Construction method and application of ratio type biofuel cell self-powered sensor

Abstract

The invention belongs to the cross technical field of energy electrochemistry and biosensing, and relates to a ratio detection sensor, in particular to a construction method and application of a ratio type biofuel cell self-energized sensor, a conductive substrate containing a loaded gold nano bowl (pAuNB) is used as an anode; a bilirubin oxidase (BOD) modified carbon nanotube / gold composite electrode is used as a cathode; tiO2 (at) Au is fixed on the surface of the electrode through a target object to form a photocatalytic channel, and light current output is regulated and controlled through an optical switch. The construction method of the ratio-type PA-EBFC self-energized sensor is provided for the first time, dynamic balance of photoelectric response and steric effect of TiO2 (at) Au is achieved by regulating and controlling the loading amount of AuNPs in TiO2 (at) Au, and construction of the ratio-type PA-EBFC self-energized sensor is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of intersection of energy electrochemistry and biosensing, and relates to a ratio detection sensor. Background Art

[0002] The photoenzymatic biofuel cell (PA-EBFC) is a novel enzymatic biofuel cell that combines photocatalysis and enzyme catalysis, directly converting chemical and light energy into electrical energy. The key advantage of the PA-EBFC-based self-powered sensor (PA-EBFC-SPB) is that it can directly convert chemical and light energy into a signal change related to the analyte concentration without requiring an external power source. This makes it portable, low-cost, and highly sensitive, and has been successfully used for the detection of cancer markers.

[0003] Traditional ratiometric electrochemical sensors achieve highly selective and sensitive detection of target analytes by monitoring the ratio of two electrochemical signals. Since the signal depends on the ratio of the two signals at different potentials, it is necessary to introduce two materials with complete discrimination to meet the output of signals at different potentials, which undoubtedly increases the difficulty of sensor design. In addition, PFC-SPB has also made some progress in the field of ratiometric sensing. Application No. CN114199967A discloses a method for constructing a ratiometric self-powered aptamer sensor based on a light-assisted fuel cell. The ratio between the two electrode signals is used to achieve quantitative detection of the target and normalize the fluctuations caused by the environment. The sensing area is divided into a detection area and a control area, and the same cathode is shared to achieve ratiometric detection. However, this design not only increases the complexity of the sensing interface, but also increases the difficulty of experimental operation. Therefore, it is very challenging to construct a simple sensor with splittable signals and clear resolution. Summary of the Invention

[0004] To solve the above technical problems, the present invention proposes a construction method and application of a ratiometric biofuel cell self-powered sensor, which achieves high-sensitivity and high-precision self-calibration detection and has the advantages of portability and low cost.

[0005] The design concept of the ratiometric biofuel cell self-powered sensor of this application is:

[0006] A conductive substrate containing loaded gold nanobowls (pAuNBs) served as the anode to catalyze glucose oxidation, while a carbon nanotube / gold composite electrode modified with bilirubin oxidase (BOD) served as the cathode for the oxygen reduction reaction. Titanium dioxide-gold nanoparticles (TiO2@Au) were immobilized on the electrode surface via a target, forming a photocatalytic channel. The photocurrent output was modulated by a photoswitch. The TiO2@Au served as the control module, with its steric hindrance inhibiting the catalytic activity of the pAuNBs. Simultaneously, the photocatalytic activity of the TiO2@Au generated a photocurrent signal.

[0007] The TiO2@Au of the present invention is composed of spherical TiO2 (size 250nm) with AuNPs uniformly loaded on the surface, wherein the loading amount of AuNPs is adjusted by adjusting the concentration ratio of TiO2 and chloroauric acid precursor to achieve a dynamic balance between the photocurrent signal and the steric effect. OCV light ) as a reference signal, and the open circuit voltage (E OCV ) and E OCV light The ratio (E OCV / E OCV light ) to construct a ratiometric detection mode. By regulating the photocatalytic material (TiO2@Au) and synergizing it with the enzyme catalytic material (pAuNB), a highly stable ratiometric biosensor without the need for an external power source is achieved. The platform can also adapt the sensitivity and dynamic range requirements of different detection scenarios by adjusting the AuNPs loading of the TiO2@Au.

[0008] The technical solution of the present invention is achieved as follows:

[0009] A method for preparing a TiO2@Au photoelectric material comprises dissolving TiO2 in ultrapure water, ultrasonically dissolving the solution, and then adding the solution to an aqueous solution containing polyvinylpyrrolidone (PVP) and ascorbic acid (AA). The solution is placed in an oil bath for reaction, followed by adding a chloroauric acid (HAuCl4) solution in two portions. After the reaction is complete, the solution is centrifuged and washed to remove unbound AuNPs, yielding TiO2@Au.

[0010] The mass ratio of the above-mentioned TiO2, polyvinyl pyrrolidone, ascorbic acid and chloroauric acid is 0.5-4:5.83:10:2.47; the oil bath reaction temperature is 60-90°C and the reaction time is 3-6h.

[0011] Preferably, the concentration of the TiO2 is 0.5-2 mg / mL, and the concentration of HAuCl4 added is 1 mM.

[0012] A method for preparing an MCH / S / pAuNB / CP electrode, comprising the following steps:

[0013] (1) Adding the nanosilver cube dispersion, hydroquinone solution, and HAuCl4 solution to the PVP solution in sequence, stirring until the solution changes color from white to gray-green, allowing it to stand and centrifuging to obtain a precipitate, then adding ammonia water to remove the nanosilver cubes, and centrifuging and washing to obtain pAuNB;

[0014] (2) Dropping the pAuNB solution prepared in step (1) onto the CP surface and drying it to obtain a pAuNB / CP electrode, then dropping the S chain solution onto the pAuNB / CP electrode surface and incubating at 4°C overnight to obtain an S / pAuNB / CP electrode, and then dropping the MCH solution and incubating at 37°C to obtain an MCH / S / pAuNB / CP electrode;

[0015] The S chain was prepared by adding the P chain, L chain, and R chain to a PBS buffer solution, maintaining the mixture at 95°C for 5 minutes, and then quenching the mixture in an ice-water bath for 15 minutes to obtain the S chain. The final concentrations of the P chain, L chain, and R chain in the PBS buffer solution were all 1.5 μM. The nucleotide sequence of the P chain is shown in SEQ ID No. 4, the nucleotide sequence of the L chain is shown in SEQ ID No. 2, and the nucleotide sequence of the R chain is shown in SEQ ID No. 1.

[0016] In the above step (1), the concentration of the nanosilver cube dispersion is 4 mg / mL, the concentration of the hydroquinone solution is 28 mM, the mass percentage of the HAuCl4 solution is 2%, and the ammonia solution is 25% ammonia water; the volume ratio of the nanosilver cube dispersion, the hydroquinone solution, and the HAuCl4 solution is 20:26:11;

[0017] The concentration of the solution containing pAuNB from step (1) in step (2) is 1-2 mg / mL.

[0018] The MCH / S / pAuNB / CP electrode was prepared using the above method.

[0019] A method for preparing a BOD / CNT@Au / CP biocathode comprises the following steps:

[0020] 1) Carboxylated CNTs were added to a PDDA aqueous solution containing NaCl and ultrasonically reacted to obtain a CNT / PDDA complex. After washing, the complex was dispersed in a 0.24 mM Au NP solution. After stirring at room temperature, the precipitate was collected by centrifugation and freeze-dried to obtain CNT@Au.

[0021] 2) CNT@Au solution was dripped onto CP. After drying, the electrode was activated with an activator to obtain a CNT@Au / CP electrode. Finally, BOD solution was dripped onto the CNT@Au / CP electrode and the BOD / CNT@Au / CP biocathode was incubated at 4°C overnight.

[0022] In the above step 1), the concentration of NaCl in the PDDA aqueous solution containing NaCl is 0.02 M, the mass percentage of PDDA is 1%, and the mass ratio of carboxylated CNT to PDDA is 1:1.

[0023] In the above step 2), the concentration of CNT@Au in the CNT@Au solution is 1 mg / mL; the activator is EDC or NHS, and the concentration of the activator solution is 1 mg / mL; the volume ratio of the CNT@Au solution to the activator solution is 1:1-3; and the concentration of the BOD solution is 10 mg / mL.

[0024] The BOD / CNT@Au / CP biocathode was prepared using the above method.

[0025] A method for constructing a ratiometric biofuel cell self-powered sensor, comprising the following steps:

[0026] a. The F chain solution was added to a solution containing the above-mentioned TiO2@Au photoelectric material, and after shaking at room temperature, NaCl solution was added in portions. The resulting reaction solution was centrifuged and washed. The resulting product was dispersed in 1 mL of PBS solution and blocked with MCH. The product was centrifuged and washed to obtain TiO2@Au-F. The nucleotide sequence of the F chain is shown in SEQ ID No. 3.

[0027] b. A mixed solution of the sample to be tested and TiO2@Au-F was dropped onto the surface of the MCH / S / pAuNB / CP electrode, incubated at 37°C, and rinsed to obtain the TiO2@Au / pAuNB / CP anode;

[0028] c. Place the TiO2@Au / pAuNB / CP anode of step b and the BOD / CNT@Au / CP biocathode according to claim 9 in an electrolyte and test the open circuit voltage under dark and light conditions, respectively.

[0029] In the above step a, the concentration of the F chain solution is 100 μM, and the concentration of the TiO2@Au solution is 2 mg / mL; the volume ratio of the F chain solution to the TiO2@Au solution is 1:100; after the NaCl solution is added in batches, the final concentration of NaCl is 0.1 M.

[0030] In the above step b, the concentration of T chain in the mixed solution is 1fM-1nM, and the concentration of TiO2@Au-F is 2mg / mL.

[0031] The electrolyte in the above step c is a 0.01M PBS buffer solution with a pH of 7.4 and containing 10 mM glucose.

[0032] Furthermore, E under dark conditions OCVAs the concentration of the target increases, it gradually decreases. Due to the balance between the TiO2@Au photocurrent and the steric effect, the E OCV light The open circuit voltage (E OCV ) and E OCV light The ratio (E OCV / E OCV light ) and the linear equation E of the logarithm of the concentration (lgc) OCV / E OCV light =0.1336-0.0563lgc, linear correlation coefficient R 2 It is 0.997, and the detection limit can reach 0.5fM.

[0033] The present invention has the following beneficial effects:

[0034] 1. This paper proposes for the first time a method for constructing a ratiometric PA-EBFC self-powered sensor. By regulating the loading amount of AuNPs in TiO2@Au, a dynamic balance between the photoelectric response and steric effect of TiO2@Au is achieved, thus realizing the construction of a ratiometric PA-EBFC biofuel cell self-powered sensor.

[0035] 2. The present invention uses a single device to achieve switching between light and dark conditions, and controls the dual-channel signal output through an optical switch. It does not require coupling with external detection platforms such as fluorescence and colorimetry, greatly reducing the complexity of the equipment and manufacturing costs.

[0036] 3. By integrating nanomaterial regulation, light-enzyme synergistic catalysis and ratio signals, the present invention breaks through the technical bottleneck of traditional sensors relying on multiple platforms and having weak anti-interference capabilities, and provides a new detection paradigm that is self-powered, highly sensitive and anti-interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 Transmission electron microscopy images of TiO2@Au with different proportions.

[0039] Figure 2 X-ray diffraction patterns of TiO2@Au with different proportions.

[0040] Figure 3Electrochemical impedance spectroscopy diagrams of TiO2@Au with different ratios.

[0041] Figure 4 The photocurrent response diagram of different TiO2@Au regulation.

[0042] Figure 5 This is the BOD catalytic performance diagram.

[0043] Figure 6 E is the ratiometric biofuel cell self-powered sensor under light conditions OCV light Change graph.

[0044] Figure 7 E of the ratiometric biofuel cell self-powered sensor under dark conditions OCV Change graph.

[0045] Figure 8 This is the linear fitting diagram of the ratiometric biofuel cell self-powered sensor. DETAILED DESCRIPTION

[0046] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. 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.

[0047] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0048] The nucleotide sequences involved in the present invention are as follows:

[0049]

[0050] Example

[0051] A method for constructing a ratiometric biofuel cell self-powered sensor, comprising the following steps:

[0052] 1. Preparation of TiO2@Au:

[0053] ① Add 1 mL of Ti(OBu)4 to EG (22.5 mL) solution and stir vigorously at room temperature for 8 hours. Subsequently, the mixed solution is quickly poured into an acetone (100 mL) solution containing deionized water (1.25 mL) and acetic acid (0.4 mL). After vigorous stirring at room temperature for 3 hours, the white precipitate is collected by centrifugation (8000 rpm, 10 minutes) and washed with ethanol, repeating this process three times. Finally, the white precipitate is added to 50 mL of water and stirred at 70°C for 8 hours to obtain TiO2 colloidal spheres. Centrifuge and wash with water, repeat this process three times, and dry for later use.

[0054] ② 1 mL of the prepared TiO2 (0.9 mg / mL) suspension was dispersed in 6 mL of a solution containing PVP (5.83 mg / mL) and AA (10 mg / mL). The solution was heated to 90°C and maintained at 90°C for 10 minutes. Subsequently, 3 mL of HAuCl4 solution (1 mM) was added and maintained at 90°C for 3 hours. Then, 3 mL of HAuCl4 solution (1 mM) was added again and maintained at 90°C for 3 hours. The TiO2@Au was centrifuged and washed to collect the collected TiO2@Au. The TiO2@Au was regulated by varying the amount of TiO2 added (0.5, 0.9, and 2 mg / mL), correspondingly labeled as TiO2@Au-1, TiO2@Au-2, and TiO2@Au-3.

[0055] The TiO2@Au with different ratios (TiO2@Au-1, TiO2@Au-2 and TiO2@Au-3) were tested by transmission electron microscopy and X-ray diffraction. The results are as follows: Figure 1 、 Figure 2 As shown in the TEM results, the surface loading of AuNPs gradually decreased with the increase of TiO2 content in TiO2@Au-1, TiO2@Au-2, and TiO2@Au-3. XRD results also showed that the surface loading of AuNPs gradually decreased with the increase of TiO2 content.

[0056] 2. Preparation of Silver Chloride Cubes (AgCl NCs)

[0057] Hydrochloric acid (37%, 1.25 mL) was slowly added dropwise to a 50 mL EG solution containing AgNO3 (8.4 mg / mL) and PVP (8.4 mg / mL). The reaction was carried out in the dark (150°C). After 20 minutes, the reaction was stopped, cooled to room temperature, and the product was collected by centrifugation (9000 rpm, 10 min). The product was washed three times with ethanol and dried (40°C) for later use.

[0058] 3. Preparation of gold nanocages (pAuNB)

[0059] A 100 μL, 4 mg / mL AgCl NTs dispersion, a 130 μL, 28 mM hydroquinone solution, and a 55 μL, 2% HAuCl₄ solution were sequentially added to a 4.5 mL, 90 mM PVP solution. Stirring was continued for 1 minute, and the solution color changed from white to gray-green. After 2 hours of static reaction, the precipitate was centrifuged and 0.5 mL, 25% NH₃H₂O (0.5 mL, 25%) was added to remove the AgCl NCs template. After a 5-minute reaction, the mixture was washed three times by centrifugation (5500 rpm, 3 minutes each) to obtain pAuNB.

[0060] 4. Preparation of CNT@Au

[0061] Carboxylated CNTs (10 mg) were added to an aqueous solution of PDDA (10.0 mL, wt%: 1%) containing 0.02 M NaCl. After sonication for 1 hour, the CNT / PDDA complex was collected by centrifugation (15,000 rpm, 10 minutes) and washed three times with ultrapure water. The resulting CNT / PDDA complex was then dispersed in a solution of Au NPs and stirred at room temperature for 6 hours. Excess Au NPs were removed by centrifugation, and the collected precipitate was freeze-dried for later use.

[0062] 5. Preparation of fuel chain F-modified TiO2@Au (TiO2@Au-F)

[0063] F chain (10 μL, 100 μM) was added to TiO2@Au-1, TiO2@Au-2, and TiO2@Au-3 (1 mL, 2 mg / mL), respectively, and shaken at room temperature for 12 hours. Subsequently, NaCl solution (1 M) was added three times over 8 hours to a final NaCl concentration of 0.1 M. After three centrifugation washes, the product was dispersed in 1 mL of PBS solution and MCH (50 μL, 10 mM) was added. After blocking for 1 hour, the product was centrifuged and washed three times before being stored at 4°C. This yielded TiO2@Au-F at different ratios.

[0064] VI. Preparation of TiO2@Au / pAuNB / CP anode

[0065] ① Add the By product strand, Report strand, and Linker strand to a PBS buffer solution at a final concentration of 1.5 μM each and incubate at 95°C for 5 minutes. Subsequently, quench in an ice-water bath for 15 minutes to obtain the substrate strand (S strand).

[0066] ② 30 μL of pAuNB solution (2 mg / mL) was dropped onto the CP surface (0.5 cm × 0.5 cm) and dried at 37°C. Subsequently, 30 μL of S (1.5 μM) was dropped onto the pAuNB / GC electrode surface and incubated at 4°C overnight to form a S / pAuNB / CP electrode through Au-S bonding. Next, 30 μL of MCH (1 mM) was added to the S / pAuNB / CP electrode and incubated for 50 min to block nonspecific adsorption sites. After rinsing the electrode three times with PBS, 50 μL of a mixed solution containing target T and TiO2@Au-F was dropped onto the MCH / S / pAuNB / CP electrode surface and incubated at 37°C for 1.5 h. After rinsing three times with PBS, the TiO2@Au / pAuNB / CP anode of the PA-EBFC-SPB was obtained and stored in a 4°C refrigerator until use.

[0067] VII. Preparation of BOD / CNT@Au / CP Biocathode

[0068] Carboxylated CNTs were added to a PDDA aqueous solution containing NaCl and sonicated to form a CNT / PDDA complex. After washing, the complex was dispersed in a 0.24 mM Au NP solution. After stirring at room temperature, the precipitate was collected by centrifugation and freeze-dried to obtain CNT@Au. A CNT@Au solution (30 μL, 1 mg / mL) was dripped onto a 1 cm × 1 cm CP and dried at 37°C. Subsequently, the electrode was activated with EDC / NHS (1 mg / mL) for 1 h. Finally, 30 μL of BOD (10 mg / mL) was dripped onto the CNT@Au / CP electrode and incubated overnight at 4°C to obtain a BOD / CNT@Au / CP biocathode. The electrode was activated with an activator to obtain a CNT@Au / CP electrode. Finally, a BOD solution was dripped onto the CNT@Au / CP electrode and incubated overnight at 4°C to obtain a BOD / CNT@Au / CP biocathode.

[0069] The catalytic performance of the prepared BOD / CNT@Au / CP biocathode was tested by cyclic voltammetry under different atmospheres. Figure 5 As shown in the figure, the reduction current gradually increases with the increase of O2 content, indicating that the BOD / CNT@Au / CP biocathode has good oxygen reduction ability.

[0070] 8. Assembly of a Ratiometric Biofuel Cell Self-Powered Sensor

[0071] A single-chamber, membrane-free EBFC-SPB was constructed based on the above-mentioned biosensor anode and biocathode at room temperature. The electrolyte was PBS (0.01M, pH=7.4) buffer solution containing 10mM glucose. The E of the EBFC composed of the S / pAuNB / CP anode and the BOD / CNT@Au / CP biocathode was first measured in the dark.OCV Then, the S / pAuNB / CP electrode was incubated with TiO2@Au-1, TiO2@Au-2 and TiO2@Au-3 and mixed solutions containing different concentrations of target substances at 37°C for 1.5h to obtain the TiO2@Au / pAuNB / CP bioanode. First, turn on the xenon lamp and measure the E of EBFC under light conditions. OCV light According to E OCV light The sensor constructed with the optimal ratio of TiO2@Au that can be used as the internal reference signal was selected. Again, under dark conditions, the E OCV .

[0072] The electrochemical impedance spectroscopy (EIS) tests were performed on TiO2@Au (TiO2@Au-1, TiO2@Au-2 and TiO2@Au-3) with different ratios. Figure 3 As shown in Figure 3, due to the good conductivity of AuNPs, the EIS of TiO2@Au gradually increases with the decrease of the surface AuNPs content.

[0073] The above-mentioned TiO2@Au with different ratios (TiO2@Au-1, TiO2@Au-2 and TiO2@Au-3) were tested for photoelectric response. The results are as follows Figure 4 As shown in the figure, the hot electrons generated by the localized surface plasmon resonance effect of AuNPs can significantly increase the photocurrent. As the TiO2 content increases, the photocurrent gradually decreases.

[0074] Implementation effect example 1

[0075] The construction method and application of a ratiometric biofuel cell self-powered sensor constructed in this application:

[0076] TiO2@Au-F in different proportions in the embodiment was taken, mixed with the target T and then dropped on the surface of the MCH / S / pAuNB / CP electrode and incubated at 37°C for 1.5h. After rinsing with PBS three times, the anode TiO2@Au / pAuNB / CP of the ratiometric self-powered sensor was obtained. Then, based on the above-mentioned biosensor anode and biocathode, a single-chamber membrane-free EBFC-SPB was constructed. The electrolyte was a PBS (0.01M, pH=7.4) buffer solution containing 10mM glucose. First, the E of the EBFC composed of the DNA / pAuNB / CP anode and the BOD / CNT@Au / CP biocathode was measured under dark conditions. OCVThen, the DNA / pAuNB / CP electrode was incubated with a mixed solution of TiO2@Au-F and a certain concentration of target at 37°C to obtain the TiO2@Au / pAuNB / CP bioanode. Then, the xenon lamp was turned on and the E of PA-EBFC was measured again under light conditions. OCV .

[0077] The results are as follows Figure 6 As shown, under light, the E of the biofuel cell self-powered sensor constructed by TiO2@Au-2 OCV The E of the biofuel cell self-powered sensor constructed by TiO2@Au-2 remained constant with the change of target T concentration. OCV As the concentration of target T increases, the E of the biofuel cell self-powered sensor constructed by TiO2@Au-3 gradually increases. OCV It gradually decreases with the increase of target T concentration. Therefore, TiO2@Au-2 can be used to construct ratiometric biofuel cell self-powered sensors.

[0078] Implementation effect example 2

[0079] The construction method and application of a ratiometric biofuel cell self-powered sensor constructed in this application:

[0080] Take the self-powered sensor constructed by TiO2@Au-2 in Example 1 and test E in the dark. OCV With the increase of target concentration, E OCV Gradually decrease, the result is Figure 7 Therefore, the self-powered sensor constructed with TiO2@Au-2 has a high energy density under the illumination condition of E OCV light is the internal reference signal, such as Figure 8 As shown, the open circuit voltage (E OCV ) and E OCV light The ratio (E OCV / E OCV light ) has good linearity with the logarithm of concentration (lgc), and the linear equation is E OCV / E OCV light =0.1336-0.0563lgc, linear correlation coefficient R 2 It is 0.997, and the detection limit can reach 0.5fM.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A TiO2@Au photoelectric material, characterized in that: The preparation steps are as follows: dissolving TiO2 in ultrapure water, adding the solution to an aqueous solution containing polyvinyl pyrrolidone and ascorbic acid, performing an oil bath reaction, adding chloroauric acid solution to the resulting reaction solution, reacting again, centrifuging, and washing to obtain TiO2@Au photoelectric material.

2. The TiO2@Au photoelectric material according to claim 1, characterized in that: The mass ratio of TiO2, polyvinyl pyrrolidone, ascorbic acid and chloroauric acid is 0.5-4:5.83:10:2.47; the oil bath reaction temperature is 60-90°C and the reaction time is 3-6 hours.

3. A method for preparing an MCH / S / pAuNB / CP electrode, characterized in that: The steps are: (1) Adding the nanosilver cube dispersion, hydroquinone solution, and HAuCl4 solution to the PVP solution in sequence, stirring until the solution changes color from white to gray-green, allowing it to stand and centrifuging to obtain a precipitate, then adding ammonia water to remove the nanosilver cubes, and centrifuging and washing to obtain pAuNB; (2) Dropping the pAuNB solution prepared in step (1) onto the CP surface and drying it to obtain a pAuNB / CP electrode, then dropping the S chain solution onto the pAuNB / CP electrode surface and incubating at 4°C overnight to obtain an S / pAuNB / CP electrode, and then dropping the MCH solution and incubating at 37°C to obtain an MCH / S / pAuNB / CP electrode; The S chain was prepared by adding the P chain, L chain, and R chain to a PBS buffer solution, maintaining the mixture at 95° C. for 5 minutes, and then quenching the mixture in an ice-water bath for 15 minutes to obtain the S chain; the final concentrations of the P chain, L chain, and R chain in the PBS buffer solution were all 1.5 μM; the nucleotide sequence of the P chain was shown in SEQ ID No. 4, the nucleotide sequence of the L chain was shown in SEQ ID No. 2, and its 5' end was modified with -SH; and the nucleotide sequence of the R chain was shown in SEQ ID No.

1.

4. The method for preparing the MCH / S / pAuNB / CP electrode according to claim 3, characterized in that: In step (1), the concentration of the nanosilver cube dispersion is 4 mg / mL, the concentration of the hydroquinone solution is 28 mM, the mass percentage of the HAuCl4 solution is 2%, and the ammonia water is 25% ammonia water; the volume ratio of the nanosilver cube dispersion, the hydroquinone solution, and the HAuCl4 solution is 20:26:11; and the concentration of the solution containing pAuNB in step (1) in step (2) is 1-2 mg / mL.

5. An MCH / S / pAuNB / CP electrode prepared by the method according to any one of claims 3 to 4.

6. A method for preparing a BOD / CNT@Au / CP biocathode, characterized in that: The steps are: 1) Carboxylated CNTs were added to a PDDA aqueous solution containing NaCl and ultrasonically reacted to obtain a CNT / PDDA complex, which was then washed and dispersed in an AuNPs solution. After stirring at room temperature, the precipitate was collected by centrifugation and freeze-dried to obtain CNT@Au. 2) CNT@Au solution was dripped onto CP. After drying, the electrode was activated with an activator to obtain a CNT@Au / CP electrode. Finally, BOD solution was dripped onto the CNT@Au / CP electrode and incubated at 4°C overnight to obtain a BOD / CNT@Au / CP biocathode.

7. The method for preparing the BOD / CNT@Au / CP biocathode according to claim 6, characterized in that: In the step 1), the concentration of NaCl in the PDDA aqueous solution containing NaCl is 0.02M, the mass percentage of PDDA is 1%, and the concentration of the AuNPs solution is 0.24mM; the mass ratio of carboxylated CNT to PDDA is 1:1; the concentration of CNT@Au in the CNT@Au solution in step 2) is 1mg / mL; the activator is EDC or NHS, and the concentration of the activator is 1mg / mL; the concentration of the BOD solution is 10mg / mL.

8. A BOD / CNT@Au / CP biocathode prepared using the method of claim 6 or 7.

9. A method for constructing a ratiometric biofuel cell self-powered sensor, characterized in that: The steps are: a. Add the F chain solution to a solution containing the TiO2@Au photoelectric material according to claim 1 or 2, shake at room temperature, and then add NaCl solution in portions. The resulting reaction solution is centrifuged and washed. The resulting product is dispersed in 1 mL of PBS solution and blocked with MCH. The product is then centrifuged and washed to obtain TiO2@Au-F; the nucleotide sequence of the F chain is shown in SEQ ID No. 3, and its 5' end is modified with -SH; b. A mixed solution of the sample to be tested and TiO2@Au-F was dropped onto the surface of the MCH / S / pAuNB / CP electrode, incubated at 37°C, and rinsed to obtain the TiO2@Au / pAuNB / CP anode; c. Place the TiO2@Au / pAuNB / CP anode of step b and the BOD / CNT@Au / CP biocathode according to claim 8 in an electrolyte and test the open circuit voltage under dark and light conditions, respectively.

10. The method for constructing a ratiometric biofuel cell self-powered sensor according to claim 9, characterized in that: In step a, the concentration of the F chain solution is 100 μM, and the concentration of the TiO2@Au solution is 2 mg / mL; the volume ratio of the F chain solution to the TiO2@Au solution is 1:100; after adding NaCl solution in batches, the final concentration of NaCl is 0.1 M; in step b, the concentration of the T chain in the mixed solution is 1 fM-1 nM, and the concentration of TiO2@Au-F is 2 mg / mL; in step c, the electrolyte is a PBS buffer solution with a pH of 7.4 and a temperature of 0.01 M containing 10 mM glucose.

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