An immobilized enzyme thin film electrode, a preparation method thereof and an application thereof

Abstract

The present invention discloses an immobilized enzyme thin-film electrode, comprising: an electrode, and an immobilized enzyme thin film covering the surface of the electrode, wherein the immobilized enzyme thin film comprises: an N-fluorenylmethoxycarbonyl diphenylalanine thin film and formate dehydrogenase and diallyl viologen dispersed and immobilized in the N-fluorenylmethoxycarbonyl diphenylalanine thin film. The immobilized enzyme thin-film electrode provided by the present invention can not only efficiently electrocatalytically reduce CO2 to produce formate, but also can be reused, has a simple preparation process, and is easy to regulate self-assembly.

Description

Technical Field

[0001] The present invention relates to the technical field of thin film electrode preparation, and particularly relates to an immobilized enzyme thin film electrode, a preparation method thereof, and an application thereof. Background Art

[0002] The generation of a large amount of greenhouse gases, especially CO2, can cause a series of abnormal climate phenomena such as global warming and acid rain, posing a threat to the development of human society. Therefore, how to reduce the content of CO2 in the atmosphere has become a common problem faced by people. Electrochemical CO2 reduction is a method that can combine solar or wind power generation with the energy storage in the chemical bonds of carbon-based fuels, enabling a cleaner energy cycle, and thus has received more attention from researchers. In order to provide better product selectivity, biocatalysts (such as enzymes) have received attention from researchers. Enzymes have the advantages of mild use conditions and high chemoselectivity, and can be used in commercial-scale applications of CO2 reduction.

[0003] In nature, formate dehydrogenase (FDH) is an enzyme that can catalyze the dehydrogenation of formic acid to generate CO2 in cooperation with the cofactor nicotinamide adenine dinucleotide (NADH). In fact, the reverse reaction of this enzymatic reaction can also occur in cooperation with the oxidized form NAD of NADH, becoming a way of electrocatalytic CO2 reduction. However, since using NADH / NAD + as a cofactor can catalyze both the forward reaction and the reverse reaction simultaneously, the CO2 reduction efficiency using this method is limited. + As a cofactor can catalyze both the forward reaction and the reverse reaction simultaneously, the CO2 reduction efficiency using this method is limited.

[0004] Viologen derivatives are a common type of redox-active substance. Due to their advantages such as good color development efficiency, cycle life, and contrast, they are widely used in fields such as information encryption and anti-counterfeiting. Researchers have found that when using the radical cation form (V ·+ ) of viologen derivatives as an artificial cofactor, its catalytic effect on the reduction of CO2 by FDH to generate formic acid is obvious.

[0005] In this regard, Jayathilake et al. (Acc. Chem. Res. 2019, 52, 676, hereinafter referred to as Document 1) disclosed a method for electrochemically reducing CO2 by formate dehydrogenase (FDH) to generate formate using an artificial cofactor methyl viologen (MV). Specifically, this document combined FDH and MV and carried out an electrochemical catalytic reaction in a liquid phase system, where both FDH and MV were present in the solution. However, the price of biocatalysts, especially FDH, is very expensive, reaching tens of thousands of yuan per gram, and the FDH used in the electrochemical catalytic reaction in the liquid phase system cannot be recovered and reused, which results in a very high cost for the method of electrochemically catalytically reducing CO2 to generate formate in the liquid phase.

[0006] Szczesny et al. (ACS Energy Lett. 2020, 5, 321 - 327, hereinafter referred to as Document 2) disclosed a carbon cloth electrode capable of electrocatalytic reduction of CO2, on which a viologen polymer, namely 1-(3-aminopropyl)-1'-methyl-4,4'-bipyridine (vio) modified poly(4-styrenesulfonate-co-glycidyl methacrylate-co-butyl acrylate) (P(SS-GMA-BA)-vio) and tungsten-dependent formate dehydrogenase (DvH-W-FDH), was immobilized. However, the process in this document involves the synthesis and modification of multiple materials, and three layers of functional materials were respectively modified on the macroporous side and the microporous side of the carbon cloth electrode with a gas diffusion layer, that is, a total of 4 layers of polymers containing vio artificial coenzymes and 2 layers of polymer films containing the enzyme FDH were co-modified. Not only is the synthesis cumbersome, but the film modification operation steps are also complex and time-consuming. Summary of the Invention

[0007] The purpose of the present invention is to provide an immobilized enzyme film electrode for CO2 reduction. Another purpose of the present invention is also to provide a preparation method of the immobilized enzyme film electrode and its application in the reduction of CO2.

[0008] To achieve the above purpose, the present invention provides the following technical solutions.

[0009] The present invention first provides an immobilized enzyme film electrode, including:

[0010] An electrode, and

[0011] An immobilized enzyme film covering the surface of the electrode, wherein the immobilized enzyme film includes: an N-fluorenylmethoxycarbonyl diphenylalanine film and formate dehydrogenase and diallyl viologen dispersed and immobilized in the N-fluorenylmethoxycarbonyl diphenylalanine film.

[0012] In some embodiments of the present invention, the mass ratio of diallyl viologen to formate dehydrogenase is 1:(0.5 - 1.5), preferably 1:(1 - 1.2).

[0013] In some embodiments of the present invention, in the immobilized enzyme film, the loading amount of diallyl viologen is 5×10 -7 -1×10 -6 mol / cm 2 , preferably 9×10 -7 -1×10 -6 mol / cm 2 , most preferably 9.1×10 -7 mol / cm 2 , and / or

[0014] The loading amount of the formate dehydrogenase is 0.1-0.4 mg / cm 2 , preferably 0.2-0.3 mg / cm 2 , most preferably 0.24 mg / cm 2 .

[0015] In some embodiments of the present invention, the electrode is selected from an ITO electrode, an FTO electrode, a glassy carbon electrode, a gold electrode, a carbon electrode or a carbon cloth electrode.

[0016] The present invention also provides a method for preparing the aforementioned immobilized enzyme thin film electrode, comprising:

[0017] Obtaining a first preparation solution containing N-fluorenylmethoxycarbonyl diphenylalanine,

[0018] Obtaining a second preparation solution containing formate dehydrogenase and diallyl viologen,

[0019] Mixing the first preparation solution with the second preparation solution, and standing to obtain a pre-gel,

[0020] Coating the pre-gel on the electrode, and standing to obtain the immobilized enzyme thin film electrode.

[0021] In some embodiments of the present invention, in the pre-gel, the mass ratio of diallyl viologen to formate dehydrogenase is 1:(1-1.5), preferably 1:(1-1.2).

[0022] In some embodiments of the present invention, in the pre-gel, the mass ratio of N-fluorenylmethoxycarbonyl diphenylalanine to formate dehydrogenase is 1:(2-3).

[0023] In some embodiments of the present invention, the second preparation solution is prepared by the following method:

[0024] Adding formate dehydrogenase and dithiothreitol to a first buffer solution at pH 7-8 and incubating, and then adding DAV diallyl viologen to obtain the second preparation solution. Preferably, the first buffer solution is selected from a phosphate buffer solution (PBS), a HEPES buffer solution, a Tris buffer solution, a Britton-Robinson buffer solution.

[0025] The present invention also provides an application of the aforementioned immobilized enzyme thin film electrode in reducing CO2.

[0026] In some embodiments of the present invention, the immobilized enzyme thin film electrode is placed in a second buffer solution and reduces CO2 at a working voltage of -0.8 V relative to SCE to produce formate;

[0027] Preferably, the second buffer solution is 50 mM PBS containing NaHCO3, where the concentration of NaHCO3 is 100 mM.

[0028] Beneficial effects

[0029] The immobilized enzyme thin film electrode provided by the present invention uses N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF) as a gelator to form a gel film, disperses and immobilizes the artificial cofactor diallyl viologen (DAV) and formate dehydrogenase (FDH) in the film, and obtains a molecular gel film with immobilized enzyme by one-step self-assembly on the electrode surface. The immobilized enzyme thin film electrode provided by the present invention can not only efficiently electrocatalytically reduce CO2 to produce formate, but also has a simple preparation process and easy regulation of self-assembly.

[0030] In addition, the present invention co-immobilizes natural FDH enzyme and diallyl viologen in the thin film phase, improves the catalyst stability through immobilized enzyme, realizes the reuse of products at the same time, and advantageously reduces the reduction cost of CO2. Brief description of the drawings

[0031] Figure 1 Shows the Fourier transform infrared spectra of each material, where (a) represents Fmoc-FF powder, (b) represents Fmoc-FF film, (c) represents FDH powder, (d) represents Fmoc-FF / FDH film, (e) represents DAV powder, (f) represents Fmoc-FF / DAV film, (g) represents the Fmoc-FF / DAV / FDH film ITO electrode prepared in Example 1;

[0032] Figure 2 Shows the cyclic voltammograms (scan rate 0.1 V / s) carried out in a pH 6.0 buffer solution, where (a) represents the Fmoc-FF / FDH film electrode, (b) represents the bare electrode (present in a solution containing 20 mM DAV), (c) represents the Fmoc-FF / DAV / FDH film ITO electrode prepared in Example 1;

[0033] Figure 3 Shows a schematic diagram of the conversion of DAV between different redox states;

[0034] Figure 4 Shows the cyclic voltammograms (scan rate 5 mV / s) of the Fmoc-FF / MV / FDH film glassy carbon electrode (a) prepared in Comparative Example 4 and the Fmoc-FF / DAV / FDH film glassy carbon electrode (b) prepared in Example 2 in a pH 6.0 buffer solution;

[0035] Figure 5AThe CV curves of the Fmoc-FF / DAV / FDH thin film ITO electrode prepared in Example 1 are shown (5 mV / s, PBS at pH 6.0, 100 mM NaHCO3), where (a) represents the N2 atmosphere and (b) represents the CO2 atmosphere;

[0036] Figure 5B The chronoamperometry curve: Detection was carried out in the gas diffusion mode at a constant voltage of -0.8 V (the black arrow indicates the bubbling of CO2);

[0037] Figure 6A The CV curves of the Fmoc-FF / DAV / FDH carbon cloth electrode prepared in Example 3 are shown (5 mV / s, PBS at pH 6.0, 100 mM NaHCO3), where: (a) represents N2 and (b) represents the CO2 atmosphere, Figure 6A The inset in is the CV curve of the Fmoc-FF / FDH thin film carbon cloth electrode prepared in Comparative Example 5 (5 mV / s, PBS at pH 6.0, 100 mM NaHCO3): where: (a) represents N2 and (b) represents the CO2 atmosphere;

[0038] Figure 6B The chronoamperometry curve: Detection was carried out in the gas diffusion mode at a constant voltage of -0.8 V (the black arrow indicates the bubbling of CO2);

[0039] Figure 7 The linear sweep voltammetry (LSV) curves of the Fmoc-FF / DAV / FDH thin film carbon cloth electrode prepared in Example 3 in PBS at pH 6.0 are shown (sweep rate 50 mV / s), where (a) represents the N2 atmosphere, (b) represents the CO2 atmosphere, (c) represents N2 + 100 mM NaHCO3; (d) represents CO2 + 100 mM NaHCO3; (e) is the LSV curve of the Fmoc-FF / FDH thin film carbon cloth electrode in a solution containing 100 mM NaHCO3 under the CO2 atmosphere;

[0040] Figure 8A The amperometry curves of the Fmoc-FF / DAV / FDH carbon cloth electrode prepared in Example 3 at different voltages are shown (CO2, PBS at pH 6.0, 100 mM NaHCO3);

[0041] Figure 8B The voltage dependence of the current signal I of the Fmoc-FF / DAV / FDH carbon cloth electrode prepared in Example 3 is shown (CO2, 100 mM NaHCO3, pH 6.0), and the error bars represent the standard deviation of three parallel experiments;

[0042] Figure 9Ion chromatograms of the following substances are shown: (a) PBS (CO2, 100 mM NaHCO3) at pH 6.0; (b) 20 μg / mL sodium formate (mixed standard, also containing sodium acetate, sodium propionate, sodium lactate, etc.); (c) electrolysis products collected after applying a constant voltage of -0.8 V to the Fmoc-FF / DAV / FDH thin film carbon cloth electrode for 2 h (CO2, PBS at pH 6.0, 100 mM NaHCO3). Detailed implementation mode

[0043] For a clearer understanding of the technical solutions, objectives and effects of the present invention, the specific implementation mode of the present invention will be described below with reference to the accompanying drawings.

[0044] It should be noted that for those not specified in the following examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0045] Example 1 Preparation of Fmoc-FF / DAV / FDH thin film ITO electrode

[0046] In this example, an immobilized enzyme thin film electrode was self-assembled on an ITO electrode (0.8 × 3.3 cm 2 ).

[0047] (1) First, the ITO electrode was ultrasonically cleaned in an ethanol solution containing 3.95% KOH, ethanol, and water for 5 min each, and then dried for later use.

[0048] (2) Fmoc-FF powder was ultrasonically dissolved in DMF (N,N-dimethylformamide) to obtain a first stock solution with a Fmoc-FF concentration of 30 mg / mL.

[0049] (3) FDH and dithiothreitol (DTT) were added to a HEPES buffer solution at pH 7.4 and mixed and incubated for 10 min, and then DAV was added to obtain a second stock solution, where the final concentrations of FDH, DTT, and DAV were 11.2 mg / mL, 100 mM, and 10 mg / mL, respectively.

[0050] (4) A volume of V1 of the first stock solution was quickly injected into a volume of V2 of the second stock solution using a pipette (V1:V2 = 2:13), and left standing to obtain a pre-gel (containing 4 mg / mL of Fmoc-FF).

[0051] (5) 40 μL of the pre-gel was drop-coated on the surface of the ITO electrode and left overnight at 4°C to form a Fmoc-FF thin film on the surface of the ITO electrode. FDH and DAV were dispersed and immobilized in this thin film, thus obtaining an immobilized enzyme thin film electrode, called the Fmoc-FF / DAV / FDH thin film ITO electrode.

[0052] Example 2 Preparation of Fmoc-FF / DAV / FDH Thin Film Glassy Carbon Electrode

[0053] Replace the ITO electrode in Example 1 with a glassy carbon electrode (GCE, d = 3 mm), and use the same method as in Example 1 to form a Fmoc-FF thin film with FDH and DAV dispersed and immobilized on the surface of the glassy carbon electrode, thereby obtaining an immobilized enzyme thin film electrode, called Fmoc-FF / DAV / FDH thin film glassy carbon electrode.

[0054] Example 3 Preparation of Fmoc-FF / DAV / FDH Thin Film Carbon Cloth Electrode

[0055] Replace the ITO electrode in Example 1 with a carbon cloth (CC, geometric area 0.8×4.0 cm 2 ) electrode, and use the same method as in Example 1 to form a Fmoc-FF thin film with FDH and DAV dispersed and immobilized on the surface of the carbon cloth electrode, thereby obtaining an immobilized enzyme thin film electrode, called Fmoc-FF / DAV / FDH thin film carbon cloth electrode.

[0056] For the Fmoc-FF / DAV / FDH thin film carbon cloth electrode prepared in this example, among them, the loading amount of DAV is 9.1×10 -7 mol / cm 2 , and the loading amount of FDH is 0.24 mg / cm 2 .

[0057] In this article, the term "loading amount of DAV" refers to the amount of substance of DAV immobilized on the unit area of the working electrode; the "loading amount of FDH" refers to the mass of FDH immobilized on the unit area of the working electrode.

[0058] Example 4 Preparation of Fmoc-FF / DAV / FDH Thin Film ITO Electrode

[0059] The difference between Example 4 and Example 1 is that in the second stock solution prepared in step (3), the final concentrations of FDH, DTT, and DAV are 5 mg / mL, 100 mM, and 10 mg / mL, respectively.

[0060] Example 5 Preparation of Fmoc-FF / DAV / FDH Thin Film ITO Electrode

[0061] The difference between Example 5 and Example 1 is that in the second stock solution prepared in step (3), the final concentrations of FDH, DTT, and DAV are 15 mg / mL, 100 mM, and 10 mg / mL, respectively.

[0062] Comparative Example 1 Preparation of Fmoc-FF Thin Film ITO Electrode

[0063] First, prepare the ITO electrode and the first stock solution according to steps (1) and (2) of Example 1.

[0064] Then, use a pipette to aspirate a volume V1 of the first stock solution and quickly inject it into a volume V2 of water (V1∶V2 = 2∶13) to form a pre-gel.

[0065] Drop 40 μL of the pre-gel onto the surface of the ITO electrode and place it at 4 °C overnight to form a Fmoc-FF film on the surface of the ITO electrode, thereby obtaining a Fmoc-FF film electrode.

[0066] Preparation of Fmoc-FF / FDH film ITO electrode in Comparative Example 2

[0067] First, prepare the ITO electrode and the first stock solution according to steps (1) and (2) of Example 1.

[0068] Then, add 11.2 mg / mL of FDH and 100 mM of dithiothreitol (DTT) to a HEPES buffer solution at pH 7.4 and mix and incubate for 10 min to obtain a second stock solution.

[0069] Prepare the pre-gel according to steps (4) and (5) of Example 1 and drop-coat it on the surface of the ITO electrode to form a Fmoc-FF film with FDH dispersed and immobilized on the surface of the ITO electrode, thereby obtaining a Fmoc-FF / FDH film electrode.

[0070] Preparation of Fmoc-FF / DAV film ITO electrode in Comparative Example 3

[0071] First, prepare the ITO electrode and the first stock solution according to steps (1) and (2) of Example 1.

[0072] Then, add DAV (10 mg / mL) to a HEPES buffer solution at pH 7.4 to obtain a second stock solution.

[0073] Prepare the pre-gel according to steps (4) and (5) of Example 1 and drop-coat it on the surface of the ITO electrode to form a Fmoc-FF film with DAV dispersed and immobilized on the surface of the ITO electrode, thereby obtaining a Fmoc-FF / DAV film electrode.

[0074] Preparation of Fmoc-FF / MV / FDH film glassy carbon electrode in Comparative Example 4

[0075] As described above, Jayathilake et al. used methyl viologen (MV) as an artificial cofactor (mediator) to assist FDH in the reduction of CO2.

[0076] In this comparative example, the preparation method of Example 1 was adopted, only replacing the ITO electrode with a glassy carbon electrode and replacing the equimolar concentration of DAV and other substances with MV to prepare a Fmoc-FF / MV / FDH thin film glassy carbon electrode.

[0077] Preparation of Fmoc-FF / FDH Thin Film Carbon Cloth Electrode in Comparative Example 5

[0078] Replace the ITO electrode in Comparative Example 2 with a carbon cloth (CC, geometric area 0.8×4.0 cm2) electrode, and use the same method as in Comparative Example 2 to prepare a Fmoc-FF / FDH thin film carbon cloth electrode.

[0079] Before use, the electrodes prepared in the above examples and comparative examples were first washed with deionized water to remove soluble reagents on the surface.

[0080] Characterization and Analysis of Immobilized Enzyme Thin Film Electrodes

[0081] (1) Characterization of Fmoc-FF / DAV / FDH thin film electrode

[0082] Fourier transform infrared spectroscopy (FTIR) was used to characterize the successful preparation of the Fmoc-FF / DAV / FDH molecular gel thin film in Example 1. As Figure 1 shown, in the infrared absorption spectrum of the DAV powder sample ( Figure 1 curve e in -1 ), the C═C and C═N stretching vibration peaks of the pyridine ring of DAV can be observed at 1600 - 1430 cm -1 . Several sharp peaks at 1560 cm -1 , 1508 cm -1 , and 1452 cm -1 present in the DAV powder sample can also be observed in the Fmoc-FF / DAV thin film and the Fmoc-FF / DAV / FDH thin film ( Figure 1 curves f, g in Figure 1 ), but not in the samples of Fmoc-FF powder, Fmoc-FF thin film, FDH powder, and Fmoc-FF / FDH thin film ( Figure 1 curves a, b, c, d in Figure 1 ). From this, it can be proved that DAV has been successfully immobilized in the Fmoc-FF / DAV / FDH thin film. The N-H stretching vibration peak of Fmoc-FF at 3306 cm -1 ( Figure 1 curve a in Figure 1 ) is blue-shifted to around 3325 cm -1 in the infrared spectra of the Fmoc-FF thin film, Fmoc-FF / FDH thin film, and Fmoc-FF / DAV / FDH ( Figure 1 curve b, d, g in Figure 1 ) thin film samples, which reflects the formation of intermolecular hydrogen bonds in the self-assembly of Fmoc-FF in the thin film.

[0083] (2) Cyclic voltammetry (CV) analysis

[0084] Cyclic voltammetry (CV) experiments were carried out using a CHI 660A electrochemical workstation from CH Instruments to further prove the successful preparation of the immobilized enzyme thin film electrode. The tests were all carried out in a typical three-electrode system, in which the thin film electrodes prepared in the examples or comparative examples, the bare ITO electrode, and the Fmoc-FF thin film electrode were used as the working electrode, the saturated calomel electrode (SCE) was used as the reference electrode, and the platinum sheet electrode was used as the counter electrode. The detection results are as follows.

[0085] When the Fmoc-FF / DAV / FDH thin film ITO electrode prepared in Example 1 was placed in a phosphate buffer solution PBS with a pH of 6.0, a pair of reversible CV peaks could be seen near -0.6V ( Figure 2 curve c in), which was very close to the peak position of the bare ITO electrode in a solution with a pH of 6.0 containing 20 mM DAV ( Figure 2 curve b in), in line with the characteristic peaks of the redox couple of DAV 2+ and DAV ·+ while the small spike appearing around -0.8V corresponded to the electron transfer of the redox couple of DAV ·+ and the neutral molecule of DAV. Both of these electrode reactions were reversible, as Figure 3 shown, where the divalent cation DAV 2+ corresponded to the oxidized state of diallyl viologen, the cation radical DAV ·+ was the first reduced state of diallyl viologen, and the neutral molecule DAV was the second reduced state of diallyl viologen. However, there was no such signal in the Fmoc-FF / FDH thin film ( Figure 2 curve a in), which strongly corroborated the successful immobilization of DAV in the thin film.

[0086] In addition, the thin film glassy carbon electrodes prepared in Example 2 and Comparative Example 4 were compared, and the results are as Figure 4 shown. As can be seen from Figure 4 , the reduction potential of the Fmoc-FF / DAV / FDH thin film glassy carbon electrode prepared in Example 2 was -0.62V, and the reduction potential of the Fmoc-FF / MV / FDH thin film glassy carbon electrode prepared in Comparative Example 4 was -0.70V. Compared with the latter, the reduction potential of the former was more positive. In other words, the electrode containing DAV could carry out CO2 reduction at a higher potential than the electrode containing MV, which could effectively avoid the occurrence of the hydrogen evolution reaction and was beneficial to improving the formate yield.

[0087] To achieve a higher equilibrium concentration of CO2 and make the buffer solution closer to the optimal pH of FDH, 100 mM NaHCO3 was added to PBS buffer at pH 6.0 to provide sufficient HCO3 - , and at this time the actual pH of PBS was approximately 7.2. When CO2 was bubbled into the buffer solution to form a CO2 atmosphere (pH approximately 6.6), it was observed that the reduction current of the Fmoc-FF / DAV / FDH thin film ITO electrode prepared in Example 1 was significantly increased compared to the current in the N2 atmosphere ( Figure 5A ), which can reflect to a certain extent the catalytic reduction of CO2 by the Fmoc-FF / DAV / FDH thin film electrode. The corresponding mechanism is as follows:

[0088] 2DAV 2+ +2e - →2DAV ·+ at the electrode (1)

[0089] FDH(Ox)+2DAV ·+ →FDH(Red)+2DAV 2+ (2)

[0090] FDH(Red)+H + +CO2→FDH(Ox)+HCOO - (3)

[0091] First, DAV in the thin film 2+ is electrochemically reduced to DAV ·+ on the electrode (Equation 1), and the formed DAV ·+ can act as an artificial cofactor to reduce FDH(Ox) in the thin film to FDH(Red). At the same time, DAV ·+ changes back to DAV 2+ (Equation 2), and FDH(Red) can catalytically reduce CO2 to obtain the product formate (Equation 3).

[0092] After that, a chronoamperometry test was carried out under the condition of a constant voltage of -0.8 V. When CO2 was bubbled into the buffer solution containing 100 mM NaHCO3, the current increased significantly and could be stably maintained for more than 10 min, indicating that the reduction reaction system could operate stably within a certain period of time ( Figure 5B ).

[0093] Furthermore, the catalytic reduction efficiency of the Fmoc-FF / DAV / FDH thin film carbon cloth electrode prepared in Example 3 was investigated. Specifically, the ITO electrode was replaced with a carbon cloth electrode (the immersed area was 0.8×2 cm 2) was used for the experiment. It can be observed that after the introduction of CO2, the reduction current of the electrode at -0.62V increased significantly ( Figure 6A ), which means that electrocatalytic reduction of CO2 can also be achieved on the surface of Fmoc-FF / DAV / FDH film carbon cloth electrode. The reduction current of Fmoc-FF / DAV / FDH film carbon cloth electrode is almost the same as the reduction current of Fmoc-FF / DAV / FDH film ITO electrode ( Figure 5A ), which may be due to the fact that the carbon cloth electrode itself can be used as a gas diffusion layer and has a higher catalytic efficiency for the gas reactant CO2. As a control, no redox peak was observed at around -0.6V for the Fmoc-FF / FDH carbon cloth electrode ( Figure 6A ). Figure 6B The chronoamperometric test of the Fmoc-FF / DAV / FDH film carbon cloth electrode under a constant voltage of -0.8 V is shown. When CO2 is bubbled into a buffer solution containing 100 mM NaHCO3, the current increases significantly, and the current density is about 10 times that of the ITO electrode, further indicating that the catalytic efficiency of CO2 is higher when the carbon cloth electrode is used ( Figure 6B ).

[0094] (3) Linear sweep voltammetry (LSV) analysis

[0095] The electrocatalytic reduction of CO2 activity of the Fmoc-FF / DAV / FDH thin film carbon cloth electrode prepared in Example 3 was evaluated by linear sweep voltammetry (LSV). Figure 7 shown.

[0096] First, the LSV curves of Fmoc-FF / DAV / FDH thin film carbon cloth electrodes were scanned in PBS at pH 6.0 under N2 and CO2 atmospheres ( Figure 7 From the curves a and b in Figure 2, it can be observed that the current increases significantly in the CO2 atmosphere. The current value of the Fmoc-FF / DAV / FDH film carbon cloth electrode obtained in the pH 6.0 buffer solution containing 100mM NaHCO3 is higher than that of the pH 6.0 buffer solution without NaHCO3 in both N2 and CO2 atmospheres ( Figure 7 Curve c, d), which means that HCO3 - The presence of does significantly improve the reaction efficiency, which may be due to the increase in CO2 equilibrium concentration and the increase in FDH activity at a relatively alkaline pH. As a control, the LSV curve of the Fmoc-FF / FDH film carbon cloth electrode was tested in a pH 6.0 buffer solution containing 100mM NaHCO3 under CO2 atmosphere ( Figure 7For curve e) in [reference], the obtained current value is much smaller than that of the Fmoc-FF / DAV / FDH thin film carbon cloth electrode, indicating that the DAV component in the Fmoc-FF / DAV / FDH thin film carbon cloth electrode plays an important role in the electrocatalytic reduction of CO2 by FDH.

[0097] (4) Amperometric test

[0098] The Fmoc-FF / DAV / FDH thin film carbon cloth electrode prepared in Example 3 was tested by amperometry at different voltages. The reduction potential of the Fmoc-FF / DAV / FDH thin film carbon cloth electrode was -0.65 V, which was set as the starting voltage, and then the voltage was gradually increased to -1.2 V. The reduction current gradually increased with the increase of the voltage. It can be observed that at a constant voltage of -0.8 V, the reduction current of the Fmoc-FF / DAV / FDH carbon cloth electrode increased significantly compared with that at a constant voltage of -0.65 V ( Figure 8A 、 Figure 8B ), which means that the electrocatalytic efficiency of CO2 is higher at -0.8 V. However, the catalytic reduction current no longer increased significantly after being greater than -1.0 V. After that, the current of the Fmoc-FF / DAV / FDH thin film carbon cloth electrode at a constant voltage of -1.2 V was much larger than that at -1.0 V and -1.1 V. The analysis may be related to the unwanted hydrogen evolution reaction. To avoid the influence of the hydrogen evolution reaction as much as possible, we set the working voltage to -0.8 V for subsequent tests.

[0099] After that, a voltage of -0.8 V was applied to the Fmoc-FF / DAV / FDH thin film carbon cloth electrode in PBS with a pH of 6.0 containing 100 mM NaHCO3 under a CO2 atmosphere for 2 h. The electrolyte after the reaction was collected, and the formate in the electrolyte was detected by ion chromatography. The test conditions are shown in Table 1, and the test results are as Figure 9 shown. The retention time of formate in the mixed standard was 7.6 min ( Figure 9 curve b) in [reference]. The product after electrolyzing the Fmoc-FF / DAV / FDH thin film carbon cloth electrode for 2 h also had an obvious peak at this retention time (curve c in the figure), while there was no peak at this retention time in the buffer before electrolysis ( Figure 9 curve a) in [reference], indicating that the formate in the product was generated by the electrocatalytic reaction. The concentration of formate in the sample was calculated by the external standard method to be about 42.4 μg mL -1 .

[0100] Table 1

[0101]

[0102] It can be seen that the present invention co - immobilizes artificial cofactor (DAV) and enzyme (FDH) into the Fmoc - FF thin film, and can obtain a very high catalytic efficiency for CO2 reduction. According to Faraday's law, the Faraday efficiency of the Fmoc - FF / DAV / FDH thin film carbon cloth electrode prepared in Example 3 during the catalytic reduction process can be estimated to be about 54.7%.

[0103] In the aforementioned Document 1, the Faraday efficiency of using MV as an artificial cofactor in the liquid phase is 61%, which is slightly higher than that of the thin film electrode of this method.

[0104] However, it should be noted that in Example 3, the DAV loading amount on the Fmoc - FF / DAV / FDH thin film carbon cloth electrode is 9.1×10 -7 mol / cm 2 (the amount of substance is 1.5×10 -6 mol).

[0105] For the technical solution disclosed in Document 1, the concentration of MV in the liquid phase system is 4.0×10 -5 mol / mL (the amount of substance is 3.0×10 -4 mol, and the solution volume reported in Document 1 is 7.5 mL). It can be seen that the molar amount of DAV used in the technical solution of the present invention is significantly lower than the molar amount of MV used in Document 1. Moreover, in the Fmoc - FF / DAV / FDH thin film carbon cloth electrode prepared in Example 3, the amount of immobilized FDH used is 0.39 mg, which is also much lower than the amount of FDH used in Document 1, which is 147 mg (reported as 2.4 μM in Document 1, and the mass is estimated according to the molecular weight of FDH provided by Roche, 74000 Da or 8140000 g / mol).

[0106] Thus, it can be seen that the Fmoc - FF / DAV / FDH thin film carbon cloth electrode provided by the present invention can still achieve a Faraday efficiency only slightly less than that of Document 1 when the usage amounts of DAV and FDH are basically two orders of magnitude lower than the usage amounts of MV and FDH in Document 1. This actually indicates that the actual catalytic efficiency of co - immobilizing artificial cofactor DAV and enzyme FDH into the thin film by the present invention is much higher than the catalytic efficiency of Document 1.

[0107] In Reference 2, as mentioned above, the synthetic viologen polymer: 1-(3-aminopropyl)-1'-methyl-4,4'-bipyridine (vio) modified poly(4-styrenesulfonate-co-glycidyl methacrylate-co-butyl acrylate) (P(SS-GMA-BA)-vio) was immobilized together with tungsten-dependent formate dehydrogenase (DvH-W-FDH) on the surface of a carbon cloth electrode for the electrocatalytic reduction of CO2. At a similar working voltage (in this article, it is -0.8V vs SCE, and in Reference 2, -0.59V vs SHE was used, which is -0.83V vs SCE after conversion), the catalytic current density J of the Fmoc-FF / DAV / FDH thin film carbon cloth electrode provided by the present invention at different electrolysis times Invention (obtained according to Figure 6B is much higher than the current density J of Reference 2 R2 , (see Table 2, the data in Reference 2 are from the data in Figure 2(b) of Reference 2, and the area of the electrode is 0.636 cm 2 ), and it is about 10 times higher.

[0108] Table 2. Comparison of current density at different times

[0109]

[0110] Although the catalytic current density of Reference 2 has been significantly improved after further modification (see Figure 3(b) of Reference 2), its process involves the synthesis and modification of multiple materials, and three layers of functional materials are respectively modified on the macroporous side and the microporous side of the carbon cloth electrode with a gas diffusion layer, that is, a total of 4 layers of polymers containing the artificial coenzyme vio and 2 layers of polymer films containing the enzyme FDH are co-modified. Not only is the synthesis cumbersome, but the film modification operation steps are also complex and time-consuming.

[0111] In addition, the loading amount of the viologen substance in Reference 2 is 1.3×10 -6 mol / cm 2 , which is higher than the DAV loading amount (9.1×10 -7 mol / cm 2 ) of the present invention mentioned above. It can be seen that the actual catalytic efficiency of immobilizing the artificial coenzyme factor DAV and the enzyme FDH together into the Fmoc-FF thin film of the present invention is much higher than the catalytic efficiency of Reference 2.

[0112] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An immobilized enzyme thin film electrode, characterized in that, Comprising: An electrode, and An immobilized enzyme film covering the surface of the electrode, wherein the immobilized enzyme film comprises: an N-fluorenylmethoxycarbonyl diphenylalanine film and formate dehydrogenase and diallyl viologen dispersed and fixed in the N-fluorenylmethoxycarbonyl diphenylalanine film, and the mass ratio of the diallyl viologen to the formate dehydrogenase is 1:(1 - 1.2).

2. The immobilized enzyme thin film electrode according to claim 1, wherein In the immobilized enzyme film, the loading amount of the diallyl viologen is 9×10 -7 -1×10 -6 mol / cm 2 , and / or The loading amount of the formate dehydrogenase is 0.2 - 0.3 mg / cm 2 .

3. The immobilized enzyme thin film electrode according to claim 2, characterized in that, In the immobilized enzyme film, the loading amount of the diallyl viologen is 9.1×10 -7 mol / cm 2 , and / or The loading amount of the formate dehydrogenase is 0.24 mg / cm 2 .

4. The immobilized enzyme thin film electrode according to claim 1, characterized in that, The electrode is selected from an ITO electrode, an FTO electrode, a glassy carbon electrode, a gold electrode, a carbon electrode or a carbon cloth electrode.

5. The preparation method of the immobilized enzyme thin film electrode according to any one of claims 1-4, characterized in that, Comprising: Obtaining a first preparation solution containing N-fluorenylmethoxycarbonyl diphenylalanine, Obtaining a second preparation solution containing formate dehydrogenase and diallyl viologen, Mixing the first preparation solution with the second preparation solution, and standing to obtain a pre-gel, Coating the pre-gel on the electrode, and standing to obtain the immobilized enzyme film electrode, wherein, in the pre-gel, the mass ratio of the diallyl viologen to the formate dehydrogenase is 1:(1 - 1.2).

6. The preparation method according to claim 5, characterized in that, In the pre-gel, the mass ratio of N-fluorenylmethoxycarbonyl diphenylalanine to formate dehydrogenase is 1:(2 - 3).

7. The preparation method according to claim 5, wherein The second preparation solution is prepared by the following method: Adding formate dehydrogenase and dithiothreitol to a first buffer solution with a pH of 7 - 8 and mixing and incubating, and then adding diallyl viologen to obtain the second preparation solution.

8. The preparation method according to claim 7, wherein The first buffer solution is selected from a phosphate buffer solution (PBS), a HEPES buffer solution, a Tris buffer solution, a Britton-Robinson buffer solution.

9. The application of the immobilized enzyme film electrode according to any one of claims 1 - 4 in the reduction of CO2.

10. The application according to claim 9, characterized in that, Placing the immobilized enzyme film electrode in a second buffer solution and reducing CO2 at a working voltage of -0.8 V relative to SCE to produce formate.

11. The application according to claim 10, characterized in that, The second buffer solution is 50 mM PBS containing NaHCO3, wherein the concentration of NaHCO3 is 100 mM.

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