carbon or nitrogen, and T is... x The terminating functional group on the surface of a material is included, but is not limited to one or more of -OH (hydroxyl), -O (oxygen), and -F (fluorine).

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

[0055] 3. Crosslinking agent A: A crosslinking agent used to immobilize redox polymers, tool enzymes, and sulfonated MXene materials in the sensing layer.

[0056] 4. Crosslinking agent B: A crosslinking agent used to crosslink nitrogen-containing heterocyclic polymers in the crosslinking restricted diffusion layer.

[0057] 5. Diffusion-restricting layer: This refers to a thin film layer coated on the outside of the sensing layer to control the diffusion rate of analytes (such as glucose) into the sensing layer. It is formed by cross-linking and curing nitrogen-containing heterocyclic polymers with cross-linking agent B.

[0058] 6. The chemical formulas are as follows: Ti3AlC2 (titanium aluminum carbide), HF (hydrofluoric acid), HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), MWCNTs (multi-walled carbon nanotubes). For other unspecified chemical formulas, the commonly used formulas shall prevail.

[0059] Reference Figure 1 and Figure 2 This invention provides a biosensor based on sulfonated MXene material, comprising a flexible substrate and an electrode layer disposed on the flexible substrate. The electrode layer includes a counter electrode, a working electrode, and a reference electrode, forming a three-electrode system. The counter electrode and the working electrode are respectively disposed on both sides of the flexible substrate, and the reference electrode is disposed on the side of the working electrode away from the flexible substrate. A dielectric layer is disposed on the side of the counter electrode away from the flexible substrate, between the working electrode and the reference electrode, and on the side of the reference electrode away from the working electrode. The surface of the working electrode has an exposed portion without the dielectric layer and the reference electrode, and the surface of the exposed portion is coated with a sulfonated MXene sensing layer. The selection of the flexible substrate and electrode materials, as well as the connection method between the electrode layer and the flexible substrate, are selected according to the conventional materials and connection methods of existing implantable biosensors, and this application does not impose any restrictions. A diffusion-limiting layer is coated on the outside of the electrode layer. This sensor is mainly used in implantable glucose monitoring systems for dynamic glucose level monitoring, and can also be used to monitor biomarkers such as lactic acid, uric acid, or cholesterol.

[0060] The sulfonated MXene sensing layer was prepared using the following method:

[0061] S1. Preparation of sulfonated MXene material: After etching the MAX phase of the MXene precursor material with an organic solvent, MXene powder material is obtained. The MXene powder material is prepared into a dispersion and then drop-coated onto a silicon wafer and dried to form an MXene film. The MXene film is subjected to plasma treatment with SO3, rinsed, and then vacuum dried to obtain sulfonated MXene material.

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

[0063] S2. Preparation of the sensing layer solution: Mixed solutions of redox polymer, tool enzyme, sulfonated MXene material and crosslinking agent A are prepared separately using solvent A. The mixed solutions are then mixed in 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 redox polymer, tool enzyme, sulfonated MXene material and crosslinking agent A is 30-50:15-30:15-30:1-10.

[0064] Solvent A is a HEPES buffer or Tris ethanesulfonic acid buffer with a pH of 7.5–8.5, preferably a 20 mmol / L HEPES buffer with a pH of 8.0; the redox polymer is a redox polymer containing a transition metal, including ruthenium, rhodium, osmium, etc., preferably a redox polymer containing osmium, with a concentration of 20–50 mg / mL, preferably 30 mg / mL; the tool enzyme is selected from one or more of glucose dehydrogenase, glucose oxidase, uricase, lactate oxidase, and cholesterol oxidase, preferably glucose oxidase, with a concentration of 10–30 mg / mL, preferably 20 mg / mL; the sulfonated MXene material is... The material prepared using S1 above has a concentration of 10–30 mg / mL, 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, tetraglycidyl-4,4-diaminodiphenylmethane, or glycerol triglycidyl ether, preferably polyethylene glycol diglycidyl ether with a molecular weight between 200 Da and 5000 Da, more preferably polyethylene glycol diglycidyl ether with a molecular weight of 200 Da, and has a concentration of 5–15 mg / mL, preferably 10 mg / mL; when preparing the sensor solution, the mass ratio of the added amounts of each mixed solution 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℃ and the time is 24h.

[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] Preparation of sulfonated MXene materials: Ti3AlC2 was selected as the precursor material. Ti3AlC2 was mixed with 40% hydrofluoric acid solution at a mass-to-volume ratio of 1 g: 20 mL. The mixture was stirred at 50 °C for 48 h, then centrifuged and washed to remove unreacted material. After drying, Ti3C2T was obtained. x Powder, Ti3C2T xThe powder was added to deionized water and ultrasonically dispersed to prepare a dispersion with a concentration of 1 mg / mL. Ti3C2T x The 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 5 minutes at a gas flow rate of 150 mL / min, a power of 100 W, and a pressure of 40 Pa. After treatment, the film was cleaned and then vacuum dried at 60 °C for 30 minutes to obtain the sulfonated MXene material.

[0079] Preparation Example 2

[0080] Preparation of sulfonated MXene materials: Ti3AlC2 was selected as the precursor material. Ti3AlC2 was mixed with 50% hydrofluoric acid solution at a mass-to-volume ratio of 1 g: 20 mL. The mixture was stirred at 40 °C for 40 h, then centrifuged and washed to remove unreacted material. After drying, Ti3C2T was obtained. x Powder, Ti3C2T x The powder was added to deionized water and ultrasonically dispersed to prepare a dispersion with a concentration of 0.5 mg / mL. Ti3C2T x The 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 3 minutes at a gas flow rate of 100 mL / min, a power of 80 W, and a pressure of 20 Pa. After treatment, the film was cleaned and then vacuum dried at 60 °C for 30 minutes to obtain the sulfonated MXene material.

[0081] Preparation Example 3

[0082] Preparation of sulfonated MXene material: Ti3AlC2 was selected as the precursor material. Ti3AlC2 was mixed with 40% hydrofluoric acid solution at a mass-to-volume ratio of 1 g: 20 mL. The mixture was stirred at 50 °C for 60 h, then centrifuged and washed to remove unreacted material. After drying, Ti3C2T was obtained. x Powder, Ti3C2T x The powder was added to deionized water and ultrasonically dispersed to prepare a dispersion with a concentration of 2 mg / mL. Ti3C2T x The 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 mixed solution of redox polymer with a concentration of 30 mg / mL, a mixed solution of tool enzyme with a concentration of 20 mg / mL, a mixed solution of sulfonated MXene material with a concentration of 20 mg / mL, and a mixed solution of crosslinking agent A 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 micro-syringe (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 2A biosensor based on sulfonated MXene material is disclosed, comprising a flexible substrate and an electrode layer disposed on the flexible substrate. The electrode layer includes a counter electrode, a working electrode, and a reference electrode. The working electrode is a working electrode with a sensing layer based on sulfonated MXene material prepared in this embodiment. The specific operation is as follows: the counter electrode, working electrode, and reference electrode are sequentially connected to form an electrode layer. The counter electrode and working electrode are respectively disposed on both sides of the flexible substrate, and the reference electrode is disposed on the side of the working electrode away from the flexible substrate. A dielectric layer is provided on the side of the counter electrode away from the flexible substrate, between the working electrode and the reference electrode, and on the side of the reference electrode away from the working electrode. The counter electrode, working electrode, and reference electrode constitute a three-electrode system. The surface of the working electrode has an exposed portion without a dielectric layer and a reference electrode. The surface of the exposed portion is coated with a sensing layer according to the above-described sensing layer preparation method. The selection of the flexible substrate and the electrode materials, as well as the connection method between the electrode layer and the flexible substrate, are selected according to the conventional materials and connection methods of existing implantable biosensors. This application does not impose any restrictions. After preparation, diffusion is restricted by dip coating on the outside of the electrode layer.

[0091] The method for preparing the confined diffusion layer is as follows:

[0092] The first step involves selecting polyvinylpyridine-styrene copolymer as the polymer containing nitrogen-containing heterocyclic groups, and polyethylene glycol diglycidyl ether with a molecular weight of 600 Da as crosslinking agent B. A mixed solution of ethanol and HEPES buffer with pH=8.0 and a concentration of 20 mmol / L at a volume ratio of 6:1 is used as the solvent to prepare polyvinylpyridine-styrene copolymer solution with a concentration of 50 mg / mL and polyethylene glycol diglycidyl ether solution with a concentration of 10 mg / mL. The polyvinylpyridine-styrene copolymer solution and polyethylene glycol diglycidyl ether solution are then mixed at a mass percentage ratio of 9:1 to obtain the outer membrane solution.

[0093] The second step involves immersing the electrode layer in the outer membrane solution obtained in the first step four times, with a 20-minute interval between each immersion. After the immersion is complete, the electrode layer is removed and cured at 45°C for 40 hours to form a biosensor with a diffusion-limiting layer. The outer membrane layer formed on the surface of the electrode layer is the diffusion-limiting layer.

[0094] 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.

[0095] Example 2

[0096] A method for preparing a sulfonated MXene sensing layer differs from Example 1 in that, in S1, the sulfonated MXene material prepared in Example 2 is used; in S2, the amounts of each mixed solution used to prepare the sensing layer solution are different; a 20 mg / mL redox polymer mixed solution, a 10 mg / mL tool enzyme mixed solution, a 10 mg / mL sulfonated MXene material mixed solution, and a 5 mg / mL crosslinking agent A mixed solution are mixed in a mass ratio of 36:30:30:4 to obtain the sensing layer solution; in S3, the crosslinking curing temperature is 30°C and the time is 28 h.

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

[0098] 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.

[0099] Example 3

[0100] A method for preparing a sulfonated MXene sensing layer differs from Example 1 in that, in S1, the sulfonated MXene material prepared in Example 3 is used; in S2, the amounts of each mixed solution used to prepare the sensing layer solution are different; a 50 mg / mL redox polymer mixed solution, a 30 mg / mL tool enzyme mixed solution, a 30 mg / mL sulfonated MXene material mixed solution, and a 15 mg / mL crosslinking agent A mixed solution are mixed in a mass ratio of 50:15:25:10 to obtain the sensing layer solution; in S3, the crosslinking curing temperature is 40°C and the time is 20 h.

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

[0102] 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.

[0103] Example 4

[0104] 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 30:30:30:10.

[0105] 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.

[0106] 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.

[0107] Example 5

[0108] 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 50:15:30:5.

[0109] 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.

[0110] 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.

[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 in a mass ratio of 45:25:25:5.

[0123] 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.

[0124] 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.

[0125] Comparative Example 3

[0126] A method for preparing a sensing layer, which differs from Comparative Example 2, is 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 in a mass ratio of 30:30:30:10.

[0127] 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 that in Comparative Example 2.

[0128] 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.

[0129] Comparative Example 4

[0130] A method for preparing a sensing layer differs from Example 1 in that sulfonated multi-walled carbon nanotubes (MWCNTs) are used instead of sulfonated MXene material. The preparation method of sulfonated MWCNTs material is as follows: Multi-walled carbon nanotubes (MWCNTs) are vacuum dried at 60°C for 24 hours to remove adsorbed water. An appropriate amount of multi-walled carbon nanotubes (MWCNTs) are added to a concentrated sulfuric acid / concentrated nitric acid mixture (volume ratio = 3:1). The mixture is refluxed in an oil bath at 120°C for 30 minutes, cooled to room temperature, diluted with ultrapure water to neutral, and then dried to obtain sulfonated MWCNTs.

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

[0132] 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.

[0133] 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.

[0134] IV. Performance Test Experiments and Results

[0135] 1. Response speed test

[0136] The response rates of the sensors prepared in Examples 1-6 and Comparative Examples 1-4 were tested using the chronoamperometry (IT) method. Glucose solutions with concentrations of 2 mmol / L, 5 mmol / L, and 15 mmol / L were prepared, and the sensors were placed in the solutions for testing until the current curves stabilized. The results of Examples 1, Comparative Examples 1, 2, and 4 are compared below. Figure 3 As shown.

[0137] Depend on Figure 3 It can be seen that the sensor without MXene material has the slowest polarization speed, a relatively slow current change curve, and it takes nearly 800 seconds for the current to reach a steady state. The MXene sensor and the sulfonated MWCNTs sensor successively improved the current response speed, while the sulfonated MXene sensor showed a very rapid current change after changing to different concentrations of sugar solution, and the current only took about 260 seconds to reach a steady state. It is evident that the better electron transfer capability of sulfonated MXene improved the sensitivity of the sensor.

[0138] 2. Structural Characterization

[0139] Based on the response speed test results, the sensor with the sulfonated MXene sensing layer had the fastest response speed. This is presumably because the enzyme molecules are immobilized on the nanosheet surface, and the active sites of the immobilized enzyme are easily accessible to the external environment, thus improving the response speed of the biosensor. Furthermore, scanning electron microscopy was used to scan the sensing layers in Example 1 and Comparative Example 1, and the results are as follows: Figure 4 As shown.

[0140] Figure 4 (a) is a scan of the sensing layer in Example 1. Figure 4 (b) is a scan of the sensing layer in Comparative Example 1, byFigure 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 exhibits a two-dimensional planar structure, resulting in 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 over 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 using the chronoamperometry (IT) method. The first test solution was a mixture of 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 glucose solution with a concentration of 10 mmol / L and ascorbic acid with a concentration of 0.15 mmol / L.

[0147] Figure 6 This is a comparison chart of the results from Example 4, Comparative Example 1, Comparative Example 2, and Comparative Example 4 in the first test solution. Figure 6 It can be seen that, in response to the interfering substance uric acid, the current value of the sulfonated MXene sensor prepared in Example 4 fluctuates less, with a current attenuation of 2.2%, while the current curve of the sensor without MXene material changes more significantly, with an attenuation of 22.6%.

[0148] Figure 7 This is a comparison chart of the results from Example 5, Comparative Example 1, Comparative Example 2, and Comparative Example 4 in the second test solution. Figure 7 It can be seen that, in response to the interfering substance ascorbic acid, the glucose sensor without MXene material showed a significant decrease in current curve after the addition of the interfering substance, with an attenuation of 26.7%, while the sulfonated MXene sensor prepared in Example 5 showed an attenuation of only 3.8%.

[0149] Furthermore, neither MXene materials nor sulfonated MWCNTs exhibited superior resistance to the metabolic products uric acid and ascorbic acid compared to sulfonated MXene. This demonstrates that the high specific surface area and conductivity of sulfonated MXene provide an efficient electron transport pathway. Simultaneously, surface charge regulation reduces the adsorption of interfering substances. The sulfonation treatment imparts a negative charge to the MXene surface, 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 Examples 1, 3, 4, and 6 were respectively applied to an implantable continuous glucose monitoring system for in vivo experiments, and the results were compared with those of a fingertip blood glucose test. The results are as follows: Figure 8 As shown.

[0152] Depend on Figure 8It was found that in the 14-day human trial, the sensor without MXene material showed low consistency with fingertip blood glucose levels in vivo, with significant discrepancies between the measured fingertip blood glucose values ​​and the actual values. Both the MXene sensor and the sulfonated MWCNTs sensor showed instances of exceeding or falling below fingertip blood glucose levels in the in vivo experiment, indicating low accuracy. In contrast, the sulfonated MXene sensor detected significant fluctuations in dynamic glucose concentration (curve), and randomly sampled fingertip blood glucose levels generally fell within the curve, demonstrating high consistency between the two methods. This collectively demonstrates that the sensor of this invention possesses high accuracy and reliability in practical applications.

[0153] In summary, the sensing layer provided in this application contains sulfonated MXene material, whose surface is covalently modified with sulfonic acid groups, exhibiting excellent electron transfer capabilities and a high specific surface area for enzyme immobilization. Its ability to catalyze H2O2 enhances sensor stability. The sensor comprises a flexible substrate, a working electrode with the sensing layer, a reference electrode, and a counter electrode. The sensor prepared using the sensing layer provided in this method possesses characteristics such as high detection accuracy, fast response speed, strong anti-interference ability, and high stability, laying the foundation for the development of implantable glucose monitoring systems and body fluid biomarker monitoring.

Claims

1. A sulfonated MXene sensing layer, characterized in that, The sensing layer contains sulfonated MXene material, which is obtained by covalently modifying sulfonic acid groups on MXene material.

2. The sensing layer according to claim 1, characterized in that, The MXene material is selected from Ti3C2T. x Ti2CT x V2CT x Nb2CT x and MoTiC2T x One or more of them, preferably Ti3C2T x .

3. The sensing layer according to claim 1, characterized in that, The materials used to prepare the sensing layer include: a redox polymer, a tool enzyme, the 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, preferably 45:25:25:

5.

4. The sensing layer according to claim 3, characterized in that: The redox polymer is a redox polymer containing a transition metal, wherein the transition metal includes ruthenium, rhodium, and osmium, and preferably a redox polymer containing osmium; The tool enzyme is selected from one or more of glucose dehydrogenase, glucose oxidase, uricase, lactate oxidase, and cholesterol oxidase, preferably glucose oxidase; 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, tetraglycidyl-4,4-diaminodiphenylmethane, or glycerol triglycidyl ether, preferably polyethylene glycol diglycidyl ether with a molecular weight between 200 Da and 5000 Da, and more preferably polyethylene glycol diglycidyl ether with a molecular weight of 200 Da.

5. A method for preparing a sulfonated MXene sensing layer according to any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Preparation of sulfonated MXene materials; S2. Prepare a sensing layer solution containing redox polymer, tool enzyme, sulfonated MXene material and crosslinking agent A using solvent A; S3. Load the sensing layer solution onto the surface of the working electrode and crosslink and cure it at 30-40°C for 20-28 hours to obtain the sensing layer.

6. The preparation method according to claim 5, characterized in that, S1 includes: Ti3AlC2 was mixed with a 40%–50% hydrofluoric acid solution and reacted at 40–50°C 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.

7. The preparation method according to claim 6, 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 gas pressure of 40 Pa. After cleaning and drying, sulfonated MXene material was obtained.

8. The preparation method according to claim 5, 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.

9. The preparation method according to claim 8, 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.

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 a sulfonated MXene sensing layer according to any one of claims 1 to 4; 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, preferably 9:1; The polymer containing nitrogen-containing heterocyclic groups is selected from one or more of polyvinylpyridine, polyvinylpyrrole, and polyvinylpyridine-styrene copolymer, preferably polyvinylpyridine-styrene copolymer; 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.

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 biomarker includes one or more of glucose, lactic acid, uric acid, or cholesterol, preferably used in dynamic glucose monitoring products.

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