A hydrogel loaded with iron oxide nanoparticles, its preparation method and application in improving bioavailability

Abstract

The present invention discloses a hydrogel loaded with iron oxide nanoparticles, its preparation method and application in improving biological effectiveness, belonging to the technical field of novel fertilizers. Preparation of ENPs-hydrogel in the present invention: Ferric chloride hexahydrate and ferrous chloride tetrahydrate are configured into a mixed solution, which is preheated to 40-60 °C, and then dropped into a NaOH solution for reaction to obtain Fe₃O₄; then Fe₃O₄ is ground and calcined to obtain Fe₂O₃ ENPs, which are then mixed with a folic acid solution and a Zn(NO₃)₂ solution to obtain ENPs-hydrogel; when this ENPs-hydrogel is used as a novel fertilizer, it can not only increase the deposition amount of the active ingredient on the leaf surface, but also, as an excellent carrier, reduce water evaporation, increase the duration of the active ingredient, improve the biological effectiveness of ENPs, and achieve the purpose of "reducing application rate and increasing efficiency".

Description

Technical Field

[0001] The present invention relates to a hydrogel loaded with iron oxide nanoparticles, a preparation method thereof, and an application in improving biological effectiveness, belonging to the technical field of novel fertilizers. Background Art

[0002] Engineered nanoparticles (ENPs) have great potential in the field of nanoagriculture, which can not only increase crop yields but also endow crops with good disease resistance. Foliar spraying is an effective method for using ENPs in agricultural production, which can avoid the fixation of ENPs by soil. Especially for iron-based ENPs, foliar spraying can effectively improve the biological effectiveness of ENPs.

[0003] However, the surface of plant leaves is covered with a hydrophobic waxy layer, resulting in droplet splashing, rolling, and bouncing. Traditional pesticide droplets cannot be targeted and deposited on the leaves, and the utilization rate is generally below 10%. Due to their small size and high rigidity, the actual deposition amount of nanoparticles may be lower than that of conventional pesticides, which will not only cause economic losses but also lead to environmental risks.

[0004] Currently, adding surfactants to droplets is the main strategy to reduce droplet bounce and promote droplet deposition on hydrophobic leaves. The addition of surfactants mainly increases the droplet deposition amount by reducing its surface tension and increasing the diffusion phase during the impact process. However, the reduction of surface tension may cause the droplets to break into smaller droplets, which will exacerbate droplet drift and evaporation, making it difficult for the active ingredients to remain on the surface of plant leaves for a long time. At the same time, surfactants are difficult to degrade in the environment and may bring new pollution. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a hydrogel loaded with iron oxide nanoparticles, a preparation method thereof, and an application in improving biological effectiveness. The iron oxide nanoparticle hydrogel uses folic acid / Zn 2+ supramolecular hydrogel as a carrier, and combines with iron oxide nanoparticles by means of vortex mixing to form an iron oxide nanoparticle hydrogel. When this hydrogel composite is applied to plants as a fertilizer, on the one hand, when the hydrogel droplets with a certain viscosity impact the leaves, the viscous dissipation with the leaf surface will increase, reducing the droplet bounce, so that most of the active ingredients are deposited on the leaf surface, improving the utilization rate of the active ingredients; on the other hand, the network structure of the hydrogel is filled with water and has good slow-release properties, which can reduce the evaporation of droplets, increase the duration of the active ingredients, and thus improve the biological effectiveness.

[0006] The first object of the present invention is to provide a method for preparing a hydrogel composite ENPs-hydrogel loaded with iron oxide nanoparticles for improving the bioavailability of iron oxide nanoparticles in plants during foliar spraying, and the method comprises the following steps:

[0007] (1) Prepare a mixed solution of ferric chloride hexahydrate and ferrous chloride tetrahydrate, preheat the mixed solution to 40-60 °C, then drop it into the preheated NaOH solution for reaction, cool to room temperature after the reaction, obtain the black precipitate, wash and dry to obtain massive Fe3O4; then grind and calcine the massive Fe3O4 to obtain Fe2O3 ENPs;

[0008] (2) Prepare a folic acid solution and a suspension of Fe2O3 ENPs; then mix the folic acid solution, Zn(NO3)2 solution and the suspension of Fe2O3 ENPs, vortex and stand to obtain ENPs-hydrogel.

[0009] In one embodiment, the molar ratio of ferric chloride hexahydrate to ferrous chloride tetrahydrate in the mixed solution in step (1) is 1:1.2-1:2.

[0010] In one embodiment, the preheating temperature of the preheated NaOH solution in step (1) is 40-60 °C.

[0011] In one embodiment, the concentration of the NaOH solution in step (1) is 1-2 mol / L.

[0012] In one embodiment, the volume ratio of the NaOH solution to the mixed solution in step (1) is 1:1-1.5.

[0013] In one embodiment, the reaction temperature in step (1) is 40-60 °C and the time is 0.5-1 h.

[0014] In one embodiment, the washing in step (1) means washing with deionized water and absolute ethanol until the pH value reaches 7.

[0015] In one embodiment, the calcination in step (1) is carried out in a muffle furnace at a temperature of 300-500 °C and a time of 2-5 h.

[0016] In one embodiment, the preparation process of the folic acid solution in step (2) is: mix folic acid with deionized water, and then add KOH solution to obtain a uniform folic acid solution.

[0017] In one embodiment, the concentration of the KOH solution in step (2) is 0.5 - 1 mol / L, and the mass-to-volume ratio of folic acid to deionized water and the KOH solution is 2 - 3:50:50, g / mL / mL.

[0018] In one embodiment, the preparation process of the Fe2O3 ENPs suspension in step (2) is as follows: Add Fe2O3 ENPs to deionized water and ultrasonicate in an ice bath (0 °C) to obtain a suspension of Fe2O3 ENPs.

[0019] In one embodiment, the molar ratio of folic acid to Zn(NO3)2 in step (2) is 1:1.6 - 1:1.9.

[0020] In one embodiment, the molar ratio of folic acid to Zn(NO3)2 in step (2) is 1:1.6.

[0021] In one embodiment, the vortex time in step (2) is 1 - 2 min.

[0022] In one embodiment, the standing time in step (2) is 30 - 60 min.

[0023] The second object of the present invention is to provide a hydrogel ENPs-hydrogel loaded with iron oxide nanoparticles prepared by the method described above.

[0024] The third object of the present invention is to provide an application of the above-mentioned hydrogel ENPs-hydrogel loaded with iron oxide nanoparticles in the preparation of agricultural fertilizers.

[0025] The fourth object of the present invention is to provide a method for improving plant photosynthesis and biomass based on the above-mentioned hydrogel ENPs-hydrogel, and the method is to directly spray the hydrogel ENPs-hydrogel on the plant surface.

[0026] In one embodiment, the plant is soybean.

[0027] In one embodiment, the concentration of the hydrogel ENPs-hydrogel is 10 - 100 mg / L.

[0028] In one embodiment, the concentration of the hydrogel ENPs-hydrogel is 50 mg / L.

[0029] In one embodiment, the spraying amount is 5 - 10 mL per plant.

[0030] In one embodiment, the spraying method is: spraying every day or spraying every other day or spraying every three days.

[0031] In one embodiment, the spraying method is as follows: once a day, continuously sprayed for 5 days, or continuously sprayed for 3 days with an interval of 1 day, or sprayed for 2 days with an interval of 3 days.

[0032] In one embodiment, the spraying method is as follows: continuously sprayed for 3 days with an interval of 1 day.

[0033] In one embodiment, the plant is soybean, and the variety is Zhonghuang 13.

[0034] In one embodiment, the spraying period is the seedling stage (the stage of three leaves and one heart).

[0035] In one embodiment, the spraying height is 5 - 25 cm.

[0036] In one embodiment, the spraying height is 25 cm.

[0037] In one embodiment, when spraying, the inclination angle of the plant leaves is 0 - 40°.

[0038] Advantages of the present invention:

[0039] (1) For the ENPs-hydrogel prepared by the present invention, on the one hand, it can increase the deposition amount of the active ingredient on the leaf surface. On the other hand, as an excellent carrier, the hydrogel can reduce water evaporation, increase the duration of the active ingredient, and improve biological effectiveness; among them, the adhesion ability of ENPs-hydrogel to the leaf surface is increased by 76.8% compared with the Fe2O3 ENPs suspension, and the leaf deposition amount is increased by 48.2% - 168.9%;

[0040] (2) Due to the improvement of the biological effectiveness of ENPs, after spraying ENPs-hydrogel on the leaf surface, compared with the Fe2O3 ENPs suspension, the application amount of ENPs can be reduced by 40%. At the same time, the photosynthesis rate of soybean can still be increased by 89.8%, the stomatal conductance is increased by 157%, and the fresh weight and dry weight are increased by 37.6% and 104.4% respectively; Loading ENPs with hydrogel can achieve the purpose of reducing application amount and increasing efficiency. Description of the drawings

[0041] Figure 1 Characterization diagrams of Fe2O3 ENPs and ENPs-hydrogel in Example 1; (A) is the TEM result diagram of Fe2O3 ENPs; (B) is the ESEM diagram of ENPs-hydrogel; (C) is the XRD diagram of ENPs-hydrogel;

[0042] Figure 2Contact angle, adhesion force curve, and viscosity effect diagrams of Fe2O3 ENPs suspension and ENPs-hydrogel; (A) Contact angles of Fe2O3 ENPs suspension and ENPs-hydrogel at blade inclinations of 0° and 40°; (B) Adhesion force curve diagram; (C) Viscosity result diagram;

[0043] Figure 3 Deposit amount data diagram of Fe2O3 ENPs suspension and ENPs-hydrogel on the blade surface;

[0044] Figure 4 Instant physical diagrams of droplets impacting the soybean leaf surface at different heights (5 cm, 10.5 cm, 25 cm) and angles (0°, 40°);

[0045] Figure 5 Growth index data diagrams of soybeans sprayed with ENPs-hydrogel at different loading concentrations; (A) Net photosynthetic rate; (B) Stomatal conductance; (C) Biomass; (D) Chlorophyll content;

[0046] Figure 6 Index data diagrams of the influence of ENPs-hydrogel on soybean growth at different volumes and frequencies of spraying; (A) Net photosynthetic rate; (B) Stomatal conductance; (C) Fresh weight; (D) Dry weight; (E) Chlorophyll content. Detailed implementation manners

[0047] The following are descriptions of the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0048] Testing methods involved in the present invention

[0049] 1. Determination of photosynthesis parameters

[0050] The photosynthetic rate and stomatal conductance of soybeans are measured by a CIRAS-3 portable gas exchange system.

[0051] 2. Determination of soybean biomass

[0052] After harvesting, the soybeans are washed and the surface moisture is dried. The fresh weight (FW) of the soybean tissue is weighed using a one-percent balance, and the soybeans are blanched in an oven at 105°C for 15 min and then dried at 75°C to a constant weight to measure their dry weight (DW).

[0053] 3. Determination of soybean chlorophyll content

[0054] The acetone extraction method was used to determine the chlorophyll content in soybean leaves. Weighed 40 mg of fresh leaf sample powder into a centrifuge tube, added 4 mL of 80% acetone (v / v), and used an ultrasonic cleaner to ultrasonically crush the sample (900 W, 20 min). The mixture was made up to 10 mL and placed in a low-temperature refrigerator (4 °C) in the dark for 72 h. Using 80% acetone as a reference, the absorbance was measured at 470 nm, 645 nm, and 663 nm.

[0055] Example 1

[0056] A preparation method of iron oxide nanoparticle hydrogel (ENPs-hydrogel) includes the following steps:

[0057] (1) Prepared a 500 mL mixed solution with 21.6 g of ferric chloride hexahydrate (FeCl3·6H2O, AR, 99%) and 9.95 g of ferrous chloride tetrahydrate (FeCl2·4H2O, AR, 99%); then, heated 100 mL of 1 mol L -1 NaOH solution to 60 °C in a three-necked flask, and preheated the mixed solution to 60 °C at the same time. Slowly added 130 mL of the mixed solution to the three-necked flask using a separatory funnel. After the addition was completed, heated the solution at 60 °C for 0.5 h and cooled it to room temperature to obtain a black precipitate; washed it several times with deionized water and absolute ethanol until the pH reached 7, dried it at 60 °C for 8 h, and obtained massive Fe3O4; finally, ground it with a ball mill for half an hour and heated it in a muffle furnace at 300 °C for 2.5 h to obtain Fe2O3 ENPs;

[0058] (2) Mixed 2.207 g of folic acid with 50 mL of deionized water, and then added 50 mL of 0.5 mol L -1 KOH solution to obtain a uniform folic acid / KOH neutralization solution; at the same time, added 15 mg of the Fe2O3 ENPs prepared in step (1) to 30 mL of deionized water and ultrasonically treated it in an ice bath (0 °C) to obtain a suspension of 500 mg L -1 of Fe2O3 ENPs; then mixed 100 mL of the folic acid / KOH neutralization solution, 80 mL of 0.1 mol L -1 Zn(NO3)2 solution and 20 mL of the Fe2O3 ENPs suspension in a bottle, vortexed for 1 min, and left to stand for 30 min to obtain ENPs-hydrogel, where the molar ratio of n(folic acid) and n(Zn 2+ ) is 1.1.6.

[0059] Example 2

[0060] The difference from Example 1 is only that in the adjustment step (2), the folic acid / KOH neutralization solution and 0.1 mol L -1 Zn(NO3)2 solutions are 100 mL and 85 mL, 100 mL and 90 mL, 100 mL and 95 mL respectively, so that the molar ratio of n(folic acid) and n(Zn 2+ ) is 1:1.7, 1:1.8 and 1:1.9 respectively; other parameters and conditions are the same as those in Example 1.

[0061] Performance characterization:

[0062] 1. Use a transmission electron microscope (TEM) to characterize the morphology and size of Fe2O3 ENPs

[0063] The results show that Fe2O3 ENPs are spherical with a diameter of 12.0 ± 2.3 nm ( Figure 1 A). Use an environmental scanning electron microscope (ESEM) and XRD to characterize the morphology and composition of ENPs-hydrogel. ENPs-hydrogel presents a regularly arranged porous morphology ( Figure 1 B), and characteristic peaks of Fe2O3 are found on ENPs-hydrogel ( Figure 1 C), proving that Fe2O3 ENPs are successfully loaded onto the hydrogel.

[0064] 2. Characterization of the adhesion parameters of ENPs-hydrogel

[0065] Use a contact angle measuring instrument to measure the contact angles of Fe2O3 ENPs suspension and ENPs-hydrogel at 6 μL when the inclination angle of soybean leaves is 0° and 40° ( Figure 2 A), and use a surface tension instrument to measure the adhesion force of Fe2O3 ENPs suspension and ENPs-hydrogel on the surface of soybean leaves ( Figure 2 B).

[0066] The results show that the viscosity of ENPs-hydrogel is much greater than that of Fe2O3 ENPs suspension. The greater viscosity causes more viscous dissipation when the droplet impacts the leaf surface and is more likely to stay on the leaf surface. The test results of the contact angle show that the contact angle on soybean leaves with an inclination angle of 40° is much smaller than that of Fe2O3 ENPs suspension. A smaller contact angle proves that the material has higher hydrophilicity, that is, it is not easy to roll off, improving the adhesion ability of the material on the leaf.

[0067] Use a rotational rheometer to measure the viscosities of ENPs-hydrogel and Fe2O3 ENPs suspension at different crosslinking ratios ( Figure 2 C), and the results show that when n(folic acid) and n(Zn2+ ) When the ratio is 1:1.6, the viscosity of ENPs-hydrogel is more suitable for spraying, and its adhesion is also higher. The results of adhesion measurement further prove that the loaded ENPs-hydrogel has a higher adhesion ability than the Fe2O3 ENPs suspension, and the adhesion ability of ENPs-hydrogel is increased by 77.4% compared with Fe2O3 ENPs.

[0068] 3. Determination of the deposition amount of ENPs-hydrogel on leaves

[0069] ENPs-hydrogel with fluorescence characteristics was prepared using rhodamine B (1 mM) as a dye. At different heights (5 cm, 10.5 cm, and 25 cm) and leaf angles (0° and 40°), 1 mL of ENPs-hydrogel with fluorescence characteristics was sprayed onto the leaf surface using a nebulizer. After natural drying, a plant in vivo imaging system was used to capture the fluorescence data on the leaf surface. The control group was the Fe2O3 ENPs suspension.

[0070] The results are as Figure 3 shown. Regardless of the changes in angle and height, the deposition amount of ENPs-hydrogel on the soybean leaf surface is higher than that of the Fe2O3 ENPs suspension. When the leaf angle is 40° and the spraying height is 25 cm, the deposition amount of ENPs-hydrogel has the highest increase ratio, which is increased by 168.9% compared with the Fe2O3 ENPs suspension. The entire falling process of a single droplet from different heights (5 cm, 10.5 cm, 25 cm) and angles (0°, 40°) was photographed using an ultra-high-speed camera. As Figure 4 shown, with the increase in the impact height and the increase in the leaf tilt angle, the droplets of the Fe2O3 ENPs suspension show phenomena such as fragmentation, rebound, and rolling, while regardless of the changes in the impact height and the leaf surface inclination angle, the droplets of ENPs-hydrogel are always completely deposited on the leaf surface. This is the microscopic mechanism that leads to a significant increase in the deposition amount of ENPs-hydrogel.

[0071] Example 3

[0072] A method for improving soybean biomass based on ENPs-hydrogel, comprising the following steps:

[0073] (1) Select the soybean variety Zhonghuang 13. Disinfect the seeds in a 5% hydrogen peroxide solution for 10 minutes, then rinse them 5 times with deionized water. After soaking for 12 hours, germinate the seeds in a controlled environment with a humidity of 60%, a temperature of 25 °C, and no light for two days. After the radicle germinates from the seed coat, transfer the seeds to a plastic container filled with 1 kg of agricultural soil. Sow three seeds in each container in a triangular arrangement and cultivate for 14 days;

[0074] (2) After 14 days of growth, retain two phenotypically similar plants in each container; from the 15th day to the 19th day, spray 10 mL of hydrogel and hydrogel containing different concentrations of Fe2O3 ENPs on each soybean plant every morning. Five concentration groups are set in the ENPs-hydrogel hydrogel: 0 (control group), 10 mg L -1 , 30 mg L -1 , 50 mg L -1 and 100 mg L -1 ; there are six parallels in each group.

[0075] The results show that the ENPs-hydrogel at 10 mg L -1 , 30 mg L -1 , 50 mg L -1 and 100 mg L -1 can all significantly promote soybean growth. The ENPs-hydrogel at 50 mg L -1 has the most obvious promotion effect on soybean growth. Compared with the blank control, the photosynthetic rate and stomatal conductance of soybeans after applying the 50 mg L -1 ENPs-hydrogel hydrogel increased significantly by 141.8% and 103.9% respectively ( Figure 5 A and Figure 5 B); compared with the blank control, the biomass increased significantly by 20.7% compared with the untreated plants ( Figure 5 C); the total chlorophyll content of the plants (Ca is chlorophyll a, Cb is chlorophyll b, CT is the total chlorophyll content, Ca + Cb = CT) increased significantly by 34.7% ( Figure 5 D).

[0076] Example 4 Application of different amounts and frequencies of ENPs-hydrogel to soybeans

[0077] A method for improving soybean biomass based on ENPs-hydrogel, comprising the following steps:

[0078] (1) Disinfect soybean seeds in a 5% hydrogen peroxide solution for 10 minutes, then rinse them 5 times with deionized water. After soaking for 12 hours, germinate them for two days in a controlled environment with a humidity of 60%, a temperature of 25°C, and no light. After the radicle germinates from the seed coat, transfer the seeds to plastic containers filled with 1 kg of agricultural soil. Sow three seeds in each container in a triangular arrangement and cultivate for 14 days;

[0079] (2) After 14 days of growth, retain two plants with similar phenotypes in each container; set up treatment groups respectively:

[0080] 50mg L -1 of ENPs-hydrogel was continuously sprayed for 5 days, 10 mL per plant each time, for a total of 50 mL;

[0081] Spray 50mg L -1 of ENPs-hydrogel for 3 days on the 1st, 3rd, and 5th days, 10 mL per day, for a total of 30 mL;

[0082] Spray 50mg L -1 of ENPs-hydrogel for 2 days on the 1st and 5th days, 10 mL per day, for a total of 20 mL;

[0083] 50mg L -1 of ENPs-hydrogel was continuously sprayed for � days, 5 mL each time, for a total of 25 mL;

[0084] 50mg L -1 of ENPs-hydrogel was continuously sprayed for 5 days, 2 mL each time, for a total of 10 mL;

[0085] Set up a control group:

[0086] 50mg L -1 of the Fe2O3 ENP suspension group was continuously sprayed for 5 days, 10 mL per plant each time, for a total of 50 mL;

[0087] The Hydrogel group was continuously sprayed for 5 days, 10 mL per plant each time, for a total of 50 mL;

[0088] The blank control group was continuously sprayed with deionized water for 5 days, 10 mL per plant each time, for a total of 50 mL.

[0089] The results showed that when sprayed for 3 days on the 1st, 3rd, and 5th days, 10 mL per day, the growth promotion effect was the best. Compared with the control group, the amount of ENPs applied was reduced by 40%, and the net photosynthetic rate was still increased by 89.8% ( Figure 6 A), and the stomatal conductance was increased by 157% ( Figure 6B), the fresh weight and dry weight increased by 37.6% and 104.4% respectively ( Figure 6 C and Figure 6 D), the chlorophyll content increased by 57.4% ( Figure 6 E).

[0090] The embodiments provided above are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit the order of their execution. Obvious improvements made by those skilled in the art in combination with the existing well-known general knowledge also fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for improving plant photosynthesis and biomass based on hydrogel ENPs-hydrogel, characterized in that, The method is to directly spray the hydrogel ENPs-hydrogel on the surface of plant leaves; The concentration of the hydrogel ENPs-hydrogel is 50 mg / L; The spraying amount is 10 mL per plant; the spraying method is: spraying at an interval of 1 day; The spraying height is 25 cm; When spraying, the inclination angle of the plant leaves is 40°; the plant is soybean; the spraying period is the seedling stage; The preparation method of the hydrogel ENPs-hydrogel includes the following steps: (1) Prepare a mixed solution of ferric chloride hexahydrate and ferrous chloride tetrahydrate, preheat the mixed solution to 40-60 °C, then drop it into the preheated NaOH solution for reaction, cool to room temperature after the reaction, obtain a black precipitate, wash and dry to obtain blocky Fe3O4; then grind and calcine the blocky Fe3O4 to obtain Fe2O3 ENPs; (2) Prepare a folic acid solution and a suspension of Fe2O3 ENPs; then mix the folic acid solution, Zn(NO3)2 solution and the suspension of Fe2O3 ENPs, vortex and let stand to obtain ENPs-hydrogel; The molar ratio of folic acid to Zn(NO3)2 is 1:1.

6.

2. The preparation method according to claim 1, characterized in that, In the mixed solution in step (1), the molar ratio of ferric chloride hexahydrate to ferrous chloride tetrahydrate is 1:1.2-1:

2.

3. The preparation method according to claim 1, characterized in that, The preheating temperature of the preheated NaOH solution in step (1) is 40-60 °C.

4. The preparation method according to claim 1, characterized in that, The concentration of the NaOH solution in step (1) is 1-2 mol / L.

5. The preparation method according to claim 1, characterized in that, The volume ratio of the NaOH solution to the mixed solution in step (1) is 1:1-1.

5.

6. The preparation method according to claim 1, characterized in that, The reaction temperature in step (1) is 40-60 °C and the time is 0.5-1 h.

7. The preparation method according to claim 1, wherein, The washing in step (1) means washing with deionized water and absolute ethanol until the pH value reaches 7.

8. The preparation method according to claim 1, wherein The calcination in step (1) is carried out in a muffle furnace at a temperature of 300-500 °C and a time of 2-5 h.

9. The preparation method according to claim 1, wherein, The concentration of the NaOH solution in step (2) is 0.5-1 mol / L, and the mass-volume ratio of folic acid to deionized water and NaOH solution is 2-3:50:50, g / mL / mL.

10. The preparation method according to claim 1, wherein, The preparation process of the Fe2O3 ENPs suspension in step (2) is: add Fe2O3 ENPs to deionized water and ultrasonicate in an ice bath (0 °C) to obtain a suspension of Fe2O3 ENPs.

11. The preparation method according to claim 1, characterized in that, The molar ratio of folic acid to Zn(NO3)2 in step (2) is 1:1.6-1:1.

9.

12. The preparation method according to claim 1, characterized in that, The vortex time in step (2) is 1-2 min.

13. The preparation method according to claim 1, wherein, The standing time in step (2) is 30-60 min.

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