CN114027296A - Nano pesticide preparation for preventing and controlling pine wood nematode and its vector insect - Google Patents
Nano pesticide preparation for preventing and controlling pine wood nematode and its vector insect Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/32—Ingredients for reducing the noxious effect of the active substances to organisms other than pests, e.g. toxicity reducing compositions, self-destructing compositions
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a novel nano pesticide preparation for preventing and treating pine wood nematodes and vector insects thereof, wherein the novel nano pesticide is prepared by preparing Dendritic Mesoporous Silica Nano (DMSNs) by a one-pot method and loading pesticide Avermectin (AVM) to obtain a DMSNs-AVM NPs pesticide preparation; DMSNs are in a dendritic porous structure, have a large specific surface area, high drug loading, good controlled release effect, uniform and stable particle size and good dispersibility; the DMSNs-AVM NPs can effectively kill pests through contact poisoning or feeding poison, and have good permeability and transmissibility. In addition, DMSNs-AVM NPs effectively penetrate the cell wall barrier of plants, transporting drugs into the bark for control of Bursaphelenchus xylophilus and its vector insects. The nano pesticide preparation prepared by the invention is prepared by a one-pot method, has low equipment requirement, low energy consumption and high yield, and is suitable for industrial production.
Description
Technical Field
The invention relates to the technical field of pesticide preparations, in particular to a novel nano pesticide preparation for preventing and treating pine wood nematodes and vector insects thereof.
Background
Pine wilt disease (also known as pine wilt disease and pine cancer) is a devastating forest disaster caused by pine nematode, which is a major foreign invasive species in China and can infect more than 40 kinds of pine, such as black pine, European red pine and Japanese red pine; in addition, the disease has strong transmission, which becomes a serious barrier for the prevention and control of the pine wood nematode disease. At present, no effective prevention and control measures are available, so that immeasurable economic losses are brought to China and even world forestry. Monochamus alternatus (Monochamus alternatus) is a vector insect of pine wood nematode, which is spread by adult insects eating pine needle, and the larvae of the pine wood nematode boring into the pine and eating the pine also cause great damage to the pine.
Currently, the control of monochamus alternatus hope against pine wood nematodes and their vector insects mainly includes biological control, chemical control and physical control. Chemical control is considered to be the most effective solution, and the chemical control process can be accelerated by the aid of other two kinds of control. Chemical control is widely researched as an efficient control means, and commercial reagents such as missible oil preparations, microcapsule suspension and the like are applied by injection drying or spraying, but from practical application, the chemical control is discovered and brings breakthrough effect. For example, Avermectin (Avermectin, AVM) is a typical biopesticide, is a high-efficiency broad-spectrum pesticide, has a good effect of preventing and treating pine wood nematodes and longicorn larvae, but has the defects of poor water solubility, short duration, low effective utilization rate and the like, so that the pesticide is large in dosage, high in prevention and treatment cost, and also inevitably causes serious environmental pollution. Therefore, the novel intelligent nano pesticide is constructed by combining the defects of the pesticide and interfering with target genes of the pine wood nematodes and the monochamus alternatus, and a stable drug-carrying slow-release system, a high-efficiency transmission and permeation system, a monitoring and imaging system with ultrahigh sensitivity and the like are urgently needed to be designed, and the mechanism and mechanism research of a new delivery means is developed.
Disclosure of Invention
In view of the prior art, the invention aims to provide a novel nano pesticide for preventing and controlling pine wood nematodes and vector insects thereof. The invention effectively solves the problems that the traditional pesticide preparation has low pesticide effect and poor controllability, can not kill pests in the trunk, and the pests are easy to generate drug resistance.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, DMSNs are provided for use as a vector in any one of 1) to 4) below;
1) loading abamectin;
2) loading fluorescein isothiocyanate;
3) preparing a nano pesticide;
4) preparing a fluorescent marker.
Preferably, the DMSNs are prepared by the following method:
(1) heating purified water in an oil bath, dropwise adding triethanolamine and stirring for 30min, then adding hexadecyl trimethyl ammonium bromide and sodium salicylate, continuously stirring for reaction for 1h, then adding tetraethyl orthosilicate, and continuously stirring for reaction for 2h to obtain a reaction solution;
(2) and (2) centrifuging the reaction solution obtained in the step (1) for the first time at 4 ℃ to obtain a precipitate, washing the precipitate, centrifuging for the second time, adding methanol and concentrated hydrochloric acid into the precipitate, performing ultrasonic dispersion, stirring and reacting for three times in a water bath, reacting for 6 hours each time, centrifuging for the third time, collecting the precipitate, adding the obtained precipitate into purified water, freezing for 24 hours at-20 ℃ or 1 hour at-80 ℃, and freeze-drying to obtain fine white powder, namely the DMSNs.
Preferably, the DMSNs have the size of 200-400 nm.
Preferably, in the step (1), the temperature of the oil bath heating is 80 ℃; the ratio of the added amounts of the purified water, the triethanol, the hexadecyl trimethyl ammonium bromide, the sodium salicylate and the tetraethyl orthosilicate is 25 mL: 60.7 μ L: 380 mg: 168 mg: 4 mL.
Preferably, in the step (2), the volume ratio of the methanol to the concentrated hydrochloric acid is 120: (0.24 to 1.54); the temperature of the water bath is 60 ℃; the speed of the first centrifugation, the second centrifugation and the third centrifugation is 11000rpm, and the centrifugation time is 10 min; the freeze-drying temperature is-56 ℃ and the freeze-drying time is 48 h.
In a second aspect of the present invention, there is provided a fluorescent marker, wherein the preparation method of the fluorescent marker comprises: dissolving FITC in purified water to obtain a FITC solution; and adding DMSNs into the FITC solution, stirring for 2h, and centrifuging at 4 ℃ to obtain DMSNs-FITC, namely the fluorescent marker.
Preferably, the concentration of the FITC solution is 0.025 mg/mL; the volume ratio of DMSNs to FITC solution is 1: 2.
In a third aspect of the invention, an avermectin-loaded nano pesticide preparation is provided, which is prepared by the following method:
dissolving abamectin in methanol to obtain an abamectin solution; and dispersing the DMSNs into the abamectin solution, stirring for 24-72h, centrifuging, collecting the precipitate, and drying to obtain the nano pesticide preparation.
Preferably, the concentration of the abamectin solution is (1-19) mg/mL; the volume ratio of the abamectin solution to the DMSNs is 1: 4.
In a fourth aspect of the invention, the invention provides the use of the nano pesticide preparation loaded with the abamectin in preventing and controlling the pine wood nematode and the vector insects thereof.
Dispersing the nano pesticide preparation into water, and spraying by using an unmanned aerial vehicle to prevent and control the pine wood nematodes and adult vector insects thereof; or dispersing the nano pesticide preparation into water, injecting the nano pesticide preparation into pine trees by adopting an injection mode, and preventing and treating the pine wood nematodes and the larvae of the vector insects thereof.
The invention has the beneficial effects that:
(1) the invention uses DMSNs as a carrier to load pesticide avermectin for the first time to prepare the nano pesticide preparation; the DMSNs used as the carrier has uniform and stable particle size and good dispersibility; moreover, the DMSNs shell is of a dendritic porous structure, has a large specific surface area and high drug loading, and has a good sustained and controlled release effect.
(2) The nano pesticide preparation prepared by the invention can penetrate through the cell wall barrier of plants due to the nano size, and the medicine is transported into the bark for preventing and controlling the pine wood nematode and the vector insects thereof.
(3) The DMSNs are synthesized by a one-pot method, the synthesis method is simple, the yield is high, complex procedures can be omitted, the method is suitable for industrial production, the used organic solvent can be recycled, the requirement on equipment is low, no environmental pollution is caused, and the method has wide market application prospect.
(4) After the abamectin is adsorbed into the DMSNs, the rapid photolysis of the abamectin can be effectively prevented, the utilization rate of the medicament is greatly improved, the application times of the medicament are reduced, the reduction of the pesticide is realized, and the environment-friendly effect is realized.
Drawings
FIG. 1 is a SEM image of DMSNs prepared in example 1.
FIG. 2 is a transmission electron microscopy topographic map of DMSNs prepared in example 1.
In fig. 3, (a) is a particle size distribution diagram of DMSNs prepared in example 2, (b) is a particle size distribution diagram of DMSNs prepared in comparative example 1, and (c) is a particle size distribution diagram of DMSNs prepared in comparative example 2.
FIG. 4 is a micropore distribution plot of DMSNs prepared in example 1.
FIG. 5 is a BET plot of DMSNs prepared in example 1.
FIG. 6 is a graph of the photodegradation of DMSNs prepared in example 1 (where A represents the photodegradation rate of AVM under 310nm UV light, B represents the photodegradation rate of AVM @ DMSNs under 310nm UV light, C represents the photodegradation rate of AVM in the dark, and D represents the photodegradation rate of AVM @ DMSNs in the dark).
FIG. 7 is a graph of sustained release rates of AVM @ DMSNs prepared in example 1 in PBS buffers of various pHs at room temperature.
FIG. 8 is a body wall fluorescence distribution (a) and a portal fluorescence distribution (b) of DMSNs-FITC prepared in example 1 in longicorn larvae.
FIG. 9 shows the intestinal fluorescence distribution (a) and bright field map (b), the intestinal fluorescence distribution map (c) and bright field map (d) of DMSNs-FITC in longicorn larvae, and the confocal image (e) of DF-1 cells under natural light and fluorescence conditions, which were prepared in example 1.
Fig. 10 is a schematic diagram of the division of the four-layer structure of the trunk.
FIG. 11 shows the distribution of avermectins in the trunk.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
The term "room temperature" as used herein means a temperature of 25 ℃ to 35 ℃.
As described in the background, the pine wood nematodes and their vector insects are all-spherical forest quarantine pests, important trunk-boring pests of pine trees, and are the vectors and potential vector insects for pine wood nematodes and other nematodes. At present, very effective prevention measures for the pine wood nematodes and the vector insects are lacked, and the pine wood nematodes and the vector insects in the trunk are difficult to kill by applying the pesticide from the outside of the pine; the pesticide is injected into the trunk in an injection mode, but most of the pesticides are hydrophobic, are difficult to transport in the trunk, and cannot achieve stomach toxicity or contact killing of the bursaphelenchus xylophilus and the vector insects thereof.
The existing research shows that the indoor bioassay method determines that the 5 percent abamectin emulsifiable solution has extremely strong poisoning property on the pine wood nematodes, and the application of the abamectin dry injection pesticide to prevent the pine wood nematode disease is an effective and simple method for controlling the pine wood nematode disease, and is an effective means for preventing pine trees with important values from infecting the pine wood nematode disease. However, abamectin is hydrophobic and susceptible to photolysis. Therefore, the technical difficulty at present is that the abamectin is prepared into which dosage form for preventing and controlling the pine wood nematode and the vector insects thereof. In order to solve the problems of avermectin hydrophobicity and photolysis, experts and scholars have prepared avermectin into a microcapsule form, but the microcapsule is easy to agglomerate, so that the application of the avermectin in prevention and treatment is restricted.
Based on the above, the invention provides a novel nano pesticide for preventing and controlling the pine wood nematodes and the vector insects thereof, which takes DMSNs as a carrier to load avermectin, can be effectively dispersed in water and can be transported in a trunk, thereby realizing stomach toxicity or contact killing of the pine wood nematodes and the vector insects thereof. The DMSNs used in the invention are mainly used as an adsorbent and a catalyst at present, and no research is related to the application of the DMSNs in the production of pesticide preparations, and no report is provided on how to control the pine wood nematodes and the vector insects thereof by loading pesticide avermectins on the DMSNs.
Due to the size, structure and charged property of the DMSNs, the drug loading rate, slow release effect and dispersion effect of the DMSNs are influenced. The invention optimizes the preparation method of the DMSNs, and the types and the mixture ratio of the reaction raw materials and the reaction solvent can influence the yield, the size, the structure and the charge of the prepared DMSNs, so the invention inspects the types and the dosage of the raw materials and the solvent for preparing the DMSNs, ensures that the yield of the DMSNs prepared by the invention is more than 98 percent, the size of the DMSNs is between 200 and 400nm, and the particle size is uniformly dispersed and stable; DMSNs are negatively charged, and each nano particle has the same charge repulsion phenomenon, so that the dispersibility of the DMSNs is good and the DMSNs do not have the agglomeration phenomenon. The Zeta potential can react and disperse, and the absolute value of the Zeta potential of the DMSNs prepared by the invention is more than 20mV, so that the DMSNs have better dispersion stability.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available.
Example 1 preparation of Avermectin-loaded NanoPesticide formulations
(1) Adding 60.7 mu L of triethanolamine into 25mL of purified water, and stirring in an oil bath kettle at 80 ℃ for 30min to obtain a triethanolamine solution;
(2) 380mg of hexadecyl trimethyl ammonium bromide and 168mg of sodium salicylate are dissolved in the triethanolamine solution prepared in the step (1), and the solution is stirred for 1 hour by oil bath at the temperature of 80 ℃;
(3) adding 4mL of tetraethyl orthosilicate (ortho) into the solution prepared in the step (2), and continuing stirring for 2h at 80 ℃; after the reaction is finished, centrifuging at 11000rpm for 10min to obtain a precipitate, washing by using ethanol to remove unreacted substances, centrifuging at 11000rpm for 10min again, and collecting the precipitate;
(4) 1.54mL of 36 wt% concentrated hydrochloric acid and 120mL of methanol were added to the precipitate obtained in step (3) and reacted three times at 60 ℃ for 6 hours each time, and centrifuged at 11000rpm for 10 minutes to remove cetyltrimethylammonium bromide. And 5mL of purified water is added into the precipitate, the mixture is frozen to-56 ℃, and then is frozen and dried for 48 hours at-56 ℃ to obtain the DMSNs, and the yield of the DMSNs is 95.0%.
The yield of DMSNs ═ (actual yield of DMSNs/theoretical calculated yield of DMSNs) × 100%.
(5) Dissolving the abamectin in methanol to obtain an abamectin solution with the concentration of 19 mg/mL.
(6) And (2) adding DMSNs into the abamectin solution with the volume ratio of 1:4, continuously stirring for 72h, centrifugally collecting precipitates, repeatedly washing with a methanol solution, and freeze-drying at-56 ℃ for 48h to obtain the novel pesticide with the DMSNs loaded with the abamectin.
The obtained novel pesticide has drug loading of 68.8%, average particle diameter of 317nm, and pore diameter of 22.3 nm.
Wherein the drug loading is the mass of the abamectin wrapped in the DMSNs/the total mass of the nano particles embedding the abamectin multiplied by 100 percent
The mass of the abamectin embedded in the DMSNs is measured by an ultraviolet spectrophotometer, then the concentration is calculated by using a standard curve, and the mass is converted by multiplying the concentration by the volume of the solution.
The scanning electron microscope and the transmission electron microscope of the DMSNs loaded with avermectins prepared in the embodiment are shown in fig. 1 and fig. 2, and the particle size distribution diagram of the DMSNs loaded with avermectins is shown in fig. 3.
Example 2:
(1) adding 60.7 μ L triethanolamine into 25mL purified water, and stirring in 80 deg.C water bath for 30 min;
(2) 380mg of hexadecyl trimethyl ammonium bromide and 168mg of sodium salicylate are dissolved in the water-triethanolamine solution prepared in the step (1), and water bath stirring at 80 ℃ is continued for 1 h;
(3) adding 4mL of tetraethyl orthosilicate into the solution prepared in the step (2), and continuously stirring in a water bath at 80 ℃ for 2 hours; after the reaction is finished, centrifuging to obtain a precipitate, and washing with ethanol to remove unreacted substances;
(4) adding 240 mu L of 36 wt% concentrated hydrochloric acid and 120mL of methanol into the precipitate obtained in the step (3), reacting for 6h and three times at the temperature of 60 ℃, centrifuging at 11000rpm for 10min to remove hexadecyl trimethyl ammonium bromide, adding 5mL of purified water into the precipitate, freezing to-56 ℃, and then freeze-drying at-56 ℃ for 48h to obtain the DMSNs, wherein the yield of the DMSNs is 98.0%.
(5) Dissolving the abamectin in methanol to obtain an abamectin solution with the concentration of 1 mg/mL.
(6) And (2) adding DMSNs into the abamectin solution with the volume ratio of 1:4, continuously stirring for 72h, centrifugally collecting precipitates, repeatedly washing with a methanol solution, and freeze-drying at-56 ℃ for 48h to obtain the novel pesticide with the DMSNs loaded with the abamectin.
The obtained novel pesticide has drug loading rate of 69.0%, average particle diameter of 319nm, and pore diameter of 19.1 nm.
Example 3
(1) Firstly, adding 25mL of purified water into a three-neck round-bottom flask, then placing a stirrer into an oil bath pot, installing a condensation reflux device above the stirrer, heating the oil bath pot to 80 ℃, taking 60.7 mu L of Triethanolamine (TEA) to drop into the three-neck flask, adjusting the rotating speed until a vortex appears, and stirring for 30 min; 380mg of hexadecyl trimethyl ammonium bromide (CTAB) and 168mg of sodium salicylate (NaSal) are added into the mixture, stirring is continuously carried out for reaction for 1 hour, 4mL of tetraethyl orthosilicate (TEOS) is added into the mixture, stirring is continuously carried out for reaction for 2 hours, then the solution is centrifuged for 10 minutes at the temperature of 4 ℃ and the speed of 11000rpm to obtain sediment, absolute ethyl alcohol is used for washing the sediment for 3 times to remove reaction substances, the sediment is centrifuged and collected under the same condition, the sediment is transferred into a beaker, a stirrer is added into the sediment, 120mL of methyl alcohol and 1.54mL of 36 wt% concentrated hydrochloric acid are added into the sediment for ultrasonic dispersion, the mixture is magnetically stirred and reacted for 6 hours under a water bath at the temperature of 60 ℃, the sediment is centrifuged and collected under the same condition to remove a template, 5mL of purified water is added into the obtained sediment, the obtained sediment is frozen by freezing and then placed into a freeze dryer for 48 hours, and fine white powder, namely DMSNs is obtained.
(2) Dissolving 5mg of FITC in 200mL of purified water, and performing ultrasonic dissolution to obtain a FITC solution; adding 5mg of DMSNs into 10mL of FITC solution, adding a stirrer, placing on a magnetic stirrer, stirring at 300rpm for 2h, and centrifuging at 4 ℃ and 11000rpm for 10min to obtain the DMSNs-FITC.
Because abamectin does not have fluorescence, DMSNs physically adsorb the abamectin and do not react, the application utilizes the fluorescent dye FITC to replace the abamectin to prepare the DMSNs-FITC, and the place with the fluorescence is the position which can be reached by the medicine. All fluorescence images in the present invention were processed with DMSNs-FITC.
The specific treatment method comprises the following steps:
1) the longicorn larvae were treated with DMSNs-FITC for 2min, then washed repeatedly, and the surface fluorescence distribution and the gas gate fluorescence distribution of the longicorn larvae were observed using a stereoscope (FIG. 8). The nano pesticide prepared by the invention has good adhesion and can quickly enter into the body of pests.
2) Intestinal tracts of DMSNs-FITC-treated Monochamus alternatus larvae were dissected out, fixed in 4% paraformaldehyde fixing solution for 24h, embedded in paraffin, sectioned into transverse sections of hindgut, and observed for fluorescence distribution under a fluorescence microscope (FIG. 9 abcd).
3) DMSNs-FITC incubates DF-1 cells for 4h, the cells were washed several times, unbound DMSNs-FITC was removed, and uptake of the cells by DMSNs-FITC was observed under a confocal laser microscope (FIG. 9 e).
Comparative example 1
(1) Adding 60.7 mu L of triethanolamine into 25mL of purified water, and stirring in an oil bath kettle at 80 ℃ for 30 min;
(2) dissolving 380mg of hexadecyl trimethyl ammonium bromide and 168mg of sodium salicylate in the water-hexadecyl trimethyl ammonium bromide-sodium salicylate-triethanolamine solution prepared in the step (1), and continuously stirring for 1h at 80 ℃;
(3) 4mL of tetraethyl (ortho) silicate and 1.6mL of 1, 2-bis (triethoxysilyl) were added to the solution prepared in step (2), and stirring was continued for 2h at 80 ℃; after the reaction is finished, centrifuging to obtain a precipitate, and washing with ethanol to remove unreacted substances;
(4) adding 240 mu L of 36 wt% concentrated hydrochloric acid and 120mL of methanol into the precipitate obtained in the step (3), and reacting for 6h three times at the temperature of 60 ℃ to remove the template to obtain DMSNs, wherein the yield of the DMSNs is 69.0%.
(5) Dissolving the abamectin in methanol to obtain an abamectin solution with the concentration of 1 mg/mL.
(6) And (3) adding DMSNs into the abamectin solution with the volume ratio of 1:4, continuing stirring for 48h, centrifuging, collecting precipitate, repeatedly washing with methanol solution, and freeze-drying to obtain the novel pesticide with DMSNs loaded with abamectin.
The obtained novel pesticide has drug loading of 37.3%, average particle diameter of 376nm, and pore diameter of 20.4 nm.
Comparative example 2
(1) DMSNs were prepared according to the method in "preparation of dendritic mesoporous silica nanoparticles and release performance test" (chemical technology and development, 9 months in 2017, vol 46, 9 th):
accurately transferring 7.5mL of Triethanolamine (TEA) into a beaker filled with 200mL of distilled water, adding 380mg of cetyltrimethylammonium bromide (CTAB), adding a certain amount of sodium trifluoroacetate (FC2), covering the upper part of the beaker with a glass sheet to isolate air, placing the beaker in a constant-temperature water bath at 80 ℃, magnetically stirring for 1h, adding a certain amount of Tetraethoxysilane (TEOS), and continuously stirring for 2h to obtain a milky suspension. After the reaction was completed, the solution was centrifuged to obtain a white solid. And (3) putting the white solid into a muffle furnace, roasting for 6h at 600 ℃, and removing the surfactant to obtain the DMSNs.
(2) Dissolving the abamectin in methanol to obtain an abamectin solution with the concentration of 19 mg/mL.
And (3) adding DMSNs into the abamectin solution with stirring, wherein the volume ratio of the abamectin solution to the DMSNs is 1:4, continuing stirring for 72 hours, centrifugally collecting precipitates, and washing with a methanol solution to obtain the novel pesticide of DMSNs loaded with abamectin.
The obtained novel pesticide has a drug loading of 25.1%, an average particle size of 310nm and a pore diameter of 14 nm.
The DMSNs prepared in the comparative example 2 has high requirements on equipment, consumes a large amount of energy, has high cost and great difficulty in temperature control, so that the yield of the product is indefinite, and the particle size is difficult to be uniform and stable due to the fact that the product cannot be stirred at high temperature.
Test example therefore the drug loading was low
1. Dispersibility test
The nano-pesticide prepared in comparative examples 1-2 and the nano-pesticide prepared in example 2 were placed in water, and the distribution in water is shown in fig. 3. As can be seen from FIG. 3, the nano-pesticide prepared by the method of the present invention has good dispersibility in water.
2. Transmission rate test
The difference between example 2 and comparative example 1 is that a solvent is added in comparative example 1, and in order to examine the effect of DMSNs prepared by using different solvents on the delivery rate of the nano-pesticide loaded with abamectin, the nano-pesticides prepared in example 2 and comparative example 1 were respectively prepared by using water according to the concentration of the abamectin of 1mg/L, 10mg/L and 50 mg/L. Then, the injection was carried out on six pine trunks of similar diameter and same health (the injection depth was the same and the injections were all made on the top layer of layer1 in fig. 10).
Note: since the nano-sized pesticides prepared in example 2 and comparative example 1 were avermectins loaded on the nano-sized carrier, the concentration of the nano-sized carrier was calculated from the concentration of avermectins at the time of injection. And configuring the corresponding solution to be injected into the trunk according to the calculated concentration of the nano-carriers. The concentration of the nano carrier is the concentration of the avermectin/the drug-loading rate of the nano carrier.
The trunk section is divided into four layers from outside to inside according to fig. 10 (i.e. Layer 1-Layer 4), and the length of each Layer in 6 pine trees is the same. After 24h, at the injection site, the cuttings were drilled according to the depths of the four layers, and the abamectin concentration in the cuttings was measured by using a high performance liquid chromatograph, and the obtained result is shown in fig. 11. As can be seen from fig. 11, at the concentration of abamectin of 50mg/L, the nano-pesticide prepared in comparative example 1 appeared in layer 2, while the nano-pesticide prepared in example 2 appeared in layer 4. The nano pesticide prepared by the invention has higher transmission speed and higher transmission efficiency in the trunk.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
- Use of DMSNs as a vector in any one of the following 1) to 4);1) loading abamectin;2) loading fluorescein isothiocyanate;3) preparing a nano pesticide;4) preparing a fluorescent marker.
- 2. The use according to claim 1, wherein the DMSNs are prepared by a process comprising:(1) heating purified water in an oil bath, dropwise adding triethanolamine and stirring for 30min, then adding hexadecyl trimethyl ammonium bromide and sodium salicylate, continuously stirring for reaction for 1h, then adding tetraethyl orthosilicate, and continuously stirring for reaction for 2h to obtain a reaction solution;(2) and (2) centrifuging the reaction solution obtained in the step (1) for the first time at 4 ℃ to obtain a precipitate, washing the precipitate, centrifuging for the second time, adding methanol and concentrated hydrochloric acid into the precipitate, performing ultrasonic dispersion, stirring and reacting for three times in a water bath, reacting for 6 hours each time, centrifuging for the third time, collecting the precipitate, adding the obtained precipitate into purified water, freezing, and freeze-drying to obtain fine white powder, namely the DMSNs.
- 3. Use according to claim 1, characterized in that in step (1), the temperature at which the oil bath is heated is 80 ℃; the ratio of the added amounts of the purified water, the triethanol, the hexadecyl trimethyl ammonium bromide, the sodium salicylate and the tetraethyl orthosilicate is 25 mL: 60.7 μ L: 380 mg: 168 mg: 4 mL.
- 4. The use according to claim 2, wherein in step (2), the volume ratio of methanol to concentrated hydrochloric acid is 120: (0.24 to 1.54); the temperature of the water bath is 60 ℃; the speed of the first centrifugation, the second centrifugation and the third centrifugation is 11000rpm, and the centrifugation time is 10 min; the freeze-drying temperature is-56 ℃ and the freeze-drying time is 48 h.
- 5. The use according to claim 2, wherein the DMSNs have a size of 200-400 nm.
- 6. A fluorescent marker is characterized in that the preparation method of the fluorescent marker comprises the following steps: dissolving FITC in purified water to obtain a FITC solution; adding the nano-carrier DMSNs as claimed in any one of claims 1 to 5 into a FITC solution, stirring for 2 hours, and centrifuging at 4 ℃ to obtain DMSNs-FITC, namely the fluorescent marker.
- 7. The fluorescent marker of claim 6, wherein the concentration of the FITC solution is 0.025 mg/mL; the volume ratio of DMSNs to FITC solution is 1: 2.
- 8. The nano pesticide preparation loaded with the abamectin is characterized by being prepared by the following method:dissolving abamectin in methanol to obtain an abamectin solution; and dispersing the nano-carrier DMSNs of any one of claims 1 to 5 into an abamectin solution, stirring for 24 to 72 hours, centrifuging, collecting the precipitate, and drying to obtain the nano-pesticide preparation.
- 9. The nano pesticide preparation loaded with abamectin of claim 8, wherein the concentration of the abamectin solution is (1-19) mg/mL; the volume ratio of the abamectin solution to the DMSNs is 1: 4.
- 10. Use of the avermectin-loaded nano pesticide formulation of claim 8 or 9 for controlling pine wood nematodes and their vector insects.
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