A method for real-time monitoring of the movement of nanopesticides in plants using in vivo imaging technology

Abstract

The present invention discloses a method for real-time monitoring of the movement of nanopesticides in plants using in vivo imaging technology. A bidirectionally conductive fluorescent nanofungicide is sprayed onto plant leaves and placed in an in vivo imaging instrument for observation. The excitation and emission wavelengths are selected according to the fluorescent molecules, and the fluorescence imaging results of the entire plant are observed to achieve real-time monitoring of the movement of the nanofungicide in the plant. The bidirectionally conductive fluorescent nanofungicide prepared by the present invention can be bidirectionally conducted in the plant body after being sprayed on the plant leaves, thereby avoiding soil application, thereby reducing the high cost, low efficacy and serious environmental problems caused by soil application. In addition, the movement of the prepared nanofungicide in the plant can be monitored in real time through in vivo fluorescence imaging technology, without having to destroy the integrity of the plant for sampling and observation, and can better reflect the actual movement of the nanofungicide in the plant body.

Description

Technical field:

[0001] The present invention belongs to the field of plant protection applications, and in particular relates to a method for real-time monitoring of the movement of nanopesticides in plants using in vivo imaging technology. Background technology:

[0002] Nanopesticides have emerged to address the problems of low pesticide utilization and severe waste and pollution. Some nanopesticides are primarily designed to improve the stability of pesticide molecules, reduce their diffusion, and achieve sustained release. However, there has been no in-depth and systematic research into their ability to enter plants and their movement within them. Some researchers have also modified nanopesticide molecules in the hope of enabling them to enter plants and achieve targeted accumulation and release at diseased plant sites, thereby further improving their utilization. However, the transport behavior of nanopesticides within plants has not been directly monitored. Instead, the determination of their reach is based on the measurement and analysis of pesticide content in different plant tissues. This significantly hinders research into the absorption and transport mechanisms of nanopesticides, hindering their potential to reduce their use and improve their efficiency.

[0003] Previous studies have used fluorescent nanocarriers to load drugs and observed their movement within animals through fluorescence imaging. However, a 2022 article published in the prestigious journal Nature Nanotechnology clearly stated that the research findings on the interaction between animal cell membranes and nanomaterials are not applicable to plant systems with cell walls.

[0004] However, there are currently no reports on real-time monitoring of nanopesticide movement within plants using in vivo imaging. Most studies investigating nanopesticide imaging in the literature use cell fluorescence imaging, which fails to accurately reflect the true state of living plants. Alternatively, methods such as fluorescence and electron microscopy, which utilize plant sections, require destructive sampling and prevent real-time monitoring of in vivo imaging results, have been used. Summary of the invention:

[0005] The purpose of the present invention is to provide a method for real-time monitoring of the movement of nanopesticides in plants using in vivo imaging technology.

[0006] The present invention utilizes fluorescent molecules with stable fluorescence properties and high quantum yield to prepare fluorescent mesoporous silica nanoparticles, and modifies their surfaces with functional molecules such as amino acids and glucosamine to load fungicide molecules to obtain small-particle bidirectionally conductive fluorescent nanofungicides. The bidirectionally conductive fluorescent nanofungicides are then sprayed onto plant leaves and placed in a living imaging instrument to achieve real-time fluorescent imaging observation of the movement behavior of the bidirectionally conductive fluorescent nanofungicides in the plant body, thereby achieving the purpose of the present invention.

[0007] The present invention utilizes in vivo imaging technology to monitor the movement of nanopesticides in plants in real time. The method comprises spraying a bidirectionally conductive fluorescent nanofungicide onto plant leaves, placing the leaves in an in vivo imaging instrument for observation, selecting excitation and emission wavelengths based on the fluorescent molecules, and observing the fluorescence imaging results of the entire plant to achieve real-time monitoring of the movement of the nanofungicide in the plant.

[0008] The bidirectionally conductive fluorescent nano-bactericide is prepared by the following method:

[0009] Fluorescent mesoporous silica nanoparticles are prepared using fluorescent molecules with stable fluorescence properties and high quantum yields, and their surfaces are modified with amino acids and glucosamine functional molecules, which are then loaded with bactericide molecules to obtain bidirectionally conductive fluorescent nanofungicides.

[0010] Preferably, the fungicide molecule is a systemic fungicide, including thiabendazole, carbendazim or thiophanate-methyl.

[0011] Preferably, the amino acids include one or more of glutamic acid, serine, glycine, histidine, lysine, tryptophan, methionine, phenylalanine, proline, threonine, and tyrosine.

[0012] Preferably, the particle size of the bidirectionally conductive fluorescent nano-bactericidal agent is in the range of 10-70 nm.

[0013] Preferably, the method for preparing fluorescent mesoporous silica nanoparticles using fluorescent molecules comprises weighing cetyltrimethylammonium bromide, poloxamer F127 and triethanolamine, dissolving them in an ethanol aqueous solution, adding the AIE solution, and stirring thoroughly in the dark; then adding tetraethyl orthosilicate, stirring vigorously, and allowing to react; after the reaction, adding the ethanol aqueous solution and shaking evenly, centrifuging and pouring out the supernatant, adding the ethanol solution again and ultrasonically dispersing, centrifuging, drying the solid, dissolving the dried solid in a hydrochloric acid methanol solution, refluxing in a water bath, centrifuging to remove the supernatant, adding methanol and ultrasonically dispersing and washing, and drying the dispersion to obtain fluorescent mesoporous silica nanoparticles.

[0014] Preferably, the surface modification with amino acid or glucosamine functional molecules comprises dispersing fluorescent mesoporous silica nanoparticles in methanol, adding (3-mercaptopropyl)trimethoxysilane, stirring and reacting to obtain thiol-modified fluorescent mesoporous silica nanoparticles; then dissolving 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt in phosphate buffer solution, adding amino acid or glucosamine, stirring and reacting to obtain activated amino acid or glucosamine solution; mixing the thiol-modified fluorescent mesoporous silica nanoparticles with the activated amino acid or glucosamine solution, stirring and reacting, and centrifuging and washing to obtain amino acid or glucosamine-modified fluorescent mesoporous silica nanoparticles.

[0015] Preferably, the loaded bactericide molecules are prepared by dispersing amino acid or glucosamine modified fluorescent mesoporous silica nanoparticles in methanol, adding bactericide molecules, mixing and stirring, centrifuging and washing, and freeze-drying to obtain a bidirectionally conductive fluorescent nano bactericide.

[0016] The excitation wavelength and emission wavelength of the fluorescent molecule should avoid the excitation wavelength (450nm) and emission wavelength (676nm) of plant chlorophyll, and have good anti-fading performance and strong quantum yield, and can be used for long-term fluorescence imaging observation.

[0017] Preferably, the fluorescent mesoporous silica nanoparticles can efficiently load fungicide molecules, including but not limited to fluorescent mesoporous silica nanoparticles.

[0018] The above-mentioned bidirectionally conductive fluorescent nanofungicide is sprayed onto the leaves of the plant and placed in a living imaging instrument for observation. The excitation and emission wavelengths are selected according to the fluorescent molecules, and the fluorescence imaging results of the whole plant are observed to achieve real-time monitoring of the movement of the nanofungicide in the plant body.

[0019] The bidirectionally conductive fluorescent nanofungicide prepared by this invention can be bidirectionally transported within the plant body after being sprayed on plant leaves, avoiding soil application, thereby reducing the high cost, low efficacy, and serious environmental problems associated with soil application. Furthermore, in vivo fluorescence imaging technology can be used to monitor the movement of the prepared nanofungicide within the plant in real time, eliminating the need to destroy the plant for sampling and observation, and better reflecting the actual movement of the nanofungicide within the plant body. Description of the drawings:

[0020] Figure 1These are in vivo fluorescence images of cucumber seedlings sprayed with a bidirectional fluorescent nanofungicide on the first and eighth days. (A) Fluorescence image of cucumber seedlings sprayed with the nanofungicide on the first day, with the white arrow pointing to the sprayed leaves; (B) Fluorescence image of cucumber seedlings sprayed with the nanofungicide on the eighth day; (C) Fluorescence image of cucumber seedlings sprayed with the nanofungicide on the first day, superimposed with a white light image; (D) Fluorescence image of cucumber seedlings sprayed with the nanofungicide on the eighth day, superimposed with a white light image. Specific implementation method:

[0021] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0022] Example 1:

[0023] 1. Preparation of fluorescent nano fungicide:

[0024] Preparation of fluorescent mesoporous silica nanoparticles: 221.1 mg of hexadecyltrimethylammonium bromide, 0.89 g of poloxamer F127, and 5.21 g of triethanolamine were weighed and dissolved in 19 mL of ethanol and 41.6 mL of water in a 250 mL flask. 5 mL of a 0.5 mg / mL solution of tetraphenylethylene in methanol was added. The mixture was placed in a magnetic rotor and stirred overnight at 500 rpm. The flask was wrapped in tin foil to protect from light. After thorough mixing, 0.85 mL of tetraethyl orthosilicate was quickly added using a pipette at room temperature. The flask was stirred vigorously at 1200 rpm for one minute and then allowed to stand for 24 hours. 66.7 mL of 95% ethanol was added and shaken thoroughly. The mixture was transferred to a 50 mL centrifuge tube and centrifuged at 10,000 rpm for 5 minutes. The supernatant was decanted, and the ethanol solution was added again for ultrasonic dispersion and centrifugation. The mixture was then dried in a 65°C oven until ready for use. The dried solid was transferred to a flask, and 3 mL of concentrated hydrochloric acid and 50 mL of methanol were added. The mixture was refluxed in an 80°C water bath for 2 hours. The mixture was then transferred to a centrifuge tube and centrifuged to remove the supernatant. The mixture was ultrasonically dispersed and washed twice with methanol and centrifuged to obtain a dispersion. The resulting dispersion was then dried to obtain fluorescent mesoporous silica nanoparticles.

[0025] Surface functional modification: 100 mg of the above-mentioned fluorescent mesoporous silica nanoparticles were dispersed in 20 mL of methanol solution, 3 mL of (3-mercaptopropyl)trimethoxysilane (MPTMS) was added, and the mixture was stirred and reacted at room temperature for 24 h to obtain thiol-modified fluorescent mesoporous silica nanoparticles; 5 mg of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt (Sulfo-SMCC) was dissolved in phosphate buffer solution, 10 mg of glucosamine was added, and the mixture was stirred in a refrigerator at 4°C for 24 h to obtain an activated glucosamine solution; the above-mentioned thiol-modified fluorescent mesoporous silica nanoparticles were mixed with the activated glucosamine solution, stirred and reacted at room temperature overnight, and then centrifuged and washed to obtain glucosamine-modified fluorescent mesoporous silica nanoparticles.

[0026] Loading fungicide: 10 mg of the above-mentioned glucosamine-modified fluorescent mesoporous silica nanoparticles were dispersed in 50 mL of methanol, 20 mg of systemic fungicide thiabendazole solution was added and stirred for 24 hours, centrifuged and washed, and freeze-dried to obtain a bidirectionally conductive fluorescent nanofungicide.

[0027] 2. Real-time fluorescence monitoring: Then spray the above-mentioned bidirectional fluorescent nano-fungicide onto the leaves of cucumber seedlings, and place the whole plant in a living imaging instrument, adjust the excitation wavelength to 365nm, and the fluorescence collection center wavelength to 450nm to observe the imaging effect. Observe the imaging results at different time points and adjust the imaging parameters to achieve real-time monitoring of the movement of the bidirectional fluorescent nano-fungicide in the plant body (such as Figure 1 shown).

[0028] The above description is only a preferred example of the present invention and is not intended to limit the present invention. The protection scope of the present invention is not limited to this. Any obvious modification, equivalent replacement, improvement, etc. within the technical principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for real-time monitoring of nanopesticide movement within plants using in vivo imaging technology involves spraying a bidirectionally conductive fluorescent nanofungicide onto plant leaves and placing them in an in vivo imaging instrument for observation. Fluorescence imaging of the entire plant is performed using excitation and emission wavelengths selected based on the fluorescent molecules, enabling real-time monitoring of the nanofungicide's movement within the plant. The preparation method of the bidirectionally conductive fluorescent nano-bactericide is to prepare fluorescent mesoporous silica nanoparticles using fluorescent molecules with stable fluorescence properties and high quantum yield, and then modify the surface of the fluorescent mesoporous silica nanoparticles with amino acid or glucosamine functional molecules, and then load the bactericide molecules to obtain the bidirectionally conductive fluorescent nano-bactericide. The fluorescent molecule is an aggregation-induced emission molecule, specifically tetraphenylethylene; The method for preparing fluorescent mesoporous silica nanoparticles using fluorescent molecules comprises weighing cetyltrimethylammonium bromide, poloxamer F127, and triethanolamine, dissolving them in an ethanol-water solution, adding the fluorescent molecule solution, and stirring thoroughly in the dark; then adding ethyl orthosilicate, stirring vigorously, and then standing to react; after the reaction, adding the ethanol-water solution and shaking evenly, centrifuging to remove the supernatant, adding the ethanol solution again, ultrasonically dispersing, centrifuging, drying the solid, dissolving the dried solid in a hydrochloric acid-methanol solution, refluxing in a water bath, centrifuging to remove the supernatant, adding methanol, ultrasonically dispersing and washing, and drying the dispersion to obtain the fluorescent mesoporous silica nanoparticles; The surface-modified amino acid or glucosamine functional molecules are prepared by dispersing fluorescent mesoporous silica nanoparticles in methanol, adding (3-mercaptopropyl)trimethoxysilane, and stirring to react to obtain thiol-modified fluorescent mesoporous silica nanoparticles; then dissolving 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt in phosphate buffer solution, adding amino acids or glucosamine, and stirring to react to obtain an activated amino acid or glucosamine solution; and mixing the thiol-modified fluorescent mesoporous silica nanoparticles with the activated amino acid or glucosamine solution, stirring to react, and centrifuging to obtain amino acid- or glucosamine-modified fluorescent mesoporous silica nanoparticles. The loaded bactericide molecules are prepared by dispersing amino acid or glucosamine modified fluorescent mesoporous silica nanoparticles in methanol, adding bactericide molecules, mixing and stirring, centrifuging and washing, and freeze-drying to obtain a bidirectionally conductive fluorescent nano bactericide.

2. The method according to claim 1, characterized in that The excitation wavelength and emission wavelength should avoid the excitation wavelength and emission wavelength of chlorophyll in plants.

3. The method according to claim 1, characterized in that The bactericide molecule is a systemic bactericide.

4. The method according to claim 3, characterized in that The systemic fungicide is thiabendazole, carbendazim or thiophanate-methyl.

5. The method according to claim 1, wherein The amino acid is one or more of glutamic acid, serine, glycine, histidine, lysine, tryptophan, methionine, phenylalanine, proline, threonine and tyrosine.

6. The method according to claim 1, characterized in that The particle size of the bidirectionally conductive fluorescent nano bactericide ranges from 10 to 70 nm.

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