A biological cathode for oxidation of glycerol to glycerol aldehyde and its preparation and use

Abstract

The present application relates to the technical field of electrochemical biological fuel cell, especially to a biological cathode for glycerol oxidation into glycerol aldehyde and its preparation method and application. The preparation method of the biological cathode comprises: S1, preparing a conductive carrier; S2, introducing a connecting molecule and constructing an interface; S3, preparing a double-enzyme modified solution; and S4, preparing an enzyme biological cathode. The present application is different from the characteristics of traditional cathode oxygen reduction reaction, and is based on the glycerol oxidation conversion mechanism of GalOx. A GalOx+HRP double-enzyme cascade catalytic interface is constructed on the surface of a functional carbon cloth. Under the synergistic action of various components, glycerol is specifically oxidized into glycerol aldehyde on the cathode side, and the in-situ generated H2O2 is reduced at the cathode, thereby generating a higher initial potential. The carbon cloth has the functions of electric conduction support and gas diffusion, and can be used as a gas-breathing electrode. Oxygen is supplied to the back surface to maintain the cathode cascade reaction under the condition of oxygen-free bulk solution. The enzyme biological cathode is simple in construction but multifunctional, and can generate high-value-added products and output electric energy.

Description

Technical Field

[0001] This invention relates to the field of electrochemical biofuel cell technology, and in particular to a biocathode for the oxidation of glycerol to glyceraldehyde, its preparation method, and its application. Background Technology

[0002] Glycerol, a major byproduct of biodiesel production, is considered a promising bio-based platform compound due to its wide availability, low price, and renewable nature. However, with the continuous expansion of the biodiesel industry, the large accumulation of byproduct glycerol has led to a gradual imbalance between supply and demand. Low-value utilization and even waste disposal not only weaken the economic viability of the biodiesel industry chain but also result in resource waste. Therefore, developing green, efficient glycerol conversion pathways that yield high-value-added products has become an important research direction in the fields of bioenergy and green chemistry.

[0003] In recent years, enzyme catalysis technology has demonstrated unique advantages in the resource utilization of glycerol due to its mild reaction conditions, high selectivity, and environmental friendliness. Galactose oxidase (GalOx) is a copper radical oxidase capable of highly regioselectively oxidizing primary alcohols to corresponding aldehydes and using molecular oxygen as the terminal electron acceptor to regenerate its active site. Therefore, it has promising applications in the specific conversion of glycerol to high-value compounds (such as glyceraldehyde). However, the industrial application of GalOx faces two major technical obstacles: first, its catalytic activity depends on a continuous oxygen supply, increasing the complexity of process control and operating costs; second, the hydrogen peroxide (H2O2) generated during the reaction easily deactivates GalOx. Existing technologies typically require the addition of excess catalase to the system to remove H2O2, further increasing operating costs and exacerbating the difficulty of downstream separation.

[0004] To address the aforementioned issues, we have attempted to immobilize GalOx using redox polymers and construct a mediator-type bioanode. This strategy utilizes the electrode as an electron acceptor to regenerate enzyme active sites, thereby reducing dependence on oxygen regeneration pathways. Simultaneously, since GalOx and the redox polymer are co-immobilized on the electrode substrate surface, separation from the reaction medium is facilitated, simplifying the product purification process. Based on this principle, a GalOx / osmium redox polymer (P...) was constructed. Os The bioanode can specifically convert glycerol into glyceraldehyde at a low overpotential. Furthermore, by coupling this bioanode with a bilirubin oxidase (BOD)-based biocathode (catalyzing the oxygen reduction reaction), an enzyme biofuel cell (EBFC) system can be constructed, enabling the simultaneous conversion of glycerol and the output of electrical energy. This transforms the conversion process, which originally required external drive, into a process that can generate electricity simultaneously, providing a novel technological path for the high-value utilization of bio-based raw materials.

[0005] However, the existing GalOx-BOD-based EBFC system still exhibits low Faradaic efficiency in the conversion of glycerol to glyceraldehyde. This is because the glycerol conversion is primarily confined to the anode side, while electrons flowing to the cathode are only used to drive the oxygen reduction reaction to form the fuel cell circuit; the cathode itself does not participate in the conversion of the glycerol substrate. Therefore, if a cathode catalytic strategy with high open-circuit potential, high catalytic selectivity, and excellent electrochemical response can be designed, enabling the cathode to not only act as an electron acceptor reduction end but also simultaneously catalyze the specific conversion of glycerol to glyceraldehyde, it is hoped that simultaneous and efficient conversion of glycerol at both the anode and cathode can be achieved, significantly improving the Faradaic efficiency of the specific conversion of glycerol to glyceraldehyde, while simultaneously using glycerol as fuel to output electrical energy.

[0006] In summary, developing a novel method with a high open-circuit potential and high-selectivity catalytic conversion of glycerol to high-value-added glyceraldehyde at the cathode is of significant research and application value for improving the resource utilization of glycerol as a byproduct of biodiesel production, reducing the cost of enzyme catalysis processes, and increasing electrochemical conversion efficiency. Summary of the Invention

[0007] The technical problem to be solved by this invention is that, in existing biofuel cell technology, glycerol is only catalytically oxidized to glyceraldehyde on the anode side and the cathode does not effectively participate in the conversion of glycerol to glyceraldehyde, resulting in low overall conversion rate of glycerol substrate and low Faraday efficiency. This invention provides a biocathode for glycerol oxidation to glyceraldehyde, its preparation method and application.

[0008] The cathode system constructed in this invention features mild reaction conditions, a green and clean process, and good environmental compatibility. This enzyme biocathode exhibits a high open-circuit potential exceeding 1 mA·cm⁻¹ in solutions containing glycerol substrates. -2 The current density is high, and it exhibits good operational stability. This scheme introduces glycerol conversion functionality at the cathode interface, expanding the cathode from a traditional single oxygen reduction function to a functional electrode with both substrate conversion and energy output capabilities. Coupled with a bioanode, it constructs a membrane-free enzyme biofuel cell, enabling simultaneous conversion of glycerol at both the cathode and anode. This improves the overall conversion of the substrate glycerol and the Faradaic efficiency of the biofuel cell. While promoting the generation of the high-value-added product glyceraldehyde, it can also provide external electrical energy, thus providing a new technical path for the high-value utilization of glycerol and the co-production of electrical energy.

[0009] The technical solution adopted by this invention to solve its technical problem is: a method for preparing a biological cathode for glycerol oxidation to glyceraldehyde, specifically including the following steps: Step S1: Preparation of conductive carrier: MWCNTs (multi-walled carbon nanotubes) were ultrasonically dispersed in ethanol (EtOH) to obtain a MWCNTs dispersion. The MWCNTs dispersion was then coated onto a functionalized carbon cloth substrate to obtain a first conductive carrier modified with MWCNTs. This step can not only effectively reduce the hydrophobicity of the substrate surface, but also significantly increase the specific surface area of ​​the electrode and enhance the electron transport capability. Step S2, Introduction of Connecting Molecules and Interface Construction: Dissolve PBSE (1-pyrenebutyrate succinimide ester) in DMF ( N,N A PBSE solution was prepared by dimethylformamide (DMF). The first conductive support obtained in step S1 was immersed in the PBSE solution to obtain the second conductive support. After immersion, the second conductive support was washed sequentially in DMF, n-hexane and PBS solution (phosphate buffer solution) to remove surface residues and obtain the third conductive support. During the immersion of the first conductive support in the PBSE solution, the PBSE solution achieved stable adsorption and fixation through the π-π interaction between the pyrene group and the surface of MWCNTs. At the same time, the succinimide ester group in the PBSE molecule can undergo covalent coupling reaction with the amino groups on the surface of subsequent enzyme molecules, thereby achieving stable fixation of enzyme molecules and providing a functional interface for the construction of subsequent enzyme cascade systems. Step S3: Prepare the dual-enzyme modified solution: HRP (horseradish peroxidase) solution and GalOx solution were mixed evenly to prepare a dual-enzyme modified solution. The dual-enzyme modified solution was thoroughly mixed before use to ensure uniform distribution of enzyme molecules in the system and efficiency of subsequent cascade catalysis. Step S4: Preparation of a dual-enzyme biocathode: The third conductive carrier obtained in step S2 is coated with the dual-enzyme modification solution prepared in step S3. After standing to allow for sufficient immobilization and self-assembly of the enzyme molecules, the finished dual-enzyme biocathode co-modified with GalOx and HRP is obtained. GalOx catalyzes the oxidation of glycerol, using O2 as an electron acceptor to generate H2O2. Subsequently, HRP uses this intermediate H2O2 as a substrate to further complete the reduction reaction under electron-donating electrode conditions, thus constructing a dual-enzyme cascade catalytic process with H2O2 as an intermediate.

[0010] Furthermore, in step S1, the concentration of MWCNTs in the MWCNTs dispersion is 2 mg / mL. At this concentration, MWCNTs exhibit excellent dispersion stability in the dispersion medium, thereby forming a uniform and dense carbon nanotube coating when dropped onto the electrode surface.

[0011] Furthermore, in step S1, each cm 2The substrate was coated with 222.3 µL of MWCNT dispersion. This ensured that the MWCNTs formed a complete and uniform conductive network on the substrate surface, effectively improving the overall conductivity and catalytic activity of the electrode.

[0012] Further, in step S1, the substrate is carbon cloth (CC) with a polytetrafluoroethylene (PTFE) integrated microporous layer (MPL) coated on the surface, and MWCNTs dispersion is coated on the PTFE / MPL surface to finally obtain an enzyme biocathode, the structure of which is represented as GalOx+HRP / MWCNTs / CC.

[0013] Furthermore, in step S2, the concentration of PBSE in the PBSE solution is 3.9 mg / mL.

[0014] Furthermore, in step S2, the first conductive carrier is immersed in the PBSE solution for 2 hours. This time ensures that the π-π conjugation between PBSE and MWCNTs reaches saturation, which is beneficial for forming a high-density and stable PBSE-modified interface, thereby satisfying the subsequent enzyme immobilization steps.

[0015] Furthermore, in step S3, the mass ratio of HRP solution to GalOx solution is 6:1. At this ratio, the GalOx-catalyzed glycerol oxidation process and the subsequent catalytic reaction involving HRP exhibit good synergistic effects, resulting in the highest catalytic current.

[0016] Further, in step S3, the HRP solution is prepared by preparing an HRP solution with an initial concentration of 20 mg / mL. The GalOx solution is prepared by preparing a GalOx solution with an initial concentration of 5 U / μL.

[0017] Furthermore, in step S4, each cm 2 The third conductive support was coated with 55.6 µL of dual-enzyme modification solution. The modified volume resulted in an enzyme layer of moderate thickness, ensuring both substrate diffusion and product transfer efficiency while shortening the electron transport path.

[0018] Furthermore, in step S4, the settling temperature is 4°C. This allows the enzyme to be fully, gently, and stably immobilized on the electrode surface while maintaining its activity.

[0019] An enzyme biocathode for oxidizing glycerol to glyceraldehyde, wherein the enzyme biocathode is prepared by the above-described preparation method.

[0020] An application of an enzyme biocathode for oxidizing glycerol to glyceraldehyde, wherein the enzyme biocathode is used in the manufacture of a membrane-free fuel cell.

[0021] The GalOx+HRP dual-enzyme cascade enzyme biocathode constructed in this invention differs from traditional cathodes, which primarily function as electrodes for reduction reactions. Based on the GalOx-catalyzed glycerol oxidation mechanism, a stable dual-enzyme cascade catalytic interface is constructed on the surface of functional carbon cloth, enabling the catalytic oxidation of glycerol at the cathode interface. This overcomes the limitation that glycerol is typically oxidized only to glyceraldehyde on the anode side. Through the synergistic effect of dual-enzyme cascade catalysis, conductive network construction, and enzyme immobilization assembly, the enzyme biocathode improves cathode catalytic activity, interfacial electron transfer efficiency, and operational stability, and exhibits a high onset potential, which is beneficial for improving the open circuit of enzyme biofuel cells. Voltage and energy output performance; furthermore, the functional carbon cloth substrate combines conductive support and gas diffusion functions, and the constructed dual-enzyme cathode can serve as a gas breathing electrode. Under low-oxygen or anaerobic conditions in the bulk solution, it can continuously obtain O2 supply from the air or oxygen through the back of the electrode to maintain the GalOx+HRP cascade reaction at the cathode. Based on the advantages of this enzyme biocathode, a membrane-free enzyme biofuel cell can be constructed with a bioanode, enabling the simultaneous occurrence of the glycerol oxidation reaction on the anode side and the glycerol cascade conversion reaction on the cathode side. This is beneficial to improving the overall conversion rate of the glycerol substrate, the generation efficiency of the target product glyceraldehyde, and the Faradaic efficiency of the battery. Therefore, this invention can balance the high-value-added conversion of glycerol with electrochemical energy output, and has the advantages of simple construction method, economical materials, mild reaction conditions, and good stability. It has good application prospects and industrialization potential in the fields of glycerol resource utilization and enzyme biofuel cells.

[0022] The working principle of the enzyme biocathode for the oxidation of glycerol to glyceraldehyde prepared in this invention is as follows: Figure 1 As shown, the electron transfer process is as follows: GalOx-catalyzed glycerol oxidation step: ; HRP electrocatalytic H2O2 reduction steps: .

[0023] Depend on Figure 1As can be seen, this invention constructs a GalOx+HRP dual-enzyme cascade catalytic interface on the surface of a functional carbon cloth electrode. The carbon cloth serves as a conductive substrate and structural support, MWCNTs as conductivity-enhancing components and enzyme loading carriers, and PBSE as an immobilization and linking component, used to improve the immobilization stability and uniform distribution of enzyme molecules at the carbon nanotube interface. Through the synergistic effect of these components, a stable composite enzyme catalytic layer can be formed on the carbon cloth surface. In this composite enzyme catalytic layer, GalOx catalyzes the oxidation of glycerol to glyceraldehyde, and uses O2 as an electron acceptor to generate H2O2; the H2O2 then serves as a substrate for HRP and is catalytically reduced by HRP at the cathode, thereby forming a GalOx+HRP dual-enzyme cascade electrocatalytic process with H2O2 as an intermediate. The construction of this cascade system allows glycerol to undergo oxidation not only at the anode but also to participate in enzyme catalytic reactions at the cathode, thus expanding the reaction pathway of glycerol in enzyme biofuel cells.

[0024] In the cathode configuration design, this invention selects functional carbon cloth as the electrode substrate. This carbon cloth possesses excellent conductivity, flexibility, and a porous gas diffusion structure, serving not only as a conductive support for the enzyme catalytic layer but also as a gas diffusion channel for O2 transport. Especially when used as a gas-diffusion-type enzyme biocathode, even under anaerobic conditions in the bulk solution, it can continuously obtain O2 from the air or oxygen through the back of the electrode, thereby maintaining the GalOx+HRP cascade catalytic reaction at the cathode. Furthermore, coupling this gas-diffusion-type enzyme biocathode with a GalOx bioanode can construct a membrane-free enzyme biofuel cell system. In this system, the GalOx bioanode typically requires substrate oxidation in an anaerobic environment to provide efficient electrons for glycerol oxidation; while the cathode reaction relies on sufficient O2 as the final electron acceptor. Therefore, the difference in oxygen requirements between the anode and cathode places special demands on the battery structure. The gas-diffusion-type cathode can continuously supply oxygen from the air side without increasing the membrane separation structure, while reducing oxygen diffusion to the anode region, which helps to achieve a synergistic match between the anaerobic reaction environment of the anode and the efficient oxygen reduction process of the cathode.

[0025] The beneficial effects of this invention are that it is rationally designed and has the following advantages: 1. This invention differs from traditional cathode electrodes that undergo reduction reactions. Based on the GalOx-catalyzed glycerol oxidation mechanism, a GalOx+HRP dual-enzyme cascade catalytic interface is constructed on the surface of a functional carbon cloth electrode. GalOx catalyzes the oxidation of glycerol and generates H2O2 in situ, while HRP further catalyzes the reduction reaction of H2O2 at the electrode interface, thus forming a GalOx+HRP dual-enzyme cascade electrocatalytic process with H2O2 as an intermediate. This cascade system allows glycerol to undergo oxidation not only at the anode but also participate in enzymatic catalysis at the cathode, thereby expanding the reaction site of glycerol in enzyme biofuel cells. Simultaneously, the constructed cathode has a high open-circuit potential, which is beneficial for improving the open-circuit voltage and energy output performance of the fuel cell.

[0026] 2. This invention introduces MWCNTs to construct a three-dimensional conductive network, improving the electrode's specific surface area, conductivity, and interfacial electron transfer efficiency. PBSE, as an immobilization and linking component, enhances the binding stability between enzyme molecules and carbon nanotubes, which helps reduce enzyme molecule loss and maintain the stability of the catalytic layer structure. Therefore, the enzyme biocathode exhibits high catalytic current response and good operational stability.

[0027] 3. This invention uses functional carbon cloth as a gas diffusion-type enzyme biocathode. Even under anaerobic conditions in the bulk solution, it can continuously obtain O2 from the air or oxygen through the back of the electrode, thereby maintaining the GalOx+HRP cascade catalytic reaction at the cathode. This design, without introducing a membrane separator structure, takes into account both the anode's requirement for an anaerobic environment and the cathode's requirement for oxygen supply, which is beneficial for improving the energy output performance of membrane-free enzyme biofuel cells and promoting the synergistic realization of high-value-added glycerol conversion and electrical energy output.

[0028] In summary, the enzyme biocathode and the membraneless enzyme biofuel cell constructed by the present invention can achieve the coupling of high-value-added conversion of glycerol with electrical energy output. It has the advantages of reasonable structural design, simple preparation method, readily available materials, and good operational stability. It has good application prospects in the fields of glycerol resource utilization, bioelectrocatalytic conversion and enzyme biofuel cells. Attached Figure Description

[0029] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0030] Figure 1 This is a diagram showing the electron transfer pathway of the dual-enzyme biocathode obtained by this invention. Figure 2The graph shows the test results of the HRP / MWCNTs / CC electrode prepared in Comparative Example 1 under different pH conditions at 25℃ and in 0.1 M PBS solution containing 3 mM H2O2, with an applied potential of 0 V vs. Ag / AgCl. Among them, (A) shows the response of the H2O2 catalytic current under different pH conditions, and (B) shows the cyclic voltammetry curves (scan rate of 10 mV / s) under different pH conditions with and without H2O2 substrate. Figure 3 The catalytic current response diagrams of the electrodes prepared in Examples 1, 2 to 4, at 25°C and in 0.1 M pH 7.4 PBS solution containing 1 M glycerol, with an applied potential of 0 V vs. Ag / AgCl; Figure 4 The graph shows the catalytic current response of the GalOx+HRP / MWCNTs / CC electrode prepared in Example 1 at different temperatures when a potential of 0 V vs. Ag / AgCl is applied in a 0.1 M pH 7.4 PBS solution containing 1 M glycerol. Figure 5 The current response of the GalOx+HRP / MWCNTs / CC electrode prepared in Example 1 to different concentrations of glycerol under air atmosphere and O2 saturation conditions is shown in the graph. Figure 6 These are the cyclic voltammetry curves of the GalOx+HRP / MWCNTs / CC electrode prepared in Example 1 under different conditions; Figure 7 The electrochemical response of the GalOx+HRP / MWCNTs / CC electrode prepared in Example 1 to different concentrations of glycerol under 0.1 M PBS (pH 7.4 and pH 8.0) conditions, with an applied potential of 0 V vs. Ag / AgCl; Figure 8 It is GalOx-P Os A schematic diagram of a test setup for a membrane-free enzyme biofuel cell constructed with a / MWCNT-NH2 / GE anode and a GalOx+HRP / MWCNTs / CC gas diffusion cathode. Figure 9 This is an electrochemical response graph of the GalOx+HRP / MWCNTs / CC gas diffusion electrode to different concentrations of glycerol in 0.1 M PBS (pH 7.4) (the solution is flushed with O2). Figure 10 This is an electrochemical response graph of the GalOx+HRP / MWCNTs / CC gas diffusion electrode to different concentrations of glycerol in 0.1 M PBS (pH 7.4) (back face O2). Figure 11This is the stability test curve of the GalOx+HRP / MWCNTs / CC gas diffusion electrode in 0.1 M PBS (pH 7.4); Figure 12 It consists of a GalOx+HRP / MWCNTs / CC gas diffusion cathode and a GalOx-P Os The electrochemical performance test results of the membrane-free enzyme biofuel cell constructed with / MWCNT-NH2 / GE anode in 0.1 M PBS (pH 7.4) solution are shown in the figure. Among them, (A) is the open circuit potential response of the constructed EBFC in the absence and presence of 1 M glycerol, and (B) is the polarization curve and power density curve of the constructed EBFC in the absence and presence of 1 M glycerol. Figure 13 These are chromatograms of the analytical results of the electrocatalytic conversion products of glycerol and standard samples by high performance liquid chromatography (HPLC); among them, (A) is the HPLC analysis result of the electrolyte sample taken at different times after electrolysis of the GalOx+HRP / MWCNTs / CC electrode prepared in Example 1 in 0.1 M pH 7.4 PBS solution containing 0.5 M glycerol at a constant potential of 0 V vs. Ag / AgCl; and (B) is the HPLC analysis result of 0.5 M glyceraldehyde standard. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to the accompanying drawings. These drawings are simplified schematic diagrams, illustrating only the basic structure of the invention, and therefore only show the components relevant to the invention.

[0032] Example 1: A method for preparing a biocathode for oxidizing glycerol to glyceraldehyde specifically includes the following steps: Step S1: Preparation of conductive carrier: 8 mg of MWCNTs were weighed and dispersed in 4 mL of anhydrous ethanol, and sonicated for 6 h to obtain a uniform and stable MWCNTs dispersion. 20 µL of the above MWCNTs dispersion was drop-coated onto carbon cloth (CC) pre-coated with a polytetrafluoroethylene / microporous layer (PTFE / MPL) (geometric area approximately 0.09 cm²). 2 On the surface of the carbon cloth, it is naturally dried to form a first conductive carrier with a MWCNTs modification layer. This step can not only effectively reduce the hydrophobicity of the carbon cloth surface, but also construct a three-dimensional conductive network structure, thereby significantly increasing the electrode specific surface area and enhancing the electron transport capability. Step S2, Introduction of Connecting Molecules and Interface Construction: Weigh 3.9 mg of 1-pyrenebutyrate succinimide ester (PBSE) and dissolve it in 1 mL of... N,NIn dimethylformamide; the carbon cloth electrode with MWCNTs drop-coated as described above was immersed in PBSE solution for about 2 h to obtain the second conductive carrier; at this time, PBSE achieves stable adsorption and fixation through the π-π interaction between the pyrene group and the surface of MWCNTs. At the same time, the succinimide ester group in the PBSE molecule can undergo a covalent coupling reaction with the amino group on the surface of the enzyme molecule, thereby achieving stable fixation of the enzyme molecule and providing a functional interface for the subsequent construction of the enzyme cascade system; after immersion, the second conductive carrier was washed in DMF, n-hexane and 5 mmol / L pH 7.4 PBS to remove residual substances on the surface, thus obtaining the third conductive carrier; Step S3: Prepare the dual-enzyme modified solution: HRP solution (20 mg·mL) -1 Dissolved in 5 mmol / L pH 7.4 phosphate buffer (PBS) and GalOx solution (5 U·µL). -1 The enzyme was dissolved in 5 mmol / L pH 7.4 PBS and mixed at a predetermined mass ratio of 6:1 to prepare a dual-enzyme modified solution. The solution was thoroughly mixed before use to ensure uniform distribution of enzyme molecules in the system and efficiency of subsequent cascade catalysis. Step S4: Preparation of a dual-enzyme biocathode: Take 5 µL of the above dual-enzyme modification solution and drop it onto the surface of the PBSE-functionalized MWCNTs / CC electrode (i.e., the third conductive carrier, modification area: 0.09 cm²). 2 The enzyme molecules were left to stand overnight at 4 °C to achieve full fixation and self-assembly, and finally an enzyme biocathode co-modified with GalOx and HRP was obtained, denoted as GalOx+HRP / MWCNTs / CC.

[0033] Comparative Example 1: The difference from Example 1 is that in steps S3 and S4 of this comparative example, specifically: Step S3: Prepare single-enzyme modification solution: Prepare an HRP solution with a concentration of 5 mg / mL; Step S4: Preparation of single-enzyme biocathode: Take 5 µL of HRP solution and drop it onto the third conductive carrier to prepare the single-enzyme biocathode HRP / MWCNTs / CC electrode.

[0034] Comparative Examples 2 to 4 The difference from Example 1 is that in step S3, preparing the dual-enzyme modified solution, the mixing mass ratio of HRP solution and GalOx solution is different. In Comparative Examples 2 to 4, the mixing mass ratio of GalOx solution to HRP solution is 1:1.25, 1:2.5, and 1:10, respectively.

[0035] Experimental Example 1 To explore the optimal pH value for the synergistic effect of GalOx and HRP in the catalysis of glycerol, it is first necessary to determine the most suitable pH value under the catalytic conditions of either GalOx or HRP alone. Therefore, the HRP / MWCNTs / CC electrode of this comparative example was first prepared.

[0036] The HRP / MWCNTs / CC electrode of this comparative example was tested at 25°C in 0.1 M PBS solution containing 3 mM H2O2 with an applied potential of 0 V vs. Ag / AgCl using a standard three-electrode system under different pH conditions. The results are as follows: Figure 2 The pH values ​​selected in this test were 6.0, 7.0, 7.4 and 8.0, respectively.

[0037] Figure 2 (A) shows the response of the HRP / MWCNTs / CC electrode (Comparative Example 1) to the catalytic current of H2O2 under different pH conditions in 0.1 M PBS solution containing 3 mM H2O2. The results indicate that the electrode exhibits a larger catalytic current response in the PBS buffer at pH 7.0, suggesting that this condition is favorable for the catalytic reduction of H2O2 by HRP. Therefore, pH conditions affect the enzyme activity of HRP and the rate of H2O2 reduction. Figure 2 (B) shows the cyclic voltammetry curves (scan rate 10 mV / s) of the HRP / MWCNTs / CC electrode (Comparative Example 1) in 0.1 M PBS solution containing 3 mM H2O2 under different pH conditions. Figure 2 As shown in (B), the position of the reduction peak and the initial reduction potential of the electrode change accordingly with the change of solution pH, indicating that pH not only affects the magnitude of the catalytic current of HRP, but also affects the potential characteristics of the cathode reaction. For enzyme biofuel cells, a more positive cathode initial reduction potential is generally more conducive to improving the open-circuit voltage and output performance of the cell.

[0038] It should be noted that the catalytic activities of both GalOx and HRP must be considered when designing the cathode for the dual-enzyme cascade. GalOx maintains its activity for glycerol oxidation in a PBS buffer at pH 8.0; while HRP exhibits a high catalytic response to H2O2 reduction near pH 7.0. Therefore, using only pH 7.0 may not fully utilize the catalytic effect of GalOx; using pH 8.0 may reduce the reduction response of HRP to H2O2 or affect the cathode current output. Considering the catalytic current response of HRP to H2O2, the cathode initial reduction potential, and the suitable pH conditions for GalOx's glycerol oxidation reaction, this invention selects a PBS buffer at pH 7.4 as the preferred electrolyte condition for the GalOx / HRP dual-enzyme cascade system. The following... Figure 7The study also demonstrated that the catalytic current of the dual-enzyme electrode in PBS buffer solution at pH 8 was significantly lower than that at pH 7.4.

[0039] Experimental Example 2 To optimize the cascade catalytic efficiency between GalOx and HRP, the effect of the mass ratio of the two enzymes on the electrode catalytic performance was further investigated. The enzyme-modified electrodes prepared in Example 1 and Comparative Examples 2–4 were used as working electrodes, and electrochemical tests were performed at 25°C in 0.1 M PBS buffer containing 1 M glycerol (pH 7.4). During the tests, 0 V vs. Ag / AgCl was applied to the working electrode, and the catalytic current response of the electrode under different GalOx to HRP mass ratios was recorded. The test results are shown below. Figure 3 As shown. By Figure 3 It can be seen that different mass ratios of GalOx to HRP have a significant impact on the catalytic current of the electrode. Specifically, when the mass ratio of GalOx to HRP is 1:6, corresponding to the conditions in Example 1, the electrode exhibits the highest cathode current response, indicating a good synergistic effect between the glycerol oxidation process catalyzed by GalOx and the subsequent catalytic reactions involving HRP at this ratio. When the HRP content is relatively insufficient, the intermediate products generated by the GalOx catalytic reaction cannot be utilized effectively and in a timely manner, potentially limiting the efficiency of the cascade reaction. Conversely, when the HRP content is too high, the enzyme loading composition on the electrode surface becomes unbalanced, which may affect the effective exposure of GalOx, substrate mass transfer, and electron transfer processes at the electrode interface, thus hindering further increases in the catalytic current. Therefore, a suitable mass ratio of GalOx to HRP needs to be maintained to achieve effective connection between the various catalytic steps in the cascade reaction.

[0040] Experimental Example 3 To explore the optimal enzyme catalytic temperature of the electrode prepared in Example 1, the HRP+GalOx / MWCNTs / CC electrode prepared in Example 1 was subjected to catalytic current response tests at different temperatures (25°C, 35°C, and 45°C) and in 0.1 M PBS solution containing 1 M glycerol at pH 7.4, with a potential of 0 V vs. Ag / AgCl. The test results are as follows. Figure 4As shown in the figure, the reaction temperature significantly affects the catalytic current response of the HRP+GalOx / MWCNTs / CC electrode. As the temperature increases from 25℃ to 35℃, the cathode catalytic current response of the electrode increases, indicating that appropriately increasing the temperature is beneficial to improving the enzyme-catalyzed reaction rate, substrate diffusion rate, and catalytic conversion efficiency in the two-enzyme cascade reaction. When the temperature is further increased to 45℃, the cathode catalytic current response of the electrode decreases, possibly because the higher temperature adversely affects the stability of the active sites of GalOx and HRP, leading to a decrease in enzyme catalytic activity and thus reducing the overall cascade catalytic efficiency. Therefore, 35℃ is the more suitable catalytic temperature under the conditions of this embodiment, at which HRP and GalOx exhibit the best catalytic response.

[0041] Test Example 4 To investigate the effects of substrate concentration and oxygen supply conditions on the catalytic performance of the HRP+GalOx / MWCNTs / CC electrode prepared in Example 1, the HRP+GalOx / MWCNTs / CC electrode was placed under air atmosphere and O2 saturation conditions, and gradient current response tests were performed in glycerol solutions of different concentrations. The test system was 0.1 M PBS buffer containing different concentrations of glycerol, the test temperature was 35°C, and the applied potential was 0 V vs. Ag / AgCl. The test results are as follows: Figure 5 As shown.

[0042] Depend on Figure 5 It can be seen that, under both air and O2 saturation conditions, the cathode catalytic current density of the HRP+GalOx / MWCNTs / CC electrode gradually increases with increasing glycerol concentration, indicating that as a substrate for the GalOx catalytic reaction, increasing glycerol concentration is beneficial to enhancing the extent of the GalOx-catalyzed glycerol oxidation reaction. When the glycerol concentration increases to approximately 1 M, the current density tends to plateau, indicating that under these test conditions, the two-enzyme cascade reaction system can gradually approach the substrate concentration-dependent catalytic saturation state. Further comparison of the current response under air and O2 saturation conditions reveals that, at the same glycerol concentration, the cathode catalytic current density under O2 saturation conditions is higher than that under air conditions. Particularly under 1 M glycerol conditions, the current density of the electrode under air conditions is approximately 800 μA·cm. -2 Under O2 saturation conditions, the current density of the electrode can be increased to approximately 1.8 mA·cm⁻¹. -2These results indicate that, in addition to glycerol concentration, the oxygen supply level is also a crucial factor affecting the catalytic performance of this dual-enzyme cascade electrode. This finding is consistent with the cascade catalytic mechanism of HRP and GalOx. During the oxidation of glycerol by GalOx, O2 acts as the electron acceptor, generating H2O2, which is then catalyzed by HRP at the electrode interface. Therefore, the mass transfer and supply of O2 affect the GalOx-catalyzed oxidation and the in-situ generation of H2O2, further influencing the HRP-mediated electrochemical reduction reaction. Thus, oxygen supply capacity is a critical factor affecting the catalytic performance of the HRP+GalOx / MWCNTs / CC enzyme biocathode, and employing an electrode structure that enhances oxygen transport is beneficial for improving the current output performance of this enzyme biocathode.

[0043] Experimental Example 5 To further verify the dual-enzyme cascade catalytic behavior of the HRP+GalOx / MWCNTs / CC electrode prepared in Example 1, the cyclic voltammetric response of this electrode under different substrate concentrations and gas atmospheres was investigated. The HRP+GalOx / MWCNTs / CC electrode prepared in Example 1 was used as the working electrode, and cyclic voltammetry was performed in 0.1 M pH 7.4 PBS buffer at a temperature of 35 °C and a scan rate of 10 mV / s. The test systems included: PBS solution without glycerol under air atmosphere, PBS solution containing 0.1 M glycerol under air atmosphere, PBS solution containing 1 M glycerol under air atmosphere, PBS solution containing 1 M glycerol under O2 saturation, and PBS solution containing 1 M glycerol under Ar atmosphere. The obtained cyclic voltammetric test results are as follows: Figure 6 As shown. By Figure 6It can be seen that, under air atmosphere conditions and without the addition of glycerol substrate, the electrode did not exhibit a significant cathode catalytic current response, indicating that the dual-enzyme cascade catalytic reaction is difficult to proceed effectively without substrate. When 0.1 M glycerol was added to the test solution, a corresponding cathode catalytic current response appeared, indicating that GalOx can catalyze the oxidation of glycerol and generate a detectable electrochemical reduction signal at the electrode interface through its cascade interaction with HRP. As the glycerol concentration increased from 0.1 M to 1 M, the cathode catalytic current of the electrode further increased, indicating that increasing the substrate concentration is beneficial to promoting the GalOx-catalyzed glycerol oxidation process and increasing the amount of intermediate products that can participate in the electrode reaction in subsequent cascade reactions. Furthermore, in the presence of 1 M glycerol, when O2 was introduced into the test solution to create an O2-saturated environment, the cathode catalytic current response of the electrode was further enhanced. This result indicates that the oxygen supply level has a significant impact on the catalytic behavior of the cathode in the dual-enzyme cascade reaction. Conversely, in the presence of 1 M glycerol, when Ar is introduced into the system to remove dissolved oxygen from the solution, the cyclic voltammetric response curve of the electrode exhibits characteristics consistent with the response without the addition of glycerol substrate. This phenomenon indicates that under anaerobic conditions, even in the presence of glycerol substrate, the oxidation of glycerol and the subsequent in-situ generation of H2O2 are limited due to the lack of O2 electron acceptors required for the GalOx catalytic reaction, resulting in a cathode lack of reduction current response.

[0044] Experimental Example 6 To further investigate the continuous catalytic response behavior of the HRP+GalOx / MWCNTs / CC electrode prepared in Example 1 to glycerol, its current response under different conditions was tested using a chronoamperometry method. The tests were conducted at 35°C and 0 V vs. Ag / AgCl; the electrolyte was 0.1 M PBS buffer at pH 7.4 or 8.0. The test results are as follows: Figure 7 As shown.

[0045] Depend on Figure 7 It can be seen that after adding 1 M glycerol, a stable cathode catalytic current is generated at the electrode; when O2 is further introduced into the solution to saturate the system with oxygen, the cathode catalytic current increases significantly, reaching a maximum of approximately 1.6 mA·cm⁻¹. - ². Subsequently, Ar was bubbled into the system to remove dissolved oxygen from the solution, and the current gradually recovered to near its initial level. This result indicates that the catalytic current response of the HRP+GalOx / MWCNTs / CC electrode is closely related to the oxygen supply in the system. This phenomenon is consistent with the dual-enzyme cascade catalytic mechanism of GalOx and HRP. Furthermore, although GalOx typically exhibits high enzymatic activity in PBS buffer at pH 8.0, Figure 7The results showed that the catalytic current obtained at pH 7.4 was higher than that at pH 8.0 in this electrode system. This indicates that the overall current response of the dual-enzyme cascade electrode depends not only on the single enzyme activity of GalOx, but also on the combined effects of HRP activity, the degree of matching between the two enzymes, electron transfer at the electrode interface, and the stability of the enzyme catalytic layer. Further verification confirms that pH 7.4 is more favorable for the HRP+GalOx / MWCNTs / CC electrode to achieve a higher overall cascade catalytic current response.

[0046] Combination Figure 5 , Figure 6 and Figure 7 It can be further seen that the results obtained by different testing methods are consistent. Figure 5 The maximum current response of 1 M glycerol under atmospheric conditions, and Figure 6 Cyclic voltammetric response of 1 M glycerol in air atmosphere and Figure 7 The corresponding chronoampere response after adding 1 M glycerin; Figure 5 The maximum current response under O2 saturation conditions is also related to Figure 6 Cyclic voltammetric response of 1 M glycerol under O2 conditions and Figure 7 The current increase phenomenon after introducing O2 is consistent with that observed after Ar saturation. Figure 6 and Figure 7 All results showed no catalytic response at the electrode, further indicating that oxygen is a crucial factor affecting the cathode catalytic current of this dual-enzyme cascade. The results also show that under ambient air conditions, due to limitations in dissolved oxygen concentration and oxygen mass transfer, the catalytic current of the electrode is lower than that under O2 saturation conditions, approximately 50% of the response under O2 saturation. This indicates that improving the oxygen supply capacity of the cathode region can effectively enhance the catalytic current output of the HRP+GalOx / MWCNTs / CC electrode.

[0047] Experimental Example 7 To further demonstrate that when functional carbon cloth is used as a gas diffusion-type enzyme biocathode, even under anaerobic conditions in the bulk solution, O2 can still be continuously supplied from the air or oxygen through the back of the electrode, thereby maintaining the GalOx+HRP cascade catalytic reaction at the cathode.

[0048] This advantage can be leveraged to combine with a GalOx-catalyzed glycerol oxidation anode to prepare enzyme biofuel cells, enhancing the Faraday effect of glycerol catalysis and improving the production of high-value-added products. Since glycerol oxidation anodes typically require anaerobic conditions to reduce the impact of oxygen on the anodic reaction and the generation of high-value-added products, while the cathode catalytic reaction relies on oxygen, a gas-diffused cathode structure can continuously supply O2 to the cathode side while maintaining an anaerobic environment in the anode region, thus achieving an effective match between the anodic glycerol oxidation reaction and the cathode oxygen reduction reaction. Therefore, the following experimental example demonstrates the catalytic response test of a cathode fabricated as a gas-diffused electrode to glycerol.

[0049] Figure 8 For GalOx-P Os A schematic diagram of a test device for an enzyme biofuel cell constructed with a / MWCNT-NH2 / GE anode and a GalOx+HRP / MWCNTs / CC gas diffusion cathode. The orange box indicates the gas diffusion electrode test device. The gas diffusion cathode is positioned between the gas and liquid phases, with one side of the carbon cloth in contact with the electrolyte and the other side in contact with air or oxygen, thus forming a gas-liquid-solid three-phase reaction interface on the electrode surface. This structure facilitates oxygen transport from the back of the electrode to the enzyme catalytic layer, improving oxygen accessibility at the cathode reaction interface.

[0050] The gas diffusion electrode is fabricated as follows: a carbon cloth with dimensions of 3.5 cm × 3.5 cm is used as the electrode substrate, and only 0.12 cm of the carbon cloth is coated with the electrode. 2 7 μL of dual-enzyme modification solution was added dropwise to a specific region to form the catalytically active region of the GalOx+HRP / MWCNTs / CC enzyme; the unmodified region of the carbon cloth served as a conductive support region and a gas transport channel. This structure ensures contact between the enzyme catalytic layer and the electrolyte, while also utilizing the porous structure of the carbon cloth to facilitate the transfer of gaseous oxygen to the reaction interface.

[0051] To investigate the catalytic response behavior of the GalOx+HRP / MWCNTs / CC electrode to glycerol, the gas diffusion cathode was subjected to chronoamperometry tests in 0.1 M PBS buffer containing different concentrations of glycerol. The pH of the PBS buffer was 7.4, the test temperature was 35℃, and the constant potential was 0 V vs. Ag / AgCl. The test results are as follows: Figure 9 and Figure 10 As shown.

[0052] Depend on Figure 9It can be seen that the gas diffusion cathode can generate corresponding cathode catalytic current responses under different glycerol concentrations, indicating that the electrode can undergo a two-enzyme cascade catalytic reaction of glycerol. When O2 is introduced into the solution to saturate the bulk solution with oxygen, the current response is further enhanced, indicating that increasing the oxygen supply is beneficial to promoting the GalOx-catalyzed oxidation of glycerol and the subsequent HRP-catalyzed electrochemical reduction process. Compared with the aforementioned ordinary submerged electrode, the current response under this condition is slightly lower, which may be related to factors such as the effective reaction interface and oxygen transport path in the gas diffusion electrode testing device. Subsequently, after introducing Ar into the solution to reduce the dissolved oxygen content in the bulk solution, the current response decreased somewhat, but approximately 600 μA·cm was still observed. -2 The cathode catalytic current was measured. This result indicates that even with reduced dissolved oxygen in the bulk solution, the gas diffusion cathode can still obtain a certain amount of oxygen supply from the gas phase side through the back of the carbon cloth, thereby maintaining part of the cathode catalytic reaction. When O2 is further introduced from the back of the electrode, i.e., the side away from the solution, the current response increases significantly again, further demonstrating that the gas diffusion electrode can improve the oxygen supply capacity of the cathode enzyme catalytic interface through back-side oxygen supply.

[0053] Depend on Figure 10 It can be further observed that, in the presence of 1 M glycerol, the current response obtained by supplying O2 from the back of the electrode is higher than that obtained by directly introducing O2 into the solution. This result indicates that, compared with bulk dissolved oxygen diffusion, back-side gas-phase oxygen supply can provide O2 to the carbon cloth / enzyme catalytic layer interface more directly, thereby increasing the local oxygen concentration at the reaction interface and alleviating the mass transfer limitations caused by low oxygen solubility and long diffusion paths in the solution.

[0054] Furthermore, even when Ar is introduced into the solution to remove dissolved oxygen from the bulk phase, the gas-diffused cathode maintains a high current response, indicating that the electrode does not completely depend on dissolved oxygen in the solution, but can utilize O2 in the gas phase on the back side of the electrode to sustain the cathode cascade catalytic process. This result further demonstrates that the constructed gas-diffused GalOx+HRP / MWCNTs / CC cathode can maintain a catalytic response to glycerol under deoxygenated conditions in the bulk solution. In summary, Figure 9 and Figure 10 The results show that O2 mass transfer and supply methods are crucial factors affecting the cathodic catalytic performance of the GalOx+HRP dual-enzyme cascade. The carbon cloth gas diffusion electrode structure enables back-side oxygen supply at the gas-liquid-solid three-phase interface, thereby improving oxygen accessibility at the cathode enzyme catalytic interface. This structure helps resolve the contradiction between the low-oxygen environment at the anode and the oxygen-demanding reaction at the cathode in membrane-free enzyme biofuel cells, providing a structural basis for improving battery output performance and operational stability.

[0055] Experimental Example 8 To investigate the operational stability of the GalOx+HRP / MWCNTs / CC gas diffusion electrode, its continuous current response under constant potential conditions was tested. The test was conducted at 35℃ and 0 V vs. Ag / AgCl. The test results are as follows: Figure 11 As shown.

[0056] Depend on Figure 11 It was found that after adding 1 M glycerol, the gas diffusion electrode produced a significant cathode catalytic current response, and maintained approximately 93% of the initial current response after 30 minutes of continuous operation, indicating that the electrode has good short-term operational stability under the test conditions. The stable current output may be attributed to the effective loading and fixation of GalOx and HRP by the MWCNTs / CC conductive network, and the improvement of the oxygen mass transfer process by the gas diffusion electrode structure. Specifically, MWCNTs / CC facilitates the provision of conductive support and enzyme fixation interface, while the carbon cloth gas diffusion structure continuously supplies O2 from the back of the electrode, maintaining a relatively stable oxygen supply at the cathode enzyme catalytic interface during continuous operation, thus contributing to the maintenance of cascade catalytic reactions and electron transfer processes. Therefore, the constructed GalOx+HRP / MWCNTs / CC gas diffusion electrode exhibits good short-term operational stability in the glycerol electrocatalytic system, indicating its suitability for the subsequent construction of enzyme biocathodes and membrane-free enzyme biofuel cell systems.

[0057] Experimental Example 9 To investigate the gas diffusion cathode composed of GalOx+HRP / MWCNTs / CC and GalOx-P Os The performance of a membrane-free enzyme biofuel cell constructed with a / MWCNT-NH2 / GE anode was tested, including its open-circuit potential and discharge performance. The test system was 0.1 M PBS buffer with a pH of 7.4, and the test temperature was 35℃. The test results are as follows: Figure 12 As shown in the diagram. During the test, Ar was introduced into the anode side to remove dissolved oxygen from the solution; the cathode used a GalOx+HRP / MWCNTs / CC gas diffusion electrode, allowing the cathode to obtain O2 from the air through the back of the electrode to maintain the cathode oxygen reduction and the dual-enzyme cascade catalytic process. This structure can maintain an oxygen-free environment in the anode region without a membrane, while simultaneously meeting the oxygen demand of the cathode, which is beneficial for achieving the synergistic operation of the anode and cathode reactions in a membrane-free enzyme biofuel cell.

[0058] Depend on Figure 12As shown in (A), the open-circuit potential of the membrane-free enzyme biofuel cell was approximately 0.35 V without the addition of glycerol substrate; upon the addition of 1 M glycerol, the open-circuit potential increased to approximately 0.60 V. This result indicates that glycerol, after participating in the catalytic reactions of the anodic and cathode-related enzymes, altered the redox state at the electrode interface, creating a larger potential difference between the anode and cathode, thereby increasing the open-circuit voltage of the cell. Furthermore, Figure 12 (B) shows the output performance of this membrane-free enzyme biofuel cell under 1 M glycerol conditions. The results indicate that the open-circuit potential of the cell is approximately 0.60 V, and the maximum short-circuit current density reaches approximately 0.99 ± 84 mA·cm⁻¹. -2 At a battery voltage of 0.2 V, the maximum power density reaches approximately 139 ± 13 μW·cm. -2 The above results indicate that GalOx-P Os The / MWCNT-NH2 / GE anode catalyzes the glycerol oxidation reaction and provides electrons, while the GalOx+HRP / MWCNTs / CC gas diffusion cathode utilizes the gas-phase supplied O2 to maintain the cathode reaction, thereby achieving glycerol substrate-driven power output.

[0059] at the same time, Figure 12 This also shows that the membrane-free enzyme biofuel cell further constructed based on the enzyme biocathode prepared in Example 1 can realize the simultaneous oxidation of glycerol to glyceraldehyde at both the cathode and anode, thereby improving the conversion rate of glycerol substrate and the Faraday efficiency of the enzyme biofuel cell, while taking into account both the generation of high-value-added products and the output of electrical energy.

[0060] Experimental Example 10 To further verify the ability of the dual-enzyme biocathode prepared in this invention to selectively convert glycerol to glyceraldehyde, the cathode electrolysis products were analyzed by HPLC. Specifically, the GalOx+HRP / MWCNTs / CC electrode prepared in Example 1 was placed in 0.1 M PBS buffer solution (pH 7.4) containing 0.5 M glycerol, and electrolysis was performed under constant potential conditions of 0 V vs. Ag / AgCl. Electrolyte samples were collected at 0 min, 30 min, 1 h, 2 h, and 3 h for analysis. Figure 13 (A) shows the HPLC analysis results of the reaction solution at different electrolysis times. The instrument background peak at retention time of 9.02 min is not used as the basis for product analysis. The chromatographic peak at retention time of 11.09 min corresponds to the target product glyceraldehyde, and the chromatographic peak at retention time of 12.70 min corresponds to the substrate glycerol. The results show that the biocathode can catalyze the selective oxidation of glycerol to glyceraldehyde. Figure 13(B) shows the HPLC analysis results of 0.5 M glyceraldehyde standard. The retention time of its characteristic peak is consistent with the chromatographic peak at 11.05 min in the electrolysis product, which further illustrates that the GalOx+HRP / MWCNTs / CC electrode can achieve the selective conversion of glycerol to the high-value-added product glyceraldehyde.

[0061] In summary, this invention utilizes the mechanism of GalOx-catalyzed glycerol conversion and employs an enzyme cascade reaction to prepare a structurally simple enzyme biocathode that can specifically convert glycerol to glyceraldehyde while exhibiting a high onset potential. This enzyme biocathode consists of a catalytic enzyme component, a conductive material component, and an immobilization and linking component. These components work synergistically to achieve the oxidative conversion of glycerol, rapid electron transfer, and stable immobilization of enzyme molecules at the cathode, thus balancing the generation of high-value-added products with electrical energy output. This solves the problem in existing technologies where glycerol can only be oxidized to glyceraldehyde at the anode and not at the cathode, further improving the Faraday efficiency of the specific conversion of glycerol substrate to glyceraldehyde.

[0062] Based on the above-described preferred embodiments of the present invention, and through the above description, those skilled in the art can make various changes and modifications without departing from the technical concept of the present invention. The technical scope of the present invention is not limited to the contents of the specification.

Claims

1. A method for preparing a biological cathode for the oxidation of glycerol to glyceraldehyde, characterized in that: Specifically, the steps include the following: Step S1: Preparation of conductive carrier: MWCNTs were placed in ethanol and ultrasonically dispersed to obtain a MWCNTs dispersion; then the MWCNTs dispersion was coated on a substrate to obtain a first conductive carrier modified with MWCNTs. Step S2, Introduction of Connecting Molecules and Interface Construction: Dissolve PBSE in DMF to prepare PBSE solution; immerse the first conductive carrier obtained in step S1 into PBSE solution to prepare the second conductive carrier; after immersion, wash the second conductive carrier in DMF, n-hexane and PBS solution in sequence to remove surface residues to prepare the third conductive carrier. Step S3: Prepare the dual-enzyme modified solution: HRP solution and GalOx solution were mixed evenly to prepare a dual-enzyme modified solution; Step S4: Preparation of enzyme biocathode: The third conductive carrier obtained in step S2 is coated with the dual-enzyme modification solution prepared in step S3, and after standing, the finished enzyme biocathode co-modified with GalOx and HRP can be obtained.

2. The method for preparing a biocathode for the oxidation of glycerol to glyceraldehyde according to claim 1, characterized in that: In step S1, the concentration of MWCNTs in the MWCNTs dispersion is 2 mg / mL.

3. The method for preparing a biocathode for the oxidation of glycerol to glyceraldehyde according to claim 1, characterized in that: In step S1, each cm 2 The substrate was coated with 222.3 µL of MWCNT dispersion.

4. The method for preparing a biological cathode for the oxidation of glycerol to glyceraldehyde according to claim 1, characterized in that: In step S1, the substrate is carbon cloth coated with PTFE / MPL, and the MWCNTs dispersion is coated on the PTFE / MPL surface.

5. The method for preparing a biocathode for the oxidation of glycerol to glyceraldehyde according to claim 1, characterized in that: In step S2, the first conductive carrier is immersed in the PBSE solution for 2 hours.

6. The method for preparing a biocathode for the oxidation of glycerol to glyceraldehyde according to claim 1, characterized in that: In step S3, the mass ratio of HRP solution to GalOx solution is 6:

1.

7. The method for preparing a biocathode for the oxidation of glycerol to glyceraldehyde according to claim 1, characterized in that: In step S4, each cm 2 The third conductive carrier was coated with 55.6 µL of dual-enzyme modification solution.

8. The method for preparing a biocathode for the oxidation of glycerol to glyceraldehyde according to claim 1, characterized in that: In step S4, the settling temperature is 4°C.

9. A biological cathode for the oxidation of glycerol to glyceraldehyde, characterized in that: The enzyme biocathode is prepared by the method described in any one of claims 1 to 8.

10. An application of a biocathode for the oxidation of glycerol to glyceraldehyde as described in claim 9, characterized in that: The biocathode is used in the manufacture of membrane-free enzyme fuel cells.

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