An enzyme bio-fuel cell anode, its preparation method and application

Abstract

This invention provides an enzyme biofuel cell anode, its preparation method, and its application. The anode is obtained using a dual-enzyme carrier layered independent immobilization strategy. The dual-enzyme carrier consists of carbonized square tubular polypyrrole as a glucose oxidase carrier material and mineralized trehalose hydrolase. Trehalose hydrolase is embedded in a metal-organic framework material using a biomimetic mineralization method, forming a bioactive mineralized trehalose hydrolase layer. Carbonized square tubular polypyrrole is used as the glucose oxidase carrier material. The glucose oxidase layer and the mineralized trehalose hydrolase layer are modified onto a glassy carbon electrode using a layered immobilization method. This method, applied to enzyme biofuel cells, avoids the problem of unexpected homo / heteroenzyme cross-linking in multi-enzyme cascade reactions and improves the utilization rate between different enzyme substrates, thereby increasing the efficiency and electron yield of multi-enzyme cascade reactions.

Description

Technical Field

[0001] This invention relates to the field of bioelectrochemical technology, and in particular to an enzyme biofuel cell anode, its preparation method, and its application. Background Technology

[0002] Enzyme biofuel cells are a novel type of green energy conversion device that uses enzymes as catalysts to convert biomass energy into electrical energy. Due to their selectivity, specificity, good biocompatibility, and mild operating environment, they are widely used in wearable devices, implantable medical devices, and environmental monitoring. Currently, besides the two major problems of low power density and short lifespan, enzyme biofuel cells also suffer from substrate limitations. Most studies report the use of monosaccharides, such as glucose, fructose, and galactose, as substrates. Reports on disaccharides as substrates are scarce, and the power densities of the reported fuel cells are generally low, typically ranging from tens to hundreds of microwatts per square centimeter. This is mainly because biofuel cell systems using disaccharides as substrates involve two enzymes catalyzing the substrate during anode construction. Furthermore, most anodes use a single enzyme carrier material. This inevitably leads to problems such as inefficient co-immobilization of the two enzymes, unintended homo / heteroenzyme crosslinking, low single-enzyme loading, and enzyme spatial competition, resulting in poor performance of the constructed two-enzyme cascade anode. Furthermore, the dual-enzyme cascade anode constructed based on this method suffers from low mass transfer efficiency between different enzyme substrates, further affecting its electrochemical performance. Therefore, there is an urgent need to develop a novel biofuel cell anode based on disaccharides as substrates to promote the further development of enzyme biofuel cells. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide an enzyme biofuel cell anode.

[0004] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned enzyme biofuel cell anode.

[0005] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned enzyme biofuel cell anode.

[0006] A method for preparing an anode for an enzyme biofuel cell employs a dual-enzyme carrier layered independent immobilization strategy. The dual-enzyme carrier consists of carbonized square tubular polypyrrole as a glucose oxidase (GOx) carrier material and mineralized trehalose hydrolase (Tre@ZIF-8). Trehalose hydrolase (Tre) is embedded in a metal-organic framework material (biomimetic mineralization carrier) using a biomimetic mineralization method, thus mineralizing the trehalose hydrolase (Tre) to form a bioactive mineralized trehalose hydrolase layer. Carbonized square tubular polypyrrole is used as the glucose oxidase (GOx) carrier material. The glucose oxidase (GOx) layer and the mineralized trehalose hydrolase (Tre@ZIF-8) layer are modified onto a glassy carbon electrode (GCE) using a layered immobilization method to obtain a trehalose biofuel cell anode with a dual-enzyme carrier configuration.

[0007] Preferably, in the above preparation method, the metal-organic framework material is ZIF-8 (Zeolitic Imidazolate Framework-8), ZIF-67, UIO-66, etc.

[0008] Preferably, in the above preparation method, the metal-organic framework material is ZIF-8, and the mineralized trehalose hydrolase Tre@ZIF-8 is prepared by an aqueous synthesis method at room temperature. The synthesized Tre@ZIF-8 enzyme activity is approximately 32 U·mg. -1 The material has a crystal size of approximately 300 nm and exhibits a dodecahedral morphology with wrinkles and internal cracks.

[0009] Preferably, in the above preparation method, trehalose hydrolase (Tre) is first immobilized in ZIF-8 (Zeolitic Imidazolate Framework-8) using a biomimetic mineralization method to form mineralized trehalose hydrolase (Tre@ZIF-8), while glucose oxidase (GOx) is separately immobilized on carbonized square tubular polypyrrole (GOx carrier) using a physical adsorption method. The current collector is selected from glassy carbon electrode (GCE) or Toray carbon paper (TCP). The glucose oxidase (GOx) layer, ferrocene (Fer) layer and mineralized enzyme (Tre@ZIF-8) layer are successively immobilized on the current collector by a layer-by-layer immobilization method. Finally, the dual-enzyme carrier layer is immobilized with chitosan (CS).

[0010] The above preparation method has significant advantages over the single-carrier modification method. The substrate trehalose is first hydrolyzed by the mineralization enzyme layer Tre@ZIF-8 to generate α-glucose, which is then converted into β-glucose after spontaneous mutarotation, and then further catalyzed by glucose oxidase (GOx) to gain electrons.

[0011] Preferably, in the above preparation method, the carbonized square tubular polypyrrole is PPy. 1200 (Subscript 1200 indicates carbonization temperature, °C), enzyme carrier materials such as carbon nanotubes and graphene, with PPy being a further preferred option. 1200 .

[0012] Preferably, in the above preparation method, the trehalose hydrolase (EC: 3.2.1.28) is a commercially available enzyme obtained through heterologous expression in *E. coli*, with an enzyme activity of 63.72 U·mg. -1 The solvent is 0.1 mol / L. -1 PBS (pH 7.2), concentration 20 mg / mL -1 .

[0013] Preferably, in the above preparation method, the glucose oxidase (EC: 1.1.3.4) is a commercially available enzyme with an enzyme activity of 270 U·mg. -1 When using, prepare a solution at a concentration of 20 mg / mL. -1 The solvent is 0.1 mol / L. -1 PBS (pH 7.2).

[0014] The above preparation method can construct a dual-enzyme cascade biosensor, wherein the detection substrate of the dual-enzyme cascade biosensor is a disaccharide and the construction process of the sensor uses two or more enzymes.

[0015] Furthermore, the above preparation method can be applied to research fields such as the construction of enzyme biofuel cell anodes using other disaccharides based on trehalose as a substrate.

[0016] An enzyme biofuel cell anode is prepared by the above method.

[0017] Preferably, the anode of the above-mentioned enzyme biofuel cell comprises, in sequence, a glassy carbon electrode (GCE) or a Toray carbon paper (TCP) substrate, and carbonized square tubular polypyrrole (PPy). 1200 The electrode configuration consists of a ferrocene (Fer) layer, a glucose oxidase (GOx) layer, a mineralizing enzyme (Tre@ZIF-8) layer, and a chitosan (CS) encapsulation layer. The electrode configuration is abbreviated as: CS / Tre@ZIF-8 / GOx / Fer / PPy / GCE.

[0018] Preferably, the above-mentioned enzyme biofuel cell anode is a trehalose-based enzyme biofuel cell anode with a dual-enzyme carrier configuration. The trehalose-based enzyme biofuel cell anode with the dual-enzyme carrier configuration can be combined with an air-diffusion cathode to construct a flexible, membrane-free enzyme biofuel cell. The enzyme biofuel cell anode uses ferrocene as the electron mediator, and its electron transfer mechanism is indirect electron transfer. The electron mediator can also be extended to other electron mediators besides ferrocene, such as ferrocene derivatives.

[0019] Preferably, in the above-mentioned enzyme biofuel cell anode, the dual enzyme carrier is a carbonized square tubular polypyrrole (PPy1200) carrier material for glucose oxidase (GOx) and a biomimetic mineralized trehalose hydrolase (Tre@ZIF-8).

[0020] Preferably, the above-mentioned enzyme biofuel cell anode exhibits a distinct boundary between two carrier materials, with the lower layer being carbonized square tubular polypyrrole (PPy). 1200 The upper layer is a distorted dodecahedral morphology Tre@ZIF-8.

[0021] The above-mentioned enzyme biofuel cell anode is used in the construction of enzyme biofuel cells.

[0022] Preferably, in the above application, the enzyme biofuel cell anode is combined with an air diffusion cathode to construct a flexible membrane-free enzyme biofuel cell. The non-conductive nature of the mineralized trehalose hydrolase (Tre@ZIF-8) layer is used as a membrane between the anode and cathode of the enzyme biofuel cell, reducing the use of proton exchange membranes or physical membranes.

[0023] An enzyme biofuel cell includes an enzyme biofuel cell anode prepared by the above method.

[0024] The enzyme biofuel cell described above can be widely used in powering electronic skin, wearable devices, flexible electronic devices, and implantable / semi-implantable electronic devices.

[0025] The above-mentioned assembly method for enzyme biofuel cells involves attaching the enzyme-modified surfaces (front sides of the anode and cathode) of the prepared anode and cathode together, and using the non-conductive nature of mineralized trehalose hydrolase (Tre@ZIF-8) as a membrane between the anode and cathode to achieve the function of disconnecting the anode and cathode.

[0026] Preferably, in the above assembly method, GCE is selected as the current collector, and a flexible air-diffusing cathode based on TCP is selected as the cathode; the modified surfaces of the prepared anode and cathode are attached to each other, a neutral filter paper is selected to absorb the battery electrolyte, a highly permeable sealing film is selected as the battery protective film, and the battery is encapsulated according to the battery structure.

[0027] The above method is limited to using insulating materials for one of the enzyme carriers in the dual-enzyme carrier configuration.

[0028] Preferably, in the above assembly method, the flexible air diffusion cathode includes biological and non-biological air diffusion electrodes.

[0029] Preferably, in the above assembly method, the battery electrolyte is 0.1 mol / L. -1 PBS (pH 6), substrate concentration 50 mmol / L -1 Trehalose concentration as fuel.

[0030] The beneficial effects of this invention are: The aforementioned method for preparing the anode of an enzyme biofuel cell avoids the problem of unexpected homo / heteroenzyme crosslinking in multi-enzyme cascade reactions, while also improving the utilization rate between different enzyme substrates, thereby increasing the efficiency and electron yield of multi-enzyme cascade reactions. The prepared enzyme biofuel cell anode, utilizing the non-conductive nature of the Tre@ZIF-8 layer, can be combined with an air-diffusion cathode to construct a membrane-free enzyme biofuel cell. It can be applied in research fields such as enzyme biofuel cell anodes using trehalose as a substrate and other disaccharides, as well as the construction of dual-enzyme cascade biosensors. Specifically, (1) The present invention provides a method for preparing an enzyme biofuel cell anode based on disaccharide substrate with a dual-enzyme carrier configuration. The principle is to use the dual-enzyme carrier to independently immobilize two enzymes in the construction of enzyme biofuel cell anode and biosensor based on disaccharide substrate. This can increase the loading sites of each enzyme and improve the single enzyme loading capacity. It can also effectively avoid the occurrence of unexpected homo / heterotype enzyme crosslinking problems in the dual-enzyme cascade reaction. At the same time, it can also improve the utilization efficiency between different enzyme substrates, thereby improving the bioelectrochemical performance of the dual-enzyme cascade anode reaction system.

[0031] (2) This invention provides a method for preparing an enzyme biofuel cell anode based on disaccharide substrate with a dual-enzyme carrier configuration. By using a biomimetic mineralization method to embed the enzyme in a metal-organic framework material, a novel catalytic layer with insulating properties and biological activity can be formed. It can replace traditional proton exchange membranes or physical membranes, effectively simplifying the battery structure and reducing the size of the device. It is suitable for the construction of wearable, implantable / semi-implantable enzyme biofuel cells.

[0032] (3) The present invention provides a method for preparing an enzyme biofuel cell anode based on disaccharide substrate with a dual-enzyme carrier configuration. The principle of this method can be extended to other enzyme immobilization methods other than biomimetic mineralization. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of an enzyme biofuel cell anode with trehalose as a substrate, based on a dual-enzyme carrier configuration according to the present invention.

[0034] Figure 2 This is a SEM image of the anode of an enzyme biofuel cell with a dual-enzyme carrier configuration and trehalose as a substrate, according to the present invention.

[0035] Figure 3 The images show the SEM (A) and TEM (B) images of the trehalose hydrolytic mineralizing enzyme (Tre@ZIF-8) in Example 2 of this invention.

[0036] Figure 4 This invention presents the bioelectrochemical performance (CV curve) of an enzyme biofuel cell anode with a dual-enzyme carrier configuration and trehalose as a substrate. Curve a corresponds to electrode CS / Tre / GOx / Fer / PPy / GCE, and curve b corresponds to electrode CS / Tre@ZIF-8 / GOx / Fer / PPy / GCE.

[0037] Figure 5 This is a schematic diagram of a membrane-free trehalose biofuel cell constructed from the anode and air diffusion cathode of an enzyme biofuel cell with a dual-enzyme carrier configuration based on trehalose as the substrate.

[0038] Figure 6 This image shows a membrane-free trehalose biofuel cell constructed using a trehalose-based anode and an air-diffusion cathode with a dual-enzyme carrier configuration, according to the present invention. In the image, (Aa) represents the substrate electrodes of the constructed trehalose biofuel cell anode and cathode; (Ab) shows the drop-coating process of the modified materials for the constructed trehalose biofuel cell anode and cathode; (Ac) shows the assembly process of the constructed trehalose biofuel cell anode and cathode; (Ad) shows the finished trehalose biofuel cell; (Ae) shows the front view of the constructed trehalose biofuel cell; and (Af) shows the back view of the constructed trehalose biofuel cell.

[0039] Figure 7 The power test results are for a membrane-free trehalose biofuel cell constructed using a trehalose-based enzyme biofuel cell anode and an air diffusion cathode with a dual-enzyme carrier configuration according to the present invention. Detailed Implementation

[0040] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0041] The following examples demonstrate the construction of an enzyme biofuel cell anode based on trehalose as a substrate. This can be further extended to enzyme biofuel cells based on trehalose and other disaccharides, and even further extended to dual-enzyme cascade biosensors.

[0042] Example 1 A method for preparing an enzyme biofuel cell anode based on trehalose as a substrate with a dual-enzyme carrier configuration, the specific steps of which are as follows: (1) The preparation of the anode involves glucose oxidase (GOx) and trehalose hydrolase (Tre). Glassy carbon electrode (GCE) is selected as the anode current collector with an electrode diameter of 3 mm.

[0043] (2) Preparation of carbonized square tubular polypyrrole (PPy) 1200 It was used as a carrier for GOx and formulated into a 5 mg / mL solution. -1 The suspension; the carbonized square tubular polypyrrole (PPy 1200 The synthesis was performed using a soft template method. The specific steps are as follows: a. Take 19.44g of FeCl3·6H2O and add it to 600mL of deionized water. Stir well and set aside for later use. b. Take 2.94g of methyl orange and add it to 1200mL of deionized water. Stir well and set aside for later use. c. Slowly add the FeCl3 solution to the methyl orange solution while stirring magnetically throughout the process. Once the mixture is homogeneous, set aside for later use. d. Slowly add 4.5 mL of pyrrole to the above mixed solution, stir magnetically for 30 min, and then let the mixed solution stand at room temperature for 24 h. e. Centrifuge the above mixture, wash the precipitate with a large amount of deionized water and anhydrous ethanol, and then place it in a 60°C oven for 24 hours to obtain a black solid product PPy. f. Place the black solid product PPy material into a high-temperature carbonization furnace and heat it at a uniform rate (2℃·min). -1 The temperature was raised to the target temperature of 1200℃, and then held at that temperature for 3 hours under a high-purity N2 protective atmosphere. The product obtained after carbonization was denoted as PPy. 1200 ; (3) A biomimetic mineralization method (see Example 2 below for specific process) was used to immobilize Tre. The carrier used was Zeolitic Imidazolate Framework-8 (ZIF-8). After mineralization, Tre@ZIF-8 was formed and prepared into 40 mg / mL solutions. -1 A suspension.

[0044] (4) After polishing the surface of the GCE electrode, add 20 μL of 5 mg / mL solution to its surface. -1 PPy 1200 After suspension, air dry at room temperature.

[0045] (5) Take 10 μL of 0.1 mol / L solution. -1 Ferrocene (Fer) ethanol solution was drop-coated onto PPy1200 Air dry at room temperature on the shelf.

[0046] (6) Take 12 μL of 20 mg / mL -1 The glucose oxidase (GOx) solution was drop-coated onto the electrode surface in step (5) and dried at 4°C.

[0047] (7) Take 16 uL of mineralized trehalose hydrolase (Tre@ZIF-8) suspension and drop it onto the electrode surface of step (6), and air dry it at 4°C.

[0048] (8) Finally, 6 μL of 0.5%wt chitosan solution (CS) was dropped onto the modified electrode surface for electrode encapsulation. The final anode abbreviation is: CS / Tre@ZIF-8 / GOx / Fer / PPy 1200 / GCE, the schematic diagram and actual morphology of this anode structure are as follows: Figure 1 and Figure 2 As shown, the constructed enzyme biofuel cell anode can serve as a biosensor for a dual-enzyme cascade.

[0049] Example 2 The preparation method of mineralized trehalose hydrolase (Tre@ZIF-8) used in Example 1 is as follows: (1) Prepare 30 mL of 0.31 mol / L solution using deionized water. -1 Zinc acetate and 1.25 mol / L -1 A solution of 2-methylimidazole (2-HmIm) is prepared for use. (2) Take out the pre-frozen Tre (20 mg / mL) from -20℃ -1 63.72 Umg -1 After thawing, store at 4℃ for later use; (3) At room temperature, add 200 μL of 0.31 mol / L solution to a 5 mL centrifuge tube. -1 Zinc acetate was slowly added to 1 mL of Tre solution with magnetic stirring at 600 rpm for 10 min, followed by the addition of 1 mL of 1.25 M 2-methylimidazole, and finally 0.1 mol / L solution was added. -1 The entire system was brought to a final volume of 3 mL with PBS (pH 6.0), and then magnetically stirred for 1 h. (4) Centrifuge the reaction solution from step (3) at 4°C and 8000 rpm for 3 min, and wash the precipitate three times with deionized water to obtain Tre@ZIF-8.

[0050] (5) Use 1 mL of 0.1 mol / L solution -1 The precipitate was resuspended in PBS (pH 7.2) in a 1.5 mL centrifuge tube to obtain the Tre@ZIF-8 suspension. (6) The residual enzyme activity of the Tre@ZIF-8 suspension was tested using a trehalase activity kit. Under the conditions of 1 standard atmosphere and 25°C, one enzyme activity unit was defined as the production of 1 μmol of glucose per minute per mg of mineralized protein. The measured enzyme activity of Tre@ZIF-8 was 32 Umg. -1 Its appearance is like Figure 3 As shown.

[0051] Example 3 A flexible membrane-free biofuel cell with a dual-enzyme carrier configuration based on trehalose as a substrate is constructed using the following specific steps: (1) First, GCE was selected as the current collector, and the dual enzyme carrier loading, enzyme loading and electrochemical reaction conditions were optimized using a three-electrode electrochemical system (see Examples 1 and 2 above for specific methods, except that the glassy carbon electrode (GCE) was replaced with Toray carbon paper (TCP)). The optimized electrode CS / Tre@ZIF-8 / GOx / Fer / PPy 1200 / TCP is applied to flexible TCP substrate electrodes; (2) A flexible air diffusion type cathode based on TCP (see the flexible air diffusion biological cathode described in Example 2 of CN116190678A) was selected as the cathode; (3) Place the prepared modified surfaces of the anode and cathode together, select a piece of neutral filter paper to absorb the battery electrolyte, and select a highly permeable sealing film as the battery protective film, according to... Figure 5 The battery structure diagram is used for packaging.

[0052] Application Example 1 The following steps were taken to test the bioelectrochemical performance of the anode of a biofuel cell based on trehalose with a dual-enzyme carrier configuration: (1) Construct the anode of the trehalose biofuel cell with a dual-enzyme carrier configuration according to the steps described in Example 1 and Example 2, and construct the control electrode in the same way. The difference is that the mineralized trehalose hydrolase (Tre@ZIF-8) in step (5) of Example 1 is replaced with free trehalose hydrolase (Tre). The control electrode is abbreviated as CS / Tre / GOx / Fer / PPy 1200 / GCE.

[0053] (2) The bioelectrochemical performance of the anode of the trehalose biofuel cell with a dual-enzyme carrier configuration was tested using a three-electrode electrochemical system. The electrolyte was 0.1 mol / L. -1 PBS (pH 6), trehalose substrate concentration of 50 mmol / L -1 The test method was cyclic voltammetry (CV), with a scan range of -0.2 to 0.6 V, Ag / AgCl as the reference electrode, and a scan rate of 10 mV / s.-1 ; (3) Test results are as follows Figure 4 As shown, curve a represents the electrode CS / Tre / GOx / Fer / PPy. 1200 The CV curve of / GCE, where the Tre loading is related to the electrode CS / Tre@ZIF-8 / GOx / Fer / PPy 1200 / GCE (curve b) indicates the optimal enzyme amount is the same as the enzyme activity.

[0054] Application Example 2 The following steps were taken to construct a flexible, membrane-free enzyme biofuel cell anode with a dual-enzyme carrier configuration based on trehalose as a substrate and to test its performance: (1) A flexible membrane-free trehalose biofuel cell anode was constructed according to Example 3 based on a dual-enzyme carrier configuration. A photograph of the actual product is shown below. Figure 6 As shown, (Aa) represents the anode and cathode substrate electrodes of the constructed trehalose biofuel cell; (Ab) represents the drop-coating process of the modified materials for the anode and cathode of the constructed trehalose biofuel cell; (Ac) represents the assembly process of the anode and cathode of the constructed trehalose biofuel cell; (Ad) represents the finished trehalose biofuel cell; (Ae) represents the front side of the constructed trehalose biofuel cell; and (Af) represents the back side of the constructed trehalose biofuel cell, where the filter paper serves as the fuel storage medium.

[0055] (2) The performance of a flexible membrane-free trehalose biofuel cell based on a dual-enzyme carrier configuration was tested using a two-electrode electrochemical system. The anode was the working electrode, the cathode was the counter electrode, and the electrolyte was 0.1 mol / L. -1 PBS (pH 6), trehalose substrate concentration 50 mmol / L -1 Trehalose was dissolved in PBS, and the test method was linear sweep voltammetry (LSV), with a scan range of -0.6 to 0 V and a scan rate of 10 mV / s. -1 .

[0056] (3) Test results are too... Figure 7 As shown, the power density of this flexible membrane-free trehalose biofuel cell is 210 μW / cm³. -2 .

[0057] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing an anode for an enzyme biofuel cell, comprising: embedding trehalose hydrolase in a metal-organic framework material using a biomimetic mineralization method to mineralize the trehalose hydrolase into a bioactive mineralized trehalose hydrolase layer; using carbonized square tubular polypyrrole as the glucose oxidase carrier material, and modifying the glucose oxidase layer and the mineralized trehalose hydrolase layer onto a glassy carbon electrode using a layered fixation method to obtain a trehalose biofuel cell anode with a dual-enzyme carrier configuration.

2. The preparation method according to claim 1, characterized in that: The metal-organic framework material is ZIF-8, and the mineralized trehalose hydrolase Tre@ZIF-8 was prepared by an aqueous synthesis method at room temperature.

3. The preparation method according to claim 1, characterized in that: First, trehalose hydrolase was immobilized in ZIF-8 using a biomimetic mineralization method to form mineralized trehalose hydrolase. Glucose oxidase was immobilized separately on carbonized square tubular polypyrrole using a physical adsorption method. Glassy carbon electrode or Toray carbon paper was used as the current collector. The glucose oxidase layer, ferrocene layer and mineralized enzyme layer were successively immobilized on the current collector by a layer-by-layer immobilization method. Finally, the dual-enzyme carrier layer was immobilized with chitosan.

4. The preparation method according to claim 1 or 3, characterized in that: The carbonized square tubular polypyrrole is PPy 1200 Carbon nanotubes or graphene.

5. An enzyme biofuel cell anode, prepared by the method described in any one of claims 1-4.

6. The enzyme biofuel cell anode according to claim 5, characterized in that: It consists of, in sequence, a glassy carbon electrode or Toray carbon paper substrate, a carbonized square tubular polypyrrole layer, a ferrocene layer, a glucose oxidase layer, a mineralization enzyme layer, and a chitosan encapsulation layer.

7. The use of the enzyme biofuel cell anode of claim 5 or 6 in the construction of an enzyme biofuel cell.

8. An enzyme biofuel cell, comprising the enzyme biofuel cell anode of claim 5 or 6.

9. The assembly method of the enzyme biofuel cell according to claim 8, characterized in that: The enzyme-modified surfaces of the prepared anode and cathode are attached to each other, and the non-conductive nature of the mineralized trehalose hydrolase is used as a membrane between the anode and cathode.

10. The assembly method according to claim 9, characterized in that: GCE was selected as the current collector, and a flexible air-diffusion cathode based on TCP was selected as the cathode. The modified surfaces of the prepared anode and cathode were attached to each other, a neutral filter paper was selected to absorb the battery electrolyte, and a highly permeable sealing film was selected as the battery protective film. The battery was then encapsulated according to the battery structure.

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