WO2020063713A1 - Kit for detecting spray droplet loss or deposition characteristics by using magnetic nano-microspheres, and detection method - Google Patents

Abstract

Provided are a kit for detecting spray droplet loss or deposition characteristics by using magnetic nano-microspheres, and a detection method; the kit of the present invention comprises magnetic nanomicrospheres connected to a fixed probe, a transition probe that may specifically bind to the fixed probe, and a biotin-labeled chromogenic probe that may specifically bind to the transition probe. The transition probe is added into a spray solution as a tracer, and after spraying, the transition probe specifically binds to the fixed probe on the magnetic nanospheres; the biotin-labeled chromogenic probe binds to the transition probe by means of hybridization technology; after chromogenic treatment, the absorbance is read out by means of an ultraviolet visible spectrophotometer, and then the loss or a deposition value of spray droplets may be accurately calculated after undergoing standard curve conversion.

Description

Kit and method for detecting spray droplet loss or deposition characteristics using magnetic nano-microspheres

This application requires that the patent application number submitted to the State Intellectual Property Office of China on September 26, 2018 is 2018111209076, and the invention name is "A kit and method for detecting the loss or deposition of spray droplets using magnetic nanospheres "Of the earlier application. The entirety of this earlier application is incorporated herein by reference.

Technical field

The invention relates to the technical field of spray spray droplet detection, in particular to a reagent box and a detection method for detecting spray spray droplet drift or deposition characteristics by using magnetic nano-microspheres.

Background technique

In agricultural production, watering, fertilizing, and pesticide application are indispensable parts to ensure a good harvest. Food safety is more stringent today, and people's requirements for food have risen from addressing food and clothing in the past to the pursuit of safe, non-toxic, and green food. From 2016, the "double reduction and synergistic effect of chemical fertilizers and pesticides" was proposed, indicating that while using fertilizers and pesticides to ensure a good harvest, we must also consider the impact of their residues in the environment on the environment. "Precise" fertilization and application of fertilizers has become one of the important "double reduction" schemes. The loss of mist droplets and the detection of deposition during agricultural spraying are one of the indicators for monitoring the entire spraying process. The traditional methods of spraying mist droplets and detecting the amount of deposition are divided into quantitative and semi-quantitative methods. Quantitative methods include direct measurement and tracer method, and semi-quantitative methods include paper card color development method.

Direct measurement method refers to the use of crops as receivers for spraying liquid droplets. After spraying, collect crop samples by sampling, and then go through extraction, purification, and enrichment steps, and finally use HPLC-MS, GC-MS And other instruments directly detect the amount of fertilizer or pesticide on the target, and finally calculate the amount of agricultural droplets lost or deposited. This method has high accuracy, but the equipment used is high, the testing process is tedious and slow, and the collected samples need to be stored at low temperature, which is not conducive to long-distance testing.

The tracer method refers to adding a tracer to an agricultural spraying solution, and collecting samples after spraying, and detecting the collected tracer after washing by an instrument, to estimate the amount of agricultural mist droplets deposited on the target. Commonly used tracers include water-soluble dyes such as lemon yellow, temptation red, and fluorescent tracers such as Brilliant sulphoflavine (BSF) and Pyranin. This method is fast and low-cost, and does not require high drug storage requirements. It is currently a pesticide droplet deposition One of the commonly used methods for detection, but the accuracy of this method is affected by the properties of the tracer, the accuracy is poor, and because the tracer is a colored dye, it is easy to cause color pollution to crops and inspectors during spraying.

Paper card color development is to use water-sensitive paper, oil-sensitive paper, carromite paper card as the droplet to receive the target, and then save the image format by scanning and taking pictures of the instrument to calculate the coverage and area on the paper card. , In order to calculate the amount of droplet loss and deposition. However, this method cannot calculate the repeated cover, bounce and roll-off of fog droplets.

Magnetic nano-microspheres refer to a class of microsphere materials with magnetic properties at the nanometer level. The core is generally composed of superparamagnetic materials such as ferric tetroxide or ferric oxide. Polystyrene is wrapped in the outer layer of the magnetic core. Polymer materials such as ethylene or dextran can be chemically bonded to modify the amino or carboxyl group on the outer layer of the polymer to couple it with biologically active macromolecules. Then, due to its superparamagnetism, the external magnetic field can be used to strengthen the magnetic field. Easily enrich the captured molecules, achieve analysis and detection of biological macromolecules, biological recognition, protein purification, etc. Magnetic nano-microspheres are also widely used in the analysis and detection of small chemical molecules, using their superparamagnetic characteristics to achieve the enrichment of small chemical molecules. The DNA binding of the characteristic sequence has high specificity, and only two single strands with perfect complementarity can perfectly combine. The single-stranded DNA with a characteristic sequence is added to the spray solution as a tracer. After spraying, a single-stranded DNA-modified magnetic nano-microsphere containing a characteristic sequence complementary to the tracer is used to collect the sample. The tracer was enriched, and after the final color development, the loss and deposition of the droplets were estimated by simple fluorescence detection.

Summary of the Invention

The invention relates to a single-stranded DNA with a characteristic sequence as a tracer, and a single-stranded DNA-modified magnetic nano-microsphere with a characteristic sequence complementary to the tracer as an enrichment material. Method for detecting drift or deposition characteristics of mist droplets.

The present invention provides a method for detecting the loss and deposition of mist droplets in agricultural spraying. The specific process is shown in Figure 1. Using a "three-stage" strategy, the "fixed probe" is first connected by chemical bonds. The method is fixed on magnetic nanospheres, and then the "transition probe" tracer is added to the spraying medicine box, and the petri dish is used as a droplet receiver. After spraying, the petri dish is eluted with deionized water. Collect the eluate from each petri dish, and then use magnetic nanospheres containing fixed probes to enrich the "transition probe" tracer, and then add the corresponding chromogenic probe for development After the color, the drift or deposition characteristics of the droplets are calculated and derived.

Specifically, the present invention provides a method for detecting the drift or deposition characteristics of sprayed mist droplets by using magnetic nano-microspheres, including the following steps:

(1) Add the transition probe to the spray solution as a tracer;

(2) The magnetic nanospheres are dispersedly placed on the surface of the target to be sprayed. After the spray liquid is sprayed, the magnetic nanospheres are used to enrich the transition probes. The magnetic nanospheres are fixed probes bonded to the magnetic nanospheres. Fixed probes can specifically bind to transition probes as tracers;

(3) The biotin-labeled chromogenic probe is combined with the corresponding transition probe by hybridization technology. After color development, the absorbance value is read by a UV-visible spectrophotometer, and the droplet is calculated after the conversion of the standard curve. Lost or deposited characteristics;

The transition probe is not modified by biotin, and has a nucleotide sequence capable of complementary pairing with the fixed probe and the chromogenic probe, respectively, and the fixed probe and the chromogenic probe do not specifically bind.

In the method of the present invention, the transition probe and the fixed probe are single-stranded DNAs with characteristic sequences; wherein the length of the transition probe is 24-50 nt (preferably 36-40 nt), and the length of the fixed probe is 12-25nt (preferably 18-20nt), one end of the fixed probe is modified by amino or carboxyl group, and the other end is bonded to the surface of the magnetic nanomicrosphere.

Among them, the complementary pairing base of the color development probe and the transition probe is 15-40nt; if the fixed probe is 5 'labeled, the color developed probe is 3' biotin labeled, and if the fixed probe is 3 'labeled, The chromogenic probe is 5 'biotin labeled.

The complementary base of the transition probe and the fixed probe is 15-25 nt. In the method of the present invention, the magnetic nano-microspheres in step (2) are treated with the following reagents to bind and fix the probe: 10-20% (preferably 15%) EDC solution (1- (3-dimethylaminopropyl) 3-ethylcarbodiimide), 0.025-0.2 μM (preferably 0.03 μM) fixed probe solution, NaCl solution (preferably 0.1M), 1-6M NaOH solution (preferably 5M), anhydrous DMF, Anhydrous DMAC, magnetic nano-microsphere modifier solution.

In the method of the present invention, the magnetic nano-microspheres in step (2) are prepared by the following methods: after the magnetic nano-microspheres are cleaned with pure water and DMF (N, N-dimethylformamide), respectively, Add 0.02-0.15M NaCl solution, magnetic nano-microsphere modifier solution, 1-6M NaOH solution, adjust the pH to 6-8, react at 25 ° C for 3-5h, wash with anhydrous DMF, and then add After water DMF and 10-20% EDC, react at 25 ° C for 3-5 hours, and then wash with anhydrous DMF and anhydrous DMAC (N, N-dimethylacetamide). Add a solution containing 0.025-0.2mM fixed probe Soak 1-3h in NaHCO ₃ solution; add 1-6M NaOH solution to soak the membrane for 1-3h, wash, and dry it to obtain magnetic nanospheres with fixed probes.

The magnetic nanosphere has a magnetic core with a superparamagnetic metal material, for example, including but not limited to any one of iron oxide, iron oxide, iron oxide, or iron-barium alloy; and its surface is modified. Is an amino or carboxyl group; the particle size of the magnetic nano-microspheres is 10-200 nm, preferably 100 nm.

The magnetic nano-microsphere modifier solution is a long-chain dicarboxylic acid anhydride solution such as succinic anhydride, glutaric anhydride, adipic anhydride, maleic anhydride, etc .; the sprayed mist is a pesticide, a liquid fertilizer Or other liquid preparations.

In the spraying liquid preparation process, the spraying liquid mainly contains: 0-60% pesticide formulation or liquid fertilizer (water can also be used directly), 0.025-0.1 μM (preferably 0.060 μM) transition probe, 0- 0.045mol / L ionic buffer solution, 0-0.15% surfactant (if pesticide or fertilizer preparation is used, because it contains surfactant and ionic buffer solution, the transition probe can be added directly; if it is directly sprayed with water , You need to add a certain amount of ionic buffer and surfactant), the main formulations are water-based preparations and oil-based preparations.

The pesticide formulation includes water, oil, wettable powder, microcapsule, water suspension, oil suspension, etc .; the pesticide types include pesticides, fungicides, herbicides, acaricides, nematicides Wait.

The liquid fertilizer includes a clear liquid type, a suspension type, a foliar fertilizer, and the like; wherein the type of the fertilizer is one or two or more compound fertilizers of nitrogen fertilizer, phosphate fertilizer, and potassium fertilizer.

The ion buffer solution is a buffer solution prepared by one or more inorganic salts and organic salts, wherein the solution anion is carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, citrate, lemon One or more kinds of acid dihydrogen and the like, and one or more kinds of cations are potassium ion, sodium ion, lithium ion, calcium ion and the like.

The surfactant is sodium alkyl sulfonate, pulverized powder, tea withered powder, saponin powder, SDS (sodium lauryl sulfate), Morwet EFW (sodium butyl naphthalene sulfonate), TERWET 1004, One or more.

In the color development treatment, the reagents used are: hybridization solution, washing solution, 0.05-0.20 μM (0.20 μM) color development probe solution, streptavidin-labeled catalase solution, 3,3 ' , 5,5'-Tetramethylbenzidine (TMB) one-component solution.

The main components of the hybridization solution are 0.02-0.045mol / L ion buffer (preferably 0.03mol / L trisodium citrate aqueous solution), 0.06-0.15% surfactant (preferably 0.12% SDS).

The main components of the washing solution are 5.0-10.0 mmol / L ionic buffer solution, and 0.02-0.20% surfactant.

The ion buffer solution is a buffer solution prepared by one or more inorganic salts and organic salts, wherein the solution anion is carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, citrate, lemon One or more kinds of acid dihydrogen and the like, and one or more kinds of cations are potassium ion, sodium ion, lithium ion, calcium ion and the like (preferably 7.5 mmol / L trisodium citrate aqueous solution).

The surfactant is sodium alkyl sulfonate, pulverized powder, tea withered powder, saponin powder, SDS (sodium lauryl sulfate), Morwet EFW (sodium butyl naphthalene sulfonate), TERWET 1004, etc. One or several (preferably 0.03% SDS).

The chromogenic probe is a single-stranded DNA with a characteristic sequence of 12-25nt (preferably 18-20nt).

The main components of the TMB single component solution are: 0.5-2.0mM (preferably 1.0mM) TMB, 0.5-2.0mM (preferably 1.0mM) oxidant, 150-300mM (preferably 200mM) ion buffer solution, 0.1 -0.5 mM stabilizer. The specific preparation process is as follows: Solution A: Weigh TMB and stabilizer, add DMSO to dissolve it; Solution B: Dissolve with deionized water to prepare an ion buffer solution. After adding an oxidant, adjust the pH to 4.0-6.0 with hydrochloric acid. After the preparation, the two liquids a and b are prepared according to a certain ratio before use to obtain a TMB single-component liquid.

The streptavidin-labeled catalase may be a streptavidin-labeled horseradish catalase.

The oxidant is one or more of hydrogen peroxide, urea hydrogen peroxide, peroxyacetic acid, tert-butyl hydroperoxide, dimethyldioxane, etc. (preferably hydrogen peroxide).

The ion buffer solution is a buffer solution prepared by one or more inorganic salts and organic salts, wherein the solution anion is carbonate, bicarbonate, phosphate, hydrogen phosphate, dihydrogen phosphate, citrate, lemon One or more kinds of acid dihydrogen and the like, and one or more kinds of cations are potassium ion, sodium ion, lithium ion, calcium ion and the like (preferably trisodium citrate).

The stabilizer is one or more of sodium borohydride, sodium cyanoborohydride, tetrabutylammonium borohydride (TBABH), lithium tri-sec-butylborohydride, lithium borohydride and the like (preferably TBABH).

As an embodiment of the present invention, each specific process of the detection method of the present invention is as follows:

1) Preparation of magnetic nano-microspheres containing fixed probes:

Preparation process of magnetic nano-microspheres: Magnetic nano-microspheres are cleaned with pure water and DMF three times respectively by ultrasonic, then 0.15M NaCl solution, magnetic nano-microsphere modifier solution are added, and the pH is adjusted to 6-8 with 3M NaOH solution (Preferably 7.5), and react at 25 ° C for 3-5h (preferably 5h). Wash 5 times with anhydrous DMF, add anhydrous DMF and EDC respectively, and react at 25 ° C for 3-5h (preferably 5h), then wash once with anhydrous DMF and 4 times with anhydrous DMAC, and add 0.125mM fixed Soak the probe in NaHCO ₃ solution for 1-3h (preferably 3h); add 1M NaOH solution to soak the membrane for 1-3h (preferably 1h), wash, and dry to obtain magnetic nanomicrospheres with fixed probe .

2) Spraying process: spraying solution preparation, adding pesticide formulation, liquid fertilizer or water to the medicine cabinet, and then adding the transition probe, the final concentration is 0.06μM, after adding water, mix them, and finally according to the pesticide formulation or The spraying equipment requires, optionally adding a surfactant and an ion buffer to prepare a transition probe spraying solution. After spraying, the spray mist droplets in the petri dish are transferred to a centrifuge tube with clean water and stored for testing.

3) Add the magnetic nanospheres prepared in step 1) to a centrifuge tube stored with spray droplets, vortex for 5 minutes, and incubate at 34-37 ° C for 60 minutes. Hold the magnetic microspheres with a magnetic stand and remove them. After removing the supernatant, the magnetic beads were washed 3 times with a washing solution, and then added to a hybridization solution containing 0.15 μM color development probe to react at 30-40 ° C. for 5-15 min (preferably 15 min); use a magnetic stand to absorb the magnetic Microspheres, after removing the supernatant, washing the magnetic beads 3 times with a washing solution, taking 15 μL of streptavidin-labeled catalase solution into the hybridization solution to configure the enzyme solution, mixing it with the magnetic beads, Enzyme-linked reaction at 37 ° C for 15-20min (preferably 20min); hold the magnetic microspheres with a magnetic stand, remove the supernatant, wash the magnetic beads 3 times with the hybridization solution, and add TMB single component to the magnetic beads The solution undergoes a color reaction. After 5 minutes, the magnetic microspheres were absorbed by a magnetic stand, and the absorbance value of the supernatant was read out using a UV-visible spectrophotometer.

4) Standard curve establishment: Take 5 centrifuge tubes, and add 1μL, 2μL, 4μL, 8μL, 16μL of streptavidin-labeled catalase to 10mL of TMB single-component solution for color reaction. After 5 min, the absorbance was read out using a UV-visible spectrophotometer. Finally, draw the standard curve with the volume of streptavidin-labeled catalase as the abscissa and the total absorbance as the ordinate, and calculate the linear equation corresponding to the volume of the enzyme solution and the absorbance. The amount of enzyme solution bound to the magnetic beads, and then the amount of transition probes on the magnetic beads is calculated, that is, the amount of the drug solution deposited on the sample to be measured.

Based on the above detection method of the present invention, the present invention also provides a kit for detecting the drift or deposition characteristics of sprayed mist droplets, comprising a magnetic nanosphere, a transition probe, and a color development probe; the magnetic nanosphere It is a magnetic nanosphere with a fixed probe bonded to it. The length of the fixed probe is 12-25nt. One end is modified with an amino or carboxyl group and the other end is bonded to the carboxyl or amino group of the magnetic nanosphere. The length is 24-50 nt; the 3 'or 5' end of the chromogenic probe is labeled with biotin, and the chromogenic probe can specifically bind to the transition probe and cannot specifically bind to the fixed probe; the fixed probe can be bound to The transition probe specifically binds.

In the present invention, the strategy of "three-stage" reverse dot hybridization technology is used to add single-stranded DNA of a characteristic sequence as a tracer to a spray solution. After the sample is sprayed and collected, the magnetic nanospheres are used to collect the sample. The transition probes obtained are enriched, and the amount of drift and deposition in the spray droplets is estimated by color development. The beneficial effects of the detection method of the present invention are mainly reflected in: (1) the present invention uses a single-stranded DNA of a characteristic sequence as a tracer, and avoids the steps of the conventional fluorescent tracer which is easy to photolyze and easily cause pollution; 2) Solve the tedious operation of the existing tracer in the pretreatment such as elution, enrichment, etc .; (3) The existing fluorescent tracer has poor accuracy during the detection process and it is difficult to achieve accurate detection. By using complementary features Sequential single-stranded DNA transfer color development can achieve high-precision detection of droplet loss and deposition in agricultural spraying.

All in all, compared with conventional methods, the present invention successfully achieves accurate detection of the loss or deposition characteristics of agricultural spray mist droplets in a pollution-free, fast, convenient, and easy-to-operate manner, helping to improve pesticide utilization and reduce the environment. Pollution, perfecting the technical system of pesticide application, has a good market application prospect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of the technical principle of detecting spray droplet loss or deposition characteristics using magnetic nano-microspheres according to the present invention.

FIG. 2 is a result of establishing a standard curve of the detection method of the present invention.

detailed description

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Modifications or replacements of the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention belong to the scope of the present invention.

Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

Embodiment 1 Method for detecting drift or deposition characteristics of spray mist

The flow chart of the method for detecting the drift or deposition characteristics of spray droplets according to the present invention is shown in FIG. 1. Specifically: using the specificity of DNA binding with different characteristic sequences, a series of single-stranded DNAs with different characteristic sequences are designed as fixed probes to be bonded to magnetic nanospheres, and will be able to interact with solid probes. A single-stranded DNA (transition probe) with a complementary paired characteristic sequence is added to the spray solution as a tracer. After spraying, the transition probe is collected by magnetic nanospheres, and after color development, it is analyzed by ultraviolet-visible spectrometry After the photometer reads the absorbance, it can obtain the information such as the droplet deposition amount of the spray solution after conversion by the standard curve. Finally, the corresponding deposition amount can be calculated by computer image processing software.

1. Determination of the probe

The length of the fixed probe is 12-25nt, preferably 18-20nt. One end of the fixed probe is modified by adding an amino or carboxyl group, and the other end is covalently bonded to the carboxyl or amino bare end of the base material.

The transition probe is a single-stranded DNA with a characteristic sequence of 24-50 nt (preferably 36-40 nt) and is not modified by biotin. The complementary base of the transition probe and the fixed probe is 15-25 nt.

The chromogenic probe is a single-stranded DNA with a characteristic sequence of 12-25 nt (preferably 18-20 nt). The complementary pairing base of the chromogenic probe and the transition probe is 15-40nt; if the fixed probe is 5 'modified, the chromogenic probe is 3' biotin-labeled, and if the fixed probe is 3 'modified, the The color probe is 5 'biotin labeled.

The chromogenic probe can specifically bind to the transition probe and cannot specifically bind to the fixed probe. The three probe sequences in Table 1 are examples. In addition to the nucleotide sequences of the probes in Table 1, any single-stranded DNA sequence that can meet the above requirements can be used for the probes in this application.

Table 1 Simple examples of 5′-amino-modified immobilized probe sequences and two matching probes

2. Preparation of magnetic nanospheres with fixed probes

Preparation process of magnetic nanospheres: Probe combination 1 in Table 1 was used. After magnetic nanospheres were cleaned with pure water and DMF three times, respectively, 0.15M NaCl solution, 1M succinic anhydride solution, and 3M NaOH were added. The solution was adjusted to pH 7.5 and reacted at 25 ° C for 5h. Wash 5 times with anhydrous DMF, add anhydrous DMF and EDC, and react at 25 ° C for 5 hours, then wash once with anhydrous DMF and 4 times with anhydrous DMAC, and add 0.125 mM fixed probe (combination 1). Soak in NaHCO ₃ solution for 3h; add 1M NaOH solution for 1h, wash, dry, and obtain magnetic nano-microspheres with fixed probe.

3. Preparation and spraying of spraying solution

In the spraying liquid preparation process, the spraying liquid composition is 30 mM trisodium citrate, 3 mM SDS, 0.06 μM transition probe (combination 1), and the spraying liquid droplets are received in a petri dish. After spraying, the petri dish The mist droplets are eluted with water and transferred to a centrifuge tube for storage.

4. Establishment of the standard curve of the present invention

Standard curve establishment: Take 5 centrifuge tubes, add 1μL, 2μL, 4μL, 8μL, 16μL of streptavidin-labeled catalase, and add 50ml TMB single-component solution for color reaction. After 5 min, the absorbance was read out using a UV-visible spectrophotometer. Finally, the catalase volume was used as the abscissa and the total absorbance value was used as the ordinate. A standard curve was drawn and the corresponding linear equation was calculated. The specific results are shown in Figure 2. The linear equation is y = 0.1198x + 0.091, and the linear correlation coefficient is 0.99, which meets the requirements of a standard curve for quantitative detection.

5, color rendering method, calculation of drift or deposition characteristics

Add the prepared magnetic nanospheres to a centrifuge tube for spray solution collection and storage. After vortexing for 5 minutes, incubate at 34 ° C for 60 minutes. Hold the magnetic microspheres with a magnetic stand, remove the supernatant, and wash with Solution (washing solution is an aqueous solution containing 7.5 mmol / L trisodium citrate, 6 mmol / L SDS), and the magnetic beads were washed three times, and then added to a hybridization solution containing 0.15 μM chromogenic probe for reaction at 34 ° C. for 15 minutes; Hold the magnetic microspheres on the magnetic stand, remove the supernatant, and wash the magnetic beads 3 times with washing solution. Then take 15 μL of catalase solution labeled with streptavidin and add it to the hybridization solution to configure the enzyme solution (hybridization solution). The composition is an aqueous solution containing 30 mmol / L trisodium citrate, 26 mmol / L SDS), and an enzyme-linked reaction at 37 ° C for 15-20 min. The magnetic microspheres are absorbed by a magnetic stand, the supernatant is removed, and the magnetic beads are washed with a hybridization solution. After 3 times, TMB single component solution was added for color reaction. Finally, the ultraviolet-visible spectrophotometer was used to read the absorbance value, and the absorbance value was brought into the standard curve to be converted into the deposition amount.

Example 2 Experiment of Determining the Deposition Amount of Spraying Pesticide

A petri dish was placed on an iron stand below the running track of the spray crane, a 9 cm diameter filter paper was placed in one petri dish, and an empty petri dish was placed at intervals. Two groups of petri dishes were placed in total. Recorded as A, B two parallel control groups. It is in the middle of the running track of the spray crane. At a pressure of 3 bar, use a spray crane (speed: 5km / h, height: 0.5m) to install a Lechler ST110-03 conventional fan spray nozzle for spraying. The composition of the spray liquid is 30 mM trisodium citrate containing 1 g / L BSF, 0.9 % SDS, 0.06 μM transition probe (combination 1) spray solution. The experimental materials were collected after spraying. The empty petri dishes of groups A and B were washed with 10 mL of deionized water (10 mL), and the washing solution was poured into a ziplock bag, and the sample was developed according to the color development method of Example 1. After reading out the corresponding absorbance value through a UV-visible spectrophotometer, the deposition amount of the two groups A and B was obtained through conversion of the standard curve. The filter paper was packed in a ziplock bag, and 10mL of deionized water was added to the ziplock bag. The BSF content was measured to obtain the deposition amount determined by the BSF. (The specific results are shown in Table 2).

The results show that compared with the traditional BSF method, magnetic nanospheres can also detect deposited droplets with high parallelism. However, the method of the present invention has high sensitivity, low detection limit and low limit of quantification, so the amount of tracer (transition probe) in the spray solution is greatly reduced, and pollution is reduced; at the same time, the magnetic nanomicrospheres adopted by the present invention The enrichment method is simpler and more convenient in operation and has a high repetition rate.

Table 2 Determining the experimental results of spraying pesticide droplets

Parallel group	A	B	BSF
Absorbance	0.37	0.36	-
Deposition amount μL / cm 2	2.29	2.23	2.15
Theoretical deposition amount μL / cm 2	2.86	2.86	2.86
Ratio (calculated value / theoretical value)	0.80	0.78	0.75

Claims (10)

1. A method for detecting the drift or deposition characteristics of spray droplets by using magnetic nano-microspheres, which comprises the following steps:

   (1) Add the transition probe to the spray solution as a tracer;

   (2) The magnetic nanospheres are dispersedly placed on the surface of the target to be sprayed. After the spray liquid is sprayed, the magnetic nanospheres are used to enrich the transition probes; a fixed probe is bonded to the magnetic microspheres, and the fixed The probe can specifically bind to a transition probe as a tracer;

   (3) The biotin-labeled chromogenic probe is combined with the corresponding transition probe by hybridization technology. After color development, the absorbance value is read by a UV-visible spectrophotometer, and the droplet is calculated after the conversion of the standard curve. Lost or deposited characteristics;

   The transition probe is not modified by biotin, and has a nucleotide sequence capable of complementary pairing with the fixed probe and the chromogenic probe, respectively, and the fixed probe and the chromogenic probe do not specifically bind.
2. The method according to claim 1, wherein the transition probe and the fixed probe are single-stranded DNAs with a characteristic sequence; wherein the length of the transition probe is 24-50 nt, and the length of the fixed probe is 12-25nt, one end of the fixed probe is modified by amino or carboxyl group, and the other end is bonded with magnetic nano-microsphere.
3. The method according to claim 1, wherein the complementary pairing base of the color development probe and the transition probe is 15-40 nt; if the fixed probe is 5 'labeled, the color development probe is 3' biotin Labeling. If the immobilized probe is 3 'labeled, the chromogenic probe is 5' biotin labeled.
4. The method according to claim 1, wherein the length of the fixed probe is 18-20 nt, and the complementary pairing base of the transition probe and the fixed probe is 15-25 nt.
5. The method according to any one of claims 1 to 4, wherein the magnetic nanospheres in step (2) are prepared by the following methods: pure water and DMF are used to clean the magnetic nanospheres, respectively. After that, add 0.02-0.15M NaCl solution, magnetic nano-microsphere modifier solution, 1-6M NaOH solution, and adjust the pH to 6-8, and react at 25 ° C for 3-5h, wash with anhydrous DMF, and then After adding anhydrous DMF and 10-20% EDC, react at 25 ° C for 3-5 hours, and then wash with anhydrous DMF and anhydrous DMAC, and add immersion in NaHCO ₃ solution containing 0.025-0.2mM fixed probe for 1-3 hours. ; Then add 1-6M NaOH solution to soak the membrane for 1-3h, wash, and dry it to obtain magnetic nanomicrospheres with fixed probes.
6. The method according to claim 5, characterized in that the magnetic core of the magnetic nano-microspheres is a metal material with superparamagnetism of iron oxide, iron oxide, iron oxide or iron-barium alloy; The surface modification is amino or carboxyl; the particle size of the magnetic nanospheres is 10-200nm, preferably 100nm.
7. The method for detecting sprayed droplet loss or deposition characteristics according to claim 5, wherein the magnetic nano-microsphere modifier solution is a long-chain dicarboxylic acid anhydride solution; the sprayed droplet is Pesticides, liquid fertilizers or other liquid preparations.
8. The method for detecting spray droplet loss or deposition characteristics according to any one of claims 1-4, wherein the final concentration of the transition probe in the spray solution in step (2) is 0.025-0.1 μM.
9. A kit for detecting the drift or deposition characteristics of spray droplets, which is characterized by containing magnetic nano-microspheres, a transition probe, and a chromogenic probe. The chromogenic probe can specifically bind to the transition probe and cannot Specific binding with fixed probe;

   The magnetic nano-microsphere is a magnetic nano-microsphere to which a fixed probe is bonded. The length of the fixed probe is 12-25 nt, one end is modified by an amino group or a carboxyl group, and the other end is bonded to the carboxyl group or amino group of the magnetic nano-microsphere;

   The length of the transition probe is 24-50 nt; the 3 'or 5' end of the color development probe is labeled with biotin.
10. The kit according to claim 9, further comprising 3,3 ', 5,5'-tetramethylbenzidine TMB single-component solution, streptavidin-labeled peroxidation Catalase.

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