Photocatalyst as well as preparation and application thereof in killing spores of orbiculaeas

Abstract

The invention belongs to the field of disease prevention and control in aquaculture, and particularly relates to a photocatalyst (ZnO / N-Cu-MOF), a preparation method thereof and an application of the photocatalyst in high-efficiency elimination and killing of testis blood egg spores under visible light. The preparation method specifically comprises the following steps: introducing polyvinylpyrrolidone to carry out N doping modification on Cu-MOF, and then compounding with ZnO through a hydrothermal synthesis method to obtain the novel Z-type heterojunction visible light photocatalyst ZnO / N-Cu-MOF. Compared with a traditional treatment method, the photocatalytic technology is short in action time and high in killing efficiency and has recoverability, half of the orbiculaeas microspores can remarkably lose motion activity when the photocatalyst with the concentration of 1 mg / L acts for 2 h, new reference is provided for prevention and treatment of the cultured crustacean epidemic diseases caused by the orbiculaeas, and the method has the advantages that the method is suitable for popularization and application. Certain research prospects and application potentials are shown.

Description

Technical Field

[0001] The present invention belongs to the field of prevention and control of aquatic diseases, and particularly relates to a photocatalyst (ZnO / N-Cu-MOF) and its preparation and application in highly efficient disinfection of Hematodinium spores under visible light. Background Art

[0002] Hematodinium is a parasitic dinoflagellate that parasitizes marine crustaceans globally and can infect more than 70 important economic marine crustaceans including Portunus trituberculatus, Callinectes sapidus, and Australian lobsters. It is the main pathogen causing the "milk disease" of Portunus trituberculatus and the "yellow water disease" of Scylla serrata, posing a huge threat to the sustainable development of the marine crustacean fishery economy. The occurrence of Hematodinium epidemics not only causes direct losses to the shrimp and crab aquaculture industry, but also due to the lack of specific prevention and control measures for Hematodinium at present, shrimp and crab farmers mostly use a large amount of antibiotics and pesticides to control during the outbreak of the epidemic, which is likely to pollute the water environment and also lead to drug residues, ultimately threatening human health. Early researchers isolated Hematodinium from Norway lobsters and Callinectes sapidus and achieved continuous culture in vitro, clarifying its complex complete life cycle. In the later stage of the life cycle development, a large number of active macrospores or microspores are released from the host into the water environment and continue to search for new hosts to complete the infection process. The active microspores have been proven to be the key stage in the spread of Hematodinium epidemics. Therefore, taking effective measures during the spore stage is expected to timely control the occurrence of Hematodinium epidemics.

[0003] As an advanced oxidation technology, photocatalysis has been widely applied in pollutant degradation, hydrogen production, etc. It is a green catalytic material with recyclable performance and high environmental compatibility. Photocatalysts have also been successfully applied to the inactivation of toxic and harmful microorganisms such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, molds, Microcystis aeruginosa, Karenia mikimotoi, etc., and have great application potential in the field of medical and health. Different from various aquaculture drugs, photocatalysts are insoluble in water and rely on various strongly oxidative and reductive free radicals generated under light catalysis to play an indirect role. Therefore, in addition to having the advantages of high algicidal activity and quick effect, photocatalysts can also achieve the effect of recycling by being loaded on floating or fixed carriers, which means that photocatalysts are environmentally friendly and reusable and will not enter the aquaculture pond water to further threaten human health, indicating that they have great application prospects.

[0004] Among numerous photocatalysts, ZnO is widely used in cosmetics, rubber, coatings, medicine and other fields due to its excellent photocatalytic activity. It is inexpensive and easily available, and is one of the commonly used photocatalysts. However, its wide band gap results in its ability to only utilize ultraviolet light for photocatalytic reactions, and specific modifications are required. Cu-MOF is a metal-organic framework material, a three-dimensional structural material formed by coordination bonds between Cu 2+ and organic ligands (usually organic molecules containing acidic functional groups), and has broad application potential in the fields of catalysis, adsorption, separation and sensing. However, as a photocatalyst, the high recombination efficiency of its photo-generated electron-hole pairs limits its application, and further performance optimization is needed. Summary of the Invention

[0005] The object of the present invention is to provide a photocatalyst (ZnO / N-Cu-MOF) and its preparation method and application in highly efficiently killing the spores of Hematodinium sp. under visible light.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A preparation method of a photocatalyst, by introducing polyvinylpyrrolidone to perform N-doping modification on Cu-MOF, and then compounding it with ZnO by hydrothermal synthesis method to obtain a novel visible-light photocatalyst ZnO / N-Cu-MOF with a Z-scheme heterojunction.

[0008] Furthermore,

[0009] Step 1: Dissolve copper nitrate in N,N-dimethylformamide (DMF) under magnetic stirring, and call it Solution I. Dissolve 1,3,5-benzenetricarboxylic acid (H3TBC) in DMF, and call it Solution II;

[0010] Step 2: Add polyvinylpyrrolidone (PVP) to Solution I and stir until completely dissolved. After dissolution, mix it with Solution II under stirring conditions to obtain Solution III;

[0011] Step 3: Add ZnO to Solution III and continuously stir until evenly mixed;

[0012] Step 4: Transfer the evenly mixed solution to a reaction kettle for reaction, and react at 80-120 °C for 20-24 h;

[0013] Step 5: After the above reaction, wait for the temperature of the reaction kettle to cool to room temperature, centrifuge the obtained reaction solution to collect the solid (5000 rpm, 10 min), wash the solid and then dry it overnight to obtain the ZnO / N-Cu-MOF composite photocatalyst.

[0014] The concentration of copper nitrate in Solution I is 0.058 - 0.06 g / mL, and the concentration of 1,3,5-benzenetricarboxylic acid in Solution II is 0.028 - 0.03 g / mL.

[0015] The concentration of polyvinylpyrrolidone in Solution I is 0.013 - 0.02 g / mL, and the volume ratio of Solution I to Solution II is 1:1; the concentration of ZnO in Solution III is 0.019 - 0.02 g / mL.

[0016] A photocatalyst is prepared by the described method, and the obtained light blue powdery photocatalyst (ZnO / N-Cu-MOF).

[0017] The composite photocatalyst shows the characteristic peaks of the crystal planes of HKUST-1 type Cu-based MOF crystals and ZnO in the X-ray diffraction pattern. The scanning electron microscope images show that the introduction of N makes the apparent morphology of MOF change from disordered to ordered, being a uniform octahedral shape, and effectively reduces the recombination efficiency of photogenerated electron-hole pairs in the MOF material. This lower recombination efficiency does not increase significantly after ZnO is loaded on N-Cu-MOF. The composite material shows the strongest absorption peak at about 700 nm in the visible light region. The accurate energy band structure proves that ZnO / N-Cu-MOF follows the Z-scheme heterojunction electron transfer mode and has excellent photocatalytic performance.

[0018] An application of the described photocatalyst, the application of the ZnO / N-Cu-MOF photocatalyst in killing the spores of the parasitic dinoflagellate Hematodinium sp.

[0019] There are two types of the spores of Hematodinium sp., namely large spores and small spores. In the examples, small spores are taken as an example, and the initial density of the spores is 10 6 cells / mL. The method for killing the small spores of Hematodinium sp. specifically includes the following steps:

[0020] Select a 6-well cell culture plate. After the small spores of Hematodinium sp. are released from the body of Hemigrapsus penicillatus into sterile seawater, dilute them to 10 6 cells / mL. Add 4 mL of cell dilution to each well of the plate, and add the photocatalyst stock solution dispersed with sterilized seawater. Place the experimental system in an incubator at 20 °C and culture it under dark conditions. At 1 h and 2 h, use a microscope to select at least 3 fields of view to record the small spore movement videos for about 5 s, observe the activity of the small spores, and calculate the half maximal effective concentration EC 50 .

[0021] Proportion of motile cells = (Number of motile cells in the field of view / Total number of cells in the field of view) × 100%

[0022] Principle of the present invention:

[0023] In the present invention, polyvinylpyrrolidone (PVP) is introduced into the photocatalyst to perform N-doping modification on the Cu-based metal-organic framework material (Cu-MOF). Without changing its crystal structure, the morphology of MOF changes from disordered to ordered. ZnO is combined with N-Cu-MOF by hydrothermal synthesis method, enabling the photocatalyst to have light absorption ability in the visible light region and forming a Z-scheme heterojunction. The photocatalyst of the present invention reduces the recombination efficiency of photogenerated electron-hole pairs. The ZnO / N-Cu-MOF photocatalyst has a significant inactivating effect on the microspores of Hematodinium sp., providing new reference and reference for the prevention and control of the epidemic diseases of cultured crustaceans caused by Hematodinium sp.

[0024] Compared with the existing treatment methods for aquaculture diseases, the present invention has the following advantages:

[0025] 1. The ZnO / N-Cu-MOF photocatalyst involved in the present invention is insoluble and relies on the strong redox radicals generated under the drive of light to play an indirect role. Therefore, fixing the photocatalyst on a specific floating carrier can achieve the purpose of secondary recovery and reuse, and it will not enter the water body of aquaculture ponds and be ingested by cultured animals.

[0026] 2. The light absorption range of the ZnO / N-Cu-MOF photocatalyst prepared in the present invention is extended to the visible light region. Traditional photocatalysts such as ZnO and TiO2 only have light absorption in the ultraviolet light spectral region. However, ultraviolet light only accounts for about 4% of the solar energy, which limits the further application of photocatalysts. The ZnO / N-Cu-MOF photocatalyst prepared in the present invention has a strong absorption peak at about 700 nm, indicating that it can utilize the visible light in the sun for photocatalytic action and has higher photocatalytic efficiency.

[0027] 3. The photocatalyst technology involved in the present invention, through experimental verification, can make half of the microspore cells of Hematodinium sp. lose their motility activity when the action time is 2 h and the concentration is 1 mg / L. Compared with the traditional treatment methods, the photocatalytic technology has a short action time and high efficiency, can slow down the diffusion rate of Hematodinium sp. spores, and further control the spread process of the epidemic diseases of Hematodinium sp. Description of the Drawings

[0028] Figure 1 Scanning electron microscope (SEM) and transmission electron microscope (TEM) pictures of five photocatalysts; among them: (a): SEM of ZnO, (b): SEM of Cu-MOF, (c): SEM of N-Cu-MOF, (d): SEM of ZnO / Cu-MOF, (e): SEM of ZnO / N-Cu-MOF, (f): TEM of N-Cu-MOF.

[0029] Figure 2The proportion of motile cells of Hematodinium sp. spores under ZnO / N-Cu-MOF photocatalysts with different concentration gradients; where: (a): the proportion of active cells of Hematodinium sp. spores at 1 h; (b): the proportion of active cells of Hematodinium sp. spores at 2 h.

[0030] Figure 3 Scanning electron microscope images of Hematodinium sp. spores; where: (a): spores in the control group; (b): 2 h EC 50 Spores at 2 h under photocatalysts with a concentration of

[0031] Figure 4 For 2 h EC 50 Changes in the total antioxidant capacity of Hematodinium sp. spore cells at 0 h, 0.5 h, 1 h, and 2 h under photocatalysts with a concentration of (Experimental group, referred to as E) and pure light irradiation conditions (Light control group, referred to as L). Specific implementation manners

[0032] The present invention will be further described in detail below in conjunction with specific implementation manners.

[0033] The present invention introduces polyvinylpyrrolidone to perform N-doping modification on Cu-MOF and composes ZnO to prepare a new visible light photocatalyst with a Z-scheme heterojunction. ZnO / N-Cu-MOF has the ability to absorb light in the visible light region, and the formed Z-scheme heterojunction further reduces the recombination efficiency of photogenerated electron-hole pairs. Compared with traditional treatment methods, the photocatalytic technology has a short action time, high disinfection efficiency, and recyclability. When 1 mg / L of the photocatalyst acts for 2 h, it can significantly make half of the Hematodinium sp. spores lose their motility. The present invention provides a new reference and reference for the prevention and control of epidemic diseases of cultured crustaceans caused by Hematodinium sp., showing certain research prospects and application potential.

[0034] Example 1

[0035] First, prepare the photocatalyst ZnO / N-Cu-MOF, and the specific steps are as follows:

[0036] Step 1: Dissolve copper nitrate in N,N-dimethylformamide (DMF) under magnetic stirring to make its final concentration 0.058 g / mL and call it Solution I. Dissolve 1,3,5-benzenetricarboxylic acid (H3TBC) in DMF to make its final concentration 0.028 g / mL and call it Solution II;

[0037] Step 2: Add polyvinylpyrrolidone (PVP) to Solution I and stir until completely dissolved. After dissolution, mix it with Solution II under stirring conditions to obtain Solution III. The concentration of PVP in Solution I is 0.02 g / mL.

[0038] Step 3: Add ZnO to Solution III to make its final concentration 0.02 g / mL, and continuously stir until evenly mixed.

[0039] Step 4: Transfer the evenly mixed solution to a reaction kettle for reaction. Set the reaction temperature to 80 °C and the time to 24 h. After the reaction, wait for the temperature of the reaction kettle to cool to room temperature.

[0040] Step 5: Centrifuge the mixed solution in the inner lining of the reaction kettle to collect the solid (5000 rpm, 10 min). After washing the solid, dry it overnight to obtain the ZnO / N-Cu-MOF composite photocatalyst.

[0041] The preparation process of Cu-MOF is the same as above, but without adding PVP and ZnO. The preparation process of ZnO / Cu-MOF is the same as above, but without adding PVP.

[0042] The scanning electron microscope images and transmission electron microscope images of the prepared photocatalysts are as Figure 1 shown. The structure of Cu-MOF is unstable during the preparation process, resulting in a relatively low degree of crystallization. Some can barely be regarded as octahedrons, and more are irregular block structures. Under the assistance of PVP in the synthesis, N-Cu-MOF ( Figure 1 c) presents a regular octahedral geometry, with small particles attached to large particles. The TEM image ( Figure 1 f) further shows the structural order from the two-dimensional internal structure image. After loading ZnO, compared with Cu-MOF, the combination of N-Cu-MOF and ZnO is denser, allowing more ZnO particles to attach.

[0043] Application Example 1

[0044] The disinfection and killing effects of ZnO / N-Cu-MOF photocatalysts with different concentration gradients on the microspores of Hematodinium sp. were tested. Using "loss of motility" as a non-lethal effect, calculate the half-effect concentration EC 50 of ZnO / N-Cu-MOF on the microspores of Hematodinium sp., specifically as follows:

[0045] (1) Microscopically examine the infected Helice tridens tientsinensis. Culture the host crabs in the pre-spore cell stage alone in the dark, and change the sterile seawater every day. After the microspores of Hematodinium sp. are released from the host crabs into the environment, use a cell counting chamber to count the cell density of the released microspores of Hematodinium sp., and dilute the cell culture solution to 10 6 cells / mL with sterile seawater according to the actual cell density.

[0046] (2) Weigh 0.01 g of ZnO / N-Cu-MOF photocatalyst and add it to 100 mL of sterile seawater. At this time, the concentration of the photocatalyst is 0.1 mg / L. Disperse it by ultrasonic wave at 40% power for 3 min to obtain a photocatalyst dispersion.

[0047] (3) Add 4 mL of microspore diluent to each well of a six-well cell culture plate. Subsequently, add 40 μL, 80 μL, 120 μL, 160 μL, and 200 μL of the photocatalyst dispersion to the experimental groups respectively, so that the concentrations of the photocatalyst in each experimental group reach the set concentrations of 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, and 5 mg / L; at the same time, use the group without adding the photocatalyst as a control.

[0048] (4) Place the six-well plate in an illumination incubator at 20 °C. At 0 h, 1 h, and 2 h at the start of the experiment, take 20 μL of the culture solution from each well and drop it on a glass slide, and record the spore movement video under an optical microscope. Randomly select at least 3 fields of view in each experimental group to record a video for more than 5 s.

[0049] (5) Count the total number of microspore cells and the number of moving cells in each video to obtain the proportion of active cells:

[0050] Proportion of motile cells = (Number of motile cells in the field of view / Total number of cells in the field of view) × 100%

[0051] Use the logarithmic regression equation to calculate the half-maximal effective concentration EC of the photocatalyst on the spores of Hematodinium 50 . Obtain the 1 h EC of the ZnO / N-Cu-MOF photocatalyst on the microspores of Hematodinium 50 is 2.8 mg / L, and the 2 h EC 50 is 1 mg / L. The results are as Figure 2 shown. The proportion of active cells of Hematodinium spores decreased significantly with the increase of the photocatalyst concentration. When the photocatalyst concentration was 3 mg / L and the action time was 2 h, the proportion of motile cells was 0%. Application Example 2

[0052] Observe the microscopic morphological changes of the microspores of Hematodinium under the action of the photocatalyst at the 2 h EC 50 concentration, and explore the damage degree and effect of the photocatalyst on the microspores. Specifically:

[0053] (1) After the spores of Hematodinium are released from the body of Hemigrapsus penicillatus into sterile seawater, dilute them to 10 6 cells / mL. According to the determination in the above application example, the 2 h EC 50Add the photocatalyst at a concentration of (3 mg / L). Collect the cells (1000 rpm, 15 min) 2 h after the start of the experiment. Additionally, set up a control group without adding the photocatalyst and collect the cells at 2 h as well.

[0054] (2) Wash the collected cells twice with PBS (1000 rpm, 15 min). After washing, transfer them to a 1.5 mL centrifuge tube.

[0055] (3) At room temperature, add 1 mL of 2.5% glutaraldehyde solution to the cell pellet and fix for 6 h.

[0056] (4) Centrifuge to collect the fixed cells (1000 rpm, 15 min) and wash them 2 - 3 times with PBS.

[0057] (5) Add 1 mL of 30% ethanol solution, mix well, let stand for 20 - 30 min, centrifuge and discard the supernatant. Repeat the gradient dehydration successively in 50%, 70%, 80%, 90%, and 100% ethanol.

[0058] (6) Replacement: Add 1 mL of isoamyl acetate to the collected cell pellet and let stand for 30 min.

[0059] (7) Drying: Break the cover glass into pieces, pick a piece with a better shape, drop the algal solution on it, and place it in a petri dish and dry it in an oven.

[0060] (8) Place the glass slide under a scanning electron microscope for observation and take pictures.

[0061] The results are as Figure 3 shown. Compared with the control group, the cell membrane structure of the microspores in the treatment group was significantly degraded, the exposed cell surface was honeycombed, and the flagella of most cells were lost, which is presumably the main reason for the loss of cell motility.

[0062] Application Example 3

[0063] A variety of antioxidants present in cells can scavenge various reactive oxygen species generated in the body to prevent the occurrence of oxidative stress induced by reactive oxygen species. Referring to the total antioxidant capacity detection kit (FRAP method), the change in the total antioxidant capacity of the microspores of Hematodinium sp. under the action of ZnO / N-Cu-MOF photocatalyst with a 2 h EC 50 concentration was measured to explore the effect of the photocatalyst on the total antioxidant capacity (TAOC) in the microspores. Since the culture environment of Hematodinium sp. is a dark environment, a separate light control group was set up to observe the effect of pure light conditions on the total antioxidant capacity of the microspores of Hematodinium sp. Specifically:

[0064] (1) After the spores of Hematodinium sp. are released from the thick crab into sterile seawater, dilute them to 106 cells / mL. Add the photocatalyst at a concentration of (3 mg / L) as determined in the above application example. Additionally, set up an experimental group under pure light irradiation conditions without adding a catalyst as a control. Collect cells (3000 rpm, 5 min) at 0 h, 0.5 h, 1 h, and 2 h at the start of the experiment. 50

[0065] (2) Add 200 μL of PBS to each tube of collected cells, add grinding beads, and break the cells in a cell disruptor (ultrasound for 5 s, pause for 10 s, repeat 3 times). Centrifuge to collect the supernatant (4 °C, 12,000 g, 5 min) for subsequent determination.

[0066] (3) Prepare an appropriate amount of FRAP working solution according to the number of samples to be measured (including the standard curve). Incubate the prepared FRAP working solution at 37 °C and it should be used within 1 - 2 h.

[0067] (4) Add 180 μL of FRAP working solution to each detection well of a 96 - well plate. Add 5 μL of distilled water or an appropriate solution such as PBS to the blank control well; add 5 μL of FeSO4 standard solutions diluted to 0.15, 0.3, 0.6, 0.9, 1.2, and 1.5 mM to the standard curve detection wells respectively; add 5 μL of various samples to the sample detection wells and mix gently.

[0068] (5) After incubating at 37 °C for 3 - 5 min, measure A 593 , and calculate the total antioxidant capacity of the sample according to the standard curve.

[0069] The results are as Figure 4 shown. Pure light irradiation conditions have no significant effect on the TAOC of microspores. Under photocatalysis, the TAOC of microspores first increases and then decreases, and finally shows a significant decrease at 2 h, indicating that photocatalysis causes oxidative damage to microspore cells.​

Claims

1. A preparation method of a photocatalyst, characterized in that: The Cu-MOF was N-doped and modified by introducing polyvinylpyrrolidone, and then compounded with ZnO by hydrothermal synthesis method to obtain a novel visible-light photocatalyst ZnO / N-Cu-MOF with Z-scheme heterojunction.

2. The preparation method of the photocatalyst according to claim 1, characterized in that: Step 1: Dissolve copper nitrate in N,N-dimethylformamide (DMF) under magnetic stirring, and call it Solution I. Dissolve 1,3,5-benzenetricarboxylic acid (H3TBC) in DMF, and call it Solution II; Step 2: Add polyvinylpyrrolidone (PVP) to Solution I and stir until completely dissolved. After dissolution, mix it with Solution II under stirring to obtain Solution III; Step 3: Add ZnO to Solution III and continuously stir until evenly mixed; Step 4: Transfer the evenly mixed solution to a reaction kettle for reaction, and react at 80-120 °C for 20-24 h; Step 5: After the above reaction, wait for the temperature of the reaction kettle to cool to room temperature, centrifuge the obtained reaction solution to collect the solid (5000 rpm, 10 min), wash the solid and then dry it overnight to obtain the ZnO / N-Cu-MOF composite photocatalyst.

3. The preparation method of the photocatalyst according to claim 2, wherein: The concentration of copper nitrate in Solution I is 0.058-0.06 g / mL, and the concentration of 1,3,5-benzenetricarboxylic acid in Solution II is 0.028-0.03 g / mL.

4. The preparation method of the photocatalyst according to claim 2, wherein: The concentration of polyvinylpyrrolidone in Solution I is 0.013-0.02 g / mL, and the volume ratio of Solution I to Solution II is 1:1; the concentration of ZnO in Solution III is 0.019-0.02 g / mL.

5. A photocatalyst obtained by the method according to claim 1, characterized in that: Obtain the light blue powdery photocatalyst (ZnO / N-Cu-MOF) according to the method described in claim 1.

6. The use of the photocatalyst according to claim 5, characterized in that: The application of the ZnO / N-Cu-MOF photocatalyst in killing the spores of the parasitic dinoflagellate Hematodinium sp.

Images