Nanometer antibody-metal hybrid photocatalyst as well as preparation method and application thereof

Abstract

The invention relates to the technical field of photocatalytic environmental protection, in particular to a nano antibody-metal hybrid photocatalyst as well as a preparation method and application thereof. The photocatalyst is prepared from biotinylated protein, macromolecules, metal nanoparticles, streptavidin and a nano antibody, the photocatalyst is obtained by taking biotinylated protein as a carrier, introducing a polymer to be coupled with the biotinylated protein, then loading metal nanoparticles, and assembling a nano antibody on the surface through streptavidin. The nano antibody-metal hybrid photocatalyst disclosed by the invention has the advantages of high photocatalytic activity, strong adsorption capacity and selectivity and high degradation rate.

Description

Technical Field

[0001] The present invention relates to the technical field of photocatalytic environmental protection, and particularly relates to a nanobody-metal hybrid photocatalyst, a preparation method thereof, and an application thereof. Background Art

[0002] In recent years, water eutrophication has become increasingly serious. Microcystins (MCs) are a type of monocyclic heptapeptide hepatotoxin produced by Microcystis, Planktothrix, Anabaena, etc. in cyanobacterial blooms. They are a class of toxic secondary metabolites produced by cyanobacteria during the algal bloom process in eutrophic waters, and have multiple toxicities such as hepatotoxicity, nephrotoxicity, neurotoxicity, and reproductive toxicity. There are various isomers of microcystins, and microcystin-LR (MC-LR) is the most common isomer. MC-LR is a liver tumor promoter and protein phosphatase inhibitor, and as a potential pollutant in aquatic ecosystems and food sources, it poses a major threat to the environment and public health.

[0003] Due to the high stability of the cyclic structure of MCs, they are difficult to volatilize, resistant to acids, bases, and high temperatures, and cannot be degraded by some common protein or polypeptide hydrolases, so they are very stable in deionized water.

[0004] Chinese Patent Application Nos. CN201710753919.1, CN202410321609.2, and CN202310535914.7 disclose that the removal methods of microcystins include physical methods, chemical methods, and biological methods. Although these methods can effectively remove microcystins, they have certain limitations in practical applications. For example, physical methods (membrane filtration method and ultrasonic method) have high costs, biological methods have low efficiency and long treatment times, and chemical oxidation methods have problems such as high chemical consumption and secondary pollution. Photocatalytic method, due to its advantages such as low cost and environmental friendliness, is a microcystin treatment technology with broad application prospects.

[0005] Silver-based materials are important visible-light-sensitive photocatalysts that can efficiently remove organic pollutants. For example, Chinese Patent Publication No. CN103408098A discloses a method for degrading microcystin-LR in water using a visible-light photocatalyst silver phosphate, and the specific steps are as follows: The silver phosphate catalyst is put into a solution containing microcystin-LR, and after dark treatment for 1 h, a continuous xenon lamp is turned on for the degradation reaction.

[0006] Doping metals (such as Ag, Fe, Pt) on silver-based photocatalysts to construct heterojunctions can significantly improve the photocatalytic efficiency and inhibit the recombination of photoinduced electron-hole pairs. For example, Chinese Patent Publication No. CN118594575A discloses a silver bromide AgBr and bismuth oxybromide Bi 4 O 5 Br 2The composite heterojunction photocatalyst can improve the stability and catalytic activity of AgBr after the two are combined, and when it is used to inactivate algae, it has stronger algae-inactivating performance.

[0007] Most photocatalysts are metal-based, but they lack selectivity. While removing microcystins, they may attack other organic molecules in eutrophic waters. Therefore, although many photocatalysts have been developed and successfully degraded MC-LR in drinking water, few photocatalysts can be efficiently applied to eutrophic water bodies.

[0008] Therefore, the existing technology still needs to be improved. It is of great significance to develop a new type of composite catalyst with high efficiency, stability and environmental protection and apply it to degrade eutrophic water bodies. Summary of the Invention

[0009] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a nanoantibody-metal hybrid photocatalyst with high photocatalytic activity, strong adsorption capacity and selectivity, and fast degradation rate, as well as its preparation method and application. Through the synergistic catalysis of nanoantibodies and metals, the present invention can efficiently remove microcystins, has good catalytic activity and stability, can be reused, and has good application prospects in the environmental field.

[0010] The present invention is realized through the following technical solutions:

[0011] The first aspect of the present invention provides a nanoantibody-metal hybrid photocatalyst, which includes biotinylated protein, polymer, metal nanoparticles, streptavidin and nanoantibody; the photocatalyst uses biotinylated protein as a carrier, introduces a polymer to couple with the biotinylated protein, then loads metal nanoparticles, and assembles nanoantibodies on the surface through streptavidin.

[0012] The present invention uses biotinylated protein as a carrier, introduces a polymer to couple with the biotinylated protein to form a confined space, controllably synthesizes metal nanoparticles, and loads nanoantibodies on the outer layer of the photocatalyst by connecting streptavidin.

[0013] As an implementation manner of the present invention, the mass ratio of biotinylated protein to polymer is 7:2 - 14:1, the mass ratio of polymer to metal nanoparticles is 1:2 - 2:1, the mass ratio of biotinylated protein to streptavidin is 7:3 - 28:3, and the mass ratio of streptavidin to nanoantibody is 3:1 - 12:1.

[0014] As an embodiment of the present invention, the protein includes at least one of bovine serum albumin, lysozyme, proteolytic enzyme, and Candida antarctica lipase B. The protein in the present invention is only used as a carrier, and the specific type of the protein is not limited. The biotinylated protein is synthesized by covalently linking biotin to the amino group of the protein.

[0015] As an embodiment of the present invention, the polymer includes at least one of branched polyethyleneimine, polyethylene glycol, and polyvinylpyrrolidone. The polymer in the present invention is used as a confinement carrier to controllably synthesize metal nanoparticles with uniform morphology and particle size, and the specific type of the polymer is not limited.

[0016] As an embodiment of the present invention, the metal nanoparticles include at least one of heterojunction particles of silver and silver chloride, and heterojunction particles of silver and silver phosphate.

[0017] As an embodiment of the present invention, the nanobody includes the amino acid sequence shown in SEQ ID NO: 1.

[0018] Preferably, the nanobody further includes a tag protein, which is used to link the nanobody and streptavidin, and the specific type of the tag protein is not limited.

[0019] More preferably, the tag protein is an SBP tag protein.

[0020] Even more preferably, the nanobody is a nanobody with an SBP tag (A2.3-SBP fusion protein), and the amino acid sequence is shown in SEQ ID NO: 2.

[0021] The nanobody with an SBP tag is synthesized from gene fragments encoding streptavidin binding peptide (SBP tag) and nanobody (Nb). In order to bind to streptavidin (SA) directionally, the SBP tag is fused to the C-terminus of Nb.

[0022] The nanobody of the present invention functions to specifically adsorb microcystin-LR, increase the concentration of microcystin-LR in the photocatalytic reaction microenvironment, thereby improving the utilization rate of photogenerated free radicals and targeting the removal of microcystin-LR. The nanobody is preferably the A2.3 gene.

[0023] As an embodiment of the present invention, the particle size of the photocatalyst is 12 - 26 nm.

[0024] The second aspect of the present invention provides a preparation method of the above-mentioned photocatalyst, including the following steps:

[0025] (1) Combine biotin with the protein to obtain a biotinylated protein;

[0026] (2) Dissolve the polymer in water, add the biotinylated protein and stir for reaction. Then add the silver nitrate solution and react in the dark. Then add the ferric chloride solution or the disodium hydrogen phosphate dodecahydrate solution and continue to react in the dark to obtain solution A containing the biotinylated protein, the polymer and the metal nanoparticles.

[0027] (3) Add the streptavidin solution to solution A and stir. Then continue to add the nanobody solution for reaction to obtain the photocatalyst.

[0028] As an embodiment of the present invention, in step (1), dissolve biotin in anhydrous dimethylformamide, dissolve the protein in a buffer solution, mix the two solutions, and stir for reaction in the dark to obtain the biotinylated protein.

[0029] Preferably, in step (1), after stirring for reaction in the dark, dialyze and purify the obtained reactant in a buffer solution.

[0030] Preferably, the buffer solution in step (1) is PB buffer solution.

[0031] More preferably, the concentration of the PB buffer solution is 0.01 M and the pH is 8.0.

[0032] Preferably, the molar ratio of biotin to protein in step (1) is 1:10 - 1:20.

[0033] Preferably, the reaction temperature in step (1) is 0 - 25 °C and the reaction time is 4 - 8 hours.

[0034] Preferably, the dialysis and purification time in step (1) is 3 days.

[0035] As an embodiment of the present invention, the concentration of the polymer dissolved in water in step (2) is 0.4 - 1.6 mg / mL.

[0036] As an embodiment of the present invention, the concentration of the biotinylated protein in step (2) is 5 - 20 mg / mL.

[0037] As an embodiment of the present invention, the concentration of the silver nitrate solution in step (2) is 0.5 - 2.5 mg / mL.

[0038] As an embodiment of the present invention, the concentration of the ferric chloride solution or the disodium hydrogen phosphate dodecahydrate solution in step (2) is 1.5 - 2.5 mg / mL.

[0039] As an embodiment of the present invention, in step (2), a biotinylated protein is added and stirred for reaction for 30 - 60 min, the stirring speed is 300 - 500 rpm, then a silver nitrate solution is added for reaction in the dark for 2 - 4 h, and then a ferric chloride solution or a disodium hydrogen phosphate dodecahydrate solution is added to continue the reaction in the dark for 30 - 60 min.

[0040] As an embodiment of the present invention, after the reaction in the dark with the ferric chloride solution or the disodium hydrogen phosphate dodecahydrate solution in step (2) is completed, the supernatant is removed by centrifugation, and the precipitate is redissolved in water with a concentration of 0.22 - 0.88 mg / mL.

[0041] As an embodiment of the present invention, in step (3), the concentration of the streptavidin solution is 0.5 - 2.5 mg / mL.

[0042] As an embodiment of the present invention, in step (3), the concentration of the nanobody solution is 0.12 - 0.2 mg / mL.

[0043] As an embodiment of the present invention, in step (3), the reaction temperature is 0 - 25 °C, and the reaction time is 1 - 3 hours.

[0044] As an embodiment of the present invention, after the addition of the streptavidin solution and stirring in step (3) is completed, the unconnected streptavidin is removed by centrifugation, and then the nanobody solution is added for reaction.

[0045] As an embodiment of the present invention, after the reaction in step (3) is completed, the supernatant is removed by centrifugation, and the precipitate is redissolved in water. The photocatalyst after redissolution is stored at 4 °C in the form of a solution with a concentration of 0.22 - 0.88 mg / mL.

[0046] The third aspect of the present invention provides an application of the above-mentioned photocatalyst or the photocatalyst prepared by the above-mentioned preparation method in the degradation of microcystin.

[0047] As an embodiment of the present invention, the application is to degrade microcystin in lake water.

[0048] As an embodiment of the present invention, the specific operation method of the application is: adding the photocatalyst to the lake water solution containing microcystin, and stirring and reacting under light to catalyze the degradation of microcystin.

[0049] After the above reaction is completed, centrifugal separation is carried out, and the obtained photocatalyst precipitate can be reused.

[0050] Preferably, the light condition is to use a continuous xenon lamp as a simulated visible light source with a power of 300 w and equipped with a 400 nm ultraviolet filter.

[0051] As an embodiment of the present invention, the application is to degrade microcystin-LR in lake water.

[0052] Preferably, when the concentration of the lake water solution containing microcystin is 250 ng / mL, the dosage of the catalyst is 0.117 mg / mL.

[0053] Preferably, the temperature of the reaction is room temperature, the rotation speed of stirring is 300 - 500 rpm, and the reaction time is 10 - 30 min.

[0054] The beneficial effects of the present invention are as follows:

[0055] (1) The degradation of microcystin-LR by the nanoantibody-metal hybrid photocatalyst of the present invention shows good degradation effect, and has potential application value in the treatment technology of photocatalytic decomposition of organic pollutants using solar energy.

[0056] (2) The nanoantibody-metal hybrid photocatalyst of the present invention comprises protein, metal nanoparticles and nanoantibodies. Using biotinylated protein as a carrier, a polymer is introduced to couple with the biotinylated protein, and then metal nanoparticles are loaded. The nanoantibodies are assembled on the surface through streptavidin, which can specifically adsorb and photocatalytically degrade microcystin-LR in lake water, and is less affected by impurities such as anions and organic matters in lake water, and has good application prospects for degrading microcystin in lake water.

[0057] (3) The nanoantibody-metal hybrid photocatalyst of the present invention has the advantages of simple synthesis method, mild conditions, high photocatalytic activity, strong adsorption capacity, fast degradation rate, etc., and is convenient for separation and can be reused multiple times. Description of the Drawings

[0058] Figure 1 Transmission electron microscope photograph and particle size distribution diagram of the Ag / AgCl / Pro photocatalyst prepared in Example 2.

[0059] Figure 2 Transmission electron microscope photograph and particle size distribution diagram of the Ag / AgCl / Pro-Nb photocatalyst prepared in Example 5.

[0060] Figure 3 Effect comparison of the photocatalysts prepared in Comparative Example 1, Example 4, Example 5, and Example 6 for degrading microcystin-LR.

[0061] Figure 4 Reusability effect of the Ag / AgCl / Pro-Nb photocatalyst prepared in Example 4.

[0062] Figure 5To compare the Ag / Ag prepared in Example 3 with different illumination times 3 PO 4 / Pro photocatalyst and the Ag / Ag prepared in Example 6 3 PO 4 / Pro-Nb photocatalyst for the catalytic efficiency of degrading microcystin-LR in lake water.

[0063] Figure 6 For the comparison of the degradation efficiency of microcystin-LR by the Ag / AgCl / Pro photocatalyst prepared in Example 2 and the Ag / AgCl / Pro-Nb photocatalyst prepared in Example 5 in ultrapure water and lake water. Detailed implementation mode

[0064] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and forms of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention.

[0065] The branched polyethyleneimine (BPEI) Mw = 25k used in each embodiment of the present invention was purchased from Thermo Fisher Scientific.

[0066] The nanobody used in Examples 4 - 6 was the nanobody Nb-SBP with an SBP tag, and the preparation process was as follows: According to the amino acid sequence SEQ ID NO:2, a recombinant plasmid was designed, and the correctly sequenced pET25b-A2.3-SBP recombinant plasmid was extracted and transformed into e.c oli Rosetta chemically competent cells. Subsequently, 3 mL of the cultured Escherichia coli Rosetta-pET25b-A2.3-SBP was inoculated into 300 mL of self-inducing medium containing 100 μg / mL ampicillin and cultured with shaking at 37 °C and 220 rpm until the OD600 reached 0.5 - 0.7. The Escherichia coli Rosetta-pET25b-A2.3-SBP cells were cultured with shaking overnight at 25 °C and 130 rpm to promote the autoinduction expression of the A2.3-SBP fusion protein. The induced cells were obtained by centrifugation (7000g, 4 °C, 10 min). After washing the cells twice with sterile PBS, the cells were resuspended in 60 mL of lysis buffer (2.6 mM KCl, 8 mM Na 2 HPO 4 2 mM KH 2 PO 4, 136 mM NaCl, 60 mg lysozyme, 1 mM PMSF) The resuspended cells were lysed by sonication and centrifuged at high speed (10,000 g, 4 °C, 15 min) to separate the supernatant containing the soluble A2.3-SBP fusion protein, and the bacterial debris was removed by filtration through a 0.45 μm membrane. According to the standard protocol, the his-tagged A2.3-SBP fusion protein was purified using a nickel column. The purity of the A2.3-SBP fusion protein was analyzed and verified by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and the concentration of the purified protein was determined using an ultra-micro spectrophotometer. The purified A2.3-SBP fusion protein was stored at -80 °C for later use.

[0067] Example 1

[0068] Preparation of a photocatalyst Ag / AgCl / Pro for degrading microcystin.

[0069] The preparation process of biotinylated bovine serum albumin solution is as follows: Biotin was dissolved in anhydrous DMF, and bovine serum albumin was dissolved in PB buffer (0.01 M, pH 8.0). The biotin solution and the bovine serum albumin solution were mixed according to a molar ratio of biotin to bovine serum albumin of 1:10. After stirring in the dark at 0 °C for 4 hours, biotinylated bovine serum albumin was obtained and dialyzed and purified in 0.01 M PB buffer for 3 days.

[0070] 10.7 mg of BPEI was dissolved in ultrapure water (26.7 mL) and placed in a 100 mL flask. Biotinylated bovine serum protein solution (5 mg / mL, 15 mL) was added to the flask. The mixture was stirred magnetically for 30 minutes at a rotation speed of 500 rpm, and the silver nitrate solution (0.5 mg / mL, 20 ml) was dropped into the mixture. The resulting mixture was stirred magnetically in the dark for 4 hours. Subsequently, the ferric chloride solution (1.5 mg / mL, 10 mL) was added dropwise, and then stirred for another 30 minutes. The precipitate was collected by centrifugation and redissolved in water to obtain Ag / AgCl / Pro (0.22 mg / mL).

[0071] Example 2

[0072] Preparation of a photocatalyst Ag / AgCl / Pro for degrading microcystin.

[0073] The preparation process of biotinylated lysozyme solution is as follows: Biotin was dissolved in anhydrous DMF, and lysozyme was dissolved in PB buffer (0.01 M, pH 8.0). The biotin solution and the lysozyme solution were mixed according to a molar ratio of biotin to lysozyme of 1:20. After stirring in the dark at 25 °C for 8 hours, biotinylated lysozyme was obtained and dialyzed and purified in 0.01 M PB buffer for 3 days.

[0074] Dissolve 21.4 mg of polyethylene glycol (average molecular weight of 5000) in ultrapure water (26.7 mL), and place it in a 100 mL flask. Add a biotinylated bovine serum albumin solution (10 mg / mL, 15 mL) to the flask. Stir magnetically for 40 minutes at a rotation speed of 400 rpm, and drip the silver nitrate solution (1 mg / mL, 20 mL) into the mixture. Magnetically stir the obtained mixture in the dark for 3 hours. Subsequently, drip the ferric chloride solution (2 mg / mL, 10 mL), stir for another 40 minutes, centrifuge to collect the precipitate, and redissolve it in water to obtain Ag / AgCl / Pro (0.44 mg / mL).

[0075] The transmission electron microscope image and particle size distribution diagram of the photocatalyst Ag / AgCl / Pro prepared in this example are as Figure 1 shown, where the left side is the transmission electron microscope image and the right side is the particle size distribution diagram. It can be seen from Figure 1 this that the average size of the photocatalyst Ag / AgCl / Pro is 21.7 nm.

[0076] Example 3

[0077] Preparation of a photocatalyst Ag / Ag 3 PO 4 / Pro for degrading microcystin.

[0078] The preparation process of the biotinylated Candida antarctica lipase B solution is as follows: Dissolve biotin in anhydrous DMF, and dissolve Candida antarctica lipase B in PB buffer (0.01 M, pH 8.0). Mix the biotin solution and the Candida antarctica lipase B solution according to a molar ratio of biotin to Candida antarctica lipase B of 1:15. After stirring magnetically at 10 °C in the dark for 6 hours, biotinylated Candida antarctica lipase B is obtained, and it is dialyzed and purified in 0.01 M PB buffer for 3 days.

[0079] Dissolve 42.8 mg of polyvinylpyrrolidone in ultrapure water (26.7 mL), and place it in a 100 mL flask. Add the biotinylated Candida antarctica lipase B solution (20 mg / mL, 15 mL) to the flask. Stir magnetically for 60 minutes at a rotation speed of 300 rpm, and drip the silver nitrate solution (2.5 mg / mL, 20 ml) into the mixture. Magnetically stir the obtained mixture in the dark for 2 hours. Subsequently, drip the disodium hydrogen phosphate dodecahydrate solution (2.5 mg / mL, 10 mL), stir for another 60 minutes, centrifuge to collect the precipitate, and redissolve it in water to obtain Ag / Ag 3 PO 4 / Pro (0.88 mg / mL).

[0080] Example 4

[0081] Preparation of a Nanoantibody-Metal Hybrid Photocatalyst Ag / AgCl / Pro-Nb for Degrading Microcystin

[0082] Mix the Ag / AgCl / Pro prepared in Example 1 (0.22 mg / mL, 4 mL) with streptavidin (0.5 mg / mL, 6 mL), incubate at 0 °C for 1 hour, centrifuge to remove the unbound streptavidin in the supernatant, and re-dissolve with water to obtain an Ag / AgCl / Pro-SA solution (0.22 mg / mL). Then, add the SBP-tagged nanoantibody Nb-SBP (0.12 mg / mL, 6 mL) to the Ag / AgCl / Pro-SA solution (0.22 mg / mL, 10 mL). Stir magnetically at 0 °C for 1 hour to obtain Ag / AgCl / Pro-Nb, and remove the unbound Nb-SBP complex by centrifugation. Finally, the mass ratio of biotinylated protein, BPEI, heterogeneous junction particles of silver and silver chloride, streptavidin, and SBP-tagged nanoantibody in Ag / AgCl / Pro-Nb is 7:2:4:3:1.

[0083] Example 5

[0084] Preparation of a Hierarchical Nanoantibody-Metal Hybrid Photocatalyst Ag / AgCl / Pro-Nb for Degrading Microcystin

[0085] Mix the Ag / AgCl / Pro prepared in Example 2 (0.44 mg / mL, 6 mL) with streptavidin (1 mg / mL, 4 mL), incubate at 10 °C for 2 hours, centrifuge to remove the unbound streptavidin in the supernatant, and re-dissolve with water to obtain an Ag / AgCl / Pro-SA solution (0.44 mg / mL). Then, add the SBP-tagged nanoantibody Nb-SBP (0.16 mg / mL, 4 mL) to the Ag / AgCl / Pro-SA solution (0.44 mg / mL, 10 mL). Stir magnetically at 10 °C for 2 hours to obtain Ag / AgCl / Pro-Nb, and remove the unbound Nb-SBP complex by centrifugation. Finally, the mass ratio of biotinylated protein, polyethylene glycol, heterogeneous junction particles of silver and silver chloride, streptavidin, and SBP-tagged nanoantibody in Ag / AgCl / Pro-Nb is 28:4:4:6:1.

[0086] The transmission electron microscope image and particle size distribution diagram of the photocatalyst Ag / AgCl / Pro-Nb prepared in this example are as Figure 2 shown. Among them, the left side is the transmission electron microscope image, and the right side is the particle size distribution diagram. From Figure 2It can be seen that the photocatalyst Ag / AgCl / Pro-Nb prepared by the present invention contains both Ag and AgCl particles, forming heterojunction particles of silver and silver chloride.

[0087] Example 6

[0088] Preparation of a hierarchical structure nanoantibody-metal hybrid photocatalyst Ag / Ag 3 PO 4 / Pro-Nb for degrading microcystin.

[0089] Mix Ag / Ag 3 PO 4 / Pro (0.88 mg / mL, 8 mL) prepared in Example 3 with streptavidin (2.5 mg / mL, 2 mL), incubate at 25 °C for 3 hours, centrifuge to remove the unbound streptavidin in the supernatant, and redissolve with water to obtain Ag / Ag 3 PO 4 / Pro-SA solution (0.88 mg / mL). Then, add the nanobody Nb-SBP with SBP tag (0.2 mg / mL, 2 mL) to Ag / Ag 3 PO 4 / Pro-SA solution (0.88 mg / mL, 10 mL). Stir magnetically at 25 °C for 3 hours to obtain Ag / Ag 3 PO 4 / Pro-Nb, and remove the unbound Nb-SBP complex by centrifugation. Finally, the mass ratio of biotinylated protein, Candida antarctica lipase B, heterojunction particles of silver and silver phosphate, streptavidin and nanobody with SBP tag in Ag / Ag 3 PO 4 / Pro-Nb is 28:2:1:3:0.25.

[0090] Comparative Example 1

[0091] Preparation of a nanoantibody-metal hybrid photocatalyst Ag / AgCl-Nb for degrading microcystin.

[0092] Dissolve 21.4 mg of polyethylene glycol (average molecular weight of 5000) in ultrapure water (26.7 mL), and place it in a 100 mL flask. Stir magnetically for 40 minutes at a rotation speed of 400 rpm, and drop the silver nitrate solution (1 mg / mL, 20 mL) into the mixture. Stir the resulting mixture magnetically in the dark for 3 hours. Subsequently, dropwise add the ferric chloride solution (2 mg / mL, 10 mL), stir for another 40 minutes, centrifuge to collect the precipitate, and redissolve it in water to obtain Ag / AgCl (0.44 mg / mL). Mix the prepared Ag / AgCl (0.44 mg / mL, 6 mL) with streptavidin (1 mg / mL, 4 mL), incubate at 10 °C for 2 hours, centrifuge to remove the unbound streptavidin in the supernatant, and redissolve it in water to obtain the Ag / AgCl-SA solution (0.44 mg / mL). Then, add the nanobody Nb-SBP with an SBP tag (0.16 mg / mL, 4 mL) to the Ag / AgCl-SA solution (0.44 mg / mL, 10 mL). Stir magnetically at 10 °C for 2 hours to obtain Ag / AgCl-Nb, and remove the unbound Nb-SBP complex by centrifugation.

[0093] Compare the catalytic efficiency of the Ag / AgCl-Nb photocatalyst prepared in Comparative Example 1 and the nanobody-metal hybrid photocatalyst (Example 4, Example 5 or Example 6) for degrading microcystin-LR.

[0094] The specific steps are as follows: Prepare four reaction solutions, each reaction solution contains 250 ng / mL of microcystin-LR, and then each reaction solution also contains 0.117 mg / mL of the photocatalysts prepared in Comparative Example 1, Example 4, Example 5, and Example 6, respectively. Stir the reaction magnetically at 500 rpm for 30 min under a device with a 300 W xenon lamp equipped with a 400 nm filter. After the reaction, centrifuge and take the supernatant to detect the concentration of MC-LR by high performance liquid chromatography and calculate the degradation efficiency. Degradation efficiency = (C 0 -C) / C 0 × 100%, where C 0 represents the concentration of microcystin-LR before catalytic degradation, and C represents the concentration of microcystin-LR measured after catalytic degradation. The results are as Figure 3 shown.

[0095] Test Example 1

[0096] Reusability effect of the nanobody-metal hybrid photocatalyst Ag / AgCl / Pro-Nb.

[0097] Microcystin-LR catalytic degradation reaction: The reaction solution consists of 250 ng / mL microcystin-LR and 0.117 mg / mL of the nanoantibody-metal hybrid photocatalyst Ag / AgCl / Pro-Nb prepared in Example 4. The reaction is carried out under a 300 W xenon lamp equipped with a 400 nm filter at 500 rpm with magnetic stirring for 10 min. After the reaction, the supernatant is obtained by centrifugation and the concentration of MC-LR is detected by high performance liquid chromatography, and the degradation efficiency is calculated. The degradation efficiency = (C 0 -C) / C 0 ×100%, where C 0 represents the concentration of microcystin-LR before catalytic degradation, and C represents the concentration of microcystin-LR measured after catalytic degradation.

[0098] After each microcystin-LR catalytic degradation reaction, the nanoantibody-metal hybrid photocatalyst Ag / AgCl / Pro-Nb is centrifuged and recovered, and then the microcystin-LR catalytic degradation experiment is repeated. In 7 consecutive cycles, the microcystin-LR solution (250 ng / mL) is repeatedly degraded with 0.117 mg / mL of Ag / AgCl / Pro-Nb, and the relative activity is calculated to verify its stability and reusability. The relative activity = degradation efficiency after cycling / degradation efficiency of the first time × 100%. The results are as Figure 4 shown. After multiple cycles, the catalytic effect of the catalyst decreases less. After 6 cycles, its catalytic effect can still reach more than 93.5%, indicating that the photocatalyst prepared by the present invention has good stability and recyclability. The photocatalysts prepared in Example 5 and Example 6 were also tested for the repeatability of the use effect according to the above method. The relative activities after 6 cycles were 95.5% and 91.5% respectively.

[0099] Test Example 2

[0100] Comparison of the degradation efficiency of photocatalyst Ag / Ag 3 PO 4 / Pro on microcystin-LR in lake water at different illumination times.

[0101] The specific steps are as follows: The reaction solution 1 consists of 250 ng / mL microcystin-LR and 0.117 mg / mL of the photocatalyst Ag / Ag 3 PO 4 / Pro prepared in Example 3. The reaction is carried out under a 300 W xenon lamp equipped with a 400 nm filter at 500 rpm with magnetic stirring for different times (0 min, 5 min, 10 min, 15 min, 20 min, 30 min). After the reaction, the supernatant is obtained by centrifugation and the concentration of MC-LR is detected by high performance liquid chromatography.

[0102] The reaction 2 solution consists of 250 ng / mL microcystin-LR and 0.117 mg / mL nanoantibody-metal hybrid photocatalyst (Example 6). Under the device of a 300 W xenon lamp equipped with a 400 nm filter, magnetic stirring reaction was carried out at 500 rpm for different times (0 min, 5 min, 10 min, 15 min, 20 min, 30 min). After the reaction, centrifugation was carried out to take the supernatant, and the concentration of MC-LR was detected by high performance liquid chromatography.

[0103] Ag / Ag 3 PO 4 / Pro composite catalyst (Example 3) and Ag / Ag 3 PO 4 / Pro-Nb nanoantibody-metal hybrid photocatalyst (Example 6) for the comparison of the catalytic efficiency of degrading microcystin-LR is as Figure 5 shown, where C 0 represents the concentration of microcystin-LR before catalytic degradation, and C represents the concentration of microcystin-LR measured after catalytic degradation. It can be seen that the nanoantibody-metal hybrid photocatalyst Ag / Ag 3 PO 4 / Pro-Nb has a better degradation effect on microcystin-LR in the lake water system.

[0104] Test Example 3

[0105] Comparison of the degradation efficiency of photocatalyst Ag / AgCl / Pro irradiated for 10 min on microcystin-LR in ultrapure water and lake water.

[0106] The specific steps are as follows: The reaction 1 solution consists of 250 ng / mL microcystin-LR and 0.117 mg / mL photocatalyst Ag / AgCl / Pro prepared in Example 2. Under the device of a 300 W xenon lamp equipped with a 400 nm filter, magnetic stirring reaction was carried out at 500 rpm for 10 min. After the reaction, centrifugation was carried out to take the supernatant, and the concentration of MC-LR was detected by high performance liquid chromatography and the degradation efficiency was calculated.

[0107] The reaction 2 solution consists of 250 ng / mL microcystin-LR and 0.117 mg / mL nanoantibody-metal hybrid photocatalyst Ag / AgCl / Pro-Nb prepared in Example 5. Under the device of a 300 W xenon lamp equipped with a 400 nm filter, magnetic stirring reaction was carried out at 500 rpm for 10 min. After the reaction, centrifugation was carried out to take the supernatant, and the concentration of MC-LR was detected by high performance liquid chromatography and the degradation efficiency was calculated.

[0108] Degradation efficiency = (C 0 - C) / C 0 × 100%, where C 0C0 represents the concentration of microcystin-LR before catalytic degradation, and C represents the concentration of microcystin-LR measured after catalytic degradation.

[0109] The degradation efficiencies of the Ag / AgCl / Pro composite catalyst (Reaction 1) and the nanoantibody-metal hybrid photocatalyst Ag / AgCl / Pro-Nb (Reaction 2) in ultrapure water and lake water are as Figure 6 shown. It can be seen that there is no obvious difference in the catalytic activities of these two catalysts in ultrapure water, but the activity value of the nanoantibody-metal hybrid photocatalyst Ag / AgCl / Pro-Nb is significantly higher in the lake water system. It shows that the nanoantibody-metal hybrid photocatalyst of the present invention is less affected by impurities such as anions and organic substances in lake water and can specifically adsorb and photocatalytically degrade microcystin-LR in lake water.

[0110] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or change made without departing from the present invention shall be included within the scope of the technical solution of the present invention.

Claims

1. A nanobody-metal hybrid photocatalyst, characterized in that: The photocatalyst comprises biotinylated protein, polymer, metal nanoparticles, streptavidin and nanoantibodies; the nanoantibodies are used for specific adsorption of microcystin-LR, the photocatalyst uses biotinylated protein as a carrier, introduces polymer and biotinylated protein for coupling, then loads metal nanoparticles, and assembles nanoantibodies on the surface through streptavidin to obtain the obtained product.

2. The photocatalyst according to claim 1, characterized in that The protein includes at least one of bovine serum albumin, lysozyme, proteolytic enzyme and Antarctic Candida lipase B; the polymer includes at least one of branched polyethyleneimine, polyethylene glycol and polyvinyl pyrrolidone; the metal nanoparticles include at least one of heterojunction particles of silver and silver chloride and heterojunction particles of silver and silver phosphate; the nanoantibody includes the amino acid sequence shown in SEQ ID NO:

1.

3. The photocatalyst according to claim 1, characterized in that The particle size of the photocatalyst is 12-26nm; the mass ratio of biotinylated protein to polymer is 7:2-14:1, the mass ratio of polymer to metal nanoparticles is 1:2-2:1, the mass ratio of biotinylated protein to streptavidin is 7:3-28:3, and the mass ratio of streptavidin to nanoantibodies is 3:1-12:

1.

4. The photocatalyst according to claim 1, characterized in that The nanobody also includes a tag protein, and the tag protein is used to connect the nanobody and streptavidin.

5. The photocatalyst according to claim 4, characterized in that The amino acid sequence of the Nanobody is shown in SEQ ID NO:

2.

6. A method for preparing the photocatalyst according to any one of claims 1 to 5, characterized in that: The steps include: (1) Binding biotin to protein to obtain biotinylated protein; (2) dissolving the polymer in water, adding the biotinylated protein and stirring for reaction, then adding a silver nitrate solution to react in the dark, and then adding a ferric chloride solution or a sodium hydrogen phosphate dodecahydrate solution to continue the reaction in the dark, to obtain a solution A containing the biotinylated protein, the polymer and the metal nanoparticles; (3) Add streptavidin solution to solution A and stir, then continue to add nanoantibody solution to react, thereby obtaining a photocatalyst.

7. The preparation method according to claim 6, characterized in that: In step (1), biotin is dissolved in anhydrous dimethylformamide, and the protein is dissolved in a buffer solution. The two solutions are mixed and stirred in the dark to obtain a biotinylated protein.

8. The preparation method according to claim 7, characterized in that: The preparation method further comprises at least one of the following conditions: (a) step (1) further comprises stirring the reaction in the dark, and then dialysis and purification of the obtained reactant in a buffer solution; (b) the buffer in step (1) is PB buffer; (c) in step (1), the molar ratio of biotin to protein is 1:10-1:20, the reaction temperature is 0-25° C., and the reaction time is 4-8 hours; (d) in step (2), the concentration of the polymer dissolved in water is 0.4-1.6 mg / mL, the concentration of the silver nitrate solution is 0.5-2.5 mg / mL, and the concentration of the ferric chloride solution or the sodium hydrogen phosphate dodecahydrate solution is 1.5-2.5 mg / mL; (e) adding the biotinylated protein in step (2) and stirring for reaction for 30-60 minutes, then adding silver nitrate solution and reacting in the dark for 2-4 hours, and then adding ferric chloride solution or disodium hydrogen phosphate dodecahydrate solution and continuing to react in the dark for 30-60 minutes; (f) In step (3), the concentration of the streptavidin solution is 0.5-2.5 mg / mL, the concentration of the nanobody solution is 0.12-0.2 mg / mL, the reaction temperature is 0-25° C., and the reaction time is 1-3 hours.

9. The preparation method according to claim 6, characterized in that: After the reaction of adding ferric chloride solution or dodecahydrate disodium hydrogen phosphate solution in step (2) is completed in the dark, the supernatant is removed by centrifugation, and the precipitate is redissolved with water to a concentration of 0.22-0.88 mg / mL; after the reaction of step (3), the supernatant is removed by centrifugation, and the precipitate is redissolved with water, and the redissolved photocatalyst is stored in the form of a solution at 4° C., and the concentration is 0.22-0.88 mg / mL.

10. Use of the photocatalyst according to any one of claims 1 to 5 or the photocatalyst prepared by the preparation method according to any one of claims 6 to 9 in degrading microcystins.

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