Nano pesticide stimulated Raman tracing probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of botanical imaging, in particular to a nano-pesticide stimulated Raman tracing probe and a preparation method and application thereof.Acetamiprid serves as a pesticide model and is loaded in cyanation mesoporous nano-silica, and on the basis of the stimulated Raman technology, the tracing probe is obtained. Raman imaging is carried out by using characteristic fingerprints of acetamiprid and cyanated mesoporous nano-silica, synchronous tracking of the pesticide and the nano-carrier can be realized, and the technology can realize tracking of the nano-pesticide complex in plants without Raman signal enhancement on the material and pesticide marking.

Description

Technical Field

[0001] The invention relates to the technical field of plant imaging, and in particular to a nano pesticide stimulated Raman tracing probe and a preparation method and application thereof. Background Art

[0002] Silica nanoparticles are one of the five most widely used nanomaterials in the world, with an annual consumption of more than 1 million tons. As one of the nanopowders with the highest production volume, mesoporous nanosilica has unique physical and chemical properties, a large specific surface area, and is easy to modify. It can be used as a pesticide sustained-release carrier and a responsive pesticide precise controlled release, and a large number of research results have been achieved. However, the absorption and transport mechanism of nanosilica-loaded nanopesticides in plants has not yet been revealed, which determines its safety in application in plants, but how to track nanopesticides in plants is an important technical challenge currently faced.

[0003] In existing research, the method for tracking nano-silica-loaded nano-pesticides is mainly fluorescent labeling. For example, some studies have tracked FITC-labeled nano-silica in tomatoes, corn, rice, and wheat. However, the background fluorescence of some endogenous substances in plants makes the fluorescence visualization of nano-silica complicated. And when used for fluorescence observation, sampling is often destructive, so fluorescent labeling usually cannot achieve in situ tracking. In addition to fluorescent labeling, scanning electron microscopy and transmission electron microscopy combined with energy spectrometry can observe unlabeled nano-silica, but the complexity of sample preparation also makes it impossible to achieve in situ tracking. In addition, the above-mentioned technology is usually only for the tracing of nano-silica carriers, while nano-silica-loaded nano-pesticides are a composite system that includes not only the carrier but also the loaded pesticide. The two may separate after entering the plant body. At present, there is no technology that can simultaneously trace pesticides and nano-silica carriers in situ.

[0004] Raman imaging technology can solve the current technical difficulties. By imaging the unique molecular vibration fingerprint of a substance to reflect the distribution of the substance, it can achieve synchronous orthogonal imaging of multiple substances with different characteristic fingerprints. However, Raman scattering is a weak effect. At present, it is mainly achieved through some enhancement methods in the fields of food safety testing and molecular imaging, such as surface enhanced Raman technology. These enhancement methods often make the materials complicated. Stimulated Raman is when the frequency difference between the pump light and the Stokes light matches the vibration frequency of the molecule, the spontaneous scattering process is transformed into a stimulated process, the intensity of the pump light is reduced, and the intensity of the Stokes light is increased, which can achieve high-sensitivity, high-resolution and high-speed biological imaging. This technology is not easily interfered by false background.

[0005] In view of the above-mentioned defects, the inventors of the present invention finally obtained the present invention after a long period of research and practice. Summary of the invention

[0006] The purpose of the present invention is to solve the problem that the existing nano-pesticide tracking technology cannot achieve synchronous tracking of pesticides and nano-carriers, and to provide a nano-pesticide stimulated Raman tracing probe and a preparation method and application thereof.

[0007] In order to achieve the above object, the present invention discloses a method for preparing a nano pesticide stimulated Raman tracer probe, comprising the following steps:

[0008] S1, Synthesis of Mesoporous Nanosilica

[0009] Deionized water was added to CTAB, and a micelle system was formed by ultrasound. The mixture was heated to 55°C in a water bath. Ammonia water, tetraethyl orthosilicate, and ethyl acetate were slowly added in sequence during stirring. The mixture was stirred in a water bath and then centrifuged. The precipitate was washed, dried, and calcined to obtain mesoporous nano-silica.

[0010] S2, Cyanolysis of Mesoporous Nanosilica

[0011] The mesoporous nano-silica obtained in step S1 is ultrasonically dispersed in anhydrous ethanol, heated to 100° C. in an oil bath, 2-cyanoethyltriethoxysilane is slowly added dropwise under stirring, reacted for 8 hours, centrifuged to obtain a precipitate, washed and dried to obtain cyanated mesoporous nano-silica;

[0012] S3, cyanated mesoporous nanosilica loaded with acetamiprid

[0013] The acetamiprid is dissolved in a 20% methanol-water solution to prepare an acetamiprid solution, and the cyanated mesoporous nano-silica obtained in step S2 is dispersed in the acetamiprid solution, shaken at room temperature, centrifuged to obtain a precipitate, washed with ultrapure water and dried to obtain the mesoporous nano-silica loaded with acetamiprid, i.e., a nano-pesticide stimulated Raman tracer probe.

[0014] In the step S1, the amount of CTAB used is 100 mg, the volume of deionized water is 120 mL, and the volume ratio of the added ammonia water, tetraethyl orthosilicate, and ethyl acetate is 0.2-3:1:5.

[0015] In step S1, the water bath stirring time is 6 hours and the centrifugal force is 8000 r / min.

[0016] In the step S1, the drying temperature is 60° C., the calcination temperature is 550° C., and the calcination time is 6 hours.

[0017] In the step S2, the amount of mesoporous nano-silica used is 100 mg, the volume of anhydrous ethanol is 100 mL, and the volume of the added 2-cyanoethyltriethoxysilane is 200 μL.

[0018] In the step S3, the concentration of the acetamiprid solution is 4 mg / mL, and the amount of cyanated mesoporous nano-silica is 40 mg.

[0019] The invention also discloses a nano pesticide stimulated Raman tracer probe prepared by the preparation method and application of the nano pesticide stimulated Raman tracer probe in plant imaging.

[0020] The nano pesticide stimulated Raman tracer probe comprises a mesoporous nano silica carrier and a loaded pesticide acetamiprid, wherein the carrier is cyanated and in the biological macromolecule Raman silent region (1800-2800cm -1 ) has a characteristic peak of cyano group. The cyano group in the molecule of the pesticide acetamiprid also has a Raman characteristic peak in the Raman silent zone. Although it is also a cyano group, the chemical environment is different, which makes its Raman characteristic peak deviate (at 2257cm -1 and 2180cm -1 It is this deviation that can realize Raman imaging through the Raman fingerprints of their respective cyano groups, and simultaneously identify the two substances to track the whereabouts of the nanopesticide carrier and the loaded pesticide in the plant body. After its application, acetamiprid will be slowly released in the plant body and separated from the carrier, so they need to be tracked separately, which is difficult to achieve with current traditional labeling and tracing technologies.

[0021] Since the spontaneous Raman spectroscopy signal is weak, effective enhancement methods are often needed to improve the temporal and spatial resolution of imaging. Currently, the imaging material itself is often enhanced, such as surface enhanced Raman spectroscopy, but this undoubtedly increases the complexity of the material. Stimulated Raman technology is when the frequency difference between the pump light and the Stokes light matches the vibration frequency of the molecule, the spontaneous scattering process is transformed into a stimulated process, the intensity of the pump light is reduced, and the intensity of the Stokes light is increased. This technology is not easily interfered by false backgrounds, and the efficiency can be increased by 4 to 6 orders of magnitude, which can achieve highly sensitive, high-resolution and high-speed stimulated Raman biological imaging. Therefore, it is possible to quickly track nanopesticide carriers and loaded pesticides in plants without enhancing the material and without macromolecular labels.

[0022] Compared with the prior art, the beneficial effect of the present invention is that: the present invention uses acetamiprid as a pesticide model, loads it in cyanated mesoporous nano-silica, and uses the stimulated Raman technology to perform Raman imaging with the characteristic fingerprints of acetamiprid and cyanated mesoporous nano-silica, which can achieve synchronous tracking of pesticides and nano-carriers. This technology does not require Raman signal enhancement of materials or labeling of pesticides, and can achieve tracking of nano-pesticide complexes in plants. . BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1The schematic diagram of the preparation process and application of the nano-pesticide stimulated Raman probe of the present invention;

[0024] Figure 2 Transmission electron microscopy images of materials prepared in Examples 1 to 3 of the present invention;

[0025] Figure 3 The infrared spectra of materials and acetamiprid prepared in Examples 1 to 3 of the present invention;

[0026] Figure 4 The Raman spectra of materials prepared in Examples 1 to 3 of the present invention and 2-cyanoethyltriethoxysilane;

[0027] Figure 5 The 3D stimulated Raman imaging diagram of the nano-pesticide stimulated Raman probe in the leaf prepared in Example 4 of the present invention;

[0028] Figure 6 The deep stimulated Raman imaging image of the nano-pesticide stimulated Raman probe in the leaf prepared in Example 4 of the present invention;

[0029] Figure 7 This is a transmission electron microscope image of the material prepared in Example 5 of the present invention;

[0030] Figure 8 The Raman spectra of cyanated mesoporous nano-silica obtained at different mesoporous nano-silica dosages in Example 6 of the present invention;

[0031] Fig. 9 The Raman spectra of cyanated mesoporous nano-silica obtained at different dosages of 2-cyanoethyltriethoxysilane in Example 7 of the present invention;

[0032] Fig.10 This is the Raman spectrum of Ace@MSN-TMPES in comparative example 1 of the invention. DETAILED DESCRIPTION

[0033] The above and other technical features and advantages of the present invention are described in more detail below in conjunction with the accompanying drawings.

[0034] Example 1

[0035] Synthesis of Mesoporous Nano-Silica

[0036] Weigh 100 mg CTAB, add 120 mL deionized water, and ultrasonicate to form a stable and uniform micelle system. Heat the water bath to 55 ° C, and slowly add 1.4 mL of ammonia water, 1 mL of tetraethyl orthosilicate, and 5 mL of ethyl acetate in sequence during stirring. Continue stirring in the water bath for 6 hours, centrifuge at 8000 r / min to obtain the precipitate, wash three times with anhydrous ethanol, and dry at 60 ° C overnight. Calcinate at 550 ° C in a muffle furnace for 6 hours to obtain mesoporous nanosilica (MSN) of about 50 nm.

[0037] Example 2

[0038] Cyanolysis of Mesoporous Nanosilica

[0039] Take 100 mg of mesoporous nano-silica, ultrasonically disperse it in 100 mL of ultrapure water, heat it to 100 ° C in an oil bath, slowly add 200 μL of 2-cyanoethyltriethoxysilane (CETES) under stirring, react for 8 hours, centrifuge to obtain the precipitate, wash it three times with anhydrous ethanol, and dry it to obtain cyanylated mesoporous nano-silica, which is recorded as MSN-CETES.

[0040] Example 3

[0041] Cyano-modified mesoporous nano-silica loaded with acetamiprid

[0042] Weigh 160 mg of acetamiprid and dissolve it in 40 mL of 20% methanol-water solution to prepare a 4 mg / mL acetamiprid solution. Weigh 40 mg of MSN-CETES and disperse it in 40 mL of 4 mg / mL acetamiprid solution, and shake it at 1000 r / min for 2 h at room temperature. Centrifuge to obtain the precipitate, wash it with ultrapure water, and then dry it to obtain the mesoporous nanosilica loaded with acetamiprid, which is recorded as Ace@MSN-CETES.

[0043] Example 4

[0044] Application of acetamiprid-loaded cyanated mesoporous nanosilica stimulated Raman probe in plant imaging

[0045] Prepare 10mg / mL of Ace@MSN-CETES, disperse in pure water, and fully sonicate. Add 100μL to the back surface of the tea leaves, apply evenly with a gun tip, and air dry naturally. After air drying, perform stimulated Raman imaging of Ace and nano-silica on the leaves to obtain 3D depth scans and representative depth cross-sections. The stimulated Raman spectroscopy test conditions are: pump light 852nm, 23mW, Stokes light 1045nm, 230mW, 40x water mirror, N A is 0.8, the number of pixels is 200×200, the single point scanning time is 10μs, and the average number of times is 5 times.

[0046] like Figure 1 As shown in the figure, CTAB is used as a template to hydrolyze ethyl orthosilicate under alkaline conditions. The silicic acid obtained by hydrolysis forms nano-silica through a condensation reaction, and then the template is removed by high-temperature calcination to obtain mesoporous nano-silica. Since nano-silica has no Raman signal, it is impossible to achieve stimulated Raman imaging in plants, so it needs to be labeled. Raman labels should preferably be selected in the Raman silent region of endogenous molecules in plants (1800-2800cm -1) has responsive alkynyl and cyano groups, and the acetamiprid molecule itself has a cyano group with a Raman signal in the silent zone. After labeling, acetamiprid is loaded to obtain a nanopesticide stimulated Raman probe. By comparison, it was found that the Raman shift variation of the alkynyl-labeled nano-silica and acetamiprid in the silent zone was small, and the two characteristic peaks could not be separated because the two substances could not be distinguished at the same time. Due to the different chemical environments, the cyano group labeled with nano-silica and the cyano group of acetamiprid have distinguishable Raman shifts, so the cyano group labeling was selected. The prepared cyanated nano-silica loaded with acetamiprid can be used as a stimulated Raman probe for tracing the nano-pesticide, and the synchronous in situ tracking of acetamiprid and nano-silica can be achieved in plants.

[0047] like Figure 2 As shown, it can be seen from transmission electron microscopy that the synthesized mesoporous nano-silica has a size of about 50nm, uniform size, good dispersion, and evenly arranged pores can be seen on the surface. The morphology of the mesoporous nano-silica has not changed after cyanidation, while most of the pores are blocked and become smaller after loading with acetamiprid, indicating that acetamiprid is loaded in the mesopores.

[0048] like Figure 3 As shown, the MSN after cyano labeling is at 2927cm -1 and 2856cm -1 There are obvious -CH2 characteristic peaks and 2255cm -1 The characteristic peak of -C≡N at 2176 cm appeared after MSN-CETES was loaded with acetamiprid. -1 and 1575cm -1 The characteristic peaks of -C≡N and -C=N of acetamiprid were detected, indicating the successful synthesis of the probe.

[0049] like Figure 4 As shown in Figure 2, Raman spectroscopy also further proves the successful synthesis of the probe. The cyano Raman peak (2257 cm -1 ) and the cyano Raman peak of acetamiprid (2180 cm -1 ), and the cyanide groups from mesoporous nanosilica and acetamiprid were distinguished in the silent zone of biological endogenous macromolecules, indicating that the nanocarriers and pesticides can be orthogonally visualized in plants in the future.

[0050] like Figures 5-6 As shown in the figure, stimulated Raman imaging technology can be used to realize in-situ non-destructive imaging of cyanated mesoporous nano-silica stimulated Raman probe loaded with acetamiprid in tea leaves, and the positional relationship between the two can be identified through Raman molecular fingerprints, among which 2187cm -1 Molecular imaging at 2250 cm -1The molecular imaging at the site represents nano-silica. Raman imaging shows that there is separate acetamiprid inside the tea leaves, and there is also unreleased acetamiprid in some carriers, indicating that the nano-carrier has a certain pesticide sustained-release effect and can carry acetamiprid to the inside of the leaves. In actual application, it can resist rain erosion, reduce wind drift of pesticides, reduce environmental pollution, and has good preventive effect on piercing-sucking pests. Its sustained-release effect means an extension of the efficacy period, which is expected to reduce the amount and frequency of pesticide use, reduce pesticide residues from the source, and improve tea quality and safety without reducing production.

[0051] Example 5

[0052] Weigh three portions of 100 mg CTAB, add 120 mL, 120 mL, and 100 mL of deionized water respectively, and ultrasonicate to form a stable and uniform micelle system. Heat to 55 ° C in a water bath, and slowly add ammonia water (0.2 mL, 1.4 mL, 3 mL), 1 mL of tetraethyl orthosilicate, and 5 mL of ethyl acetate in the stirring process. Continue stirring in a water bath for 6 hours, centrifuge at 8000 r / min to obtain the precipitate, wash three times with anhydrous ethanol, and dry at 60 ° C overnight. Calcinate at 550 ° C in a muffle furnace for 6 hours to obtain three types of mesoporous nano-silica, which are respectively denoted as MSN D1 , MSN D2 , MSN D3 .like Figure 7 As shown, under the above three conditions, nano-silica with uniform particle size, good dispersion and clear mesopores can be obtained, but the proportions are different and the sizes will be different.

[0053] Example 6

[0054] The difference between this embodiment and embodiment 2 is that the dosage of mesoporous nano-silica in this embodiment is 50 mg, 100 mg, and 150 mg respectively.

[0055] like Figure 8 As shown, when the amount of mesoporous silica is large, the nanoparticles in the dispersed system are easy to flocculate and settle, and the surface area is reduced, resulting in poor cyanation surface modification effect and low intensity of the cyano Raman characteristic peak. Therefore, when the amount is 150 mg, the Raman characteristic peak of the cyano group is not obvious; when the amount is reduced to 50 mg, the characteristic peak of the cyano group is more obvious, but the condensation efficiency may be lower than that of 100 mg of mesoporous silica due to the low concentration, so the cyano intensity is slightly lower, so the final mesoporous silica amount is selected to be 100 mg.

[0056] Example 7

[0057] The difference between this embodiment and embodiment 2 is that the dosage of 2-cyanoethyltriethoxysilane in this embodiment is 100, 200, and 300 μL, respectively. Fig. 9As shown in the figure, the amount of 2-cyanoethyl triethoxysilane will also affect the modification effect of cyanated silica. The higher the condensation efficiency and degree of polymerization, the more cyano groups are introduced to the surface, and the greater the Raman intensity. When the amount is 100 μL, the cyano Raman intensity is low, and when the amount is 200 μL and 300 μL, the cyano intensity increases. Considering the saving of 2-cyanoethyl triethoxysilane and the convenience of removing free 2-cyanoethyl triethoxysilane in the later stage, the amount of 2-cyanoethyl triethoxysilane is selected as 200 μL.

[0058] Comparative Example

[0059] Alkynylation of Mesoporous Nanosilica

[0060] Take 100 mg of mesoporous nano-silica, ultrasonically disperse it in 100 mL of ultrapure water, heat it to 100°C in an oil bath, slowly add 200 μL of trimethoxy(phenylethynyl)silane (TMPES) under stirring, react for 8 hours, centrifuge to obtain the precipitate, wash it three times with anhydrous ethanol, and dry it to obtain acetylated mesoporous nano-silica, which is recorded as MSN-TMPES. Weigh 160 mg of acetamiprid and dissolve it in 40 mL of 20% methanol-water solution to prepare a 4 mg / mL acetamiprid solution. Weigh 40 mg of MSN-TMPES and disperse it in 40 mL of 4 mg / mL acetamiprid solution, and oscillate it at 1000 r / min for 2 hours at room temperature. Centrifuge to obtain the precipitate, wash it with ultrapure water, and dry it to obtain mesoporous nano-silica loaded with acetamiprid, which is recorded as Ace@MSN-TMPES. By Fig.10 From the Raman spectrum of Ace@MSN-TMPES, it can be seen that the characteristic peaks of acetamiprid and acetylenic mesoporous nano-silica overlap in the silent zone, and the two substances cannot be distinguished. Therefore, Ace@MSN-TMPES is not suitable for the stimulated Raman probe of the nanopesticide to simultaneously track acetamiprid and the carrier in plants.

[0061] The above description is only a preferred embodiment of the present invention, which is only illustrative and not restrictive of the present invention. Those skilled in the art understand that many changes, modifications, and even equivalences may be made to the present invention within the spirit and scope defined by the claims of the present invention, but all of them will fall within the scope of protection of the present invention.

Claims

1. A nanopesticide stimulated Raman tracer probe, characterized in that: The following steps are involved: S1, Synthesis of Mesoporous Nanosilica Deionized water was added to CTAB, and a micelle system was formed by ultrasound. The mixture was heated to 55°C in a water bath. Ammonia water, tetraethyl orthosilicate, and ethyl acetate were slowly added in sequence during stirring. The mixture was stirred in a water bath and then centrifuged. The precipitate was washed, dried, and calcined to obtain mesoporous nano-silica. S2, Cyanolysis of Mesoporous Nanosilica The mesoporous nano-silica obtained in step S1 is ultrasonically dispersed in anhydrous ethanol, heated to 100° C. in an oil bath, 2-cyanoethyltriethoxysilane is slowly added dropwise under stirring, reacted for 8 hours, centrifuged to obtain a precipitate, washed and dried to obtain cyanated mesoporous nano-silica; S3, cyanated mesoporous nanosilica loaded with acetamiprid The acetamiprid is dissolved in a 20% methanol-water solution to prepare an acetamiprid solution, and the cyanated mesoporous nano-silica obtained in step S2 is dispersed in the acetamiprid solution, shaken at room temperature, centrifuged to obtain a precipitate, washed with ultrapure water and dried to obtain the cyanated mesoporous nano-silica loaded with acetamiprid, i.e., a nano-pesticide stimulated Raman tracer probe.

2. A nanopesticide stimulated Raman tracer probe as claimed in claim 1, characterized in that: In the step S1, the amount of CTAB used is 100 mg, the volume of deionized water is 120 mL, and the volume ratio of the added ammonia water, tetraethyl orthosilicate, and ethyl acetate is 0.2-3:1:

5.

3. A nanopesticide stimulated Raman tracer probe as claimed in claim 1, characterized in that: In step S1, the water bath stirring time is 6 hours and the centrifugal force is 8000 r / min.

4. A nanopesticide stimulated Raman tracer probe as claimed in claim 1, characterized in that: In the step S1, the drying temperature is 60° C., the calcination temperature is 550° C., and the calcination time is 6 hours.

5. The nanopesticide stimulated Raman tracing probe according to claim 1, characterized in that: In the step S2, the amount of mesoporous nano-silica used is 100 mg, the volume of anhydrous ethanol is 100 mL, and the volume of the added 2-cyanoethyltriethoxysilane is 200 μL.

6. A nanopesticide stimulated Raman tracer probe as claimed in claim 1, characterized in that: In the step S3, the concentration of the acetamiprid solution is 4 mg / mL, and the amount of cyanated mesoporous nano-silica is 40 mg.

7. A nanopesticide stimulated Raman tracing probe prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the nanopesticide stimulated Raman tracer probe as claimed in claim 7 in plant imaging.

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