Insecticide scavenger as well as preparation method and application thereof

Abstract

The invention discloses an insecticide scavenger as well as a preparation method and application thereof, the scavenger MTSM is prepared by continuously modifying polyphenol tannic acid and locust cell membranes on mesoporous silicon, the preparation method is simple and efficient, and complex process conditions and procedures are not needed. The scavenging agent particles have a dual-mode scavenging function, and by means of pi-pi accumulation of the insecticide and TA and specific binding with acetylcholin esterase, nicotinic acetylcholine receptors or voltage-gated sodium channels on locust cell membranes, the removal effect on various insecticides such as organic phosphorus, pyrethroid and neonicotine is remarkably improved. Compared with particles which are not modified by locust cell membranes, the MTSM has the advantages that the non-specific removal of digestive enzymes in gastrointestinal tracts is obviously reduced, and the gastrointestinal functions of pollinating insects are favorably maintained. The scavenger disclosed by the invention is high in safety and convenient to prepare, can efficiently remove different insecticides in a broad-spectrum manner, has relatively large transformation potential in the aspect of detoxification of pollinating insects, and is expected to assist sustainable agricultural development.

Description

Technical Field

[0001] The present invention relates to a preparation method and application of an insecticide scavenger, and specifically to a preparation method of mesoporous silicon microparticles modified by natural molecule tannic acid and locust cell membrane, and application of the microparticles in the field of broad-spectrum insecticide removal. Background Art

[0002] Pesticides are an indispensable means of production in modern agricultural production. They are used in large quantities around the world every year, but the actual effective utilization rate is very low. About 60-70% of pesticides remain in the environment (Xiang QS CriticalReviews in Food Science and Nutrition 2023, 63, 4431-4449). Today, the global pesticide market is diversified, mainly including neonicotinoids, pyrethroids and organophosphorus insecticides. Compared with carbamates and organochlorine insecticides, these insecticides require smaller doses during use but are more toxic. If used improperly, they will cause more serious environmental problems (Eng ML Science 2019, 365, 1177-1180). Organophosphorus insecticides inhibit the activity of acetylcholinesterase (AChE), causing acetylcholine to accumulate in bees, affecting the conduction of bee nerve signals. Neonicotinoids can selectively bind to nicotinic acetylcholine receptors, while pyrethroids can specifically bind to voltage-sensitive sodium channels, and their binding to insect receptors is much stronger than that to mammalian nerve receptors (Ihara M. Current Opinion in Insect Science 2018, 30, 86-92). More seriously, due to their wide targeting and neurotoxicity, the pesticides remain in the environment, directly leading to a sudden decrease or even extinction of various pollinating insects, especially bees, posing a great threat to the ecological balance (Siviter H. Nature 2021, 596, 389-392.). The methods currently developed to remove pesticides through physical and chemical oxidation technologies require a large amount of chemical reagents to achieve a strong oxidizing environment, which not only poses a safety hazard, but also is difficult to implement in agricultural production due to high costs and complex operating processes. In addition, the use of pesticides in modern agricultural production tends to be a mixed application of multiple types of pesticides to achieve better pest control effects. This is a huge challenge to the removal of pesticides. Therefore, it is necessary to develop an efficient, low-cost, broad-spectrum removal method to cater to the current trend of pesticide use. Summary of the invention

[0003] The purpose of the present invention is to provide a method for preparing an efficient and broad-spectrum insecticide remover, such as a bee, and the remover prepared by the method has a good application prospect in the field of agricultural insecticide management. The present invention uses a micro-colony breeding cage of a bumblebee living environment, and simulates insecticide poisoning and detoxification of bumblebees in nature by contaminating pollen grains with insecticides. The remover can effectively remove organophosphorus insecticides, neonicotinoid insecticides, and pyrethroid insecticides from bumblebees.

[0004] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is: A pesticide remover comprises cell membrane modified mesoporous silicon; the mesoporous silicon is tannic acid modified mesoporous silicon.

[0005] The invention discloses a preparation method of the cell membrane modified mesoporous silica, comprising the following steps: mixing tannic acid modified mesoporous silica with a cell membrane suspension to obtain the cell membrane modified mesoporous silica.

[0006] The present invention uses the cell membrane modified mesoporous silica as a pesticide scavenger, or the cell membrane modified mesoporous silica as an active ingredient and a conventional carrier to form a pesticide scavenger.

[0007] In the present invention, amino-modified mesoporous silicon particles and tannic acid are dispersed in a buffer solution to obtain a mixed suspension, and then reacted to obtain tannic acid-modified mesoporous silicon. Preferably, the mass ratio of amino-modified mesoporous silicon particles to tannic acid is (1-10):1, and more preferably, the mass ratio of amino-modified mesoporous silicon particles to tannic acid is (4-6):1. Preferably, the reaction is room temperature reaction for 10-15 hours. More preferably, the amino-modified mesoporous silicon particles are linear amino-modified mesoporous silicon particles.

[0008] Preferably, the tannic acid modified mesoporous silica is washed and then mixed with a cell membrane suspension to obtain cell membrane modified mesoporous silica. Further preferably, the tannic acid modified mesoporous silica is washed and then mixed with a cell membrane suspension, and subjected to low temperature ultrasound to obtain cell membrane modified mesoporous silica.

[0009] In the present invention, the buffer solution includes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid hemisodium salt (HEPES) buffer solution, tris(hydroxymethylaminomethane) (Tris) buffer solution, etc. During cleaning, the cleaning solution is phosphate-buffered saline (PBS) buffer solution.

[0010] In the present invention, the mesoporous silica is mesoporous silica particles.

[0011] In the present invention, the cell membrane is a locust cell membrane, which is obtained by freezing, grinding, crushing and separating living locusts.

[0012] In the present invention, the mass ratio of tannic acid modified mesoporous silica to cell membrane is 10: (0.05-0.25), preferably, the mass ratio of tannic acid modified mesoporous silica to cell membrane is 10: (0.1-0.15), wherein the mass of the cell membrane is calculated based on the protein on the cell membrane.

[0013] In the present invention, the low temperature is an ice water bath; the ultrasonic time is 10 to 20 minutes, and the power is 50 to 150W.

[0014] The invention discloses the application of the cell membrane modified mesoporous silica as or in the preparation of an insecticide scavenger.

[0015] The invention discloses the application of the insecticide remover in removing insecticides or in preparing an insecticide removing agent.

[0016] The invention discloses the use of the above-mentioned insecticide scavenger in reducing the toxicity of insecticides to insects, or in preparing an agent for reducing the toxicity of insecticides to insects; preferably, the insect is a pollinating insect.

[0017] The invention discloses a method for removing pesticides, comprising the following steps: removing the pesticides by using the pesticide remover.

[0018] In the present invention, the insecticide includes one or more of carbamate insecticides, organochlorine insecticides, neonicotinoid insecticides, pyrethroid insecticides, and organophosphorus insecticides.

[0019] In the present invention, the removal of insecticides can be achieved in vivo or in vitro. In particular, the present invention provides a highly efficient and broad-spectrum insecticide remover for removing insecticides from bees, which has a good application prospect in the field of agricultural insecticide management.

[0020] The present invention further discloses the use of the above-mentioned insecticide scavenger in removing organophosphorus insecticides from bumblebees; the use of the above-mentioned insecticide scavenger in removing neonicotinoid insecticides from bumblebees; and the use of the above-mentioned insecticide scavenger in removing pyrethroid insecticides.

[0021] The microparticles for removing pesticides from bumblebees disclosed in the present invention have excellent biosafety and will not cause health problems to bumblebees after feeding; the present invention also discloses a micro-colony that simulates the living environment of bumblebees. In the simulated living micro-environment of bumblebees, after bumblebees accidentally eat pollen grains contaminated by pesticides, under the detoxification action of the scavenger, the detoxification effect is concentration-dependent, which can improve the survival rate of bumblebees in the pesticide-contaminated environment.

[0022] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: 1. The preparation method of the scavenger particles disclosed in the present invention is simple and efficient, and does not require complicated process conditions and procedures; after modification, the mesoporous structure with high surface area and high pore volume is still retained; in particular, the constituent materials used in the present invention are low-cost and widely exist in nature, and have good biosafety. Feeding bumblebees will not cause health and safety problems.

[0023] 2. The scavenger particles disclosed in the present invention have a dual-mode scavenging function. On the one hand, the tannic acid molecules can bind to the insecticide through π-π stacking, and on the other hand, the relevant receptors on the locust cell membrane can specifically bind to the insecticide, thereby achieving efficient scavenging of the insecticide.

[0024] 3. The scavenger particles prepared by the present invention have the ability to remove pesticides with broad spectrum and high efficiency, and can be used in environmental management after the application of mixed pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the infrared spectra of mesoporous silicon, tannic acid, and tannic acid-modified mesoporous silicon particles in Example 1.

[0026] Figure 2 This is a characterization diagram of the mass fraction of TA in TSM at different TA / SM ratios in Example 1.

[0027] Figure 3 This is a confocal image of the DiD-labeled MTSM microparticles in Example 3.

[0028] Figure 4 This is a characterization of the number (A) and activity (B) of receptors on the surface of locust cell membrane modified microparticles in Example 3.

[0029] Figure 5 This is the effect of the composition of the particles on the clearance effect in Example 4, where A is an unmodified cell membrane and B is a modified cell membrane.

[0030] Figure 6 1 is a characterization diagram of the removal effect of the microparticles on the organophosphorus insecticide in Example 4, wherein A is the influence of the microparticle concentration, B is the influence of the TZP concentration, C is the influence of the mixing time, and D is a characterization diagram of the removal efficiency of the microparticles on different organophosphorus insecticides.

[0031] Figure 7 : This is a characterization diagram of the removal effect of the microparticles on neonicotinoid insecticides in Example 5, wherein A is the effect of the microparticle concentration, B is the effect of the IMI concentration, C is the effect of the mixing time, D is the effect of the pH environment, and E is a characterization diagram of the removal effect of the microparticles on different neonicotinoid insecticides.

[0032] Figure 8This is a characterization diagram of the removal effect of the microparticles on pyrethroid insecticides in Example 5, wherein A is the effect of the microparticle concentration, B is the effect of the IMI concentration, and C is the effect of the mixing time.

[0033] Fig. 9 3 is a characterization diagram of the inhibitory effect of the microparticles on nonspecific adsorption in Example 6, wherein A is a schematic diagram of the inhibitory effect of the microparticles on nonspecific adsorption, B, C, and D are characterization diagrams of the inhibitory effect of the microparticles on the nonspecific adsorption of gastrointestinal digestive enzymes of pollinating insects, and E is a characterization diagram of the selective removal of TZP in the microparticle GI enzyme environment.

[0034] Fig.10 1 is the distribution diagram of FITC-labeled MTSM in the gastrointestinal tract of honey bees tracked by fluorescence imaging at different times in Example 7, wherein A is the distribution diagram of FITC-labeled MTSM particles in the gastrointestinal tract at 0 hours after eating, B is at 4 hours, C is at 12 hours, and D is at 48 hours.

[0035] Fig.11 It is a diagram showing the effect of organophosphorus insecticide removal in microparticles in Example 8, wherein A is a diagram showing the biosafety of organophosphorus insecticide removal in microparticles, B is a diagram showing the toxicity of TZP, C is a diagram showing the effect of organophosphorus insecticide removal in microparticles at sublethal concentrations of TZP, and D is a diagram showing the effect of organophosphorus insecticide removal in microparticles at acute poisoning concentrations of TZP.

[0036] Fig.12 This is a diagram showing the effect of removing neonicotinoid insecticides from microsomes in Example 8, wherein A is at a sublethal concentration of IMI, and B is a diagram showing the effect of removing organophosphorus insecticides from microsomes at an acute poisoning concentration of IMI. DETAILED DESCRIPTION

[0037] The present invention specifically discloses a preparation method of a locust cell membrane modified mesoporous silicon microparticle insecticide scavenger, comprising the following steps: dispersing amino-modified mesoporous silicon microparticles and natural molecule tannic acid in a buffer to obtain a mixed suspension; then reacting at room temperature for 12 hours; then washing the microparticle suspension and adding it to a locust cell membrane suspension; and then performing low-temperature ultrasound to obtain locust cell membrane modified mesoporous silicon microparticles.

[0038] In the above technical solution, the mass ratio of amino-modified mesoporous silica particles to tannic acid is 5:1.

[0039] In the above technical solution, the buffer solution includes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid hemisodium salt (HEPES) buffer solution, tris(hydroxymethylaminomethane) (Tris) buffer solution, etc.; and washing is performed with phosphate-buffered saline (PBS) buffer solution.

[0040] In the above technical solution, the locust cell membrane suspension is obtained by freezing, grinding, crushing and separating living locusts.

[0041] In the above technical solution, the particle concentration in the particle suspension after washing is 20 mg / mL; in the locust cell membrane suspension, the concentration of locust cell membrane is quantified based on the total concentration of protein on the cell membrane, which is 0.25 mg / mL.

[0042] In the above technical solution, low-temperature ultrasound is mixed ultrasound in an ice water bath, the time is 15 min, and the power is 100 W.

[0043] In the present invention, linear amino-modified mesoporous silicon (mesoporous silica) is used as a substrate, and tannic acid with a polyphenol structure is oxidized to quinone under normal oxygen, and then reacts with amino groups to be attached to mesoporous silicon; as an example, the nitrogen content in mesoporous silicon is 1.3%, and the content of amino groups per 100 mg of mesoporous silicon is 0.0929 mmol / L. The modified content of tannic acid in mesoporous silicon can be changed by adjusting the reaction feed ratio. When the mass ratio of mesoporous silicon particles to tannic acid is 5:1, the mass fraction of tannic acid in the obtained particles is 12.4%.

[0044] In the present invention, the concentration of total protein on locust cell membrane is used as a standard to measure the number of cell membranes; the content of cell membrane modified on the microparticles can be changed by adjusting the feed ratio of microparticles to cell membranes during ultrasound; in the preferred technical solution, the concentration of total cell membrane protein added is 0.25 mg / mL.

[0045] The invention discloses locust cell membrane modified mesoporous silicon particles prepared according to the method, which are used as insecticide scavengers.

[0046] The present invention is further described below in conjunction with the accompanying drawings and examples; the raw materials used are all existing products, and the specific preparation operations and performance tests are all conventional techniques. Amino-modified mesoporous silica particles were purchased from UniSil-NH2 normal phase silica gel chromatography filler of Suzhou Nanotech Co., Ltd.

[0047] Example 1 Preparation and Characterization of Tannic Acid Modified Mesoporous Silica Microparticles (TSM) In air environment, amino-modified mesoporous silica (100 mg, 0.10 mmol NH2, SM) was dispersed in HEPES (20 mL, 5 mM, pH=7.4) solution, and then tannic acid (20 mg, 0.02 mmol, TA) was added and mixed, and reacted at room temperature for 12 h; after the end, the obtained product was centrifuged (6000 rpm, 5 min) and washed three times with phosphate buffer (PBS, 10 mM, pH= 7.4) to obtain a crude product suspension; sodium borohydride (NaBH4, 0.20 mmol) was mixed with the crude product suspension at room temperature for 4 h, and then centrifuged and washed three times; the obtained product was placed in a vacuum constant temperature drying oven at 35 ℃ and dried for 1 d to obtain the final product. Yield: 95.6%. By comparing the infrared spectra of SM, TA, and TSM, it can be clearly seen that after the introduction of tannic acid, TSM has a wavelength of 1717 cm -1 The characteristic peak caused by the stretching vibration of carbonyl (C=O) appeared at 1623 cm -1 and 1520 cm -1 The characteristic peak at 3460 cm -1 and 1445 cm -1 The characteristic peaks at the bottom are caused by the stretching vibration of the hydroxyl group (-OH) and the aromatic ring CC ( Figure 1 ). The above characteristic peaks come from TA, indicating that TA was successfully introduced into SM and TSM particles were prepared.

[0048] Similarly, a series of different TSMs were prepared by adjusting the TA / SM mass ratio (0.01, 0.10, 0.50, 1.0). The TA content in TSM particles can be determined by thermogravimetric analysis ( Figure 2 ), the results show that when the mass ratio of TA / SM increases from 0.01 to 0.2, the mass fraction of TA in TSM increases from 0.76 wt.% to 11.4 wt.%; when the mass ratio of TA / SM is higher than 0.2, the mass fractions of TA are 11.8 wt.% and 12.0 wt.%, respectively, which is obviously close to the maximum mass fraction of TA in TSM. Unless otherwise specified, TSM particles prepared under the condition of TA / SM mass ratio of 0.2 were selected for subsequent experiments.

[0049] Example 2 Extraction of locust cell membrane The living locusts were frozen in liquid nitrogen and then ground in a mortar to crush the locust tissue. The locust tissue suspension was then filtered through a 70 μm cell filter to obtain a cell suspension. The resulting cell suspension was then centrifuged at 4 °C (2000 rpm, 5 min), resuspended in PBS (10 mM, pH = 7.4) buffer, and counted using a cell counter. According to the counting results, the membrane and cytoplasm protein extraction kit was used to extract the locust cell membrane: first, membrane protein extraction solution (1.0 mL / 20 million locust cells) and protease inhibitor (PMSF, 1.0 mM) were added to the locust cells in sequence, and then the mixture was mixed in an ice water bath for 30 min. After the end, the mixture was frozen in liquid nitrogen and thawed at room temperature. The above freeze-thaw process was repeated, and the freezing and thawing of locust cells was observed under an optical microscope; when about 80% of the locust cells in the field of view were broken, the freezing and thawing was stopped, and the mixture was centrifuged (1000g, 10 min). After the centrifugation, the cell contents at the bottom were removed, and the supernatant was centrifuged again (12000g, 30 min), the cell membrane fragments at the bottom were collected, and the protein content on the membrane fragments was determined using the Pierce BCA protein assay kit, and then placed in a -80 ℃ refrigerator for storage and diluted to 0.25 mg / mL when used.

[0050] Example 3 Preparation and Characterization of Locust Cell Membrane Modified Microparticles (MTSM) The prepared TSM (10 mg / mL) and locust cell membrane suspension (0.5 mL, 0.25 mg / mL (membrane protein concentration)) were mixed in an ice water bath and sonicated (100 W, 15 min). The mixture was then centrifuged (6000 rpm, 5 min, 4°C) and the supernatant was removed to obtain MTSM.

[0051] In order to observe the cell membrane modification in the microparticles, 0.5 wt.% DiD was added to the locust cell membrane solution, and then DiD was coated on the microparticles by ultrasound to prepare DiD-stained MTSM (DiD-MTSM), which was finally observed and photographed by CLSM.

[0052] MTSM microparticles can be easily prepared by mixing locust cell membranes and TSM microparticles at low temperature and ultrasonication. Cell membrane far-infrared fluorescent probe (DiD) is a common cell membrane fluorescent dye that binds to the cell membrane and emits far-infrared fluorescence after excitation. DiD-labeled MTSM (DiD-MTSM) was prepared here. Red halos can be observed around DiD-MTSM microparticles using a confocal fluorescence microscope ( Figure 3 ), which indicates that the locust cell membrane was successfully coated onto the particle surface.

[0053] When preparing locust cell membrane modified microparticles, the feed ratio of TSM and locust cell membrane can be controlled to prepare locust cell membrane modified microparticles with different acetylcholinesterase (AChE) contents. According to the insect acetylcholinesterase ELISA kit, the membrane solution to be tested is first added to the multi-well plate, incubated at 37 °C for 30 min, then the detergent is added, and the developer is added after repeated washing for 5 times. After the color development is completed, the absorbance at 450 nm is detected using a multifunctional microplate reader. When feeding, the total protein concentration on the cell membrane is used as the standard for cell membrane content. The results showed that when the cell membrane protein concentration reached 0.25 mg / mL, the AChE content on the prepared locust cell membrane modified microparticles was 0.29 μg / mg. When the cell membrane protein concentration continued to increase to 1.0 mg / mL, the AChE content only increased by 0.02 μg / mg, and when the cell membrane protein concentration was 0.1 mg / mL, the AChE content was only 0.14 μg / mg ( Figure 4 A).

[0054] In addition, the effect of the preparation process on the activity of AChE on the cell membrane was studied. First, potassium chloride (KCl, 20mM), thioacetylcholine (ACh, 2.0 mM), and 5,5'-dithio-2-nitrobenzoic acid (DTNB, 2.0 mM) were added to tris-HCl (Tris-HCl, 100 mM) buffer to prepare the working solution required for the activity test. Then the working solution (100 μL) and an equal volume of cell membrane solution were added to a 96-well plate, and a multifunctional microplate reader was used to record the ultraviolet absorption value at 412 nm once a minute for 20 min to determine the activity of AChE. The results showed that there was no statistical difference in the change of acetylcholinesterase activity before and after preparation ( Figure 4 B). Comparison Example

[0055] Amino-modified mesoporous silica particles SM (10 mg / mL) and locust cell membrane suspension (0.5 mL, 0.25 mg / mL) were mixed and sonicated (100 W, 15 min) in an ice water bath. The mixture was then centrifuged (6000 rpm, 5 min, 4°C) and the supernatant was removed to obtain locust cell membrane-modified SM (MSM).

[0056] Example 4 Study on the removal of organophosphorus pesticides by MTSM in vitro First, KH2PO4 (6.8 g, 50 mM) and NaOH (0.9 g, 22.5 mM) were dissolved in 1.0 L of distilled water to prepare simulated intestinal fluid (SIF, pH = 6.5). Taking triazophos (TZP) as an example, MTSM (20 mg / mL) and TZP (50 μg / mL) were added to SIF and incubated in a 37°C constant temperature shaker (100 rpm, 60 min) to simulate the MTSM removal environment. After mixing, centrifugation was performed at room temperature (6000 rpm, 5 min) and the supernatant was collected. Finally, the concentration of residual TZP in the supernatant was determined by HPLC. MTSM was replaced with other microparticles as a control group.

[0057] The results are as follows Figure 5 As shown in A (unmodified cell membrane), SM itself does not have a strong scavenging ability for TZP, but the scavenging efficiency is improved with the introduction of TA. When the TA / SM mass ratio reaches 0.1, the scavenging efficiency of TSM for TZP can reach 50.5%. Continuing to increase the TA / SM mass ratio to the range of 0.2-1.0, the scavenging efficiency increased from 76.4% to 78.2%. Unless otherwise specified, TSM particles with a TA / SM mass ratio of 0.2 were selected to explore the effect of locust cell membrane modification on TZP clearance.

[0058] Using the total protein concentration of locust cell membrane as a standard, locust cell membrane modified microparticles with different locust cell membrane modification contents were prepared. The clearance results are shown in Figure 5 As shown in Figure B, the locust cell membrane modification content can only improve the TZP removal efficiency of locust cell membrane modified particles within a certain range. When the total locust cell membrane protein concentration reaches 0.25 mg / mL, the removal efficiency can reach 83.1%, and when the locust cell membrane protein concentration is further increased to 1.0 mg / mL, the TZP removal efficiency is increased to 84.9%.

[0059] MTSM was constructed with a TA / SM mass ratio of 0.2 and a locust cell membrane protein concentration of 0.25 mg / mL, and the effects of clearance time, particles, and insecticide concentration on the clearance results of MTSM were systematically evaluated. For better comparison, locust cell membrane-modified SM (MSM) and TSM were used as control groups.

[0060] First, the effect of particle concentration on TZP clearance was investigated ( Figure 6A), the TZP concentration in the scavenging system was 50 μg / mL, and the scavenging time was 1 h. MTSM was concentration-dependent in the scavenging of TZP. When the MTSM concentration was 2.0 mg / mL, the scavenging efficiency was 51.6%. As the MTSM concentration continued to increase, the TZP scavenging efficiency continued to increase. When the MTSM concentration reached 40 mg / mL, the scavenging efficiency reached 87.1%. In the control group, the scavenging efficiency of TSM was lower than that of MTSM but was also concentration-dependent. When the TSM particle concentration was 40 mg / mL, the scavenging efficiency was 79.5%. In contrast, the scavenging efficiency of MSM was not ideal, with a maximum of 22.5%.

[0061] We then investigated the effect of pesticide concentration on TZP removal ( Figure 6 B), the particle concentration in the removal system was 20 mg / mL, and the removal time was 1 h. The results showed that with the increase of TZP concentration, the removal efficiency of TZP decreased, but the amount of TZP removed increased. When the TZP concentration was 0.01 mg / mL, the removal efficiency of MTSM for TZP was 82.3%. As the TZP concentration increased to 0.50 mg / mL, the removal efficiency of MTSM for TZP was still as high as 63.3%. This shows that MTSM particles have a high absorption capacity and can handle the removal of pesticides at various concentrations. In the control group, the removal efficiency of the MSM and TSM groups was lower than that of the MTSM group, and under the condition of higher TZP concentration (0.5 mg / mL), the removal efficiency dropped significantly to 2.8% and 44.1%, respectively.

[0062] Finally, the effect of clearance time on TZP clearance was investigated, and the concentrations of particles and TZP in the clearance system were 20 mg / mL and 50 μg / mL, respectively. Figure 6 As shown in Figure C, in the first 5 minutes, the removal efficiency of TSM and MTSM for TZP can reach 53.0% and 57.3%, respectively. This shows that TSM and MTSM particles have a very high removal rate. As time goes on, the removal efficiency of TSM and MTSM gradually increases. When the time is extended to 240 minutes, the removal efficiency of TSM for TZP reaches a maximum of 79.4%. The removal efficiency of MTSM continues to increase with the extension of time, until 720 minutes, the removal efficiency can reach 95.0%. This shows that MTSM has both a faster removal rate in a short period of time and the ability to clear for a long time. The control group MSM has reached the maximum removal efficiency of about 17.2% when the time is extended to 60 minutes.

[0063] Then, two common organophosphorus insecticides, MPT (methyl parathion) and CP (chlorpyrifos), were selected to further evaluate the removal ability of the microparticles against organophosphorus insecticides. The results showed that both TSM and MTSM can effectively remove a variety of organophosphorus insecticides ( Figure 6 D). Compared with MSM and TSM, MTSM has higher removal efficiency for MPT and CP, which are 80.8% and 77.3%, respectively.

[0064] Example 5 Study on MTSM in vitro broad-spectrum insecticide removal The effect of particle concentration on the removal of IMI (imidacloprid) was investigated. The IMI concentration in the removal system was 50 μg / mL, the removal time was 1 h, and the pH of the removal environment was 6.5. These conditions were used in the following sections unless otherwise specified. The results showed that ( Figure 7 A) The clearance of IMI by MTSM is concentration-dependent. When the concentration of MTSM is 5.0 mg / mL, the clearance efficiency is 37.4%. With the continuous increase of MTSM concentration, the clearance efficiency of TZP increases continuously. When the concentration of MTSM reaches 40 mg / mL, the clearance efficiency reaches 63.1%. In the control group, the clearance efficiency of TSM is lower than that of MTSM. When the concentration of TSM particles is 40 mg / mL, the clearance efficiency is 42.5%. The clearance efficiency of MSM is not ideal, not exceeding 20%.

[0065] Figure 7 B explored the effect of insecticide concentration on IMI removal, showing that MTSM still maintained a high removal efficiency for higher concentrations of IMI, with a particle concentration of 20 mg / mL. When the IMI concentration was 0.01 mg / mL, the removal efficiency reached 61.5%, and as the IMI concentration increased to 0.5 mg / mL, the removal efficiency of MTSM was 42.1%, indicating that MTSM has a high absorption capacity and can cope with the removal of high concentrations of insecticides.

[0066] The effects of different clearance times on IMI clearance were investigated, with a particle concentration of 20 mg / mL and an IMI concentration of 50 μg / mL. Figure 7 As shown in Figure C, the clearance efficiency of MTSM can reach 60.7% within 1 hour. As time goes by, the clearance efficiency will increase and reach 73.7% within 48 hours. However, the clearance efficiency of TSM and MSM in the control group did not change much with the extension of clearance time and was much lower than that of the MTSM group. Therefore, MTSM is more efficient in clearing IMI.

[0067] According to the pH environment of the bee digestive tract, the effect of pH on the removal of IMI by MTSM was investigated. SIF simulated solutions with pH values ​​of 4.8 and 6.5 were prepared according to conventional methods. The concentrations of particles and IMI in the removal system were 20 mg / mL and 50 μg / mL, respectively, and the removal time was 1 h. The removal efficiency of MTSM in the environments of pH 4.8 and 6.5 was more than 60%, and there was no statistical difference ( Figure 7D). Therefore, MTSM can fully adapt to the pH environment of the bee's gastrointestinal tract to complete the clearance work.

[0068] Afterwards, the in vitro clearance ability of MTSM on ACM (acetamiprid) and TMX (thiamethoxam) was investigated. The clearance results showed that MTSM had a certain clearance ability on a variety of neonicotinoid insecticides, and compared with MSM and TSM, MTSM's clearance efficiency was more efficient.

[0069] Cyfluthrin (CFT) is a pyrethroid insecticide widely used in agricultural production. Taking it as an example, the removal of pyrethroid insecticides by MTSM was explored. Similar to IMI, the removal ability of MTSM on CFT was evaluated by conditions such as removal time, particles and CFT concentration. The removal of CFT (50 μg / mL) by MTSM is also concentration-dependent. When the MTSM concentration increases from 5.0 mg / mL to 40 mg / mL, the removal efficiency increases from 22.4% to 53.1% ( Figure 8 A, the clearance time is 1 h). Moreover, the clearance efficiency of MTSM (20 mg / mL) for various concentrations of CFT remained at a high level. When the CFT concentration reached 0.5 mg / mL, the clearance efficiency of MTSM could still reach 42.4% ( Figure 8 B, the clearance time is 1 h). The clearance time also affects the clearance of CFT to a certain extent, and the clearance efficiency can reach 63.1% within 48 h.

[0070] Example 6 Study on the anti-nonspecific adsorption of MTSM in vitro The inhibitory effect of MTSM on nonspecific adsorption was evaluated using sucrase, protease and amylase, which are commonly found in the gastrointestinal digestive system of honey bees. Fig. 9 A). Specifically, MTSM solution (20 mg / mL) and different enzymes (1-100 μg / mL) were mixed at 37°C for 1 h, then centrifuged at 6000 rpm for 6 min, and the enzyme content in the supernatant was determined using the corresponding enzyme kit. An ideal insecticide scavenger should not only have a strong scavenging ability, but also minimize the impact on the gastrointestinal system of pollinating insects. Sucrase, amylase and protease are important digestive enzymes for pollinating insects, and they are widely present in the gastrointestinal system of pollinating insects. Fig. 9 B. Fig. 9 C and Fig. 9D. TSM has a high absorption of sucrase, protease and amylase. When the enzyme concentration is 1.0 μg / mL, the absorption efficiency of TSM for sucrase, amylase and protease is 46.2%, 72.1% and 62.1%, respectively. In sharp contrast, the absorption of the same three enzymes by MTSM is significantly reduced, which is 17.4%, 23.1% and 21.1%, respectively. This shows that the introduction of locust cell membrane significantly inhibits the nonspecific removal of digestive enzymes by microparticles. Then, a mixture of TZP and digestive enzymes was prepared to simulate the gastrointestinal environment of pollinating insects and explore the ability of microparticles MTSM to remove insecticides ( Fig. 9 E). Compared with TSM, MTSM significantly enhanced the removal effect of TZP, while the adsorption effect on sucrase (15% VS 27%), amylase (17% VS 48%) and protease (14% VS 42%) was significantly reduced. The high efficiency of insecticide removal and minimal digestive enzyme adsorption (<20%) confirmed the excellent removal efficiency and safety of MTSM; TSM microparticles were used as a control, and the experimental conditions were microparticle and TZP concentrations of 20 mg / mL and 50 μg / mL, respectively, and the concentration of various enzymes was 10 μg / mL.

[0071] Example 7 Distribution of MTSM in vivo To facilitate the observation and tracking of MTSM in the digestive tract of bumblebees, FITC-labeled MTSM (FITC-MTSM) was prepared. The specific method is as follows: TSM (100 mg) and FITC (3.89 mg, 0.01 mmol) were dispersed in PBS (10 mM, pH = 7.4) buffer and stirred at room temperature for 1 h. After the stirring reaction, the mixture was centrifuged (6000 rpm, 5 min) and washed with PBS to remove excess FITC. FITC-labeled TSM was prepared; then MTSM was prepared, and finally FITC-MTSM was obtained.

[0072] The FITC-MTSM obtained above was used to investigate the distribution of microparticles in the gastrointestinal tract of bumblebees. The brief description is as follows: First, 20 bumblebees were placed in ventilated plastic cups and starved for 2 h, and then FITC-MTSM dispersed in sucrose solution (2 g / mL) was fed to the bumblebees. After 30 min of feeding, the bumblebees were randomly divided into 4 groups, anesthetized after 0, 4, 12, and 48 h, respectively, and the gastrointestinal tract of the bumblebees was removed and placed on a slide. The samples were analyzed for FITC fluorescence under an inverted microscope. FITC-labeled MTSM (FITC-MTSM) was used to track the distribution and metabolism of microparticles in the gastrointestinal tract of pollinating insects. After feeding pollinating insects with FITC-MTSM for 30 minutes, it was observed that MTSM passed through the crop, forestomach, midgut, and hindgut over time. In the early stages of digestion, most of the microparticles were concentrated in the crop, and the distribution of microparticles in the midgut and hindgut was negligible ( Fig.10 A). As the digestion time was extended to 4 h, most of the particles had been removed from the crop ( Fig.10 B), indicating that MTSM can be successfully filtered by the forestomach. From 4 to 12 h, a large number of particles were present in the midgut and hindgut of the gastrointestinal tract ( Fig.10 C). It is reported that OP (organophosphorus insecticides) are usually present in high levels in pollen, and pollen grains take about 12 hours to digest in the gastrointestinal tract, which promotes the interaction between MTSM and digested pollen in the gastrointestinal tract of bumblebees. This maximizes the detoxification function of MTSM. After 48 hours, no fluorescent particles were observed in the crop, forestomach, midgut, and hindgut, indicating that all particles can be completely excreted within 48 hours ( Fig.10 D). MTSM will not accumulate in the digestive tract of bumblebees and has good biosafety.

[0073] Example 8 Study on the removal of pesticides from MTSM To evaluate the ability of MTSM to remove organic phosphorus in vivo, microcolony cages suitable for bumblebees were constructed, each containing 20 bumblebees. When preparing pollen balls, TZP (0.3 mg / mL, 2 mL) was thoroughly mixed with pollen grains (10 g) until a uniform slurry was formed, and then placed at room temperature for 10 min to allow TZP to be completely absorbed by the pollen. The TZP-contaminated pollen was then crushed in a mortar and rolled into pollen balls of equal size by hand. The scavenger was prepared by dispersing TSM and MTSM in sucrose (2 g / mL). One pollen ball and 5 mL of scavenger were placed in each microcolony cage. The mortality of the small colony was monitored every 12 h until all bumblebees died or the time reached 10 days.

[0074] A microcolony cage suitable for bumblebee survival was constructed to evaluate the ability of MTSM to remove organophosphates. Before studying the detoxification ability of MTSM, the biosafety of MTSM in bumblebees was first evaluated. Each group of 20 bumblebees was fed with MTSM sucrose solution with a MTSM concentration of 0.1 to 50 mg / mL. The results showed that ( Fig.11 A) All groups of bumblebees remained alive within 10 days, indicating that MTSM has excellent biosafety. Then, 20 bumblebees in each group were treated with TZP-contaminated pollen balls and MTSM-sucrose to determine the effectiveness of MTSM-sucrose in reducing mortality under TZP exposure. LD of TZP on bumblebees 500.098 μg per bumblebee. This data was used as the basis for setting the TZP dosage, and a pollen ball containing 3 μg / g TZP was formed. It was found that there was no deterioration in the health of bumblebees within 10 days. Subsequently, through repeated experiments, the concentration of TZP-contaminated pollen was greatly increased. The results showed that pollen containing 60 μg / g TZP caused a 100% mortality rate among bumblebees within 4 days, and pollen containing 120 μg / g TZP caused a 90% mortality rate among bumblebees within 12 hours, and all died within 1 day ( Fig.11 B).

[0075] The two concentrations of pollen contamination were used for moderate and acute toxicity assessments, respectively. At moderate toxicity levels, TSM and MTSM treatments significantly reduced mortality ( Fig.11 C).

[0076] When the MTSM-sucrose concentration was 1.0 mg / mL, the survival rates of the TSM and MTSM groups within 10 days were 60% and 70%, respectively, while the survival rate of the untreated group dropped to 0% on the 4th day. Due to the good safety of the microparticles, the MTSM concentration was increased to 20 mg / mL, showing a better detoxification effect, and the survival rate within 10 days could reach 90%. Under acute toxicity conditions, both TSM and MTSM prolonged the survival time of bumblebees. All bumblebees in the TSM group died after 7 days, while bumblebees in the MTSM group still had a survival rate of 15% after 10 days. When the concentration of the microparticles was increased to 20 mg / mL, the survival rate of bumblebees within 10 days could be further increased to 60% ( Fig.11 D). The above results show that TSM and MTSM have excellent ability to remove the toxicity of TZP in bumblebees, and MTSM has a stronger detoxification ability.

[0077] Pollen balls with different levels of IMI contamination were prepared for in vivo clearance experiments of bumblebee sublethal poisoning (5 μg IMI / g in pollen) and acute poisoning (15 μg IMI / g in pollen). At sublethal poisoning levels, both high and low concentrations of MTSM particles can reduce the mortality of bumblebees ( Fig.12 A). After treatment with 1.0 mg / mL MTSM-sucrose solution, the survival rate of 20 bumblebees in the cage was 55% within 10 days. The survival rate of high concentration MTSM treatment can be maintained at 95% within 10 days, and the health of bumblebees will not deteriorate even if the time is extended to 30 days. In contrast, the survival rate of bumblebees in the untreated group dropped to 0 within 3 days. Under the acute poisoning level, the survival rate of the untreated group dropped sharply to 10% within 12 hours, and all died within 1 day ( Fig.12B). In contrast, the MTSM-treated group had a higher survival rate within 10 days. When the microparticle concentration was increased to 20 mg / mL, the survival rate within 10 days was 60%. In the IMI poisoning prevention experiment, the survival rate after MTSM treatment reached 40% within 10 days. The above results show that MTSM has an excellent ability to remove the toxicity of IMI in bumblebees and has a broad-spectrum removal function.

Claims

1. A pesticide scavenger, comprising cell membrane modified mesoporous silica; the modified mesoporous silica is tannic acid modified mesoporous silica.

2. The insecticide remover according to claim 1, characterized in that: The mesoporous silica is mesoporous silica particles; and the cell membrane is locust cell membrane.

3. The method for preparing the cell membrane modified mesoporous silica according to claim 1 comprises the following steps: mixing the tannic acid modified mesoporous silica with the cell membrane suspension to obtain the cell membrane modified mesoporous silica.

4. The method for preparing cell membrane modified mesoporous silica according to claim 3, characterized in that: The amino-modified mesoporous silica particles and tannic acid are dispersed in a buffer solution to obtain a mixed suspension, which is then reacted to obtain tannic acid-modified mesoporous silica; the tannic acid-modified mesoporous silica is washed and mixed with a cell membrane suspension to obtain cell membrane-modified mesoporous silica.

5. The method for preparing cell membrane modified mesoporous silica according to claim 4, characterized in that: The buffer solution includes 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid hemi-sodium salt buffer solution or tris(hydroxymethyl)aminomethane buffer solution; and the cleaning solution during cleaning includes phosphate buffer solution.

6. The method for preparing cell membrane modified mesoporous silica according to claim 3, characterized in that: The mass ratio of tannic acid modified mesoporous silica to cell membrane is 10:(0.05-0.25), wherein the mass of the cell membrane is calculated based on the protein on the cell membrane.

7. Use of the cell membrane modified mesoporous silica according to claim 1 as or in the preparation of an insecticide scavenger.

8. Use of the insecticide scavenger according to claim 1 in removing insecticides, or in preparing an insecticide removing agent.

9. Use of the insecticide scavenger according to claim 1 in reducing the toxicity of insecticides to insects, or in the preparation of an agent for reducing the toxicity of insecticides to insects.

10. A method for removing pesticides, comprising the following steps: removing the pesticides using the pesticide remover according to claim 1.

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