3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecular nanoezyme, working electrode and biofuel cell

Abstract

The application belongs to the technical field of bio-fuel cell construction, and discloses 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nano-enzyme, a bio-fuel cell electrode prepared by the nano-enzyme, and a high-performance novel bio-fuel cell. The 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nano-enzyme can be used as glucose oxidase or / and hydrogen peroxidase. The 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nano-enzyme PD is used to prepare a cell working electrode PD / CNT, and the PD / CNT is used as an anode and a cathode to prepare a self-cascading nano-enzyme bio-fuel cell BFC. The bio-fuel cell BFC has high output power and outstanding stability.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bio-fuel cell construction, and particularly relates to a 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nano-enzyme PD, a bio-fuel cell electrode prepared therefrom, and application of the bio-fuel cell electrode in construction of a high-performance novel bio-fuel cell. BACKGROUND

[0002] Nano-enzymes are a kind of artificial enzymes synthesized based on inorganic nanomaterials, which have both the properties of nanomaterials and the properties of enzymes. Compared with natural enzymes, nano-enzymes with the properties of artificial enzymes have the advantages of high catalytic stability, good adjustability, and feasible preparation process, which have attracted great attention. Since Fe3O4 nanoparticles were reported to have peroxidase-like activity in 2007, more and more metal-based nanomaterials have been found to exhibit enzyme-like activity, such as peroxidase, catalase, oxidase, and superoxide dismutase. Nano-enzymes have been widely used in the fields of biosensing, antibacterial therapy, and disease diagnosis. Although nano-enzymes have been pushed onto the historical stage as substitutes for natural enzymes, improving selectivity, biocompatibility, and multifunctionality remains a challenge.

[0003] Supramolecular chemistry has become a powerful tool for preparing multifunctional nanomaterials through simple reversible non-covalent interactions. Supramolecular nano-enzymes with dynamic structures and complex hierarchical structures have attracted great attention due to their potential as enzyme mimics in practical applications. Supramolecular strategies can easily manipulate the morphology and structure of materials constructed by self-assembly, providing a convenient method for improving catalytic activity. Various self-assembled materials, such as peptides, dendrimers, and coordination polymers, have shown potential in constructing effective nano-enzymes. Although many studies have investigated the activity and catalytic mechanism of supramolecular nano-enzymes, the behavior of nano-enzymes in the aggregation state of the material itself has been relatively unexplored.

[0004] Conjugated molecules have been widely used in energy storage, gas separation, sensing design, and catalysis due to their high π-electron delocalization properties, high chemical / thermal stability, structural diversity, and excellent carrier mobility. However, conjugated molecules are mainly used as precursors for synthesizing nano-enzymes.

[0005] Biofuel cells (BFCs) are simple and efficient energy supply devices that can directly convert chemical energy into electrical energy, and have been widely applied in implantable power sources, self-powered sensors, etc. However, due to the limitations of biological enzymes and anodic oxidation by-products (H2O2), BFCs still face several key challenges of low output power and poor stability. Meanwhile, different electrode materials have different optimal reaction conditions in two separate catalytic reactions, resulting in complex analysis process. In view of the above limitations, it is of great significance to find a new type of self-cascading nanoenzyme catalyst, and to construct a high-performance biofuel cell with excellent output power and stability based on the same nanoenzyme material for both anode and cathode. SUMMARY

[0006] Therefore, the present application aims to provide 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoenzyme, a biofuel cell electrode prepared therefrom, and application thereof in constructing a high-performance novel biofuel cell. The 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoenzyme has high glucose oxidase-like activity, hydrogen peroxidase-like activity and electrocatalytic activity.

[0007] The present application provides 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoenzyme, which can be used as glucose oxidase-like enzyme or / and hydrogen peroxidase-like enzyme.

[0008] Further, the 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoenzyme exhibits irregular rod-like structure under a microscope; the average length of the 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoenzyme is 10-15 μm.

[0009] The present application provides a preparation method of 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoenzyme, which comprises the following steps: dispersing 3,4,9,10-perylenetetracarboxylic dianhydride powder in water, and ultrasonic treatment to form a uniform system, thereby obtaining a red 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoenzyme solution.

[0010] Further, in the preparation method, 0.5-2 mg of 3,4,9,10-perylenetetracarboxylic dianhydride powder is dispersed in 2-10 mL of water; the ultrasonic treatment time is 10-20 min.

[0011] The present application provides a biofuel cell electrode, which comprises at least one of the above-mentioned 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoenzyme or at least one of the 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoenzyme prepared by the above-mentioned preparation method, and an enzyme immobilization material and a cell substrate.

[0012] Further, the enzyme immobilization material is carbon nanotubes; and the battery substrate is an ITO electrode.

[0013] The preparation method of the bio-fuel cell electrode comprises the following steps,

[0014] The 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nano-enzyme solution and the carbon nanotube solution are mixed in a volume ratio of 1:1 under stirring, and stirring is performed at room temperature; the mixture is centrifuged to remove unbound carbon nanotubes, and is resuspended in an aqueous solution to obtain a mixed suspension; finally, the prepared mixed suspension is dropped onto the surface of the ITO electrode, and drying is performed to obtain the bio-fuel cell electrode PD / CNT electrode.

[0015] The bio-fuel cell electrode provided by the application is used in the preparation of a bio-fuel cell or a biosensor.

[0016] The application provides a dual-chamber bio-fuel cell, and the above bio-fuel cell electrode PD / CNT electrode is used as an anode or / and a cathode.

[0017] Finally, the application further provides a construction method of the dual-chamber bio-fuel cell, which comprises the following steps:

[0018] 1) The bio-fuel cell electrode PD / CNT electrode is prepared according to the preparation method of the bio-fuel cell electrode provided by the application; wherein the PD / CNT electrode with glucose oxidase-like activity and hydrogen peroxidase-like activity is used as a cascade anode, and the PD / CNT electrode with electrocatalytic activity is used as a cathode;

[0019] 2) The solution in the anode chamber is 5-10 mL of HAC-NaAC buffer solution with a pH of 4.0, and 0-40 mmol / L glucose is contained in the solution;

[0020] 3) The solution in the cathode chamber is 5-10 mL of HAC-NaAC buffer solution with a pH of 4.0, and the cathode chamber is in an air-saturated condition;

[0021] 4) A proton semi-permeable membrane is used to separate the anode chamber and the cathode chamber, and a dual-chamber bio-fuel cell BFC with a self-cascade nano-enzyme is constructed.

[0022] The application provides a 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nano-enzyme, which can be used as glucose oxidase-like enzyme or / and hydrogen peroxidase-like enzyme.

[0023] Firstly, the 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecular nanozyme has glucose oxidase-like activity or / and catalase-like activity, and is involved in the construction of an electrode, in the anode, the nanozyme PD is a cascade anode, which can catalyze glucose oxidation to produce gluconic acid and H2O2 by simulating the activity of glucose oxidase, and the generated H2O2 is consumed in situ by the catalase-like activity of PD, avoiding the interference of H2O2 generated by glucose oxidation on the battery. In the cathode, the nanozyme PD electrocatalytic activity makes the cathode receive the electrons generated by the anode and undergoes an oxygen reduction reaction, which can construct a new type of high-performance BFC.

[0024] Secondly, compared with the BFCs in which the anode and the cathode are composed of different electrodes, the self-cascading nanozyme BFCs of the application are based on PD / CNT, and the optimal conditions of the double enzyme activity on the PD / CNT electrode are similar, which can be adjusted to achieve the optimal state at the same time, promoting the application of cascade reaction in BFC.

[0025] Finally, compared with the BFCs constructed by traditional natural enzymes, the BFCs based on PD / CNT of the application avoid the use of natural enzymes which are expensive, difficult to store, harsh reaction conditions, and have the advantages of simple preparation process, low cost, good stability, mild reaction conditions, not easy to be disturbed by environmental factors, etc. The biological fuel cell BFC prepared based on PD / CNT has higher output power and outstanding stability, and the output power is nearly 10 times that of the BFC based on natural enzymes. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The controllable synthesis route map of PD in different aggregation states;

[0027] Figure 2 The SEM imaging map of PD (a), PD-AH (b), and PD-RS (c) provided in Example 1 of the application;

[0028] Figure 3 The TEM imaging map of PD (a), PD-AH (b), and PD-RS (c) provided in Example 1 of the application;

[0029] Figure 4 The XRD map of PD, PD-AH, and PD-RS provided in Example 1 of the application;

[0030] Figure 5 The infrared spectrum of PD, PD-AH, and PD-RS provided in Example 1 of the application;

[0031] Figure 6 The enzyme-like activity map of PD, PD-AH, and PD-RS provided in Example 2 of the application;

[0032] Figure 7 CV plot of PD and glucose provided for Example 3 of the present application;

[0033] Figure 8 Cascade-like enzyme activity plot of PD provided for Example 4 of the present application;

[0034] Figure 9 Anodic CV plot (a) and cathodic CV plot (b) provided for Example 5 of the present application;

[0035] Figure 10 Polarization curve plot (a) and output power plot (b) provided for Example 6 of the present application. DETAILED DESCRIPTION

[0036] The present application discloses 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecular nanoscale enzyme, working electrode and biofuel cell, and those skilled in the art can refer to the content herein, and appropriately improve process parameters to realize. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are regarded as included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0037] The present application provides 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecular nanoscale enzyme PD, which can be used as glucose oxidase or / and hydrogen peroxidase.

[0038] The 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecular nanoscale enzyme PD provided by the present application shows irregular rod-like structure; the average length of the 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecular nanoscale enzyme is 10-15 μm.

[0039] The present application provides a preparation method of 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecular nanoscale enzyme PD, which comprises the following steps: dispersing 3,4,9,10-perylenetetracarboxylic dianhydride powder in water, ultrasonic treatment to form a uniform system, and obtaining a red 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecular nanoscale enzyme solution.

[0040] The above preparation method is preferably prepared according to the following method: dispersing 0.5-2 mg of 3,4,9,10-perylenetetracarboxylic dianhydride powder in 2-10 mL of secondary water; the ultrasonic treatment time is 10-20 min.

[0041] The 3,4,9,10-perylenetetracarboxylic dianhydride mentioned in the preparation method of the nanometer enzyme PD of the application is also known as perylene-3,4,9,10-tetracarboxylic dianhydride or 3,4,9,10-perylenetetracarboxylic dianhydride, and its molecular structure is as follows:

[0042]

[0043] The 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecular nanometer enzyme PD is in an aggregated form. Other different aggregation states form products in different states, wherein PD-AH is an alkaline hydrolysis product of PD synthesized by an alkaline hydrolysis reaction, and PD-RS is a recombination self-condensation product of PD-RS synthesized by an acid-assisted condensation reaction.

[0044] Specifically, the synthesis method of PD-AH and PD-RS can specifically include the following steps:

[0045] PD-AH: 0.1-0.5 g of PD powder is dissolved in 2-10 mL of potassium hydroxide solution (5%), and then ultrasonic treatment is performed for 30-60 minutes to obtain a PD-AH solution. The PD-AH solution is dried in a vacuum oven at 90-120℃ overnight, and a brown powder is collected and ground to form a PD-AH powder.

[0046] PD-RS: The PD-AH solution is stirred at 60-90℃ for 60-120 minutes to form a uniform slurry, and after the temperature of the system is reduced to room temperature, the impurities are removed by filtration, and the filtrate is acidified to 1.0-2.0 pH with dilute hydrochloric acid to obtain an orange sticky precipitate, which is centrifuged and washed with water for 3 times. The obtained precipitate is dried in a vacuum drying oven at 100-120℃ overnight, and a red-brown powder is collected and ground to obtain a PD-RS powder.

[0047] The application also provides monitoring of the glucose oxidase-like enzyme activity of PD, PD-AH and PD-RS in different aggregation states: PD, PD-AH and PD-RS are added to the same reaction system, and the change in absorbance is monitored to reflect the enzyme activity. The enzyme activity detection is preferably carried out according to the following steps:

[0048] PD, PD-AH and PD-RS are dispersed in water, and the reaction system includes a PD solution (PD-AH or PD-RS), 50 mmol / L glucose, 50 μg / mL HRP, 0.5 mmol / L TMB and NaAc-HAc buffer with a pH of 4.0. Each solution is incubated at 37℃ for 30 minutes, and the absorbance at 400-800 nm is monitored.

[0049] The test results show that the PD in the aggregated form exhibits good enzyme activity, and the aggregation level is directly related to the catalytic ability. Compared with the PD, the PD-AH has no glucose oxidase enzyme activity, and the PD-RS has reduced glucose oxidase enzyme activity; the breakage of the acid anhydride bond destroys the aggregation state of the PD-AH, resulting in no enzyme activity, and the PD-RS may be aggregated into large amorphous aggregates due to the disordered self-assembly, resulting in reduced enzyme catalytic activity.

[0050] Meanwhile, the application provides a catalytic mechanism of the PD with simulated enzyme activity. By comparing the oxidation potentials of the PD, glucose and PD / glucose, it is shown that the potential interaction between the PD and glucose leads to the electron transfer behavior.

[0051] Another application purpose of the application is to provide various applications of the PD non-metal carbon-free nanoscale enzyme.

[0052] The application provides a bio-fuel cell electrode, which comprises at least one of the above 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoscale enzyme or the 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoscale enzyme prepared by the above preparation method, and an enzyme immobilization material and a cell substrate.

[0053] The enzyme immobilization material can be specifically selected from carbon nanotubes, and the cell substrate can be preferably an ITO electrode.

[0054] The preparation method of the above bio-fuel cell electrode comprises the following steps:

[0055] The 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoscale enzyme solution and the carbon nanotube solution are mixed in a volume ratio of 1:1 under stirring, and stirring is maintained at room temperature; the mixture is centrifuged to remove the unbound carbon nanotubes, and is resuspended in an aqueous solution to obtain a mixed suspension; finally, the prepared mixed suspension is dropped onto the surface of the ITO electrode, and is dried to obtain the bio-fuel cell electrode PD / CNT electrode.

[0056] Specifically, the preparation method of the bio-fuel cell electrode is preferably prepared according to the following steps:

[0057] 1-10 mg / L PD and 1-10 mg / L CNT are mixed in a volume ratio of 1:1 under stirring, and stirring is maintained at room temperature for 2 hours. The mixture is centrifuged at 10000-12000 rpm for 10-15 minutes to remove the unbound CNT, and is resuspended in an aqueous solution, and the above operation is repeated three times. Then, 20-30 μL of the prepared PD / CNT suspension is dropped onto the surface of the above ITO electrode (0.5×4.0 cm), and is dried in a vacuum oven at 30-40°C overnight to obtain the PD / CNT electrode.

[0058] In addition, the application also provides the use of the above-mentioned bio-fuel cell electrode in the preparation of a bio-fuel cell or a biosensor.

[0059] The application provides a double-chamber bio-fuel cell, which uses the above-mentioned bio-fuel cell electrode PD / CNT electrode as an anode or / and a cathode.

[0060] The application preferably constructs the double-chamber bio-fuel cell BFC according to the following steps:

[0061] 1) preparing a bio-electrode PD / CNT electrode according to the preparation method of the bio-fuel cell electrode of the application; wherein the PD / CNT electrode with glucose oxidase-like activity and hydrogen peroxidase-like activity is used as a cascade anode, and the PD / CNT electrode with electrocatalytic activity is used as a cathode;

[0062] 2) the solution in the anode chamber is 5-10 mL of HAC-NaAC buffer with pH of 4.0, and 0-40 mmol / L glucose is contained in the solution;

[0063] 3) the solution in the cathode chamber is 5-10 mL of HAC-NaAC buffer with pH of 4.0, and the cathode chamber is under air saturation condition;

[0064] 4) a proton semi-permeable membrane is used to separate the anode chamber and the cathode chamber, thereby constructing the double-chamber bio-fuel cell BFC with self-cascade nanoscale enzyme.

[0065] The application is based on the preparation of the double-chamber bio-fuel cell BFC with self-cascade nanoscale enzyme by using PD / CNT. In the anode, the PD / CNT can catalyze the oxidation of glucose to produce gluconic acid and H2O2 by simulating the glucose oxidase activity, and the generated H2O2 is consumed in situ by the hydrogen peroxidase-like activity of PD, thereby avoiding the interference of H2O2 on the cell. The cathode receives the electrons generated by the anode and undergoes an oxygen reduction reaction, thereby constructing a novel high-performance BFC.

[0066] In order for those skilled in the art to better understand the application, the application will be further described in detail below in combination with specific examples, but they should not be understood as limiting the protection scope of the application.

[0067] Example 1 Synthesis of PD in different aggregation states

[0068] 1.1 dispersing 0.5-2 mg of 3,4,9,10-perylenetetracarboxylic dianhydride powder in 2-10 mL of water; ultrasonic treatment for about 10-20 min to obtain 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecular nanoscale enzyme PD.

[0069] This example specifically prefers dispersing 1 mg of 3,4,9,10-perylenetetracarboxylic dianhydride powder in 7 mL of water.

[0070] 1.2 Dissolve 0.1-0.5 g of PD powder in 2-10 mL of potassium hydroxide solution (5%), then ultrasonic treatment for about 30-60 min to obtain PD-AH solution. The PD-AH solution is dried in a vacuum oven at 90-120 °C overnight, collect the brown powder, the powder is placed in a clean agate mortar, grinding to form PD-AH powder.

[0071] This embodiment is particularly preferred 0.3 g of PD powder dissolved in 6 mL of potassium hydroxide solution with a molar concentration of 1 mol / L

[0072] 1.3 10 mL of PD-AH solution prepared in 1.2 is stirred at 60-90 °C for about 60-120 min to form a uniform slurry, after the temperature of the system is reduced to room temperature, the impurities are removed by filtration, and the brownish green fluorescent filtrate is collected. The filtrate is acidified to pH about 1.0-2.0 with dilute hydrochloric acid to obtain orange sticky precipitate, centrifuged at 8000 revolutions per minute, washed with water twice. The obtained precipitate is dried in a vacuum drying oven at 100-120 °C overnight, and the red-brown powder is collected. The powder is placed in a mortar, ground to obtain PD-RS powder.

[0073] The morphology and size of PD, PD-AH and PD-RS were studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images. As shown in Figure 2 a and Figure 3 a, due to π-π stacking and hydrogen bonding, PD exhibits irregular rod-like structure with a length of about 10-15 μm. As shown in Figure 2 b and Figure 3 b, due to the breaking of anhydride bond under alkaline conditions caused by KOH treatment, the rod-like structure of PD is destroyed, resulting in the length of PD-AH reduced to 2-6 μm. As shown in Figure 2 c and Figure 3 c, after acid-assisted treatment, the broken anhydride bond reassembles into blocky structure with a size of about 20-30 μm. In addition, the semiconductor crystal properties and structural composition of PD, PD-AH and PD-RS were characterized by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). As shown in Figure 4 and Figure 5 It can be observed that the characteristic peaks of C-O-C and C=O of PD and PD-RS, indicating the presence of anhydride in PD and PD-RS. In PD-AH, the C-O-C absorption peak completely disappears, and the C=O absorption peak shifts, indicating the destruction of anhydride and the formation of carboxyl. In summary, the above results strongly verify the successful synthesis of PD-AH and PD-RS with different morphology and size.

[0074] Example 2 Comparison of PD, PD-AH and PD-RS with simulated glucose oxidase activity

[0075] Experimental system a: the catalytic reaction system contains 0.5 mmol / L TMB solution, 50 μg / mL HRP solution, 50 mmol / L glucose solution, HAC-NaAC buffer solution with pH of 4.0 and PD prepared in Example 1, and the absorbance value in 400-800 nm is detected by ultraviolet-visible spectrophotometer after reaction for 30 minutes at 37℃;

[0076] Experimental system b: the catalytic reaction system contains 0.5 mmol / L TMB solution, 50 μg / mL HRP solution, 50 mmol / L glucose solution, HAC-NaAC buffer solution with pH of 4.0 and PD-AH prepared in Example 1, and the absorbance value in 400-800 nm is detected by ultraviolet-visible spectrophotometer after reaction for 30 minutes at 37℃;

[0077] Experimental system c: the catalytic reaction system contains 0.5 mmol / L TMB solution, 50 μg / mL HRP solution, 50 mmol / L glucose solution, HAC-NaAC buffer solution with pH of 4.0 and PD-RS prepared in Example 1, and the absorbance value in 400-800 nm is detected by ultraviolet-visible spectrophotometer after reaction for 30 minutes at 37℃;

[0078] In addition, three control experiments: in the catalytic reaction of control experiment d, no PD is added, and the absorbance value is detected after reaction for 30 minutes under the same conditions as experimental system a; in the catalytic system of control experiment e, no PD and HRP are added, and the absorbance value is detected after reaction for 30 minutes under the same conditions as experimental system a; the catalytic system of control experiment f is TMB solution (0.5 mmol / L) in HAC-NaAC (pH 4.0) buffer, and the absorbance value is detected after reaction for 30 minutes under the same conditions as the above experimental systems.

[0079] Figure 6 The enzyme activity of PD, PD-AH and PD-RS is shown in Figure Figure 6 As shown in Figure, experimental system a shows ultraviolet absorption peak at 652 nm, indicating that the aggregated PD shows glucose oxidase-like enzyme activity (curve 10). Experimental system b shows no ultraviolet absorption peak at 652 nm, indicating that the breakage of acid anhydride bond destroys the aggregation state of PD-AH, resulting in no glucose oxidase-like enzyme activity (curve 8). Compared with PD, experimental system c shows reduced ultraviolet absorption peak at 652 nm, indicating that PD-RS is possibly due to disordered self-assembly into large amorphous aggregates, resulting in reduced enzyme catalytic activity (curve 9). The aggregation level is directly related to the catalytic ability. The aggregated PD shows enzyme activity, while the PD-HA and PD-RS with changed aggregation state lose or reduce enzyme activity, indicating that the aggregation level is directly related to the catalytic ability of PD.

[0080] Example 3: Glucose oxidation potential analysis of conjugated molecular nanozyme PD

[0081] Cyclic voltammetry (CV) tests were performed on a CHI 660E electrochemical workstation using a Pt wire electrode as the counter electrode, Ag / AgCl as the reference electrode, and a glassy carbon electrode (GCE) as the working electrode. The CV tests were conducted in a deoxyacetonitrile solution containing 0.10 M tetrabutylhexafluorophosphonate ammonium as the supporting electrolyte under a N2 atmosphere.

[0082] Experimental System A: Glucose was dissolved in a deoxyacetonitrile solution containing 0.10 M tetrabutylhexafluorophosphonate ammonium as the supporting electrolyte. Nitrogen was bubbled into the solution for 30–60 minutes to remove oxygen, and the system was saturated with nitrogen during the experiment. The CV measurement scan rate was 100 mV / s, and the measurement scan range was 0–1.6 V. The entire measurement was performed at room temperature.

[0083] Experimental System B: The PD described in Example 1 was dissolved in a deoxyacetonitrile solution containing 0.10 M tetrabutylhexafluorophosphonate ammonium as the supporting electrolyte. Nitrogen was bubbled into the solution for 30–60 minutes to remove oxygen, and the system was saturated with nitrogen during the experiment. The CV measurement scan rate was 100 mV / s, and the measurement scan range was 0–1.6 V. The entire measurement was performed at room temperature.

[0084] Experimental System C: Glucose and the PD described in Example 1 were dissolved in a deoxyacetonitrile solution containing 0.10 M tetrabutylhexafluorophosphonate ammonium as the supporting electrolyte. Nitrogen was bubbled into the solution for 30–60 minutes to remove oxygen, and the system was saturated with nitrogen during the experiment. The CV measurement scan rate was 100 mV / s, and the measurement scan range was 0–1.6 V. The entire measurement was performed at room temperature.

[0085] Figure 7 The CV diagram of PD and glucose provided in Embodiment 3 of the present invention is shown below. Figure 7 As shown, in the glucose / PD system, the PD oxidation peak (curve 2) is located at approximately +1.4 V, while the glucose oxidation peak is located at approximately +0.8 V (curve 1). When PD and glucose are present simultaneously, the glucose oxidation peak shifts to the left to approximately +0.7 V (curve 3). This indicates that the potential interaction between PD and glucose leads to electron transfer behavior.

[0086] Example 4: Verification of the tandem catalytic activity of conjugated molecular nanozyme PD

[0087] Experimental system g: the catalytic reaction system comprises 0.5 mmol / L TMB solution, 50 mmol / L glucose solution, HAC-NaAC buffer solution with pH of 4.0 and PD, and the absorbance value in 400-800 nm is detected by using a UV-visible spectrophotometer after reaction at 37℃ for 30 minutes;

[0088] Experimental system h: the catalytic reaction system comprises 0.5 mmol / L TMB solution, 50 μg / mL HRP solution, 50 mmol / L glucose solution, HAC-NaAC buffer solution with pH of 4.0 and PD, and the absorbance value in 400-800 nm is detected by using a UV-visible spectrophotometer after reaction at 37℃ for 30 minutes;

[0089] Experimental system i: the catalytic reaction system comprises 0.5 mmol / L TMB solution, 0.5 mmol / L H2O2, HAC-NaAC buffer solution with pH of 4.0 and PD, and the absorbance value in 400-800 nm is detected by using a UV-visible spectrophotometer after reaction at 37℃ for 30 minutes;

[0090] Experimental system j: the catalytic reaction system comprises 0.5 mmol / L TMB solution, 50 μg / mL HRP solution, 50 μg / mL GOx solution, 50 mmol / L glucose solution and HAC-NaAC buffer solution with pH of 4.0, and the absorbance value in 400-800 nm is detected by using a UV-visible spectrophotometer after reaction at 37℃ for 30 minutes;

[0091] Four control experiments: in the catalytic reaction of control experiment k, no glucose is added, and the absorbance value is detected after reaction for 30 minutes under the same conditions of experimental system g; in the catalytic system of control experiment l, no glucose is added, and the absorbance value is detected after reaction for 30 minutes under the same conditions of experimental system h; in the catalytic system of control experiment m, no PD is added, and the absorbance value is detected after reaction for 30 minutes under the same conditions of experimental system i; the catalytic system of control experiment n is that PD is in HAC-NaAC (pH of 4.0) buffer solution, and the absorbance value is detected after reaction for 30 minutes under the same conditions of the above experimental systems; the catalytic system of control experiment o is TMB solution (0.5 mmol / L) in HAC-NaAC (pH of 4.0) buffer solution, and the absorbance value is detected after reaction for 30 minutes under the same conditions of the above experimental systems.

[0092] Figure 8 The cascade enzyme activity diagram of the PD provided for example 4 of the present application is as shown in Figure 8As shown, PD exhibited two broad absorption peaks in the wavelength range of 400-800 nm, which was attributed to the π-π* transition between aromatic groups (curve 1). Meanwhile, when only horseradish peroxidase (HRP) was added, there was neither color change nor appearance of the absorption peak of oxidized TMB at 652 nm during the catalysis of PD (curve 7). When H2O2 and TMB were present in the system, the solution color changed from red (or colorless) to blue under the catalysis of PD, with the maximum absorption peak at 652 nm, indicating that PD had catalase activity (curve 5). When PD (or GOx), glucose and horseradish peroxidase (HRP) coexisted in the system, the solution color changed from red (or colorless) to blue, with the maximum absorption peak at 652 nm, indicating that PD (or GOx) could catalyze the oxidation of glucose and accompany the formation of H2O2 (curves 8 and 9). Meanwhile, when glucose was added, an absorption peak appeared at 652 nm during the catalysis of PD, indicating that PD could catalyze the oxidation of glucose to produce H2O2, and further catalyze the decomposition of H2O2 to promote the oxidation of TMB to develop color (curve 6).

[0093] Example 5 Preparation of PD / CNT electrode and feasibility test

[0094] 5.1 Preparation of PD / CNT electrode

[0095] 5 mg / L PD and 5 mg / L CNT were mixed at a volume ratio of 1:1 under stirring, and stirring was maintained at room temperature for 2 hours. The mixture was centrifuged at 10,000-12,000 rpm for about 10-15 min to remove unbound CNT, and resuspended in an aqueous solution. The above operation was repeated three times. Then, about 20-30 μL of the prepared PD / CNT suspension was dropped onto the surface of the above ITO electrode (0.5 x 4.0 cm). Vacuum drying was performed in a vacuum oven at about 30-40 °C overnight to obtain a PD / CNT electrode.

[0096] 5.2 Feasibility test of PD / CNT as anode and cathode

[0097] Anode: Cyclic voltammetry test was performed in a three-electrode system of an electrochemical workstation, using a Pt wire as a counter electrode, Ag / AgCl as a reference electrode, and the PD / CNT electrode prepared in Example 5.1 as a working electrode. CV test was performed in a 0-40 mmol / L glucose solution from -0.8 to -0.1 V, with a scan rate of 100 mV / s.

[0098] Cathode: cyclic voltammetry test was performed in a three-electrode system of electrochemical workstation, using Pt wire as counter electrode, Ag / AgCl as reference electrode, and PD / CNT of Example 5.1 as working electrode. CV test was performed from -1 to 0.6 V in solution with different dissolved oxygen content, with a scan rate of 100 mV / s.

[0099] Figure 9 Provided are an anode CV graph (a) and a cathode CV graph (b) for Example 5 of the present application; as shown in Figure 9 a, the reduction peak current significantly decreases with the increase of glucose concentration in the concentration range of 0-40 mmol / L of glucose concentration, mainly due to the consumption of O2 by anode glucose oxidation, indicating that PD / CNT can effectively catalyze glucose oxidation. As shown in Figure 9 b, compared with N2 saturation condition, the cathode current increases under air saturation condition, and further significantly increases under O2 saturation condition, indicating that PD / CNT can effectively catalyze oxygen reduction.

[0100] Example 6 Construction and performance test of double-chamber biofuel cell BFC

[0101] 1) The PD / CNT electrode with glucose oxidase-like and catalase-like activities prepared in Example 5 is used as a cascade anode of the fuel cell, and the PD / CNT electrode with electrocatalytic activity prepared in Example 5 is used as a cathode of the fuel cell at the same time;

[0102] 2) The solution in the anode chamber is about 5-10 mL of HAC-NaAC buffer with a pH of about 4.0, containing about 0-40 mmol / L of glucose;

[0103] 3) The solution in the cathode chamber is about 5-10 mL of HAC-NaAC buffer with a pH of about 4.0, and the cathode chamber is under air saturation condition;

[0104] 4) The anode chamber and the cathode chamber are separated by a proton semi-permeable membrane, and a double-chamber biofuel cell BFC from cascade nanoscale enzyme is constructed.

[0105] The double-chamber biofuel cell BFC from cascade nanoscale enzyme is operated at room temperature. Open circuit test is performed in the concentration range of 0-40 mmol / L of glucose concentration. The polarization curve of the BFC is measured by linear sweep voltammetry at a scan rate of 100 mV / s, and is operated at room temperature. According to the polarization curve, the relationship between power output and current is calculated by the formula P=UI.

[0106] Figure 10 Provided are a polarization curve graph (a) and an output power graph (b) for the present application. As shown in Figure 10 , the open circuit voltage (E OCV) and maximum power output (P max ) both increase with increasing glucose concentration. When the glucose concentration is 40 mmol / L, the E OCV and P max can reach 0.32 V and 1.16 μW cm -2 The output power of the double-chamber biofuel cell BFC provided by the present application is nearly 10 times that of a natural enzyme-based BFC, and the stability of the double-chamber biofuel cell BFC is better.

[0107] The optimum temperature of the glucose oxidase-like activity of PD / CNT is 37-50℃, and the optimum pH is 4.0-5.0; the optimum temperature of the hydrogen peroxide oxidase-like activity of PD / CNT is 37-60℃, and the optimum pH is 4.0-6.0. Both the anode and the cathode of the fuel cell BFC are based on the PD / CNT electrode, and the optimum conditions of the double-enzyme activity on the PD / CNT electrode are similar, so the optimum state can be achieved by adjusting the temperature and the pH at the same time, and the cascade reaction in the BFC is promoted.

[0108] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecular nanoszyme, characterized in that: The 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoscale enzyme can be used as glucose oxidase or / and catalase; the 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoscale enzyme exhibits irregular rod-like structure; the average length of the 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoscale enzyme is 10-15 μm, and the 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoscale enzyme PD exists in an aggregated form; The preparation method of the 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoscale enzyme is as follows: 3,4,9,10-perylenetetracarboxylic dianhydride powder is dispersed in water, and a uniform system is formed by ultrasonic treatment to obtain a red 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoscale enzyme solution.

2. The method for preparing 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecular nanoszyme according to claim 1, characterized in that: The preparation steps are as follows: 3,4,9,10-perylenetetracarboxylic dianhydride powder is dispersed in water, and a uniform system is formed by ultrasonic treatment to obtain a red 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoscale enzyme solution.

3. The production method according to claim 2, characterized by: 0.5-2 mg of 3,4,9,10-perylenetetracarboxylic dianhydride powder is dispersed in 2-10 mL of water; the ultrasonic treatment time is 10-20 min.

4. A biofuel cell electrode characterized by: The 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoscale enzyme of claim 1 or the preparation method of any one of claims 2-3, and an enzyme immobilization material and a battery substrate.

5. The biofuel cell electrode of claim 4, wherein: The enzyme immobilization material is a carbon nanotube; and the battery substrate is an ITO electrode.

6. The biofuel cell electrode of claim 5, wherein: The preparation method of the biological fuel cell electrode comprises the following steps, The 3,4,9,10-perylenetetracarboxylic dianhydride conjugated molecule nanoscale enzyme solution and the carbon nanotube solution are mixed at a volume ratio of 1:1 under stirring, and stirring is performed at room temperature; the mixture is centrifuged to remove unbound carbon nanotubes, and is resuspended in an aqueous solution to obtain a mixed suspension; finally, the prepared mixed suspension is dropped onto the surface of the ITO electrode, and is dried to obtain a biological fuel cell electrode PD / CNT electrode.

7. Use of the biological fuel cell electrode of any one of claims 4-6 in the preparation of a biological fuel cell or a biosensor.

8. A dual-chamber biofuel cell characterized by: The biological fuel cell electrode of claim 6 is used as an anode or / and a cathode.

9. The method for constructing a dual-chamber bio-fuel cell according to claim 8, wherein: The preparation method comprises the following steps, 1) a biological fuel cell electrode PD / CNT electrode is prepared according to the preparation method of the biological fuel cell electrode of claim 6; wherein the PD / CNT electrode with glucose oxidase and catalase activities is used as a cascade anode, and the PD / CNT electrode with electrocatalytic activity is used as a cathode; 2) the solution in the anode chamber is 5-10 mL of HAC-NaAC buffer with a pH of about 4.0, and contains 0-40 mmol / L of glucose, excluding the end point value 0; 3) the solution in the cathode chamber is 5-10 mL of HAC-NaAC buffer with a pH of about 4.0, and the cathode chamber is saturated with air; 4) a proton semi-permeable membrane is used to separate the anode chamber and the cathode chamber to construct a two-chamber biological fuel cell BFC with a cascade nanoscale enzyme.

Images