Enzyme-free biological fuel cell cathode and anode as well as preparation method and application of enzyme-free biological fuel cell cathode and anode

Abstract

The invention belongs to the technical field of biological fuel cells, and relates to an enzyme-free glucose biological fuel cell and a preparation method thereof. The enzyme-free glucose biofuel cell is constructed by taking an electrode loaded with the PtNPs at bacteriophage as a biological anode and taking an electrode loaded with NiOOH NFs with laccase activity as a biological cathode, and the electricity generation performance of the biofuel cell is improved by utilizing the electrooxidation activity of the PtNPs at bacteriophage to glucose and the excellent catalytic oxygen reduction capability of NiOOH NFs. The fuel cell is divided into an anode chamber and a cathode chamber through the Dupont Nafion117 membrane, so that different electrolyte requirements of the cathode and the anode are met. The biological anode and the biological cathode prepared by the invention not only can greatly improve the performance and the stability of the biological fuel cell, but also have the advantages of low cost, simplicity in operation and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biofuel cells, and in particular relates to a cathode and anode of an enzyme-free biofuel cell, and a preparation method and application thereof. Background Art

[0002] In the field of glucose electrooxidation, the most studied non-biological catalysts are generally precious metals and their alloys, especially platinum-based catalysts. However, platinum nanoparticles (Pt NPs) often face problems such as poor stability, high cost, and susceptibility to poisoning. To address these problems, researchers have tried to use various nanomaterials with a certain specific surface area as carriers to load platinum to improve its stability and catalytic performance, such as carbon nanotube-loaded platinum, graphene-loaded platinum, etc., to make supported catalysts. However, traditional artificial nanomaterials have problems such as complex synthesis and high cost, so there is an urgent need for a new catalyst material.

[0003] A glucose biofuel cell is a device that uses glucose as fuel and relies on enzymes to perform chemical reactions to generate electrical current. Laccase is widely used in the field of biofuel cells because of its low overpotential for catalyzing oxygen reduction. However, laccase has drawbacks such as poor stability and environmental tolerance, is easily inactivated and denatured, and has harsh reaction conditions. It only maintains high activity at neutral pH and near room temperature, and is permanently inactivated in strong acid / base or high temperature environments. In addition, laccase has high extraction purity requirements, and the cost of industrial production is significantly higher than that of synthetic catalysts. Laccase-like nanozymes are a class of nanomaterials with catalytic activity similar to that of laccases. They can mimic the structure and function of natural laccases and catalyze oxygen reduction reactions under specific conditions. Moreover, laccase-like nanozymes avoid the drawbacks of laccases and have the potential to replace laccases as cathode oxygen reduction catalysts.

[0004] Based on this, the present invention develops a biofuel cell based on a phage-templated platinum nanoparticle (Pt NPs) anode and a laccase-mimicking nanozyme cathode. Summary of the Invention

[0005] The present invention aims to address the aforementioned issues in the prior art and proposes an enzyme-free biofuel cell cathode and anode, as well as their preparation and application. The present invention develops a phage-templated Pt NPs@phage bioanode and a laccase-mimicking nickel oxyhydroxide nanoflower (NiOOH NFs) cathode. This biofuel cell, fueled by glucose, exhibits excellent power output and operational stability.

[0006] The technical solution of the present invention is:

[0007] The present invention constructs a dual-chamber biofuel cell with glucose as fuel, a Pt NPs@bacteriophage modified electrode as an anode, and a laccase mimetic enzyme (NiOOH NFs) modified electrode as a cathode.

[0008] The bioanode fabricated using a Pt NPs@phage-modified electrode exhibits excellent electrooxidation, capable of oxidizing glucose at relatively low potentials, meeting the low potential requirement of the anode. This also prevents aggregation of the platinum nanoparticles and increases the adhesion of the modified electrode. The cathode catalyst used is a NiOOH NFs nanomaterial, a transition metal-based laccase-like nanozyme. NiOOH NFs are inexpensive, structurally stable, have a large specific surface area, and possess numerous catalytic sites, which are beneficial for the long-term development of biofuel cells. More importantly, NiOOH NFs, as cathode catalysts, can provide a suitable oxygen reduction potential.

[0009] The present invention provides an enzyme-free glucose biofuel cell, which uses a PtNPs@bacteriophage-modified electrode as a bioanode, and the anolyte is a PBS buffer solution containing 0.1-10 mM glucose; an electrode modified with nickel oxyhydroxide nanoflowers (NiOOH NFs) having laccase activity as a biocathode, and the catholyte is a PBS buffer solution containing 1 mM 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid).

[0010] like Figure 14 The figure shows the principle diagram of the enzyme-free glucose biofuel cell constructed by the present invention. The preparation method of the anode material is to first synthesize platinum nanoparticles and load a layer of Cu on the surface of the platinum nanoparticles. 2+ , PtNPs@bacteriophage was obtained by electrostatic attraction, and the cathode material was synthesized laccase-like nanozyme NiOOHNFs, which was assembled to obtain an enzyme-free glucose biofuel cell.

[0011] Furthermore, the enzyme-free biofuel cell is a dual-chamber H-type biofuel cell, wherein the anode chamber and the cathode chamber are separated by a proton exchange membrane;

[0012] The bioanode is a glassy carbon electrode (GCE) loaded with PtNPs@bacteriophage, and the anolyte is a PBS buffer containing 5-10 mM glucose;

[0013] The biocathode is a glassy carbon electrode (GCE) loaded with NiOOHNFs, the cathode electrolyte is a PBS buffer solution containing 1mM ABTTS, and the pH value of the electrolyte is 7-8.

[0014] The present invention also provides a method for preparing the enzyme-free biofuel cell, which is characterized by comprising the following steps:

[0015] The synthesized NiOOHNFs were ground into powder and then made into a suspension, which was then dropped onto the electrode to prepare the biocathode.

[0016] The synthesized PtNPs@bacteriophage was freeze-dried in a vacuum chamber, resuspended in deionized water to prepare a suspension, and then added dropwise to the electrode to prepare a bioanode.

[0017] Specifically, the bioanode is prepared by loading PtNPs@bacteriophage on GCE. The steps include: ultrasonicating the polished GCE in anhydrous ethanol and water for 0.5 to 1.5 minutes respectively, then drop-coating 5 to 10 μL of PtNPs@bacteriophage with a concentration of 1 to 2 mg / mL onto the GCE. After drying, 3 to 10 μL of 0.05% Nafion117 solution is added to prevent the material from falling off, thereby preparing the PtNPs@bacteriophage / GCE bioanode.

[0018] Specifically, the biocathode is GCE, 5-10 μL of NiOOH NFs with a concentration of 2-3 mg / mL is loaded on the polished GCE surface, and 3-10 μL of 0.05% Nafion 117 solution is added dropwise to prevent the material from falling off, thereby obtaining a NiOOH NFs / GCE biocathode.

[0019] Furthermore, the synthesis method of NiOOHNFs comprises the following steps:

[0020] Under stirring conditions in a water bath at 60-70° C., adding aqueous ammonia to the nickel nitrate solution at a molar ratio of 1:2 to obtain a nickel hydroxide precipitate, which was then centrifuged, washed, dried, and then ground into powder;

[0021] A certain amount of nickel hydroxide powder was weighed and a 6% to 14% sodium hypochlorite aqueous solution was added thereto to oxidize the nickel hydroxide to black. The mixture was stirred at room temperature for 0.5 to 1.5 hours and centrifuged until the supernatant was neutral. The black precipitate was collected and dried to obtain NiOOH NFs.

[0022] Furthermore, the synthesis method of PtNPs@bacteriophage comprises the following steps:

[0023] (1) Cultivation of wild-type phage: Escherichia coli was inoculated into liquid culture medium and cultured at 37°C with shaking until the solution was slightly turbid; then wild-type phage was added and the reaction was shaken at 37°C for 4-5 hours. The supernatant was collected by centrifugation, and the phage was precipitated and resuspended. This was repeated several times and then centrifuged to obtain the supernatant, which was the wild-type phage. The phage titer was then measured;

[0024] (2) Synthesis of PtNPs: Under 70-80℃ water bath conditions, dilute 0.1M hexachloroplatinic acid aqueous solution with water to a final concentration of 1mM, stir evenly, and wait for 1min; add a certain amount of 0.5M trisodium citrate aqueous solution while stirring, where trisodium citrate aqueous solution is used as a stabilizer, dilute the hexachloroplatinic acid aqueous solution to a final concentration of 0.1mM, and stir for 30min; then quickly add 0.1M sodium borohydride aqueous solution dropwise, stir in a 70-80℃ water bath, and after the temperature drops to room temperature, take the prepared PtNPs, mix with 0.1mM CuCl2 in a volume ratio of 1:1, incubate with shaking for 30min, and collect the precipitate after centrifugation and washing;

[0025] (3) Preparation of Pt NPs@phage nanorods: Add phage solution to the washed Pt NPs, shake overnight, and collect the precipitate by centrifugation to obtain PtNPs@phage.

[0026] Furthermore, in step (1), ER2738 Escherichia coli is inoculated into LB liquid culture medium and cultured at 37°C with shaking until the solution becomes slightly turbid; then wild-type M13 phage is added, and the reaction is shaken at 37°C for 4 to 5 hours. The supernatant is collected by centrifugation, and 5 to 8 mL of a PEG8000 / NaCl mixture is added to precipitate the phage. The supernatant is removed by centrifugation and resuspended with TBS. The phage is repeatedly precipitated with a PEG8000 / NaCl mixture, and after a second precipitation, the phage is resuspended in TBS and centrifuged to obtain the supernatant, which is pure wild-type M13 phage.

[0027] Furthermore, in step (2), 100 μL of 0.1 M hexachloroplatinic acid aqueous solution was added to 10 mL of ultrapure water at a final concentration of 1 mM in a 75°C water bath, stirred moderately, and waited for 1 minute; 20 μL of 0.5 M trisodium citrate aqueous solution was added while stirring to a final concentration of 0.1 mM as a stabilizer, and stirred for 30 minutes; then 150 μL of 0.1 M sodium borohydride aqueous solution was quickly added dropwise, stirred in a 75°C water bath, and after the temperature was lowered to room temperature, 500 μL of the prepared PtNPs was taken and mixed with 0.1 mM CuCl2 at a volume ratio of 1:1, incubated with shaking for 30 minutes, and the precipitate was collected after centrifugal washing.

[0028] The present invention also provides the use of any of the above-mentioned enzyme-free glucose biofuel cells or the enzyme-free glucose biofuel cells prepared by any of the above-mentioned preparation methods in the preparation of sensors using glucose as fuel.

[0029] Furthermore, the sensor is a portable sensor.

[0030] The biofuel cell can operate stably under neutral conditions and has the potential to develop portable self-powered energy.

[0031] Beneficial effects of the present invention:

[0032] (1) The enzyme-free glucose biofuel cell proposed in this invention utilizes PtNPs@bacteriophage bioanode to electrocatalyze the oxidation of glucose, generating electrons, which are then transferred to the cathode NiOOH NFs to catalyze oxygen reduction, accompanied by current generation. Based on this, an enzyme-free biofuel cell (BFCs) was constructed using PtNPs@bacteriophage / GCE as the anode and NiOOH NFs / GCE as the oxygen reduction cathode. In a 10 mM glucose solution, the BFCs had an output potential of 550 mV and a maximum power density of 47.5 μW·cm -2 , and has good stability.

[0033] (2) The NiOOH NFs used in this invention were developed to mimic the activity of natural laccase. The NiOOH NFs nanomaterial-modified cathode exhibits excellent electrocatalytic oxygen reduction performance, low overpotential, stable performance, and a simple modification process. Biofuel cells based on laccase-resistant nanozymes and glucose electrooxidation catalysts can significantly improve the performance and stability of biofuel cells while also offering advantages such as low cost and ease of operation.

[0034] (3) The present invention utilizes the strong oxidizing effect of sodium hypochlorite to synthesize nanomaterials containing high-valent nickel, and investigates the laccase activity of the synthesized materials. The obtained nanozyme K m 0.064mM, V max 0.3 μM s -1 , which is far superior to natural laccase (V max =0.025 μM s -1 ), the excellent enzyme activity further improved the power generation performance of BFCs. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A is a scanning electron microscope image of NiOOH NFs prepared in the present invention, Figure 1 B is the corresponding EDS mapping image of Ni and O elements in the NiOOH NFs.

[0036] Figure 2 This is the X-ray diffraction (XRD) spectrum of the NiOOHNFs prepared in the present invention.

[0037] Figure 3 The XPS full spectrum of NiOOH NFs prepared in the present invention and the fine spectrum of related elements, where A is the XPS full spectrum, B is the Ni 2p XPS spectrum, and C is the O1s XPS spectrum.

[0038] Figure 4 A is a scanning electron micrograph of the Pt NPs@bacteriophage prepared in the present invention. Figure 4 B is the XRD spectrum of Pt NPs@bacteriophage, Figure 4 C is the XPS fine spectrum of Pt 4f in PtNPs@phage.

[0039] Figure 5 The UV absorption peak at 510 nm with (line a) and without (line b) NiOOH NFs is used to verify the laccase simulation activity of NiOOH NFs.

[0040] Figure 6 Double reciprocal curves for the kinetic analysis of enzyme activity of NiOOHNFs.

[0041] Figure 7 Cyclic voltammograms (CVs) showing the electrooxidation activity of PtNPs@bacteriophage bioanode for glucose and comparison with commercial Pt / C catalyst.

[0042] Figure 8 Cyclic voltammograms (CV) of the PtNPs@bacteriophage bioanode at different scan rates in PBS buffer containing 10 mM glucose were obtained to verify the electrochemical behavior of glucose on the electrode surface.

[0043] Figure 9 A is the current response of Pt NPs@phage / GCE and 20% Pt / C / GCE at +0.3 V when different concentrations of glucose were continuously added using chronoamperometry (it). Figure 9 B is the calibration curve showing the change of response current with glucose concentration.

[0044] Figure 10 Electrochemical characterization diagram of NiOOHNFs / GCE cathode catalyzed oxygen reduction.

[0045] Figure 11 Power density curve (a) and polarization curve (b) of a fuel cell assembled with a Pt NPs@phage / GCE anode and a NiOOH NFs / GCE cathode, and power density curve (c) and polarization curve (d) of a fuel cell assembled with a 20% Pt / C / GCE anode and a NiOOH NFs / GCE cathode. Glucose concentration was 10 mM.

[0046] Figure 12 To construct the power density curve of the glucose biofuel cell, the anode electrolyte of the BFC was 0.1 M PBS solution containing different concentrations (0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM) of glucose.

[0047] Figure 13This is a stability test of the biofuel cell constructed by the present invention.

[0048] Figure 14 This is a schematic diagram of the principle of the enzyme-free glucose biofuel cell constructed in the present invention. DETAILED DESCRIPTION

[0049] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0050] In order to further understand the present invention, the present invention will be further described with reference to the accompanying drawings and embodiments.

[0051] Example 1

[0052] Synthesis and Characterization of Nickel Oxyhydroxide Nanoflowers (NiOOH NFs) and PtNPs@Bacteriophage

[0053] (1) The specific steps for the synthesis of NiOOH NFs are:

[0054] In a 65°C water bath and with stirring at 860 rpm, 10 mL of 2 M aqueous ammonia was added to 10 mL of a 1 M nickel nitrate solution to produce a green precipitate of nickel hydroxide. The precipitate was centrifugally washed, dried, and then ground into powder. 100 mg of nickel hydroxide powder was weighed and added to a conical flask. A 6% to 14% aqueous sodium hypochlorite solution was then added to the flask until the solution rapidly turned black. The solution was stirred at room temperature for 1 h and centrifuged until the supernatant was neutral. The black precipitate was collected and dried in vacuo at 60°C.

[0055] The synthesized materials were characterized by scanning electron microscopy (SEM), XPS, and XRD.

[0056] Figure 1 A and Figure 1 B shows the SEM image of the synthesized NiOOH NFs and the corresponding EDS mapping images of Ni and O, respectively. As can be seen from the figure, the NiOOH NFs exhibit a flower-like shape and are present in sheets. EDS analysis reveals that Ni and O are uniformly distributed within the NiOOH NFs.

[0057] from Figure 2It can be seen from the XRD that strong and sharp diffraction peaks are observed at 2θ = 17.07°, 33.0°, 38.42°, 51.83° and 59.13°, corresponding to the (006), (100), (101), (102) and (110) lattice planes, respectively, indicating that the crystallinity is high and the grains are large.

[0058] Figure 3 The XPS full spectrum of NiOOH NFs and the fine spectrum of related elements are shown. The element composition and valence state of NiOOH NFs are analyzed by XPS. The XPS full spectrum ( Figure 3 The electron energy spectrum peaks of Ni and O elements appear in A), which once again proves that NiOOHNFs are mostly composed of Ni and O elements. Figure 3 In B), the two peaks at 855.0 and 858.9 eV are attributed to Ni 2+ and Ni 3+ Ni 2p 3 / 2 The binding energy, the two peaks at 870.6 and 874.4 eV are attributed to Ni 2+ and Ni 3+ Ni 2p 1 / 2 Binding energy, so it may contain two valence forms of nickel, including Ni(OH)2 (855.0eV) and NiOOH (858.9eV). 3+ The existence of Ni 2+ Successfully oxidized to Ni by sodium hypochlorite 3+ In the O1s XPS spectrum ( Figure 3 In Figure 3 (C), O 1s shows a broad peak at 532.0 eV, which can be attributed to the existence of oxygen in NiOOH NFs in the form of NiOOH.

[0059] The above characterization results show that NiOOHNFs were successfully prepared.

[0060] (II) The specific steps of PtNPs@phage synthesis are:

[0061] First, culture wild-type phage: inoculate ER2738 E. coli into a flask containing 30 mL of LB liquid medium and culture at 37°C with shaking until the solution is slightly turbid. Then add 20 μL of purchased wild-type M13 phage (2×10 13PFU / mL), shaken at 37°C for 4-5 hours, the supernatant was collected by centrifugation, and then about 6 mL of PEG8000 / NaCl mixture was added to precipitate the phage, followed by a washing and concentration step. Specifically, after the phage was precipitated, the supernatant was removed by centrifugation, and then resuspended with TBS. The phage was repeatedly precipitated with PEG8000 / NaCl mixture. After the second precipitation, it was resuspended in 300 μL TBS and centrifuged to remove residual cells. The supernatant was pure wild-type M13 phage, and the phage titer was measured (2×10 13 PFU / mL).

[0062] Subsequently, PtNPs were synthesized using the sodium borohydride reduction method. The specific steps are as follows: In a 75°C water bath, 100μL of a 0.1M hexachloroplatinic acid aqueous solution was added to 10mL of ultrapure water to a final concentration of 1mM, stirred moderately, and waited for 1 minute. Under magnetic stirring, 20μL of a 0.5M trisodium citrate aqueous solution was added to a final concentration of 0.1mM as a stabilizer and stirred for 30 minutes. After this step, 150μL of a 0.1M sodium borohydride aqueous solution (NaBH4) was quickly added dropwise, stirred in a 75°C water bath, and after the temperature dropped to room temperature, 500μL of the prepared PtNPs was mixed with 500μL of 0.1mM CuCl2, incubated with shaking for 30 minutes, and then centrifuged. Washed three times to remove free copper ions, the precipitate was collected.

[0063] 1 mL of 2×10 8 The phage solution with a concentration of PFU / mL was shaken overnight, and the positive charge of the copper ions on the surface of the platinum nanoparticles was used to bind to the phages. The precipitate was then collected by centrifugation to obtain PtNPs@phages.

[0064] The synthesized materials were characterized by SEM, XPS, and XRD.

[0065] From the SEM images, it was observed that Pt NPs@phage existed in the form of rods ( Figure 4 A). The crystal structure of PtNPs@bacteriophage was further analyzed by XRD patterns ( Figure 4 B). The crystal structure of PtNPs@phage was characterized by XRD. The diffraction peaks at 39.76°, 46.24°, 67.45°, 81.29° and 85.71° correspond to the (111), (200), (220), (311) and (222) crystal planes of Pt (JCPDS PDF#04-0802), respectively. In the high-resolution Pt 4fXPS spectrum, two pairs of peaks can be found, corresponding to the binding energy of Pt(0) and Pt(Ⅱ) ( Figure 4 C).

[0066] Example 2

[0067] Method for preparing cathode and anode in biofuel cell

[0068] The NiOOHNFs synthesized in Example 1 were ground into powder in a mortar, 2 mg was weighed, dispersed in 1 mL of ultrapure water, and ultrasonicated for 30 min to prepare a uniformly dispersed nanomaterial suspension, which was stored in a refrigerator at 4°C for later use.

[0069] A glassy carbon electrode (GCE, inner diameter 3 mm) was polished with 0.3 μm alumina powder and then ultrasonicated in anhydrous ethanol and water for 1 min respectively to remove residual alumina particles. Subsequently, 8 μL of NiOOH NFs suspension (2 mg / mL) was drop-coated on the cleaned GCE and dried under an infrared lamp. After the electrode was dried, 5 μL of 0.05% Nafion 117 solution was added to prevent the material from falling off, thereby preparing a NiOOH NFs / GCE biocathode.

[0070] The Pt NPs@phage synthesized in Example 1 was freeze-dried and vacuum-dried to collect the solid, which was then resuspended in deionized water to prepare a 2 mg / mL suspension. A GCE with an inner diameter of 3 mm was polished using 0.3 μm alumina powder, and then sonicated in anhydrous ethanol and water for 1 minute to remove residual alumina particles. Subsequently, 8 μL of the Pt NPs@phage suspension (2 mg / mL) was drop-coated onto the cleaned GCE and dried under an infrared lamp. After the electrode was dried, 5 μL of a 0.05% Nafion 117 solution was added to prevent the material from falling off, thereby preparing a Pt NPs@phage / GCE bioanode.

[0071] Example 3

[0072] Preparation of enzyme-free glucose biofuel cell

[0073] The enzyme-free biofuel cell in this example uses a dual-chamber H-type biofuel cell (BFC) consisting of an anode chamber, a cathode chamber, and a proton exchange membrane. The proton exchange membrane is Nafion 117, which is used to separate the cell into an anode chamber and a cathode chamber. The volume of each chamber is 10 mL.

[0074] The NiOOH NFs / GCE prepared in Example 2 was used as the cathode, and the catholyte was PBS buffer (pH = 7.0) containing 1 mM ABTS;

[0075] The Pt NPs@phage / GCE prepared in Example 2 was used as the anode, and the anolyte was PBS buffer (pH=7.0) containing 10 mM glucose.

[0076] Test Example 1

[0077] Laccase mimicking activity test of NiOOH NFs

[0078] Laccase activity was verified using a classic laccase activity assay. Specifically, 2,4-DP, as a substrate for laccase, can be oxidized by laccase into quinones. In the presence of 4-AP, a quinoneimine complex, quinoneimine (QI), is generated. It exhibits an ultraviolet absorption peak at 510 nm, and the molar extinction coefficient of QI is ε = 12600 L / mol·cm -1 .

[0079] The steps are as follows: 2,4-dichlorophenol (2,4-DP) (10 mM, 30 μL) and 4-aminoantipyrine (4-AP) (10 mM, 30 μL), and NiOOH NFs synthesized in Example 1 (1 mg / mL, 30 μL) were ultrasonically mixed in phosphate buffer (pH 7.0, 210 μL), and incubated at 35°C for 15 min. A control group without NiOOH NFs was used. 200 μL was taken and transferred to a 96-well plate, and its absorbance at 510 nm (A 510nm ).

[0080] The verification results of NiOOH NFs laccase mimicking activity are as follows Figure 5 As shown in the figure, compared with the control group (curve b), only when NiOOH NFs exist (curve a) does an obvious absorption peak exist at 510 nm, indicating that NiOOH NFs have laccase mimetic activity.

[0081] Figure 6 This is the double reciprocal curve of the enzyme activity kinetics analysis of NiOOH NFs under the optimal experimental conditions (35°C, pH 7.0, 120μg / ml NiOOH NFs). The K of NiOOH NFs was obtained from the figure. m 0.064mM, V max 0.3 μM s -1 .

[0082] Steady-state kinetic studies are based on the following Michaelis-Menten equation:

[0083] V=V max [S] / ([S]+K m )

[0084] Where V is the initial reaction rate, V max is the initial maximum reaction rate, [S] is the substrate concentration, K m is the Michaelis-Menten constant.

[0085] Test Example 2

[0086] Verification of the performance of anodic glucose electrooxidation catalysts in enzyme-free biofuel cells.

[0087] The performance of the biofuel cell anode electrooxidation catalyst was verified using a standard three-electrode system, with a glassy carbon electrode modified with Pt NPs@bacteriophage as the working electrode, a saturated calomel electrode as the reference electrode, a platinum sheet as the counter electrode, and a phosphate buffered saline solution (PBS, pH = 7.0) containing 10 mM glucose as the electrolyte.

[0088] The catalytic performance and process of PtNPs@phage catalyst in glucose oxidation reaction were investigated by cyclic voltammetry (CV) and chronoamperometry (IT). In order to demonstrate the advantages of the electrocatalyst of the present invention, a series of comparisons were made with a commercial 20wt% platinum-carbon catalyst.

[0089] Figure 7 The CV curves of Pt NPs@phage and a commercial 20wt% Pt-on-carbon catalyst for the electrooxidation of 10 mM glucose are shown. The results show that Pt NPs@phage exhibits superior catalytic performance compared to the commercial 20wt% Pt-on-carbon catalyst. This is primarily attributed to the unique structure of the phage template, which not only improves the dispersion of the Pt nanoparticles but also increases the exposure of the active sites, thereby enhancing the catalytic efficiency.

[0090] Figure 8 This is an investigation of the electrochemical kinetics of glucose electrocatalytic oxidation on PtNPs@phage-modified platinum carbon electrode. From the CV diagram of scan rate and glucose oxidation current, it can be seen that with the increase of scan rate, the redox current of glucose increases. Figure 8 A. Oxidation current around 0.1V (i pa at 0.1V) shows a linear relationship proportional to the square root of the scan rate, see Figure 8 B. This proves that the electrooxidation process of glucose on the catalyst surface is a typical diffusion-controlled process.

[0091] Figure 9 A is the response of the PtNPs@phage and 20% commercial Pt / C modified electrodes to glucose. At a potential of +0.3V, both the PtNPs@phage and 20% commercial Pt / C electrodes responded, but the current change of the commercial platinum carbon modified electrode was not as obvious as that of the PtNPs@phage modified electrode. It only had a rapid and stable current effect when the glucose concentration was 0.5mM. The change of the PtNPs@phage modified electrode was very obvious, and a clear and stable current signal was generated when the glucose concentration was only 0.1mM. Figure 9 B is the linear relationship between the catalytic current response and glucose concentration. In the range of glucose concentration from 0.1 to 3 mM, the sensitivity of PtNPs@phage / GCE is 0.645 μA·mM-1 cm -2 , which is higher than 0.555 μA·mM of 20% Pt / C / GCE -1 cm -2 , which indicates that PtNPs@phage has better ability to electrocatalytically oxidize glucose than commercial platinum carbon.

[0092] Test Example 3

[0093] Verification of the electrocatalytic oxygen reduction activity of a laccase-mimicking nanozyme biocathode in a biofuel cell

[0094] The electrocatalytic oxygen reduction performance of the laccase-mimicking nanozyme biocathode of the biofuel cell was tested using a standard three-electrode system, with a glassy carbon electrode modified with NiOOH NFs (i.e., the laccase-mimicking nanozyme biocathode) as the working electrode, a saturated calomel electrode as the reference electrode, a platinum sheet as the counter electrode, and a phosphate buffer solution (PBS, pH = 7) containing 1 mM ABTS as the electrolyte. All electrochemical tests were performed in an air-saturated environment at room temperature.

[0095] Firstly, the laccase-like nanozyme biocathode was analyzed using linear sweep voltammetry (LSV). Figure 10 As shown in the figure, the onset potential of oxygen reduction catalyzed by NiOOH NFs was studied under the optimal pH conditions, namely, in a PBS buffer solution (pH 7.0) containing 1 mM ABTS, under argon-saturated and air-saturated conditions. The results showed that the onset potential of oxygen reduction of NiOOH NFs / GCE was 640 mV, indicating that NiOOH NFs / GCE has a relatively low overpotential for oxygen reduction catalysis and can be used as the cathode of biofuel cells.

[0096] Figure 11 The anode and cathode are connected using a dual-chamber electrolytic cell to form a biofuel cell. The polarization current curve (line b) of the biofuel cell BFCs is obtained using linear sweep voltammetry at a scan rate of 10mV / s. The power density curve (line a) is obtained by calculation. For comparison, a commercial platinum-carbon modified anode was made and assembled with the cathode in the same way to obtain glucose BFCs. The polarization current curve of the BFCs is line d, and the power density curve is line c. The output potential of the BFCs assembled with Pt NPs@bacteriophage anode is 550mV, and the maximum power density is 47.5μW cm -2 The output potential and maximum power density are significantly better than those of commercial platinum-carbon assembled biofuel cells.

[0097] Figure 12The power density curves under different glucose concentrations (PBS buffer containing 0.1mM, 0.5mM, 1mM, 5mM, and 10mM glucose, respectively) were measured. The maximum power output density and open circuit voltage of the biofuel cell increased with the increase of glucose concentration.

[0098] Figure 13 The operating stability of BFC was further explored. Within 6 hours of continuous operation, the power density decayed by only about 10%. After 8 to 10 hours of operation, the power density decay became larger. This may be due to the increase in the adsorption of glucose on the electrode surface, which affects the further oxidation of glucose.

[0099] The above studies show that the biofuel cell constructed by the present invention using a platinum nanoparticle anode with a phage as a template and a laccase-like nanozyme cathode has good potential output and power output.

[0100] This biofuel cell based on glucose electrooxidation catalyst and laccase-like nanozyme can not only significantly improve the performance and stability of the biofuel cell, but also has the advantages of low cost and simple operation.

[0101] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, and modifications made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An enzyme-free glucose biofuel cell, characterized in that: The PtNPs@phage modified electrode was used as the bioanode, and the anolyte was a phosphate buffer containing 0.1-10 mM glucose; the electrode modified with nickel oxyhydroxide nanoflowers with laccase activity was used as the biocathode, and the catholyte was a phosphate buffer containing 1 mM 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid).

2. The enzyme-free glucose biofuel cell according to claim 1, characterized in that The enzyme-free biofuel cell is a dual-chamber H-type biofuel cell, wherein the anode chamber and the cathode chamber are separated by a proton exchange membrane; The bioanode is a glassy carbon electrode loaded with PtNPs@bacteriophage, and the anolyte is a phosphate buffer containing 5-10 mM glucose; The biological cathode is a glassy carbon electrode loaded with nickel oxyhydroxide nanoflowers, the cathode electrolyte is a phosphate buffer solution containing 1mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and the pH value of the electrolyte is 7-8.

3. A method for preparing the enzyme-free biofuel cell according to claim 1, characterized in that: The following steps are involved: The synthesized nickel oxyhydroxide nanoflowers were ground into powder and then made into a suspension, which was then dropped onto the electrode to prepare a biocathode. The synthesized PtNPs@bacteriophage was freeze-dried in a vacuum chamber, resuspended in deionized water to prepare a suspension, and then added dropwise to the electrode to prepare a bioanode.

4. The preparation method according to claim 3, characterized in that The method for synthesizing the nickel oxyhydroxide nanoflowers comprises the following steps: Under stirring conditions in a water bath at 60-70° C., adding aqueous ammonia to the nickel nitrate solution at a molar ratio of 1:2 to obtain a nickel hydroxide precipitate, which was then centrifuged, washed, dried, and then ground into powder; A certain amount of nickel hydroxide powder was weighed, and a sodium hypochlorite aqueous solution with a concentration of 6% to 14% was added thereto. The mixture was stirred at room temperature for 0.5 to 1.5 hours, and centrifuged and washed until the supernatant was neutral. The black precipitate was collected and dried to obtain nickel oxyhydroxide nanoflowers.

5. The preparation method according to claim 3, characterized in that The synthesis method of Pt NPs@bacteriophage comprises the following steps: (1) Escherichia coli was inoculated into a liquid culture medium and cultured at 37°C with shaking until the solution was slightly turbid; then wild-type phage was added and the reaction was shaken at 37°C for 4-5 hours. The supernatant was collected by centrifugation, and the phage was precipitated and resuspended. This was repeated several times and then centrifuged to obtain the supernatant, which was the wild-type phage. The phage titer was then measured; (2) Dilute 0.1M hexachloroplatinic acid aqueous solution to a final concentration of 1mM in a 70-80℃ water bath, stir evenly, and wait for 1 minute; add 0.5M trisodium citrate aqueous solution while stirring to a final concentration of 0.1mM, and stir for 30 minutes; then quickly add 0.1M sodium borohydride aqueous solution dropwise, stir in a 70-80℃ water bath, and after the temperature drops to room temperature, take the prepared PtNPs, mix them with 0.1mM CuCl2 in a volume ratio of 1:1, incubate with shaking for 30 minutes, and collect the precipitate after centrifugation and washing; (3) Add the phage solution to the washed Pt NPs, shake overnight, and collect the precipitate by centrifugation to obtain Pt NPs@phage.

6. The preparation method according to claim 5, characterized in that In the step (1), ER2738 Escherichia coli is inoculated into LB liquid culture medium and cultured with shaking at 37°C until the solution becomes slightly turbid; then wild-type M13 phage is added, and the reaction is shaken at 37°C for 4-5 hours. The supernatant is collected by centrifugation, and 5-8 mL of a PEG8000 / NaCl mixture is added to precipitate the phage. The supernatant is removed by centrifugation and resuspended with TBS. The phage is repeatedly precipitated with the PEG8000 / NaCl mixture, and the phage is resuspended in TBS after a second precipitation. The supernatant is obtained by centrifugation, which is pure wild-type M13 phage.

7. The preparation method according to claim 5, characterized in that In the step (2), 100 μL of 0.1 M hexachloroplatinic acid aqueous solution was added to 10 mL of ultrapure water at a final concentration of 1 mM in a 75°C water bath, stirred moderately, and waited for 1 minute; 20 μL of 0.5 M trisodium citrate aqueous solution was added while stirring to a final concentration of 0.1 mM as a stabilizer, and stirred for 30 minutes; then 150 μL of 0.1 M sodium borohydride aqueous solution was quickly added dropwise, stirred in a 75°C water bath, and after the temperature was lowered to room temperature, 500 μL of the prepared PtNPs was taken and mixed with 0.1 mM CuCl2 at a volume ratio of 1:1, incubated with shaking for 30 minutes, and the precipitate was collected after centrifugal washing.

8. Use of the non-enzyme glucose biofuel cell according to any one of claims 1 to 2 or the non-enzyme glucose biofuel cell prepared by the preparation method according to any one of claims 3 to 7 in the preparation of a sensor using glucose as fuel.

9. The use according to claim 8, characterized in that The sensor is a portable sensor.

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