Bilirubin oxidase / modified carbon cloth cathode material and preparation method and application thereof

Abstract

The invention relates to the technical field of cathode materials, in particular to a bilirubin oxidase / modified carbon cloth cathode material as well as a preparation method and application thereof, the modified carbon cloth cathode material is prepared by modifying an aminated multi-walled carbon nanotube loaded with 2, 2 '-biazole-bis-3-ethylbenzothiazoline-6-sulfonic acid on carbon cloth, and modifying the aminated multi-walled carbon nanotube loaded with 2, 2'-biazole-bis-3-ethylbenzothiazoline-6-sulfonic acid on the carbon cloth by using 2, 2 '-biazole-bis-3-ethylbenzothiazoline-6-sulfonic acid. 2, 2 '-biazo-bis-3-ethylbenzothiazoline-6-sulfonic acid and the aminated multi-walled carbon nanotubes are combined by adopting pi-pi stacking and electrostatic interaction, and the aminated multi-walled carbon nanotubes and the carbon cloth are combined through hydrophobicity, so that the active specific surface area of the carbon cloth is increased; in addition, bilirubin oxidase is loaded on the modified carbon cloth cathode material, and due to the fact that the hydrophobicity of the aminated multi-walled carbon nanotubes is lower than that of the carbon cloth, adhesion of the bilirubin oxidase is allowed, the loading capacity of the bilirubin oxidase is increased, and the catalytic oxygen reduction efficiency of the biological cathode is improved.

Description

Technical Field

[0001] The invention relates to the technical field of cathode materials, in particular to a bilirubin oxidase / modified carbon cloth cathode material and a preparation method and application thereof. Background Art

[0002] Over the past few decades, enzymatic biofuel cells have attracted increasing attention due to their ability to operate under mild conditions and use renewable biomass as fuel, which makes them particularly suitable for a variety of applications including self-powered biosensors, point-of-care devices, portable or wearable power sources, and even for high-performance sustainable energy conversion systems.

[0003] In most enzymatic biofuel cells, O2 is introduced as the oxidant for the biocathode reaction due to its high abundance and accessibility to air. The typical cathode catalyst in enzymatic biofuel cells is multi-copper oxidase, which exhibits a high turnover rate for reducing molecular oxygen to water. Bilirubin oxidase can catalyze the direct reduction of oxygen to water molecules with 4 electrons under extremely low overpotential conditions, and compared with other types of multi-copper oxidases, it can also show high catalytic activity under neutral conditions, which has a wider range of adaptability when used to construct the biocathode of enzymatic biofuel cells. However, the solubility of O2 is only 0.3mmol / L, and its low solubility leads to limited substrate content reaching the biocathode in the electrolyte, which limits the current generated by the biocathode. Therefore, one of the key challenges in the development of high-performance enzymatic biofuel cells is to increase the local concentration of O2 at the biocathode. The traditional method is to achieve forced convection by using a rotating disk electrode to overcome the limitation of mass transport, but this method requires more complex equipment, thus hindering its integration into technology-related miniaturized devices.

[0004] In addition, the prior art discloses a gas breathing electrode constructed based on bilirubin oxidase. The surface of the carbon cloth currently on the market is extremely hydrophobic, which makes it difficult to fix and modify the bilirubin oxidase on its surface, and it is also difficult to establish an effective electron transfer channel between the bilirubin oxidase and the substrate surface. Therefore, it is difficult to construct an effective biological cathode for oxygen reduction, let alone construct an effective enzymatic biofuel cell. Summary of the invention

[0005] In view of the deficiencies in the above-mentioned prior art, the present invention uses inexpensive and readily available carbon cloth as a raw material, provides a bilirubin oxidase / modified carbon cloth cathode material and a preparation method and application thereof. The present invention first loads 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid on an aminated multi-walled carbon nanotube to obtain a 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid / aminated multi-walled carbon nanotube cathode material, and then modifies the surface of the carbon cloth to obtain a modified carbon cloth cathode material. Then, using the modified carbon cloth cathode material as a substrate, bilirubin oxidase is loaded on the modified carbon cloth cathode material to obtain a bilirubin oxidase / modified carbon cloth cathode material. Since the hydrophobicity of the aminated multi-walled carbon nanotubes is lower than that of the carbon cloth, the bilirubin oxidase is allowed to attach, thereby increasing the enzyme loading and improving the catalytic current of the bilirubin oxidase / modified carbon cloth cathode material, thereby overcoming the problem that the bilirubin oxidase in the prior art cannot attach to the surface of the carbon cloth; the present invention also constructs the bilirubin oxidase / modified carbon cloth cathode material into a gas breathing electrode, and then the gas breathing electrode and the anode electrode are jointly constructed into an enzymatic biofuel cell, in which an anaerobic bioanode electrode can be used, while increasing the local concentration of O2 at the biocathode, thereby overcoming the problem that the enzymatic biofuel cell can only be assembled with an aerobic bioanode electrode.

[0006] The present invention is achieved through the following technical solutions: The preparation method of bilirubin oxidase / modified carbon cloth cathode material comprises the following steps: 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid is dissolved in water to prepare a 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid aqueous solution.

[0007] Aminated multi-walled carbon nanotubes were added to an aqueous solution of 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid, mixed evenly, and then allowed to stand, 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid was adsorbed by the aminated multi-walled carbon nanotubes, and then centrifuged and dried to obtain a 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid / aminated multi-walled carbon nanotube cathode material.

[0008] The 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid / aminated multi-walled carbon nanotube cathode material is dispersed in ethanol, then coated on a carbon cloth, and then dried at room temperature to obtain a modified carbon cloth cathode material.

[0009] A PBS buffer solution containing bilirubin oxidase is coated on the modified carbon cloth cathode material, and then dried to obtain a bilirubin oxidase / modified carbon cloth cathode material.

[0010] Preferably, the mass ratio of the aminated multi-walled carbon nanotubes to 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid is 5:2.2×10 -3 ~41×10 -3 A ratio greater than this value will result in too little 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid fixed on the amino-modified multi-walled carbon nanotubes, which cannot well realize the function of electron transfer mediator. A ratio less than this value will result in an increase in the amount of free 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid not bound to the amino-modified multi-walled carbon nanotubes, resulting in a waste of samples.

[0011] Preferably, the loading amount of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid / aminated multi-walled carbon nanotube cathode material on the carbon cloth is 0.12 mg / cm 2 ~0.96mg / cm 2 A loading amount greater than this will result in the thickness of the modified carbon cloth cathode material being too large and the electrode stability being poor; a loading amount less than this will reduce the loading amount of bilirubin oxidase per unit area, resulting in a low cathode catalytic current.

[0012] Preferably, the PBS buffer solution is a mixture of NaH2PO4 solution and Na2HPO4 solution, and the pH of the PBS buffer solution is 7.0.

[0013] Preferably, the mass ratio of the modified carbon cloth cathode material to the bilirubin oxidase is 12:2 to 30. If the ratio is greater than this ratio, the bilirubin oxidase loading will be insufficient, resulting in low catalytic efficiency of oxygen reduction; if the ratio is less than this ratio, the bilirubin oxidase will be overloaded. Since the bilirubin oxidase is a non-conductive substance, it will reduce the conductivity of the constructed biological cathode, thereby causing low catalytic efficiency of oxygen reduction. In addition, the use of excessive bilirubin oxidase will increase the manufacturing cost of the cathode electrode.

[0014] The present invention also protects the bilirubin oxidase / modified carbon cloth cathode material prepared by the above preparation method. The modified carbon cloth cathode material is prepared by modifying the surface of the carbon cloth with amino multi-walled carbon nanotubes loaded with 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid.

[0015] The present invention also protects the use of the above-mentioned bilirubin oxidase / modified carbon cloth cathode material in the preparation of an enzymatic biofuel cell cathode electrode piece. The enzymatic biofuel cell comprises a bilirubin oxidase / modified carbon cloth cathode material, a biological anode electrode piece and an anode fuel. The biological anode electrode piece is selected from an aerobic biological anode electrode piece or an anaerobic biological anode electrode piece.

[0016] Preferably, the bioanode electrode piece is selected from an aerobic bioanode electrode piece or an anaerobic bioanode electrode piece.

[0017] Preferably, when the biological anode electrode is selected from an aerobic biological anode electrode, the application method is: using bilirubin oxidase / modified carbon cloth cathode material as the cathode electrode, immersing one end of the cathode electrode and the aerobic biological anode electrode in an electrolyte containing an anode fuel, and electrically connecting the other ends to a power source to construct an enzymatic biofuel cell.

[0018] Preferably, when the biological anode electrode is selected from an anaerobic biological anode electrode, the application method is: using bilirubin oxidase / modified carbon cloth cathode material as the cathode electrode, constructing the cathode electrode into a gas breathing electrode, immersing one end of the anaerobic biological anode electrode in an electrolyte containing an anode fuel, and electrically connecting the other end of the anaerobic biological anode electrode and the cathode electrode to a power source to construct an enzymatic biofuel cell.

[0019] The assembly method is as follows: two beakers with connecting ports are symmetrically arranged, and a cathode electrode is placed between the two connecting ports. After the two beakers are clamped and stabilized, anode fuel is added to one of the beakers, and one end of the anaerobic biological anode electrode is immersed in the anode fuel. At this time, one beaker contains anode fuel and the other beaker does not contain anode fuel. The modified surface of the cathode electrode is in contact with the anode fuel, and its back is exposed to the air. The other end of the anaerobic biological anode electrode and the cathode electrode are electrically connected to a power source to construct an enzymatic biological fuel cell.

[0020] Preferably, when the anode electrode is galactose oxidase / redox polymer / glassy carbon electrode, the anode fuel is galactose; when the anode electrode is glucose oxidase / redox polymer / glassy carbon electrode, the anode fuel is glucose.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention adopts a simple method to prepare a modified carbon cloth cathode material. First, 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid is loaded on an amination multi-walled carbon nanotube. The 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid and the amination multi-walled carbon nanotube are stably combined due to π-π stacking to obtain a 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid / amination multi-walled carbon nanotube cathode material; then, 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid is loaded on an amination multi-walled carbon nanotube. Ethylbenzothiazoline-6-sulfonic acid / aminated multi-walled carbon nanotube cathode material is modified on the surface of carbon cloth. Aminated multi-walled carbon nanotubes and carbon cloth are stably combined due to their hydrophobicity, forming a substrate with high stability, low cost, high specific surface area, and easy surface binding of biocatalyst-bilirubin oxidase, thereby obtaining a modified carbon cloth cathode material. Then, using the modified carbon cloth cathode material as a substrate, bilirubin oxidase is loaded on the modified carbon cloth cathode material, thereby obtaining a bilirubin oxidase / modified carbon cloth cathode material.

[0022] 2. The present invention uses aminated multi-walled carbon nanotubes as raw materials because: multi-walled carbon nanotubes are highly hydrophobic, and compared with multi-walled carbon nanotubes, the presence of -NH2 reduces the hydrophobicity of aminated multi-walled carbon nanotubes; in addition, the electrostatic interaction between aminated multi-walled carbon nanotubes and 2,2'-azo-bis-3-ethylbenzothiazoline-6-sulfonic acid is more stable than the combination of multi-walled carbon nanotubes and 2,2'-azo-bis-3-ethylbenzothiazoline-6-sulfonic acid. Therefore, the present invention uses aminated multi-walled carbon nanotubes as carrier materials. On the one hand, because aminated multi-walled carbon nanotubes are hydrophobic, they can be stably combined with carbon cloth due to hydrophobic interaction, thereby increasing the active specific surface area; on the other hand, aminated multi-walled carbon nanotubes can be stably combined with 2,2'-azo-bis-3-ethylbenzothiazoline-6-sulfonic acid through π-π stacking and electrostatic forces.

[0023] In order to improve the performance of enzymatic biofuel cells and improve their output power, the present invention optimizes the cathode geometry by using aminated multi-walled carbon nanotubes to increase the active specific surface area. Aminated multi-walled carbon nanotubes are highly porous and provide a large specific surface area, allowing a high amount of enzyme loading, and are excellent carrier materials for enzyme immobilization. In addition, compared with carbon cloth, the hydrophobicity of the modified carbon cloth cathode material is greatly reduced, allowing bilirubin oxidase to attach and modify the surface of the modified carbon cloth cathode material, thereby increasing the loading amount of bilirubin oxidase on the cathode surface, overcoming the technical defect that the carbon cloth surface is difficult to modify with bilirubin oxidase due to its strong hydrophobicity, and improving the catalytic performance of the biological cathode.

[0024] 3. For bilirubin oxidase modified on the substrate surface, the electron transfer mechanism between it and the electrode is divided into direct electron transfer mechanism or mediated electron transfer mechanism. Under the direct electron transfer mechanism, the T1 site of bilirubin oxidase directly transfers electrons with the electrode surface, thereby performing an electrocatalytic oxygen reduction reaction. However, since the electron transfer rate decreases exponentially with the increase of the electron transfer distance, electron transfer can no longer be performed after the distance exceeds 14Å. Only the bilirubin oxidase with the T1 site facing the electrode in the electrode surface monolayer can effectively transfer electrons with the electrode surface. Even if other bilirubin oxidases are stably present on the substrate surface, they cannot perform their electrocatalytic oxygen reduction function. Therefore, it is very important to control the directional modification of bilirubin oxidase on the electrode substrate surface.

[0025] In the bilirubin oxidase / modified carbon cloth cathode material constructed by the present invention, the bilirubin oxidase presents a negative charge as a whole under neutral conditions, while the T1 site region presents a positive charge locally. The negatively charged 2,2'-azo-bis-3-ethylbenzothiazoline-6-sulfonic acid on the surface of the modified carbon cloth cathode material is electrostatically combined with the positively charged region of the T1 site of the bilirubin oxidase, thereby achieving uniform orientation modification of the bilirubin oxidase on the surface of the modified carbon cloth cathode material. In addition, here, 2,2'-azo-bis-3-ethylbenzothiazoline-6-sulfonic acid not only serves as an auxiliary agent for assisting the uniform orientation modification of the bilirubin oxidase, but also can serve as an electron transfer mediator to accelerate the interface electron transfer kinetics between the T1 site of the bilirubin oxidase and the surface of the modified carbon cloth cathode material. Therefore, the present invention overcomes the technical defect that the prior art cannot achieve uniform orientation modification of the bilirubin oxidase on the electrode surface, and also overcomes the technical defect that the electron transfer rate between the bilirubin oxidase and the electrode surface is slow. The bilirubin oxidase / modified carbon cloth cathode material prepared based on the present invention has high-efficiency catalytic oxygen reduction performance.

[0026] 4. The bilirubin oxidase / modified carbon cloth cathode material of the present invention is a gas breathing electrode, which has the advantages that: with the modified carbon cloth cathode material as the substrate, the carbon cloth can achieve back breathing, and the back breathing method allows oxygen in the air to reach the vicinity of the bilirubin oxidase, overcoming the mass transfer limitation of oxygen reaching the electrode surface caused by the low solubility of oxygen in the electrolyte, and further increasing the catalytic current of the bilirubin oxidase / modified carbon cloth cathode material. In addition, the back breathing method can also use an oxygen-free electrolyte in the process of constructing a biofuel cell, which expands the range of choices for anode materials that can be assembled with the bilirubin oxidase / modified carbon cloth cathode material for enzymatic biofuel cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The cyclic voltammograms of ABTS / MCNT-NH2 / carbon cloth in Example 1 after 2, 50, 100, 150, 200 and 250 cycles in a PC solution with a pH of 7.0; ABTS / MCNT-NH2 / carbon cloth is a modified carbon cloth cathode material.

[0028] Figure 2 The cyclic voltammograms of ABTS / MCNT-NH2 / carbon cloth of Example 1 in PC solutions with pH values ​​of 5, 6, 7, and 8, respectively.

[0029] Figure 3Cyclic voltammograms of electrocatalytic reduction of O2 by BOD / carbon cloth, BOD / MCNT-NH2 / carbon cloth, ABTS / MCNT-NH2 / carbon cloth of Example 1 and BOD / ABTS / MCNT-NH2 / carbon cloth in PC solution of pH 7.0 filled with O2; BOD / carbon cloth is bilirubin oxidase / carbon cloth, BOD / MCNT-NH2 / carbon cloth is bilirubin oxidase / aminated multi-walled carbon nanotubes / carbon cloth, and BOD / ABTS / MCNT-NH2 / carbon cloth is bilirubin oxidase / modified carbon cloth cathode material.

[0030] Figure 4 Cyclic voltammograms of BOD / ABTS / MCNT-NH2 / carbon cloth in PC solutions with pH values ​​of 5, 6, 7, and 8 filled with O2 and in PC solution with pH value of 8 filled with Ar.

[0031] Figure 5 The figure is a diagram of the device and a schematic diagram of the reduction of O2 in the air, where: Figure 5 Figure (a) is a physical picture of the three-electrode system device in which BOD / ABTS / MCNT-NH2 / carbon cloth is used as a conventional electrode for measurement; Figure 5 Figure (b) shows a physical picture of the device in which BOD / ABTS / MCNT-NH2 / carbon cloth is tested as a gas breathing electrode; Figure 5 Figure (c) is a schematic diagram of BOD / ABTS / MCNT-NH2 / carbon cloth used as a gas breathing electrode to reduce O2 in the air.

[0032] Figure 6 The cyclic voltammetry curves of BOD / ABTS / MCNT-NH2 / carbon cloth as a conventional electrode immersed in a PC solution with a pH of 5.0 for O2 reduction measurement are compared with the cyclic voltammetry curves of BOD / ABTS / MCNT-NH2 / carbon cloth as a gas breathing electrode for O2 reduction measurement in a PC solution with a pH of 5.0.

[0033] Figure 7 This is a comparison of the cyclic voltammetry curves of BOD / ABTS / MCNT-NH2 / carbon cloth as a gas breathing electrode in an oxygen-free pH 5.0 PC solution, and the back side of BOD / ABTS / MCNT-NH2 / carbon cloth in air atmosphere and oxygen atmosphere respectively.

[0034] Figure 8 This is a chronoamperometric curve of O2 reduction stability measurement using BOD / MCNT-NH2-ABTS / carbon cloth as a conventional electrode immersed in a PC solution with a pH of 5.0, and a comparison chart of the chronoamperometric curve of O2 reduction stability measurement using BOD / ABTS / MCNT-NH2 / carbon cloth as a gas breathing electrode in a PC solution with a pH of 5.0.

[0035] Fig. 9 is the SEM picture, where Fig. 9 Figure a is the SEM image of bare carbon cloth. Fig. 9 Figure b is the SEM image of MCNT-NH2 / carbon cloth. Fig. 9 Figure c is the SEM image of ABTS / MCNT-NH2 / carbon cloth in Example 1, and MCNT-NH2 / carbon cloth is amino-modified multi-walled carbon nanotube / carbon cloth. DETAILED DESCRIPTION

[0036] The specific implementation of the present invention is described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific implementation. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Taking into account the technical defects of the prior art using rotating disk electrodes, the present invention has conducted research based on gas diffusion electrodes. The gas diffusion electrode ensures the formation of a three-phase boundary on the electrode surface, namely the electrolyte / catalyst / gas boundary, and effectively increases the O2 flux through gas breathing on the back of the electrode, thereby overcoming the limitation of the mass transfer rate and increasing the catalytic current of the biocathode. At the same time, the gas breathing electrode developed based on the gas diffusion electrode can also be assembled without a diaphragm with the bioanode that needs to work in an oxygen-free environment, and has a wider range of applications.

[0038] Considering that the surface of carbon cloth used for fuel cell construction in the prior art is extremely hydrophobic, it is difficult to fix and modify bilirubin oxidase on its surface, which makes it difficult to establish an effective electron transfer channel. In the present invention, the surface of carbon cloth is modified by aminated multi-walled carbon nanotubes loaded with 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid. ABTS not only serves as an auxiliary agent to assist bilirubin oxidase in achieving uniform orientation modification, but also serves as an electron transfer mediator to accelerate the interfacial electron transfer kinetics between the T1 site of bilirubin oxidase and the surface of the ABTS / MCNT-NH2 / carbon cloth cathode material substrate. The aminated multi-walled carbon nanotubes increase the active specific surface area, and because the aminated multi-walled carbon nanotubes are less hydrophobic than the carbon cloth, the attachment of bilirubin oxidase is allowed, the enzyme loading amount is increased, and the catalytic current of the biological cathode is improved.

[0039] Using bilirubin oxidase / modified carbon cloth cathode material as the oxygen reduction cathode, efficient catalytic reduction of O2 was achieved and a high current density was obtained.

[0040] In the present invention, MCNT represents multi-walled carbon nanotubes, MCNT-NH2 represents amino-modified multi-walled carbon nanotubes, ABTS represents 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid, and BOD represents bilirubin oxidase.

[0041] The technical solution of the present invention is further explained by using embodiments below, which are specifically as follows: Example 1 The preparation method of BOD / ABTS / MCNT-NH2 / carbon cloth cathode material comprises the following steps: S1. Preparation of ABTS / MCNT-NH2 cathode material: 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid was prepared into a 5 mmol / L ABTS aqueous solution with pure water, 5 mg of MCNT-NH2 was weighed, and MCNT-NH2 was placed in 5 mL, 5 mmol / L ABTS aqueous solution, stirred with a magnetic stirrer for 2 h, and then allowed to stand for 22 h. At this time, the mass ratio of aminated multi-walled carbon nanotubes to 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid was 5:13.72×10 -3 , and then centrifuged for 5 minutes at a speed of 6000 rpm. After removing the supernatant, the precipitate was dissolved in pure water, shaken, centrifuged again and the supernatant was removed to remove the loosely bound ABTS. Finally, the precipitate was dried at room temperature to prepare a dry ABTS / MCNT-NH2 cathode material.

[0042] S2. Preparation of ABTS / MCNT-NH2 / carbon cloth cathode material: The ABTS / MCNT-NH2 cathode material was dissolved in ethanol and then ultrasonically dispersed for 3 hours to prepare a 2 mg / mL ABTS / MCNT-NH2 cathode material dispersion.

[0043] The carbon cloth was first rinsed with ethanol, then rinsed with pure water, dried at room temperature, and then cut into a size of 0.5 cm×1.5 cm to obtain a treated carbon cloth.

[0044] Take 60 μL of ABTS / MCNT-NH2 cathode material dispersion and apply it on the treated carbon cloth. The coating area is 0.5 cm × 0.5 cm. Dry it at room temperature. At this time, the loading amount of ABTS / MCNT-NH2 cathode material on the carbon cloth is 0.48 mg / cm 2 , and obtain ABTS / MCNT-NH2 / carbon cloth cathode material, referred to as ABTS / MCNT-NH2 / carbon cloth.

[0045] Preparation of S3, BOD / ABTS / MCNT-NH2 / carbon cloth cathode material: The purchased bilirubin oxidase powder was dissolved in 5 mmol / L PBS buffer solution with a pH of 7.0 to prepare a 10 mg / mL bilirubin oxidase solution.

[0046] The preparation method of 5mmol / L PBS buffer solution with a pH of 7.0 is as follows: 5mmol / L NaH2PO4 solution and 5mmmol / L Na2HPO4 solution are mixed to prepare a PBS buffer solution with a pH of 7.0.

[0047] 5 μL of 10 mg / mL bilirubin oxidase solution was added to the ABTS / MCNT-NH2 / carbon cloth cathode material, and then placed in a 4°C refrigerator to dry. At this time, the mass ratio of ABTS / MCNT-NH2 / carbon cloth cathode material to bilirubin oxidase was 12:5, and BOD / ABTS / MCNT-NH2 / carbon cloth cathode material was obtained, referred to as BOD / ABTS / MCNT-NH2 / carbon cloth.

[0048] Example 2 The preparation method of BOD / ABTS / MCNT-NH2 / carbon cloth cathode material comprises the following steps: S1. Preparation of ABTS / MCNT-NH2 cathode material: 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid was prepared into 0.8 mmol / L ABTS aqueous solution with pure water, 5 mg of MCNT-NH2 was weighed, MCNT-NH2 was placed in 5 mL, 0.8 mmol / L ABTS aqueous solution, stirred with a magnetic stirrer for 2 h, and then allowed to stand for 22 h. At this time, the mass ratio of aminated multi-walled carbon nanotubes to 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid was 5:2.2×10 -3 , and then centrifuged for 5 minutes at a speed of 6000 rpm. After removing the supernatant, the precipitate was dissolved in pure water, shaken, centrifuged again and the supernatant was removed to remove the loosely bound ABTS. Finally, the precipitate was dried at room temperature to prepare a dry ABTS / MCNT-NH2 cathode material.

[0049] S2. Preparation of ABTS / MCNT-NH2 / carbon cloth cathode material: The ABTS / MCNT-NH2 cathode material was dissolved in ethanol and then ultrasonically dispersed for 3 hours to prepare a 2 mg / mL ABTS / MCNT-NH2 cathode material dispersion.

[0050] The carbon cloth was first rinsed with ethanol, then rinsed with pure water, dried at room temperature, and then cut into a size of 0.5 cm×1.5 cm to obtain a treated carbon cloth.

[0051] Take 60 μL of ABTS / MCNT-NH2 cathode material dispersion and apply it on the treated carbon cloth. The coating area is 0.5 cm × 0.5 cm. Dry it at room temperature. At this time, the loading amount of ABTS / MCNT-NH2 cathode material on the carbon cloth is 0.12 mg / cm 2, and obtain ABTS / MCNT-NH2 / carbon cloth cathode material.

[0052] Preparation of S3, BOD / ABTS / MCNT-NH2 / carbon cloth cathode material: The purchased bilirubin oxidase powder was dissolved in 5 mmol / L PBS buffer solution with a pH of 7.0 to prepare a 10 mg / mL bilirubin oxidase solution.

[0053] The preparation method of 5mmol / L PBS buffer solution with a pH of 7.0 is as follows: 5mmol / L NaH2PO4 solution and 5mmmol / L Na2HPO4 solution are mixed to prepare a PBS buffer solution with a pH of 7.0.

[0054] 5 μL of 10 mg / mL bilirubin oxidase solution was added to the ABTS / MCNT-NH2 / carbon cloth cathode material, and then placed in a 4°C refrigerator to dry. At this time, the mass ratio of ABTS / MCNT-NH2 / carbon cloth cathode material to bilirubin oxidase was 12:2, and BOD / ABTS / MCNT-NH2 / carbon cloth cathode material was obtained.

[0055] Example 3 The preparation method of BOD / ABTS / MCNT-NH2 / carbon cloth cathode material comprises the following steps: S1. Preparation of ABTS / MCNT-NH2 cathode material: 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid was prepared into a 15 mmol / L ABTS aqueous solution with pure water, 5 mg of MCNT-NH2 was weighed, and MCNT-NH2 was placed in 5 mL, 15 mmol / L ABTS aqueous solution, stirred with a magnetic stirrer for 2 h, and then allowed to stand for 22 h. At this time, the mass ratio of aminated multi-walled carbon nanotubes to 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid was 5:41×10 -3 , and then centrifuged for 5 minutes at a speed of 6000 rpm. After removing the supernatant, the precipitate was dissolved in pure water, shaken, centrifuged again and the supernatant was removed to remove the loosely bound ABTS. Finally, the precipitate was dried at room temperature to prepare a dry ABTS / MCNT-NH2 cathode material.

[0056] S2. Preparation of ABTS / MCNT-NH2 / carbon cloth cathode material: The ABTS / MCNT-NH2 cathode material was dissolved in ethanol and then ultrasonically dispersed for 3 hours to prepare a 2 mg / mL ABTS / MCNT-NH2 cathode material dispersion.

[0057] The carbon cloth was first rinsed with ethanol, then rinsed with pure water, dried at room temperature, and then cut into a size of 0.5 cm×1.5 cm to obtain a treated carbon cloth.

[0058] Take 60 μL of ABTS / MCNT-NH2 cathode material dispersion and apply it on the treated carbon cloth. The coating area is 0.5 cm × 0.5 cm. Dry it at room temperature. At this time, the loading amount of ABTS / MCNT-NH2 cathode material on the carbon cloth is 0.96 mg / cm 2 , and obtain ABTS / MCNT-NH2 / carbon cloth cathode material.

[0059] Preparation of S3, BOD / ABTS / MCNT-NH2 / carbon cloth cathode material: The purchased bilirubin oxidase powder was dissolved in 5 mmol / L PBS buffer solution with a pH of 7.0 to prepare a 10 mg / mL bilirubin oxidase solution.

[0060] The preparation method of 5mmol / L PBS buffer solution with a pH of 7.0 is as follows: 5mmol / L NaH2PO4 solution and 5mmmol / L Na2HPO4 solution are mixed to prepare a PBS buffer solution with a pH of 7.0.

[0061] 5 μL of 10 mg / mL bilirubin oxidase solution was added to the ABTS / MCNT-NH2 / carbon cloth cathode material, and then placed in a 4°C refrigerator to dry. At this time, the mass ratio of ABTS / MCNT-NH2 / carbon cloth cathode material to bilirubin oxidase was 12:30, and BOD / ABTS / MCNT-NH2 / carbon cloth cathode material was obtained.

[0062] Examples 1 to 3 of the present invention all prepared BOD / ABTS / MCNT-NH2 / carbon cloth cathode materials. The BOD / ABTS / MCNT-NH2 / carbon cloth of Example 1 is used as an example for research. The specific research method and results are as follows: 1. Preparation of working electrode: Taking the BOD / ABTS / MCNT-NH2 / carbon cloth of Example 1 as an experimental example, and taking BOD / carbon cloth, BOD / MCNT-NH2 / carbon cloth and ABTS / MCNT-NH2 / carbon cloth as control examples, a comparative study was conducted as working electrodes. BOD / carbon cloth and BOD / MCNT-NH2 / carbon cloth were prepared according to the following steps: 1. Preparation of BOD / carbon cloth: S1. Dissolve the purchased bilirubin oxidase powder in 5 mmol / L PBS buffer solution with a pH of 7.0 to prepare a 10 mg / mL bilirubin oxidase solution.

[0063] The preparation method of the 5mmol / L PBS buffer solution with a pH of 7.0 is as follows: 5mmol / L NaH2PO4 solution and 5mmmol / L Na2HPO4 solution are mixed to prepare a PBS buffer solution with a pH of 7.0.

[0064] S2. The carbon cloth is first rinsed with ethanol, then rinsed with pure water, dried at room temperature, and then cut into a size of 0.5 cm×1.5 cm to obtain a treated carbon cloth.

[0065] S3. Add 5 μL of 10 mg / mL bilirubin oxidase solution to the treated carbon cloth, with a coating area of ​​0.5 cm×0.5 cm, and place in a 4°C refrigerator to dry. At this time, the mass ratio of carbon cloth to bilirubin oxidase is 12:5, and a BOD / carbon cloth cathode material is obtained, referred to as BOD / carbon cloth.

[0066] 2. Preparation of BOD / MCNT-NH2 / carbon cloth: S1. Dissolve MCNT-NH2 solid in ethanol and then disperse it by ultrasonic for 3 hours to prepare a 2 mg / mL MCNT-NH2 dispersion.

[0067] S2. The carbon cloth is first rinsed with ethanol, then rinsed with pure water, dried at room temperature, and then cut into a size of 0.5 cm×1.5 cm to obtain a treated carbon cloth.

[0068] S3. Take 60 μL of MCNT-NH2 dispersion and apply it on the treated carbon cloth. The coating area is 0.5 cm×0.5 cm. Then dry it at room temperature to obtain MCNT-NH2 / carbon cloth.

[0069] S4. Dissolve the purchased bilirubin oxidase powder in 5 mmol / L PBS buffer solution with a pH of 7.0 to prepare a 10 mg / mL bilirubin oxidase solution.

[0070] The preparation method of the 5mmol / L PBS buffer solution with a pH of 7.0 is as follows: 5mmol / L Na2HPO4 solution and 5mmmol / L NaH2PO4 solution are mixed to prepare a PBS buffer solution with a pH of 7.0.

[0071] S5. Add 5 μL of 10 mg / mL bilirubin oxidase solution to MCNT-NH2 / carbon cloth, and then place it in a 4°C refrigerator to dry. At this time, the mass ratio of MCNT-NH2 / carbon cloth cathode material to bilirubin oxidase is 12:5, and BOD / MCNT-NH2 / carbon cloth cathode material is obtained, referred to as BOD / MCNT-NH2 / carbon cloth.

[0072] 2. Performance Research: Assemble a three-electrode system: use a platinum electrode as the counter electrode, an Ag / AgCl electrode as the reference electrode, and BOD / ABTS / MCNT-NH2 / carbon cloth, BOD / carbon cloth, ABTS / MCNT-NH2 / carbon cloth, and BOD / MCNT-NH2 / carbon cloth as working electrodes, respectively. Immerse one end of the working electrode, reference electrode, and counter electrode in the PC solution, and connect the other end to the electrochemical workstation to assemble a three-electrode system. The PC solution is selected from a PC solution with a pH of 7.0 for testing.

[0073] PC solution with a pH of 7.0 was prepared using 0.2 mol / L Na2HPO4 solution and 0.1 mol / L citric acid solution as raw materials.

[0074] In air atmosphere, at a scan rate of 10 mVs -1 250 cycles were performed under the condition of 0.7V~0V voltage, and cyclic voltammetry test was performed using the ABTS / MCNT-NH2 / carbon cloth of Example 1. Figure 1 It can be seen that after obtaining the cyclic voltammetry curves of 2 cycles, 50 cycles, 100 cycles, 150 cycles, 200 cycles, and 250 cycles, ABTS / MCNT-NH2 / carbon cloth has a pair of obvious ABTS redox peaks, and the peak current of the redox peaks does not decrease after 250 cycles, indicating that ABTS and MCNT-NH2 have been well combined through π-π stacking and electrostatic interaction, and the constructed ABTS / MCNT-NH2 / carbon cloth has excellent stability; at the same time, Figure 1 As can be seen from the figure, the peak potential difference between the oxidation peak and the reduction peak is 10 mV, indicating that the oxidation-reduction process is a surface adsorption process, which further verifies that ABTS and MCNT-NH2 have been well combined.

[0075] PC solutions with pH values ​​of 5.0, 6.0, 7.0 and 8.0 were prepared using 0.2 mol / L Na2HPO4 solution and 0.1 mol / L citric acid solution as raw materials. PC solutions with pH values ​​of 5.0, 6.0, 7.0 and 8.0 were respectively prepared in an air atmosphere at a scan rate of 10 mVs -1 , the scanning range is 0.8V~0V, the ABTS / MCNT-NH2 / carbon cloth of Example 1 is used as the working electrode, and the cyclic voltammetry test under different pH conditions is performed using a three-electrode system. At pH 8.0, after testing the cyclic voltammetry curve in the air environment, the cyclic voltammetry curve in the anaerobic PC solution is also tested. The anaerobic environment is to fill the three-electrode system with argon until the air is completely discharged. Figure 2It can be seen that the peak potential and peak current of the redox peak of ABTS in ABTS / MCNT-NH2 / carbon cloth do not change with the change of pH. At the same time, the oxygen reduction peak starts from 0.3V. Due to the reduction of O2 by MCNT on ABTS / MCNT-NH2 / carbon cloth, it shows that ABTS itself has little effect on mediating oxygen reduction.

[0076] via Figure 3 It can be seen that in the PC solution with a pH of 7.0 filled with O2, for the electrocatalytic O2 reduction reaction, BOD / carbon cloth has almost no catalytic current. This is because the strong hydrophobic effect of carbon cloth hinders its adsorption of BOD, resulting in almost zero catalytic current of BOD / carbon cloth. The catalytic current of BOD / MCNT-NH2 / carbon cloth is -1050μA / cm 2 , much higher than BOD / carbon cloth. On the one hand, MCNT-NH2 is hydrophobic and can stably combine with carbon cloth due to hydrophobic effect, thereby increasing the active specific surface area of ​​the carbon cloth surface; on the other hand, MCNT-NH2 is less hydrophobic than carbon cloth, which allows BOD to adhere to its surface, increasing the loading amount of BOD on the surface of MCNT-NH2 / carbon cloth, thereby increasing the catalytic current of the biocathode. The catalytic current of BOD / ABTS / MCNT-NH2 / carbon cloth is as high as -2320μA / cm 2 , which is twice the catalytic current of BOD / MCNT-NH2 / carbon cloth, and has excellent oxygen reduction ability. At the same time, the cathode catalytic current of BOD / ABTS / MCNT-NH2 / carbon cloth increases rapidly before 0.3V, reaching 78% of the total current, indicating that BOD / ABTS / MCNT-NH2 / carbon cloth constructed by MCNT-NH2 after combining with ABTS has a faster electron transfer rate. The results show that ABTS not only serves as an auxiliary agent to assist bilirubin oxidase in achieving uniform orientation modification, but also as an electron transfer mediator to accelerate the interface electron transfer kinetics between the T1 site of bilirubin oxidase and the surface of the ABTS / MCNT-NH2 / carbon cloth substrate. In addition, from Figure 3 It can be seen that ABTS / MCNT-NH2 / carbon cloth has no obvious oxygen reduction peak, indicating that ABTS has no obvious catalytic reduction effect on oxygen, proving that the increase in the catalytic current of BOD / ABTS / MCNT-NH2 / carbon cloth and BOD / MCNT-NH2 / carbon cloth is due to the catalytic effect of BOD.

[0077] Figure 3The conclusion that BOD / ABTS / MCNT-NH2 / carbon cloth has the best catalytic O2 reduction performance has been given, and the present invention also explores its catalytic effect under different pH conditions. BOD / ABTS / MCNT-NH2 / carbon cloth was first immersed in PC solutions of pH 5.0, pH 6.0, pH 7.0, and pH 8.0 for 5 minutes, and then cyclic voltammetry was performed using a three-electrode system in a saturated oxygen atmosphere at a scan rate of 10mV / s and a voltage of 0.8V~0V; at pH 8.0, after testing the cyclic voltammetry curve in an air environment, the cyclic voltammetry curve in an anaerobic PC solution was also tested, and the anaerobic environment was filled with argon gas into the three-electrode system until the air was completely discharged.

[0078] like Figure 4 As shown in Figure 2, the catalytic current decreases with the increase of the pH value of the PC solution, but the catalytic current is still maintained. Figure 2 The conclusion is drawn that the redox potential of ABTS in ABTS / MCNT-NH2 / carbon cloth does not change with pH, ​​indicating that the change of BOD / ABTS / MCNT-NH2 / carbon cloth catalytic current with pH is caused by the effect of pH on BOD. Therefore, the constructed BOD / ABTS / MCNT-NH2 / carbon cloth can catalyze O2 reduction in the pH range of 5-8, with a wide range of applications, and the best effect is at pH 5.0. Figure 4 It can also be seen that the peak potential of the O2 reduction catalytic current also shifts negatively with the increase of pH value.

[0079] like Figure 5 As shown in Figure (a), a conventional three-electrode system test device is constructed with BOD / ABTS / MCNT-NH2 / carbon cloth as conventional electrodes, and one end of the working electrode, counter electrode and reference electrode are immersed in the PC solution for testing. Since the amount of oxygen dissolved in the PC solution in the air environment is low, the current density of O2 reduction in the biocathode is relatively low; in addition, for some bioanodes that are sensitive to O2, they need to be tested under anaerobic conditions, which limits the assembly of membrane-free and miniaturized biofuel cells with the biocathode.

[0080] like Figure 5As shown in Figure (b), considering the technical defects of the conventional three-electrode system, the present invention uses BOD / ABTS / MCNT-NH2 / carbon cloth as a gas breathing electrode to construct a gas diffusion system. The gas diffusion system is as follows: two beakers with connecting ports are symmetrically arranged, and BOD / ABTS / MCNT-NH2 / carbon is arranged between the two connecting ports. After the two beakers are clamped and stabilized, PC solution is added to one of the beakers, and one end of the counter electrode and the reference electrode are immersed in the PC solution together. At this time, one beaker contains PC solution and the other beaker does not contain PC solution, so that the modified surface of the working electrode is in contact with the PC solution and its back is exposed to the air. The constructed gas breathing electrode can prevent the PC solution from leaking from the carbon cloth due to the existence of gas-solid-liquid three-phase equilibrium, and at the same time greatly improves the adhesion of oxygen on the surface of the carbon cloth, so that the catalytic reduction of O2 by BOD / ABTS / MCNT-NH2 / carbon cloth is carried out efficiently and is no longer limited by the mass transfer of O2. At the same time, O2 only exists near the carbon cloth and will not affect the oxygen content in the main body of the PC solution. Therefore, the gas breathing electrode BOD / ABTS / MCNT-NH2 / carbon cloth can also carry out the catalytic reduction reaction of O2 in an electrolyte that does not contain O2, which greatly improves the application scope of BOD / ABTS / MCNT-NH2 / carbon cloth.

[0081] like Figure 5 As shown in Figure (c), it shows the working principle of BOD / ABTS / MCNT-NH2 / carbon cloth as a gas breathing electrode, reducing oxygen from the air on its back side into water molecules.

[0082] BOD / ABTS / MCNT-NH2 / carbon cloth was tested as a conventional electrode and a gas breathing electrode, and compared. The test method is: when used as a conventional electrode, BOD / ABTS / MCNT-NH2 / carbon cloth was immersed in a PC solution with a pH of 5.0 for 5 minutes, and a cyclic voltammetry scan was performed at a scan rate of 10mV / s and a voltage of 0.8V~0V; when used as a gas breathing electrode, the modified surface of the carbon cloth was contacted with a PC solution with a pH of 5.0, and the back was exposed to the air, and a cyclic voltammetry scan was performed at a scan rate of 10mV / s and a voltage of 0.8V~0V.

[0083] via Figure 6 It can be seen that after testing under the same conditions, the oxygen reduction catalytic current of BOD / ABTS / MCNT-NH2 / carbon cloth as a gas breathing electrode is higher, indicating that the constructed BOD / ABTS / MCNT-NH2 / carbon cloth gas breathing electrode has better oxygen reduction ability.

[0084] To further illustrate the advantages of BOD / ABTS / MCNT-NH2 / carbon cloth gas breathing electrode, BOD / ABTS / MCNT-NH2 / carbon cloth was assembled according to the gas diffusion system, and then cyclic voltammetry test was carried out in oxygen-free PC solution, and the back of BOD / ABTS / MCNT-NH2 / carbon cloth was exposed to air and oxygen flow respectively. The test method is: BOD / ABTS / MCNT-NH2 / carbon cloth was assembled according to the gas diffusion layer system, so that its modified surface was in contact with the PC solution, and the back was in contact with air or oxygen flow, and BOD / ABTS / MCNT-NH2 / carbon cloth was immersed in a PC solution with a pH of 5.0 for 5 minutes. The PC solution was deoxygenated with argon gas. While exposing the back of BOD / ABTS / MCNT-NH2 / carbon cloth to air and oxygen flow respectively, cyclic voltammetry scans were carried out at a scan rate of 10mV / s and a voltage of 0.8V~0V, and the two were compared. Figure 7 It can be seen that even if there is no O2 in the PC solution, the BOD / ABTS / MCNT-NH2 / carbon cloth gas breathing electrode can use the O2 on its back as cathode fuel to work normally, catalyze oxygen reduction and generate cathode current. When the back is charged with O2, the cathode current density of oxygen reduction can reach 5mA / cm 2 This also shows that the gas diffusion system constructed by the present invention can enable the biocathode and the anaerobic bioanode to construct a membrane-free enzymatic biofuel cell.

[0085] The present invention also investigates the stability of BOD / ABTS / MCNT-NH2 / carbon cloth catalyzing oxygen reduction, and tests the BOD / ABTS / MCNT-NH2 carbon cloth as a conventional electrode and a gas breathing electrode, respectively, and performs a stability test using a chronoamperometry method, wherein the test method is as follows: when the BOD / ABTS / MCNT-NH2 / carbon cloth is used as a conventional electrode, the BOD / ABTS / MCNT-NH2 / carbon cloth as a whole is placed in a PC solution with a pH of 5.0 for a chronoamperometry test of 13 hours, and an applied voltage of 0 V; when used as a gas breathing electrode, the modified surface of the BOD / ABTS / MCNT-NH2 / carbon cloth is placed in a PC solution with a pH of 5.0, and the back side is exposed to the air, and a chronoamperometry test of 13 hours is performed, and an applied voltage of 0 V is performed.

[0086] via Figure 8It can be seen that under the same conditions, the catalytic current of BOD / ABTS / MCNT-NH2 / carbon cloth as a gas breathing electrode is higher, the oxygen reduction effect is better, and the current density retention rate after 13 hours of testing is 70.6%. When tested as a conventional electrode in PC solution, the current density retention rate after 13 hours is 61.2%. This shows that when the constructed BOD / ABTS / MCNT-NH2 / carbon cloth is used as a gas breathing electrode, the electrode stability is better and it has better practical value.

[0087] Preparation of MCNT-NH2 / carbon cloth: 5 mg of MCNT-NH2 was placed in ethanol, and then ultrasonically dispersed for 3 hours to prepare a 2 mg / mL MCNT-NH2 dispersion; the carbon cloth was first rinsed with ethanol, then rinsed with pure water, dried at room temperature, and then cut into 0.5 cm × 1.5 cm sizes to obtain treated carbon cloth; 60 μL of MCNT-NH2 dispersion was drop-coated on the treated carbon cloth, with a drop-coating area of ​​0.5 cm × 0.5 cm, and dried at room temperature. At this time, the loading amount of MCNT-NH2 on the carbon cloth was 0.45 mg / cm 2 , and obtain MCNT-NH2 / carbon cloth.

[0088] from Fig. 9 Comparison of Figures a, b, and c shows that both MCNT-NH2 and ABTS / MCNT-NH2 can be evenly distributed on the carbon cloth. Fig. 9 Comparing Figure b and Figure c, we can see that there is no significant difference in MCNT-NH2 before and after binding with ABTS, indicating that the binding of ABTS will not form crystals and will not cause any damage to MCNT-NH2.

[0089] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a bilirubin oxidase / modified carbon cloth cathode material, characterized in that: The steps include: Dissolving 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid in water to prepare a 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid aqueous solution; Adding aminated multi-walled carbon nanotubes to an aqueous solution of 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid, mixing the mixture evenly, and then standing the mixture, using the aminated multi-walled carbon nanotubes to adsorb 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid, and then centrifuging and drying the mixture to obtain a 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid / aminated multi-walled carbon nanotube cathode material; Dispersing 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid / aminated multi-walled carbon nanotube cathode material in an organic solvent, coating it on carbon cloth, and drying it at room temperature to obtain a modified carbon cloth cathode material; A PBS buffer solution containing bilirubin oxidase is coated on the modified carbon cloth cathode material, and then dried to obtain a bilirubin oxidase / modified carbon cloth cathode material.

2. The method for preparing the bilirubin oxidase / modified carbon cloth cathode material according to claim 1, characterized in that: The mass ratio of amino-modified multi-walled carbon nanotubes to 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid is 5:2.2×10 -3 ~41×10 -3 .

3. The method for preparing the bilirubin oxidase / modified carbon cloth cathode material according to claim 1, characterized in that: The loading amount of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid / amino-modified multi-walled carbon nanotube cathode material on carbon cloth is 0.12 mg / cm 2 ~0.96mg / cm 2 .

4. The method for preparing the bilirubin oxidase / modified carbon cloth cathode material according to claim 1, characterized in that: The mass ratio of the modified carbon cloth cathode material to the bilirubin oxidase is 12:2~30.

5. The method for preparing the bilirubin oxidase / modified carbon cloth cathode material according to claim 1, characterized in that: The PBS buffer solution is a mixture of NaH2PO4 solution and Na2HPO4 solution, and the pH of the PBS buffer solution is 7.

0.

6. A bilirubin oxidase / modified carbon cloth cathode material prepared by the preparation method according to any one of claims 1 to 5, characterized in that: The modified carbon cloth cathode material is prepared by modifying the surface of the carbon cloth with amino multi-walled carbon nanotubes loaded with 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid.

7. Use of the bilirubin oxidase / modified carbon cloth cathode material according to claim 6 in preparing cathode plates of enzymatic biofuel cells, characterized in that: The enzymatic biofuel cell includes a bilirubin oxidase / modified carbon cloth cathode material, a biological anode electrode and an anode fuel.

8. The use of the bilirubin oxidase / modified carbon cloth cathode material according to claim 7 in the preparation of an enzymatic biofuel cell cathode electrode, characterized in that: The biological anode electrode piece is selected from an aerobic biological anode electrode piece or an anaerobic biological anode electrode piece.

9. The use of the bilirubin oxidase / modified carbon cloth cathode material according to claim 8 in the preparation of cathode plates of enzymatic biofuel cells, characterized in that: When the biological anode electrode is selected from an aerobic biological anode electrode, the application method is: using bilirubin oxidase / modified carbon cloth cathode material as the cathode electrode, immersing one end of the cathode electrode and the aerobic biological anode electrode in an electrolyte containing an anode fuel, and electrically connecting the other ends to a power source to construct an enzymatic biological fuel cell.

10. The use of the bilirubin oxidase / modified carbon cloth cathode material according to claim 8 in preparing cathode plates of enzymatic biofuel cells, characterized in that: When the biological anode electrode is selected from the anaerobic biological anode electrode, the application method is: using bilirubin oxidase / modified carbon cloth cathode material as the cathode electrode, constructing the cathode electrode into a gas breathing electrode, immersing one end of the anaerobic biological anode electrode in an electrolyte containing an anode fuel, and electrically connecting the other end of the anaerobic biological anode electrode and the cathode electrode to a power source to construct an enzymatic biological fuel cell.

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