A nanopesticide Raman probe, preparation method and application thereof

Abstract

The invention discloses a nano pesticide Raman probe, a preparation method and an application thereof, which belongs to the field of SERS detection technology, and includes the following steps: S11: synthesizing negatively charged nSiO2 and GNPs; S12: modifying nSiO2 to be positively charged by a silane coupling agent, i.e. obtaining A‑nSiO2; S13: combining A‑nSiO2 and negatively charged GNPs by electrostatic adsorption force, so that a layer of GNPs is uniformly adsorbed on the surface of A‑nSiO2 to obtain GNPs‑A‑nSiO2; S14: after GNPs‑A‑nSiO2 adsorbs ferbam, the ferbam molecules fall between two adjacent GNPs particles, generating SRES signals, thereby tracking and locating nSiO2 by SRES signals. The present invention can utilize SERS to detect the signal of this Raman probe in the tea tree, thereby in situ non-destructively determining the penetration of nano pesticides in the plant body; providing help and new ideas for the subsequent research and development of pesticide preparations and the study of the interaction mechanism between nano pesticides and plants, and is worthy of being promoted and used.

Description

Technical Field

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

[0002] Surface-Enhanced Raman Scattering (SERS) greatly improves the detection sensitivity of Raman spectroscopy, and can qualitatively and quantitatively analyze samples at super-levels through unique molecular vibration fingerprints. With the development of nanomaterial technology, the application of Raman spectroscopy is becoming more and more extensive. So far, SERS has been widely used in the fields of pesticide detection, biomolecules, food safety, molecular imaging, cancer cell recognition, etc. SERS has the advantages of fast detection speed and high sensitivity, and its application in pesticide residue detection is becoming more and more extensive. In recent years, there have been many reports on the application of SERS in pesticide residue detection. In agriculture, using nanomaterials as carriers to load pesticides can protect pesticides from the influence of harsh environments, and play a role in slow-release pesticides and transporting pesticides to designated sites, while improving the chemical stability of pesticides and reducing the loss of pesticides, thereby improving the efficacy of pesticides, reducing the amount of pesticides used and reducing the burden on the environment.

[0003] The absorption and transport of nanoparticles in plants is a complex process. At present, there is no public technology for using SERS to detect nano-gold silica Raman probes loaded with pesticides to track the penetration of nanopesticides in plants. Therefore, a nanopesticide Raman probe, a preparation method and its application are proposed. Summary of the invention

[0004] The technical problem to be solved by the present invention is: how to provide assistance for the research and development of pesticide preparations and the study of the interaction mechanism between nanopesticides and plants, and provides a method for preparing a nanopesticide Raman probe.

[0005] like Figure 1 As shown, the present invention solves the above technical problems through the following technical solutions, and the present invention comprises the following steps:

[0006] S11: Synthesis of negatively charged nSiO2 and GNPs;

[0007] S12: modifying nSiO2 to be positively charged by a silane coupling agent to obtain A-nSiO2;

[0008] S13: A-nSiO2 and negatively charged GNPs are combined together through electrostatic adsorption force, so that a layer of GNPs is uniformly adsorbed on the surface of A-nSiO2 to obtain GNPs-A-nSiO2;

[0009] S14: After GNPs-A-nSiO2 adsorbs ferbam, the ferbam molecules fall between two adjacent GNPs particles, generating SRES signals, thereby tracking and locating nSiO2 through the SRES signals.

[0010] Furthermore, in step S1, the particle sizes of nSiO2 include 50nm, 100nm, and 150nm, and the synthesis process of nSiO2 with different particle sizes is as follows:

[0011] Synthesis of 50nm nSiO2: 50mL of anhydrous ethanol, 1mL of 1680ppm lithium hydroxide monohydrate, 2mL of ammonia water, and 1mL of TEOS were added to a beaker in sequence, and stirred at room temperature for 6h. After the reaction was completed, it was centrifuged and washed three times with deionized water, and dried at 60℃ overnight to obtain 50nm solid nSiO2;

[0012] Synthesis of 100nm nSiO2: 50mL of anhydrous ethanol, 1mL of 1680ppm lithium hydroxide monohydrate, 2.5mL of ammonia water, and 1mL of TEOS were added to a beaker in sequence, and stirred at room temperature for 6h. After the reaction was completed, it was centrifuged and washed three times with deionized water, and dried at 60℃ overnight to obtain 100nm solid nSiO2;

[0013] Synthesis of 150nm nSiO2: Add 50mL of anhydrous ethanol, 1mL of 2100ppm lithium hydroxide monohydrate, 3mL of ammonia water, and 1mL of TEOS into a beaker in sequence, and stir at room temperature for 6h. After the reaction is completed, wash it with deionized water by centrifugation for 3 times, and dry it at 60℃ overnight to obtain 150nm solid nSiO2.

[0014] Furthermore, in the step S11, the synthesis process of GNPs is as follows: under vibration stirring, 1 mL of citrate solution is added to 98.9 mL of ultrapure water, heated on a heating table, 100 μL of chloroauric acid solution is added, and after cooling on ice, GNPs particles with a diameter of 15 nm are obtained.

[0015] Furthermore, in the step S12, the method of modifying nSiO2 to be positively charged by a silane coupling agent is as follows: 5 mg of nSiO2 of different particle sizes are ultrasonically dispersed in 50 mL of ethanol, 50 μL of APTMS is added, and the mixture is heated to reflux in an oil bath under set temperature conditions for a set time. After the reaction of each solution is completed, the mixture is centrifuged and washed twice with deionized water and ethanol respectively, and then dried at 40°C overnight to obtain A-nSiO2 of different particle sizes; A-nSiO2 of different particle sizes are dispersed in deionized water, and the zeta potential and particle size are measured using a particle size analyzer.

[0016] Furthermore, in the step S13, the specific process of obtaining GNPs-A-nSiO2 is as follows: 5 mg A-nSiO2 is ultrasonically dispersed in 2 mL of ethanol, and the solution is added dropwise into 20 mL of a 15 nm GNPs solution that is constantly stirred. After the addition is completed, the solution is stirred for 2 hours, centrifuged at 14000 r / min for 10 minutes, washed three times by centrifugation with deionized water, and dispersed in 300 μL of deionized water to obtain GNPs-A-nSiO2 of different particle sizes.

[0017] Furthermore, in the step S14, the specific process of GNPs-A-nSiO2 adsorbing ferbam is: accurately weigh 2.5 mg of ferbam standard, dilute it with deionized water in a 50 mL volumetric flask to obtain a 50 ppm ferbam solution; take 1 mL of GNPs-A-nSiO2 and 1 mL of ferbam solution and shake them in a constant temperature oscillator for 2 hours to obtain a nano-gold silica Raman probe loaded with ferbam.

[0018] The present invention also provides a nano pesticide Raman probe, which is prepared by the above-mentioned nano pesticide Raman probe preparation method.

[0019] The present invention also provides an application of a nanopesticide Raman probe, using SERS to detect the signal of the Raman probe in the tea plant, and then determining the penetration of the nanopesticide in the plant. The specific application steps are as follows:

[0020] S21: The synthesized GNPs and GNPs-A-nSiO2 loaded with ferbam were dripped onto the leaves and roots of tea plants for Raman testing, and multiple acquisition times were set to acquire Raman scanning spectra;

[0021] S22: 20 μL of ferbam-loaded nano-gold silica Raman probe solutions of different particle sizes were dripped on the upper and lower surfaces of young and old leaves of living tea trees, respectively, and completely dried after waiting for 1 hour at room temperature, which was recorded as 0 hours; the two-dimensional surface signals of the ferbam-loaded nano-gold silica Raman probe solutions dripped on the upper and lower surfaces of tea leaves at 0 hours and the two-dimensional deep signals on the tea leaves after 0, 1, 2, 3, 4, and 5 hours were respectively measured in situ to observe its penetration in the tea leaves;

[0022] S23: The newborn roots of tea trees were placed in 200 μL of nano-gold silica Raman probe solutions loaded with ferbam of different sizes, and the roots of the same part were taken at 0, 1, 2, 3, 4, and 5 h, respectively. After freeze-drying for 48 h, the two-dimensional deep signals of the roots of the tea trees were measured at 0, 1, 2, 3, 4, and 5 h to observe its penetration in the roots of the tea trees;

[0023] S24: Take microscopic images of the tea leaves before and 5 hours after the measurement in step S22; drop 20 μL of 100nm of nano-gold silica Raman probe solution loaded with ferbam on the lower surface of the living tea tree leaves, and wait for 1 hour at room temperature to completely dry, marked as 0h, and measure the two-dimensional deep signal on the tea leaves 5 hours later to observe its penetration in the tea leaves; drop 20 μL of 100nm of nano-gold silica Raman probe solution loaded with ferbam on the lower surface of the living tea tree leaves, and wait for 1 hour at room temperature to completely dry, marked as 0h, and wash off the surface nanoprobe solution after 5 hours, then measure the two-dimensional deep signal on the tea leaves to observe its penetration in the tea leaves.

[0024] Furthermore, in step S22, the young leaves are the first leaves and the old leaves are the third leaves.

[0025] Compared with the prior art, the present invention has the following advantages: the nanopesticide Raman probe, preparation method and application can use SERS to detect the signal of the Raman probe in the tea plant, thereby in situ non-destructively measuring the penetration of nanopesticides in the plant. It provides help and new ideas for the subsequent research and development of pesticide preparations and the study of the interaction mechanism between nanopesticides and plants, and is worthy of promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the process of preparing the nano-pesticide Raman probe of the present invention;

[0027] Figure 2 A is a TEM image of 50 nm nSiO2 in an embodiment of the present invention;

[0028] Figure 2 B is a TEM image of 100 nm nSiO2 in an embodiment of the present invention;

[0029] Figure 2 C is a TEM image of 150 nm nSiO2 in an embodiment of the present invention;

[0030] Figure 3 A is the TEM image of 50 nm GNPs-A-nSiO2 in an embodiment of the present invention;

[0031] Figure 3 B is a TEM image of 100 nm GNPs-A-nSiO2 in an embodiment of the present invention;

[0032] Figure 3 C is a TEM image of 150 nm GNPs-A-nSiO2 in an embodiment of the present invention;

[0033] Figure 4A is the particle size distribution diagram of GNPs-A-nSiO2 before centrifugation 3 times at 14000 r / min for 10 min in the embodiment of the present invention;

[0034] Figure 4 B is the particle size distribution diagram of GNPs-A-nSiO2 after centrifugation at 14000 r / min for 10 min three times in the embodiment of the present invention;

[0035] Figure 5 A is a comparative Raman spectrum of ferbam enhancement by 15 nm GNPs in the embodiment of the present invention and commercially synthesized 15 nm AuNPs;

[0036] Figure 5 B is a comparative Raman spectrum of ferbam enhancement by 15 nm GNPs and GNPs-A-nSiO2 of different sizes in the embodiment of the present invention;

[0037] Figure 6 A is a comparative Raman spectrum of ferbam enhancement by 50 nm GNPs-A-nSiO2 on gold plate, tea leaves and tea roots in the embodiment of the present invention;

[0038] Figure 6 B is a comparative Raman spectrum of 100 nm GNPs-A-nSiO2 on gold plate, tea leaves and tea roots for the enhancement of ferbam in an embodiment of the present invention;

[0039] Figure 6 C is a comparative Raman spectrum of ferbam enhancement by 150 nm GNPs-A-nSiO2 on gold plate, tea leaves and tea roots in the embodiment of the present invention;

[0040] Figure 7 A is a comparative Raman spectrum of ferbam enhancement on tea leaves by GNPs-A-nSiO2 with 50 nm, 100 nm and 150 nm in the embodiment of the present invention;

[0041] Figure 7 B is a comparative Raman spectrum of ferbam enhancement by GNPs-A-nSiO2 of 50 nm, 100 nm and 150 nm in the embodiment of the present invention at the root of tea tree;

[0042] Figure 8 is the Raman spectra of Ferbam@GNPs-A-nSiO2 in tea leaves at different collection times in the embodiment of the present invention;

[0043] Fig. 9 A1 is the 2D surface signal diagram of 50nm Ferbam@GNPs-A-nSiO2 on the surface of tea tree tender leaves at 0h and the 2D deep signal diagram at 0, 1, 2, 3, 4, and 5h in the embodiment of the present invention (point a is the point selected by the 2D deep scan);

[0044] Fig. 9 A2 is the 2D surface signal diagram of 50 nm Ferbam@GNPs-A-nSiO2 on the lower surface of tea leaves at 0 h and the 2D deep signal diagram at 0, 1, 2, 3, 4, and 5 h (point a is the point selected for the 2D deep scan) in the embodiment of the present invention;

[0045] Fig. 9 A3 is the 2D surface signal diagram of 50 nm Ferbam@GNPs-A-nSiO2 on the surface of old leaves at 0 h and the 2D deep signal diagram at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention (point a is the point selected for the 2D deep scan);

[0046] Fig. 9 A4 is the 2D surface signal diagram of 50 nm Ferbam@GNPs-A-nSiO2 on the lower surface of old leaves at 0 h and the 2D deep signal diagram at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention (point a is the point selected for the 2D deep scan);

[0047] Fig. 9 B1 is the 2D surface signal diagram of 100 nm Ferbam@GNPs-A-nSiO2 on the surface of tea tree tender leaves at 0 h and the 2D deep signal diagram at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention (point a is the point selected by the 2D deep scan);

[0048] Fig. 9 B2 is the 2D surface signal diagram of 100 nm Ferbam@GNPs-A-nSiO2 on the lower surface of tea leaves at 0 h and the 2D deep signal diagram at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention (point a is the point selected by the 2D deep scan);

[0049] Fig. 9 B3 is the 2D surface signal diagram of 100 nm Ferbam@GNPs-A-nSiO2 on the surface of old leaves at 0 h and the 2D deep signal diagram at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention (point a is the point selected for the 2D deep scan);

[0050] Fig. 9 B4 is the 2D surface signal diagram of 100 nm Ferbam@GNPs-A-nSiO2 on the lower surface of old leaves at 0 h and the 2D deep signal diagram at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention (point a is the point selected for the 2D deep scan);

[0051] Fig. 9C1 is the 2D surface signal diagram of 150 nm Ferbam@GNPs-A-nSiO2 on the surface of tea tree tender leaves at 0 h and the 2D deep signal diagram at 0, 1, 2, 3, 4, and 5 h (point a is the point selected by the 2D deep scan) in the embodiment of the present invention;

[0052] Fig. 9 C2 is the 2D surface signal diagram of 150nm Ferbam@GNPs-A-nSiO2 on the lower surface of tea leaves at 0h and the 2D deep signal diagram at 0, 1, 2, 3, 4, and 5h in the embodiment of the present invention (point a is the point selected by the 2D deep scan);

[0053] Fig. 9 C3 is the 2D surface signal diagram of 150 nm Ferbam@GNPs-A-nSiO2 on the surface of old leaves at 0 h and the 2D deep signal diagram at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention (point a is the point selected for the 2D deep scan);

[0054] Fig. 9 C4 is the 2D surface signal diagram of 150 nm Ferbam@GNPs-A-nSiO2 on the lower surface of old leaves at 0 h and the 2D deep signal diagram at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention (point a is the point selected for the 2D deep scan);

[0055] Fig.10 A1 is the Raman spectrum of the strongest point of the two-dimensional deep signal of 50nm Ferbam@GNPs-A-nSiO2 on the surface of tea leaves at 0, 1, 2, 3, 4, and 5h in the embodiment of the present invention;

[0056] Fig.10 A2 is the Raman spectrum of the strongest point of the two-dimensional deep signal of 50nm Ferbam@GNPs-A-nSiO2 on the lower surface of tea leaves at 0, 1, 2, 3, 4, and 5h in the embodiment of the present invention;

[0057] Fig.10 A3 is the Raman spectrum of the strongest point of the two-dimensional deep signal of 50nm Ferbam@GNPs-A-nSiO2 on the upper surface of old leaves at 0, 1, 2, 3, 4, and 5h in the embodiment of the present invention;

[0058] Fig.10 A4 is the Raman spectrum of the strongest point of the two-dimensional deep signal of 50 nm Ferbam@GNPs-A-nSiO2 on the lower surface of old leaves at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention;

[0059] Fig.10B1 is the Raman spectrum of the strongest point of the two-dimensional deep signal of 100 nm Ferbam@GNPs-A-nSiO2 on the upper surface of tea leaves at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention;

[0060] Fig.10 B2 is the Raman spectrum of the strongest point of the two-dimensional deep signal of 100 nm Ferbam@GNPs-A-nSiO2 on the lower surface of tea leaves at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention;

[0061] Fig.10 B3 is the Raman spectrum of the strongest point of the two-dimensional deep signal of 100 nm Ferbam@GNPs-A-nSiO2 on the upper surface of old leaves at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention;

[0062] Fig.10 B4 is the Raman spectrum of the strongest point of the two-dimensional deep signal of 100 nm Ferbam@GNPs-A-nSiO2 on the lower surface of old leaves at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention;

[0063] Fig.10 C1 is the Raman spectrum of the strongest point of the two-dimensional deep signal of Ferbam@GNPs-A-nSiO2 with a diameter of 150 nm on the surface of tea leaves at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention;

[0064] Fig.10 C2 is the Raman spectrum of the strongest point of the two-dimensional deep signal of Ferbam@GNPs-A-nSiO2 with a diameter of 150 nm on the lower surface of tea leaves at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention;

[0065] Fig.10 C3 is the Raman spectrum of the strongest point of the two-dimensional deep signal of Ferbam@GNPs-A-nSiO2 with a diameter of 150 nm on the upper surface of old leaves at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention;

[0066] Fig.10 C4 is the Raman spectrum of the strongest point of the two-dimensional deep signal of 150nm Ferbam@GNPs-A-nSiO2 on the lower surface of old leaves at 0, 1, 2, 3, 4, and 5h in the embodiment of the present invention;

[0067] Fig.11 A is a Raman spectrum scan of 50nm 5h Ferbam@GNPs-A-nSiO2 on the lower surface of tea leaves in the embodiment of the present invention;

[0068] Fig.11B is a Raman spectrum scan of 100nm 5h Ferbam@GNPs-A-nSiO2 on the lower surface of tea leaves in the embodiment of the present invention;

[0069] Fig.11 C is a Raman spectrum scan of the lower surface of tea tree leaves of Ferbam@GNPs-A-nSiO2 with a diameter of 150 nm and a length of 5 h in the embodiment of the present invention;

[0070] Fig.11 D is a Raman spectrum scan of a blank leaf in an embodiment of the present invention;

[0071] Fig.12 A is a two-dimensional deep signal graph of 50 nm Ferbam@GNPs-A-nSiO2 in the root of tea tree at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention;

[0072] Fig.12 B is a two-dimensional deep signal diagram of 100 nm Ferbam@GNPs-A-nSiO2 in the root of tea tree at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention;

[0073] Fig.12 C is a two-dimensional deep signal graph of 150 nm Ferbam@GNPs-A-nSiO2 in the tea tree root at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention;

[0074] Fig.13 A is the Raman spectrum of the strongest point of the two-dimensional deep signal of 50nm Ferbam@GNPs-A-nSiO2 in the root of tea tree at 0, 1, 2, 3, 4, and 5h in the embodiment of the present invention;

[0075] Fig.13 B is the Raman spectra of the strongest point of the two-dimensional deep signal of 100 nm Ferbam@GNPs-A-nSiO2 in the tea tree root at 0, 1, 2, 3, 4, and 5 h in the embodiment of the present invention;

[0076] Fig.13 C is the Raman spectrum of the strongest point of the two-dimensional deep signal of 150nm Ferbam@GNPs-A-nSiO2 in the root of tea tree at 0, 1, 2, 3, 4, and 5h in the embodiment of the present invention;

[0077] Fig.14 A is a Raman spectrum scan of Ferbam@GNPs-A-nSiO2 with a thickness of 50 nm and 5 h at the root of tea tree in the embodiment of the present invention;

[0078] Fig.14B is a Raman spectrum scan of 100nm 5h Ferbam@GNPs-A-nSiO2 in the root of tea tree in the embodiment of the present invention;

[0079] Fig.14 C is a Raman spectrum scan of Ferbam@GNPs-A-nSiO2 with a size of 150 nm and 5 h in the root of tea tree in the embodiment of the present invention;

[0080] Fig.14 D is a Raman spectrum layer scan of a blank root in an embodiment of the present invention;

[0081] Fig.15 A is a microscopic image of tea leaves before Raman deep scanning in an embodiment of the present invention;

[0082] Fig.15 B is a microscopic image of a tea leaf after 5 hours of Raman deep scanning in an embodiment of the present invention;

[0083] Fig.15 C is a Raman spectrum scan of 100 nm Ferbam@GNPs-A-nSiO2 in the embodiment of the present invention after penetration into the lower surface of tea leaves for 5 hours;

[0084] Fig.15 D is a Raman spectrum scan of Ferbam@GNPs-A-nSiO2 in an embodiment of the present invention after 100 nm Ferbam@GNPs-A-nSiO2 penetrates into the lower surface of tea leaves for 5 hours and then washes away the surface Ferbam@GNPs-A-nSiO2. DETAILED DESCRIPTION

[0085] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0086] This embodiment provides a technical solution: a method for preparing a nano-pesticide Raman probe, that is, the preparation and characterization process of a nano-gold silica Raman probe loaded with ferbam (Ferbam@GNPs-A-nSiO2), comprising the following steps: firstly synthesizing negatively charged nSiO2 and GNPs, then modifying nSiO2 to a positive charge (A-nSiO2) by a silane coupling agent, and acting together with negatively charged GNPs through electrostatic adsorption force, so that a layer of GNPs is uniformly adsorbed on the surface of A-nSiO2 to obtain GNPs-A-nSiO2. After GNPs-A-nSiO2 adsorbs ferbam, the ferbam molecule falls between two adjacent GNPs particles, generating an SRES signal, so that nSiO2 can be tracked and located by the SRES signal.

[0087] In this embodiment, the synthesis process of nSiO2 with different particle sizes is as follows:

[0088] ① Synthesis of 50nm nSiO2: Add 50mL of anhydrous ethanol, 1mL of 1680ppm lithium hydroxide monohydrate, 2mL of ammonia water, and 1mL of TEOS into a beaker in sequence and stir at room temperature (25℃) for 6h.

[0089] ② Synthesis of 100nm nSiO2: Add 50mL of anhydrous ethanol, 1mL of 1680ppm lithium hydroxide monohydrate, 2.5mL of ammonia water, and 1mL of TEOS into a beaker in sequence and stir at room temperature (25℃) for 6h.

[0090] ③Synthesis of 150nm nSiO2: Add 50mL of anhydrous ethanol, 1mL of 2100ppm lithium hydroxide monohydrate, 3mL of ammonia water, and 1mL of TEOS into a beaker in sequence and stir at room temperature (25℃) for 6h.

[0091] After the reaction of the above solutions was completed, they were washed three times by centrifugation with deionized water and dried at 60°C overnight to obtain solid nSiO2 with different particle sizes.

[0092] In this embodiment, the modification conditions of nSiO2 are shown in Table 1 below:

[0093] Table 1 Different modification conditions of nSiO2

[0094]

[0095] After consulting the literature, it was found that APTMS, APTES and KH590 are the most commonly used silane coupling agents for modifying silica nanoparticles. First, the modification effects of these three silane coupling agents on the synthesized silica nanoparticles were compared. Under the same conditions, the modification effect of APTMS was the best, so APTMS was used as the modification material in the subsequent reaction. Weigh the above 5 mg of nSiO2 with different particle sizes and ultrasonically disperse them in 50 mL of ethanol, add 50 μL of APTMS, and heat and reflux them in a heat-collecting constant temperature heating magnetic stirrer oil bath at 30, 50, 70, 90, and 110 ° C for 2, 4, 6, and 8 hours. After the reaction of the above solutions is completed, they are centrifuged and washed twice with deionized water and ethanol (14000r / min, 10min), and then dried at 40 ° C overnight to obtain modified nSiO2 (A-nSiO2) with different particle sizes. Modified nSiO2 with different particle sizes were dispersed in deionized water, and the zeta potential and particle size were measured using a ZETA potential and nanoparticle size analyzer.

[0096] In this embodiment, the preparation process of gold nanoparticles (GNPs) is as follows:

[0097] Under vibration stirring, add 1 mL of citrate solution (30 mg mL -1 ), heated on a heating table, quickly added 100 μL of chloroauric acid solution (100 mM), and quickly cooled on ice to obtain gold nanoparticles with a diameter of 15 nm.

[0098] In this embodiment, the synthesis process of the nano-gold silicon dioxide composite material (GNPs-A-nSiO2) is as follows:

[0099] Take the above 5 mg A-nSiO2 and ultrasonically disperse it in 2 mL of ethanol, and add it drop by drop into 20 mL of 15 nm GNPs solution which is constantly stirred. After the addition is completed, stir for 2 hours, centrifuge at 14000 r / min for 10 minutes, wash it by centrifugation with deionized water for 3 times, and disperse it in 300 μL of deionized water to obtain nano-gold silica composites (GNPs-A-nSiO2) with different particle sizes.

[0100] In this embodiment, the nSiO2 and GNPs-A-nSiO2 structures were characterized by observing the sample structure using a Hitachi HT7700 transmission electron microscope (TEM) at a voltage of 80 kV.

[0101] In this embodiment, the specific process of GNPs-A-nSiO2 adsorbing ferbam is as follows:

[0102] Accurately weigh 2.5 mg of ferbam standard and dilute it to a 50 mL volumetric flask with deionized water to obtain a 50 ppm ferbam solution. Take 1 mL of GNPs-A-nSiO2 and 1 mL of ferbam solution and shake them in a constant temperature oscillator (30°C, 300r) for 2 h to obtain a nano-gold silica Raman probe loaded with ferbam (Ferbam@GNPs-A-nSiO2).

[0103] In this embodiment, the SERS characterization process of Ferbam@GNPs-A-nSiO2 is as follows:

[0104] The synthesized GNPs and GNPs-A-nSiO2 loaded with ferbam were dropped onto a gold plate and the samples were collected under the following conditions: laser: 785 nm-Edge, objective: ×50-VIS-LVD, acquisition time: 5 s, acquisition times: 5, laser power: 5%, acquisition range: 400-2000 cm -1 Raman tests were performed on it under the conditions of

[0105] This embodiment also provides an application of a nano-silica Raman probe loaded with ferbam, which mainly measures the absorption and penetration of the nano-silica Raman probe loaded with ferbam (Ferbam@GNPs-A-nSiO2) in a tea plant, specifically including the following process:

[0106] S1: SERS characterization of Ferbam@GNPs-A-nSiO2 on the surface of tea tree

[0107] The synthesized GNPs and GNPs-A-nSiO2 loaded with ferbam were added dropwise to the leaves and roots of tea plants. The laser was 785 nm-Edge, objective lens was ×50-VIS-LVD, acquisition time was 5 s, acquisition times was 5, laser power was 5%, acquisition range was 400-2000 cm -1 Raman tests were performed on it under the conditions of

[0108] S2: Selection of Raman deep scanning time

[0109] In order to shorten the scanning time while measuring the deep scanning signal of plant tissue, 14 acquisition times (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5s) were set to collect Raman scanning spectra. Laser: 785nm-Edge, objective: ×50-VIS-LVD, acquisition times: 1, laser power: 5%, acquisition range: 400-2000cm -1 Raman tests were performed on it under the conditions of

[0110] S3: Absorption and penetration of Ferbam@GNPs-A-nSiO2 in tea leaves

[0111] 20 μL of Ferbam@GNPs-A-nSiO2 of different sizes were dripped on the upper and lower surfaces of young leaves (the first leaf that just unfolded) and old leaves (the third leaf) of living tea trees, and waited for 1 hour at room temperature to completely dry, which was recorded as 0h. The two-dimensional surface signal of Ferbam@GNPs-A-nSiO2 dripped on the upper and lower surfaces of tea leaves at 0h and the two-dimensional deep signal on the tea leaves after 0, 1, 2, 3, 4, and 5h were measured in situ to observe its penetration in tea leaves. Raman parameters: laser: 785nm-Edge, objective lens: ×50-VIS-LVD, acquisition time: 1s, acquisition times: 1, laser power: 5%, acquisition range: 400-2000cm -1 . A 2D surface map was generated using 100 scan points (10 points each on the X-axis and Y-axis), with each image containing 100 points and each point at a distance of 10 μm. A 2D depth map was generated by five scan points on the X-axis and 30 scan points on the Z-axis (depth: 0-290 μm), with each image containing 150 points and each point at a distance of 10 μm.

[0112] S4: Absorption and penetration of Ferbam@GNPs-A-nSiO2 in tea tree roots

[0113] The new roots of tea trees were placed in 200 μL of Ferbam@GNPs-A-nSiO2 solutions of different sizes. The roots of the same part were taken at 0, 1, 2, 3, 4, and 5 hours. After freeze-drying for 48 hours, the two-dimensional deep signals of the roots of tea trees at 0, 1, 2, 3, 4, and 5 hours were measured to observe its penetration in the roots of tea trees. Raman parameters: laser: 785nm-Edge, objective: ×50-VIS-LVD, acquisition time: 1s, acquisition times: 1, laser power: 25%, acquisition range: 400-2000cm -1 The 2D depth map was generated by five scanning points on the X-axis and 30 scanning points on the Z-axis (depth: 0–290 μm), and each image contained 150 points with a distance of 10 μm for each point.

[0114] S5: Ferbam@GNPs-A-nSiO2 Raman probe penetration tracing verification experiment in tea trees

[0115] Take microscopic images of tea leaves before and 5 hours after the measurement in step S3. Add 20 μL of 100 nm Ferbam@GNPs-A-nSiO2 to the lower surface of the young leaves of a living tea tree (the first leaf that has just unfolded), and wait for 1 hour at room temperature to completely dry, marked as 0 hours, and measure the two-dimensional deep signal on the tea leaves 5 hours later to observe its penetration in the tea leaves. Add 20 μL of 100 nm Ferbam@GNPs-A-nSiO2 to the lower surface of the young leaves of a living tea tree (the first leaf that has just unfolded), and wait for 1 hour at room temperature to completely dry, marked as 0 hours, and wash off the surface nanoprobes after 5 hours. Measure the two-dimensional deep signal on the tea leaves and observe its penetration in the tea leaves, where the Raman parameters are the same as step S3.

[0116] Results and analysis in this embodiment:

[0117] 1. Characterization of ferbam-loaded nano-gold silica Raman probe (Ferbam@GNPs-A-nSiO2):

[0118] 1.1. Structural characterization of nSiO2 with different particle sizes

[0119] Agglomeration of nano-silicon dioxide is a common problem in the synthesis of nano-silicon dioxide. Agglomeration will make the actual application size of nano-silicon dioxide larger than the size of a single material, thus failing to exert the expected size effect. Three different sizes of nSiO2, 50nm, 100nm and 150nm, were synthesized, and TEM images of the synthesized nSiO2 were measured ( Figure 2The three sizes of nSiO2 synthesized all have good uniformity and dispersion, without agglomeration and adhesion, and can represent the size characteristics of 50, 100, and 150 nm silica, and can be used for the next adsorption tracer experiment.

[0120] 1.2 Modification of nSiO2

[0121] Zeta potential can be used to characterize the stability of colloidal dispersion. The higher the absolute value of zeta potential, the stronger the stability of the system. Since the synthesized gold nanoparticles are negatively charged, the higher the charge of the modified nSiO2, the better the gold adsorption effect. The zeta potential of the synthesized GNPs is -17.8mV and the diameter is 15nm. The modification results of nSiO2 by different modification methods are shown in Table 2. The modified nSiO2 after adding 50μL APTMS and heating and refluxing in a 90℃ oil bath for 6h has the highest zeta potential and the lowest radius, indicating that the nSiO2 obtained by this modification method has the best dispersibility and adsorption effect on gold nanoparticles. Therefore, the modified nSiO2 obtained by this modification method was used for adsorption and tracing experiments.

[0122] Table 2 Modification results of nSiO2 by different modification methods

[0123]

[0124] 1.3 Structural characterization of GNPs-A-nSiO2 with different particle sizes

[0125] TEM results of synthesized 50nm, 100nm and 150nm GNPs-A-nSiO2 were measured ( Figure 3 ), 15nm GNPs can be adsorbed on these three sizes of A-nSiO2 by electrostatic adsorption, and these three sizes of GNPs-A-nSiO2 have good dispersibility.

[0126] Since GNPs are tightly bound to the surface of nSiO2, and GNPs have a Raman enhancement effect on ferbam, the position of the detected ferbam Raman signal represents the position of nSiO2, so the nanopesticide can be located by Raman tracking. However, if there are free GNPs in the system, free gold will also bind to ferbam, thereby generating Raman signals and interfering with the results. Therefore, centrifugation was used to remove free gold to ensure the accuracy of the experimental results. The sample was centrifuged 3 times with deionized water (14000r / min, 10min) before ( Figure 4 A) After Figure 4B) for comparison. It can be seen from the figure that the particle size distribution of GNPs-A-nSiO2 before centrifugation is around 15nm, indicating that there are free GNPs in the system. After centrifugation, the particle size is only distributed around 100nm, indicating that centrifugation can effectively remove free GNPs in the system.

[0127] 1.4. SERS characterization of Ferbam@GNPs-A-nSiO2 with different particle sizes

[0128] The mixed fluorescence intensity of ferbam and gold nanoparticles was 448, 551, 945, 1139 and 1371 cm -1 There is a Raman characteristic peak at 1371cm -1 The highest peak at 1371 cm comes from the mixed vibration of CN and C=S stretching, as well as the symmetrical CH3 deformation. -1 The peak intensity of the Raman characteristic peak at is used to indicate the Raman signal enhancement effect of the material on ferbam.

[0129] The Raman enhancement effect of synthesized GNPs and GNPs-A-nSiO2 on ferbam was determined and compared. The Raman signal enhancement results of synthesized 15nm GNPs and commercial 15nm AuNPs on ferbam were compared ( Figure 5 A), Ferbam@15nmGNPs at 1371cm -1 The Raman peak intensity at 397.195 is Ferbam@15nm AuNPs at 1371cm -1 The Raman peak intensity at is 313.016. From the results, it can be seen that compared with ferbam, both the synthesized 15nm GNPs and the commercial 15nm AuNPs have Raman enhancement effects on ferbam, and the enhancement effect of the synthesized 15nm GNPs on ferbam is better than that of the commercial 15nm AuNPs.

[0130] Comparison of Raman signal enhancement of ferbam by synthesized GNPs-A-nSiO2 of different sizes ( Figure 5 B), 15nm GNPs and 50nm, 100nm and 150nm GNPs-A-nSiO2 at 1371cm -1The Raman peak intensities at the locations are 397.195, 261.241, 287.135 and 249.467 respectively. The results show that the synthesized GNPs-A-nSiO2 of different sizes have Raman enhancement effects on ferbam, and are weaker than 15nm GNPs. The enhancement effects are from large to small, 100nm, 50nm, and 150nm, and the difference in enhancement effects is not large. The analysis reasons are as follows: SERS active nanostructures can be divided into three generations of SERS hotspots. The first generation of SERS hotspots is generated by a single nanomaterial, the second generation of SRES hotspots is generated in the coupled nanostructure of nanomaterial gaps or cross-nanogaps, and the third generation of SRES hotspots is generated by mixed structures. The GNPs adsorbed on the surface of GNPs-A-nSiO2 are not very uniform and dense, so the gap structure of nanogold becomes less, resulting in a reduction in the second generation of SRES hotspots, which makes the Raman enhancement effect of GNPs-A-nSiO2 weaker than that of 15nm GNPs. A-nSiO2 and GNPs are adsorbed by electrostatics. The surface charge of 50nm, 100nm and 150nm A-nSiO2 is roughly 5.07mV, so the number of GNPs adsorbed by A-nSiO2 of different sizes should be roughly the same. The size of 150nm nSiO2 is larger, so when the same number of GNPs are adsorbed on the surface, the distance between gold and gold will also be larger, resulting in fewer gap structures of nano-gold, resulting in fewer second-generation SRES hotspots and weakened Raman signals. The size difference between 50nm nSiO2 and GNPs is small, so the number of GNPs adsorbed on the surface will also be relatively reduced, resulting in a weakening of the Raman signal.

[0131] 2. Penetration tracing of ferbam-loaded nano-silica Raman probe (Ferbam@GNPs-A-nSiO2) in tea plants:

[0132] 2.1. SERS characterization of Ferbam@GNPs-A-nSiO2 on the surface of tea plants

[0133] The differences in the composition and structure of tea leaf and root tissues will cause differences in the Raman signal intensity of Ferbam@GNPs-A-nSiO2, and the tea tree contains a large number of Raman interfering substances. For example, Raman spectroscopy can be used to detect carotenoids, so tea leaves and roots have strong Raman interference. Therefore, before the penetration tracer experiment, it is necessary to measure the Raman signals of the synthesized Ferbam@GNPs-A-nSiO2 Raman probes of different sizes in tea leaves and roots ( Figure 6 ). Ferbam@GNPs-A-nSiO2 Raman probes of different sizes can detect Raman signals in tea leaves and roots. The 50 nm Ferbam@GNPs-A-nSiO2 Raman probes on the gold plate have a Raman signal of 1371 cm -1The Raman peak intensity is 261.241 at the blade, 95.857 at the root, and 120.682 at the root. The 100nm Raman peak on the gold plate is 1371cm -1 The Raman peak intensity is 287.135 at the blade, 100.737 at the root, and 124.845 at the root. The 150nm Raman peak on the gold plate is 1371cm -1 The Raman peak intensity at the root is 249.467, on the blade is 83.890, and at the root is 116.161.

[0134] The Raman signals of Ferbam@GNPs-A-nSiO2 in tea leaves and roots are significantly different from those of blank tea leaves and roots ( Figure 7 ), the enhancement effect of Ferbam@GNPs-A-nSiO2 on tea leaves and roots is from large to small 100nm, 50nm, 150nm, which is consistent with the trend on the gold plate, and the enhancement effect is not much different. Therefore, the synthesized Ferbam@GNPs-A-nSiO2 Raman probes of different sizes can be used for subsequent penetration tracer detection.

[0135] 2.2. Selection of Raman deep scanning time

[0136] In order to establish a fast in-situ Raman deep scanning method, a shorter method should be selected under the premise of satisfying the good Raman signal. A total of 14 acquisition times (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5s) were set to collect the Raman scanning spectra of Ferbam@GNPs-A-nSiO2 on the surface of tea leaves ( Figure 8 . The results show that as the scanning time increases, the Raman signal increases. At 0.1s, the noise of the leaves is large, the interference is strong, and the Raman signal is very weak. From 0.4s, the Raman peak gradually becomes obvious. The sampling interval is 1h, and the interference in the deep layer of the tea tree tissue is large, which will cause the Raman signal to be weaker than the surface of the tissue. The longer the scanning time, the better the Raman signal. Therefore, it is necessary to control the total acquisition time within 1h and select the time scan with stronger Raman signal. In summary, in order to shorten the scanning time of each layer and make the Raman signal better, the acquisition time is 1s, and the total acquisition time of 150 points is 55min.

[0137] 2.3. Penetration tracing of Ferbam@GNPs-A-nSiO2 in tea leaves

[0138] The penetration tracing results of Ferbam@GNPs-A-nSiO2 Raman probe in tea leaves were determined by Raman spectroscopy. Fig. 9). At the same time, in order to verify the accuracy of the measured two-dimensional deep scanning results, the Raman spectrum of the strongest point of the two-dimensional deep signal of Ferbam@GNPs-A-nSiO2 was selected ( Fig.10 ), it can be seen from the Raman spectrum that the measured signals are all SERS signals of ferbam, indicating that Fig. 9 The penetration results are the penetration tracing results of the Raman probe in tea leaves, which verifies the correctness of the experimental results.

[0139] Fig. 9 A1 shows that the 50nm Ferbam@GNPs-A-nSiO2 Raman probe has penetrated to 250μm after being dripped on the upper surface of the young leaf for 5h. A2 shows that the 50nm Ferbam@GNPs-A-nSiO2 Raman probe has penetrated to 300μm after being dripped on the lower surface of the young leaf for 4h. B1 shows that the 100nm Ferbam@GNPs-A-nSiO2 Raman probe has penetrated to 150μm after being dripped on the upper surface of the young leaf for 5h. B2 shows that the 100nm The Ferbam@GNPs-A-nSiO2 Raman probe penetrated to 300μm after being dropped on the lower surface of young leaves for 5 hours. C1 and C2 showed that the 150nm Ferbam@GNPs-A-nSiO2 Raman probe had no penetration after being dropped on the upper and lower surfaces of young leaves for 5 hours. A3-C4 showed that the 50nm, 100nm and 150nm Ferbam@GNPs-A-nSiO2 Raman probes had no penetration after being dropped on the upper and lower surfaces of old tea leaves for 5 hours.

[0140] Ferrobam itself has no penetration ability, but after applying Ferbam@GNPs-A-nSiO2 on tea leaves, the SERS signal of ferobam can be detected inside, indicating that nano-silica can change the penetration behavior of ferobam, turning it from a non-systemic pesticide to a systemic pesticide. Fig. 9It can be seen that 50nm and 100nm Ferbam@GNPs-A-nSiO2 can penetrate downward on the upper and lower surfaces of the young leaves of tea trees over time, and the penetration rate of 50nm Ferbam@GNPs-A-nSiO2 is faster than that of 100nm, and the penetration rate of the lower surface is faster than that of the upper surface. 150nm Ferbam@GNPs-A-nSiO2 has no penetration in the young leaves of tea trees, and Ferbam@GNPs-A-nSiO2 of three sizes has no penetration in the old leaves of tea trees. The reason for this may be that there are stomata on the lower surface of the leaves, and the nanomaterials enter the interior of the leaves through the stomata, thereby accelerating the absorption and penetration of the nanomaterials. Both 50nm and 100nm Ferbam@GNPs-A-nSiO2 Raman probes can enter the young leaves of tea trees, while the 150nm material cannot detect signals inside the young leaves, indicating that the 150nm Ferbam@GNPs-A-nSiO2 cannot enter the young leaves of tea trees. This shows that whether nanomaterials can enter tea leaves and the penetration rate in the leaves are related to the size of the nanomaterials. The larger the size, the more difficult it is to enter the leaves and the slower the penetration rate in the leaves. Regardless of the size of Ferbam@GNPs-A-nSiO2, no signal can be measured in the old leaves of tea trees, indicating that nanomaterials above 50nm cannot penetrate in the old leaves of tea trees. The difference in the penetration results of old and young tea leaves may be due to the different thickness of the wax layer of the leaves. The first leaf that has just unfolded is soft and has a thin wax layer. Nanomaterials can easily penetrate the surface of the leaves and enter the inside of the leaves. The third leaf is thick and has a thick wax layer. It is difficult for nanomaterials to penetrate the surface and therefore cannot enter the inside of the leaves.

[0141] Will Fig. 9 The Raman two-dimensional deep signal image of Ferbam@GNPs-A-nSiO2 of different sizes infiltrating the lower surface of tea leaves for 5 hours. The Raman image of each layer is taken at the position of the red line to obtain the Raman layer scanning spectrum ( Fig.11 ). The results show that the SERS signal of ferbam can be measured in every layer of 50nm and 100nm Ferbam@GNPs-A-nSiO2, while the SERS signal of ferbam can only be measured in the surface layer of 150nm Ferbam@GNPs-A-nSiO2, and the Raman spectrum of the deep layer is consistent with the signal of the blank leaf. It further shows that after dripping on the lower surface of the tea tree young leaves for 5h, 50nm and 100nm Ferbam@GNPs-A-nSiO2 penetrate into the deep layer of the leaves, while the penetration of 150nm Ferbam@GNPs-A-nSiO2 only stays on the surface of the leaves and does not penetrate downward, verifying the accuracy of the Raman two-dimensional deep signal map results.

[0142] 2.4. Penetration tracing of Ferbam@GNPs-A-nSiO2 in tea tree roots

[0143] The penetration tracing results of Ferbam@GNPs-A-nSiO2 Raman probe in the roots of tea trees were determined by Raman spectroscopy. Fig.12 ). At the same time, in order to verify the accuracy of the measured two-dimensional deep scanning results, the Raman spectrum of the strongest point of the two-dimensional deep signal of Ferbam@GNPs-A-nSiO2 was selected ( Fig.13 ), it can be seen from the Raman spectrum that the measured signals are all SERS signals of ferbam, indicating that Figure 8 The penetration results are the penetration tracing results of the Raman probe in the roots of tea trees, which verifies the correctness of the experimental results.

[0144] Fig.12 The 50nm, 100nm and 150nm Ferbam@GNPs-A-nSiO2 Raman probes shown in the figure all stayed on the surface after being dripped onto the roots of tea trees for 5 hours without penetration. This indicates that no matter what size of Ferbam@GNPs-A-nSiO2, it cannot enter the interior of the roots of tea trees. The reason may be related to the root structure of tea trees and the sample preparation method. The new roots of tea trees were selected. Once the roots leave the water environment, they will quickly lose water and shrink, destroying the original structure. Raman must ensure that the samples can be focused and measured in a dry environment. Therefore, the roots can only be immersed in the nanomaterial solution, absorbed for a certain period of time, and then freeze-dried to retain the original structure for measurement. Therefore, unlike the in-situ measurement of leaves, the roots can only be measured by selecting similar structures and sampling points, which also creates certain difficulties for point selection.

[0145] Will Fig.12 The Raman two-dimensional deep signal map of Ferbam@GNPs-A-nSiO2 of different sizes infiltrated into the roots of tea trees for 5 hours. The Raman map of each layer is taken at the position of the red line to obtain the Raman layer scanning spectrum map ( Fig.14 ). The results show that the SERS signal of ferbam can only be measured on the surface layer of 50nm, 100nm and 150nm Ferbam@GNPs-A-nSiO2, and the deep Raman spectrum is consistent with the signal of the blank root. It further shows that after dripping on the roots of tea trees for 5h, 50nm, 100nm and 150nm Ferbam@GNPs-A-nSiO2 only stays on the surface of the roots and does not penetrate downward, verifying the accuracy of the Raman two-dimensional deep signal map results.

[0146] 3. Ferbam@GNPs-A-nSiO2 Raman probe penetration tracer verification experiment in tea trees

[0147] Raman spectroscopy measures signals by irradiating the surface of a sample with laser light. Tea leaves are biological samples with a relatively soft surface. Repeated in-situ measurements may damage the tissue, thus affecting the results. Therefore, microscopic images of tea leaves were taken before and 5 hours after Raman deep scanning ( Fig.15 A, B), it can be seen from the figure that the leaves before and after Raman deep scanning are not damaged and have the same morphology, indicating that Raman deep scanning will not cause damage to tea tree tissue. Then reselect the point to directly measure the Raman two-dimensional deep signal image and layer scanning image after 100nm Ferbam@GNPs-A-nSiO2 penetrated the lower surface of tea tree tender leaves for 5 hours ( Fig.15 C), the results show that the penetration SERS signal of ferbam can be measured at 0-300μm, eliminating the influence of repeated in-situ scanning on the penetration results. The Raman two-dimensional deep signal image and layer scan image of the nanomaterial after 100nm Ferbam@GNPs-A-nSiO2 penetrated the lower surface of tea leaves for 5h and then cleaned the surface ( Fig.15 D), the deep SERS signal can still be detected, eliminating the influence of the surface Raman probe on the deep Raman signal, verifying the feasibility of the experiment and the accuracy of the results.

[0148] Fig.15 Microscopic images of tea leaves before Raman deep scanning (A) and after 5 hours of scanning (B), and Raman spectrum layer scanning images of 100nm Ferbam@GNPs-A-nSiO2 infiltrating the lower surface of tea leaves for 5 hours (C) and after 5 hours of infiltration and washing away the surface Ferbam@GNPs-A-nSiO2 (D).

[0149] In summary, the non-systemic pesticide ferbam itself has no penetration ability, but after combining with nano-gold silica composite particles, the SERS signal of ferbam was detected inside the young leaves of tea trees. This shows that the nano-gold silica composite particles have changed the penetration behavior of ferbam. In addition, the penetration rate of the nano-gold silica Raman probe loaded with ferbam is very fast. Within 5 hours, the Raman probe quickly penetrated into the young leaves of tea trees, with a depth of about 150-300μm. The penetration rate depends on the size of the Raman probe. Compared with the 100nm Raman probe, the 50nm Ferbam@GNPs-A-nSiO2 has a faster penetration rate, the 150nm Raman probe cannot penetrate into the young leaves of tea trees, and the three sizes of Raman probes cannot penetrate into the old leaves and roots of tea trees; the results show that SERS is an effective method to study the interaction between pesticides and nanomaterials and the penetration and transport of nanopesticides in plants, and Raman can non-destructively detect the interaction inside and outside plant tissues in situ; secondly, the nano-gold silica composite material changed the non-systemic pesticide ferbam. The system behavior allows it to penetrate into the tender leaves of the tea tree; the size of the nanomaterial and the morphology of the plant tissue affect the penetration behavior of the nanopesticide in the plant; small-sized nanomaterials are easier to enter the plant body and have a faster penetration rate, and the more mature the plant tissue, the more difficult it is for the nanopesticide to penetrate; the nano-gold silica Raman probe loaded with ferbam described in the present invention can use SERS to detect the signal of this Raman probe in the tea tree body, thereby in situ non-destructively determining the penetration of nanopesticides in the plant body; providing help and new ideas for the subsequent research and development of pesticide formulations and the study of the interaction mechanism between nanopesticides and plants.

[0150] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A nanopesticide Raman probe, characterized in that: The following steps are involved: S11: Synthesis of negatively charged nSiO2 and GNPs; S12: modifying nSiO2 to be positively charged by a silane coupling agent, thus obtaining A-nSiO2; In the step S12, the method of modifying nSiO2 to be positively charged by a silane coupling agent is as follows: 5 mg of nSiO2 with different particle sizes are weighed and ultrasonically dispersed in 50 mL of ethanol, 50 μL of APTMS is added, and the mixture is heated and refluxed in an oil bath under set temperature conditions for a set time. After the reaction of each solution is completed, the mixture is centrifuged and washed twice with deionized water and ethanol, and then dried at 40° C. overnight to obtain A-nSiO2 with different particle sizes; A-nSiO2 with different particle sizes is dispersed in deionized water, and the zeta potential and particle size are measured using a particle size analyzer; S13: A-nSiO2 and negatively charged GNPs are combined together through electrostatic adsorption force, so that a layer of GNPs is uniformly adsorbed on the surface of A-nSiO2 to obtain GNPs-A-nSiO2; In the step S13, the specific process of obtaining GNPs-A-nSiO2 is as follows: 5 mg A-nSiO2 is ultrasonically dispersed in 2 mL ethanol, and dripped dropwise into 20 mL of a 15 nm GNPs solution that is constantly stirred. After the dropwise addition is completed, the mixture is stirred for 2 h, centrifuged at 14000 r / min for 10 min, washed by centrifugation with deionized water for 3 times, and dispersed in 300 μL deionized water to obtain GNPs-A-nSiO2 of different particle sizes; S14: After GNPs-A-nSiO2 adsorbs ferbam, the ferbam molecules fall between two adjacent GNPs particles, generating SRES signals, thereby tracking and locating nSiO2 through the SRES signals.

2. A nanopesticide Raman probe according to claim 1, characterized in that: In step S1, the particle sizes of nSiO2 include 50nm, 100nm, and 150nm. The synthesis process of nSiO2 with different particle sizes is as follows: Synthesis of 50nm nSiO2: 50mL of anhydrous ethanol, 1mL of 1680ppm lithium hydroxide monohydrate, 2mL of ammonia water, and 1mL of TEOS were added to a beaker in sequence, and stirred at 25℃ for 6h. After the reaction was completed, it was centrifuged and washed three times with deionized water, and dried at 60℃ overnight to obtain 50nm solid nSiO2; Synthesis of 100nm nSiO2: 50mL of anhydrous ethanol, 1mL of 1680ppm lithium hydroxide monohydrate, 2.5mL of ammonia water, and 1mL of TEOS were added to a beaker in sequence, and stirred at 25℃ for 6h. After the reaction was completed, it was centrifuged and washed three times with deionized water, and dried at 60℃ overnight to obtain 100nm solid nSiO2; Synthesis of 150nm nSiO2: Add 50mL of anhydrous ethanol, 1mL of 2100ppm lithium hydroxide monohydrate, 3mL of ammonia water, and 1mL of TEOS into a beaker in sequence, stir at 25℃ for 6h, wash three times with deionized water by centrifugation after the reaction is completed, and dry at 60℃ overnight to obtain 150nm solid nSiO2.

3. A nanopesticide Raman probe according to claim 2, characterized in that: In step S11, the synthesis process of GNPs is as follows: under vibration stirring conditions, 1 mL of citrate solution is added to 98.9 mL of ultrapure water, heated on a heating table, 100 μL of chloroauric acid solution is added, and after cooling on ice, GNPs particles with a diameter of 15 nm are obtained.

4. A nanopesticide Raman probe according to claim 3, characterized in that: In step S14, the specific process of GNPs-A-nSiO2 adsorbing ferbam is as follows: weigh 2.5 mg of ferbam standard, dilute it with deionized water in a 50 mL volumetric flask to obtain a 50 ppm ferbam solution; take 1 mL of GNPs-A-nSiO2 and 1 mL of ferbam solution and shake them in a constant temperature oscillator for 2 hours to obtain a nano-gold silica Raman probe loaded with ferbam.

5. A nanopesticide Raman probe, characterized in that: The nanopesticide Raman probe is prepared by the method for preparing the nanopesticide Raman probe as described in any one of claims 1 to 4.

6. An application of a nanopesticide Raman probe, characterized in that: The SERS is used to detect the signal of the nanopesticide Raman probe as claimed in claim 5 in the tea plant, and then the penetration of the nanopesticide in the plant is determined. The specific application steps are as follows: S21: The synthesized GNPs and GNPs-A-nSiO2 loaded with ferbam were dripped onto the leaves and roots of tea plants for Raman testing, and multiple acquisition times were set to acquire Raman scanning spectra; S22: 20 μL of ferbam-loaded nano-gold silica Raman probe solutions of different particle sizes were dripped on the upper and lower surfaces of young and old leaves of living tea trees, respectively, and completely dried after waiting for 1 hour at 25°C, which was recorded as 0 hours; the two-dimensional surface signals of the ferbam-loaded nano-gold silica Raman probe solutions dripped on the upper and lower surfaces of tea leaves at 0 hours and the two-dimensional deep signals on the tea leaves after 0, 1, 2, 3, 4, and 5 hours were measured in situ, respectively, to observe its penetration in the tea leaves; S23: The newborn roots of tea trees were placed in 200 μL of nano-gold silica Raman probe solutions loaded with ferbam of different sizes, and the roots of the same part were taken at 0, 1, 2, 3, 4, and 5 h, respectively. After freeze-drying for 48 h, the two-dimensional deep signals of the roots of the tea trees were measured at 0, 1, 2, 3, 4, and 5 h to observe its penetration in the roots of the tea trees; S24: Take microscopic images of the tea leaves before and 5 hours after the measurement in step S22; drop 20 μL of 100 nm gold-silica Raman probe solution loaded with ferbam on the lower surface of the living tea leaves, and wait for 1 hour at 25°C to completely dry, marked as 0 h, and measure the two-dimensional deep signal on the tea leaves 5 hours later to observe its penetration in the tea leaves; drop 20 μL of 100 nm gold-silica Raman probe solution loaded with ferbam on the lower surface of the living tea leaves, and wait for 1 hour at 25°C to completely dry, marked as 0 h, and wash off the surface nanoprobe solution after 5 hours, then measure the two-dimensional deep signal on the tea leaves to observe its penetration in the tea leaves.

7. The use of a nano-pesticide Raman probe according to claim 6, characterized in that: In step S22, the young leaves are the first leaves and the old leaves are the third leaves.

Images