Thiamethoxam up-conversion fluorescence detection method based on magnetic separation

Abstract

The invention provides a fluorescent biosensor detection technology for up-conversion nanoparticles and magnetic nanoparticles. In combination with the inherent optical characteristic of the upconversion nanoparticles and the advantage that the magnetic nanoparticles can be rapidly enriched, an aptamer and a complementary chain are used as biological recognition elements to respectively modify the magnetic nanoparticles and the upconversion nanoparticles to obtain a capture probe and a signal probe, and the capture probe and the signal probe are combined through a base complementary effect to form a biological sensing system. The fluorescence in the system is the strongest. The target detection object can be preferentially combined with the capture probe, so that the fluorescence intensity in the system is reduced. Quantitative detection can be achieved through the linear relation between the change value of the fluorescence intensity and the concentration of a target detection object. The invention realizes quantitative detection application in thiamethoxam residue, and has the advantages of strong specificity, high sensitivity, simple, rapid and accurate detection, low cost, wide application range and easy popularization and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of pesticide residue detection, and particularly relates to a fluorescence biosensor detection technology for upconversion nanoparticles and magnetic nanoparticles. Background Art

[0002] Neonicotinoid insecticides are a class of insecticides developed since the 1980s. Due to their unique action mechanism and high-efficiency and broad-spectrum insecticidal effects, neonicotinoid insecticides have become one of the fastest-developing classes of insecticides in recent years. As a second-generation neonicotinoid insecticide, thiamethoxam has a novel structure and good stomach toxicity, contact toxicity and systemic toxicity activities, and is one of the most profitable and widely used neonicotinoid insecticides. However, due to the continuous accumulation of thiamethoxam and its metabolites in plant parts and soil, its safety is becoming a worrying issue. The US Environmental Protection Agency released a final biological assessment report on three neonicotinoid insecticides, thiamethoxam, imidacloprid and clothianidin, in June 2022. These three insecticides may have an adverse impact on 77% of species and 81% of important habitats, and endanger the vast majority of endangered species (fish, birds and mammals). Therefore, it is necessary to develop a simple, rapid and reliable method to detect thiamethoxam residues in food and the environment to protect consumers and life from its harm.

[0003] The detection methods of thiamethoxam mainly include instrumental methods such as gas chromatography, liquid chromatography, liquid chromatography-tandem mass spectrometry, gas chromatography-tandem mass spectrometry, etc. Instrumental methods have the advantages of high sensitivity, strong anti-interference ability and accurate quantification, but the sample pretreatment is complex, the detection cost is high, and it cannot meet the rapid detection requirements of large-volume samples. At present, the commercially mature ones are mainly pesticide residue rapid test cards and rapid detectors based on the enzyme inhibition method. However, these rapid pesticide detection methods generally have disadvantages such as easy false positives, inability to specifically identify pesticide types, poor sensitivity, and difficult quantitative analysis. The repeatability of enzyme sensor detection is poor, the activity and stability of enzymes are easily interfered by the environment, and the preparation process of antibodies is complex. Summary of the Invention

[0004] The purpose of the present invention is to rapidly detect thiamethoxam based on the luminescence characteristics of upconversion particles and the magnetic enrichment ability of magnetic nanoparticles, combined with the specific recognition ability of aptamers for target analytes, so as to solve the problems existing in the above technologies. By constructing an upconversion / magnetic nanoparticle fluorescence biosensing system, rapid detection of thiamethoxam in food is realized, and this method is superior to traditional instrumental methods in terms of detection time.

[0005] In order to achieve the above purpose, the present invention provides the following solutions:

[0006] An upconversion fluorescence detection method for thiamethoxam based on magnetic separation, comprising the following steps:

[0007] (i) React alendronic acid with oil-soluble upconversion nanoparticles to prepare water-soluble upconversion nanoparticles;

[0008] (ii) Add the water-soluble upconversion nanoparticles to a glutaraldehyde solution, stir, then add a complementary strand, and react to obtain complementary strand-modified upconversion fluorescent nanoparticles;

[0009] (iii) Add amino-modified magnetic nanoparticles to the glutaraldehyde solution, stir, then add a streptavidin solution and an aptamer for reaction, and then retain the precipitate after separation by an external magnetic field (magnetic field strength: 5000 gs) to obtain aptamer-modified magnetic nanoparticles;

[0010] (iv) Mix the aptamer-modified magnetic nanoparticles and the complementary strand-modified upconversion fluorescent nanoparticles for a conjugation reaction to obtain a detection solution. Then prepare thiamethoxam solutions with different concentrations, add the thiamethoxam solutions with different concentrations to the detection solution respectively, measure the fluorescence signal values of the thiamethoxam solutions with different concentrations and the fluorescence signal value of the solution without thiamethoxam added, and establish a standard curve of the linear relationship between the thiamethoxam solution concentration and the fluorescence intensity based on the measured fluorescence intensities.

[0011] In a preferred embodiment, the preparation method of the oil-soluble upconversion nanoparticles includes: dissolving yttrium chloride hexahydrate, gadolinium chloride hexahydrate, ytterbium chloride hexahydrate, and holmium chloride hexahydrate in a methanol solution, then adding oleic acid and 1-octadecene and mixing. Under nitrogen protection, continuously stir until completely dissolved, add a methanol solution of sodium hydroxide and ammonium fluoride, heat until methanol is completely removed, then quickly heat to 300 °C to form a crystalline phase unique to upconversion, and then cool to room temperature. After washing, the oil-soluble upconversion nanoparticles are obtained.

[0012] In a preferred embodiment, the preparation method of the water-soluble upconversion nanoparticles further includes: dissolving alendronic acid and the oil-soluble upconversion particles in deionized water and chloroform respectively, mixing the two, adding ethanol, then performing ultrasonic treatment until completely dissolved, adjusting the pH to 2-3 with a hydrochloric acid solution, stirring until the reaction ends, and washing with a mixed solution of ethanol and water to obtain the water-soluble upconversion nanoparticles.

[0013] In another embodiment of the present invention, the preparation method of the complementary strand-modified upconversion fluorescent nanoparticles further includes: adding the water-soluble upconversion nanoparticles to a phosphate buffer solution and stirring evenly, then adding a glutaraldehyde solution, stirring until the reaction is complete, adding a complementary strand, reacting at room temperature, and centrifuging after the reaction is complete to retain the precipitate to obtain the complementary strand-modified upconversion fluorescent nanoparticles.

[0014] In another embodiment of the present invention, the preparation method of the amino-modified magnetic nanoparticles includes: dissolving anhydrous sodium acetate, 1,6-hexanediamine, and ferric chloride hexahydrate in an ethylene glycol solution, heating to a clear and transparent solution, baking at 60 degrees Celsius for 12 hours, cooling to room temperature, and obtaining the amino-modified magnetic nanoparticles through magnetic separation by an external magnetic field.

[0015] In another embodiment of the present invention, the preparation method of the aptamer-modified magnetic nanoparticles further includes: adding the amino-modified magnetic nanoparticles into a phosphate buffer solution and stirring evenly, then adding a glutaraldehyde solution, stirring until the reaction is complete, adding a streptavidin solution, and then adding an aptamer for reaction. After the reaction is complete, the precipitate is retained through separation by an external magnetic field to obtain the aptamer-modified magnetic nanoparticles. Preferably, the magnetic field strength used is 5000 gs.

[0016] In another embodiment of the present invention, in the preparation method of the oil-soluble upconversion nanoparticles, the mass ratio of yttrium chloride hexahydrate, gadolinium chloride hexahydrate, ytterbium chloride hexahydrate, and holmium chloride hexahydrate is: 0.1164 g∶0.0892 g∶0.062 g∶0.0006 g, and the methanol∶oleic acid∶octadecene = 2 mL∶6 mL∶14 mL; the mass ratio of ammonium fluoride and sodium hydroxide is: 0.2944 g∶0.2 g.

[0017] In another embodiment of the present invention, the alendronic acid∶deionized water = 50 mg∶6 mL, and the oil-soluble upconversion particles∶chloroform∶ethanol = 200 mg∶10 mL∶4 mL.

[0018] In another embodiment of the present invention, the anhydrous sodium acetate∶1,6-hexanediamine∶ferric chloride hexahydrate∶ethylene glycol = 2 g∶6.4 g∶1.0 g∶45 mL.

[0019] In another embodiment of the present invention, the water-soluble upconversion nanoparticles∶glutaraldehyde = 4 mg∶0.7 mL, the added complementary strand is 30 μL, and the concentration is 100 μM / L; the amino-modified magnetic nanoparticles∶glutaraldehyde solution = 4 mg∶1 mL; the streptavidin is 0.5 mL, and the concentration is 1 mg / mL; the aptamer is 40 μL (100 μM); the aptamer-modified magnetic nanoparticles∶the complementary strand-modified upconversion fluorescent nanoparticles = 50 μL (concentration 2 mg / mL)∶140 μL (concentration 2 mg / mL); the different concentrations of thiamethoxam solutions are 0 - 102.4 ng / mL.

[0020] In a possible embodiment, the present invention further provides a method for detecting thiamethoxam upconversion fluorescence based on magnetic separation, including the following steps:

[0021] Step 1, preparation of oil-soluble upconversion nanoparticles: Dissolve yttrium chloride hexahydrate, gadolinium chloride hexahydrate, ytterbium chloride hexahydrate, and holmium chloride hexahydrate in methanol solution (99.5 wt%). Then mix the mixture with oleic acid and 1-octadecene. Under nitrogen protection, heat the mixture and continuously stir magnetically until completely dissolved. Then, add a methanol solution of sodium hydroxide and ammonium fluoride, perform water bath heating, and then remove the sealing film and completely volatilize methanol in the water bath. After completely removing methanol, quickly heat the mixture and then cool it to room temperature. Wash the synthesized product with a mixture of cyclohexane and ethanol to obtain oil-soluble upconversion nanoparticles (UCNPs), and bake them in an oven and store them in powder form.

[0022] Step 2, preparation of water-soluble upconversion nanoparticles: Dissolve alendronic acid and the oil-soluble upconversion nanoparticles obtained in Step 1 in deionized water and chloroform respectively. After mixing the two, add ethanol and ultrasonically treat until completely dissolved. Adjust the pH of the mixed solution with hydrochloric acid solution (1 mol / L), and then react under magnetic stirring. After the reaction is completed, wash the synthesized product with a mixed solution of ethanol and water (concentration ratio: 3:1) to obtain water-soluble upconversion nanoparticles, bake them in an oven and store them in powder form.

[0023] Step 3, preparation of complementary strand-modified upconversion fluorescent nanoparticles: Dissolve the water-soluble upconversion fluorescent nanoparticles obtained in Step 2 in phosphate buffer solution (PBS, concentration 0.01 mol / L, pH = 7.2) and then ultrasonicate. Then add glutaraldehyde solution and react under magnetic stirring. Add complementary strand (cDNA) to the reacted solution and react overnight at room temperature. Subsequently, centrifuge the solution and retain the precipitate, and wash it with PBS. Finally, disperse the washed cDNA-UCNPs into PBS buffer solution and store;

[0024] Step 4, preparation of amino-modified magnetic nanoparticles (MNPs): Disperse anhydrous sodium acetate, 1,6-hexanediamine, and ferric chloride hexahydrate in ethylene glycol solution, heat in a water bath at 50 °C for 1 hour until a clear and transparent solution is obtained. Subsequently, transfer the solution to a polytetrafluoroethylene inner liner and bake it in an oven. After baking is completed, cool it to room temperature, magnetically separate with an external magnetic field to obtain a black solid, and wash it with deionized water to obtain amino-modified magnetic nanoparticles, bake them in an oven and store them in powder form.

[0025] Step 5, Preparation of aptamer-modified magnetic nanoparticles: The amino-modified magnetic nanoparticles obtained in Step 4 are dissolved in PBS and then ultrasonicated (60 KHz, for 30 minutes), and then glutaraldehyde solution (concentration 25 wt%) is added and reacted under magnetic stirring. After the reaction ends, streptavidin solution is added and reacted under magnetic stirring. Subsequently, aptamer (apt) is added to the reacted solution and reacted overnight at room temperature. Subsequently, the solution is centrifuged and the precipitate is retained, and washed with PBS. Finally, the washed apt-MNPs are dispersed into PBS buffer solution and stored.

[0026] Step 6, Construction of upconversion / magnetic nanoparticle fluorescence biosensor system: The aptamer-modified magnetic nanoparticles and complementary strand-modified upconversion nanoparticles are connected, and then thiamethoxam is added, and the fluorescence signal values before and after the addition of thiamethoxam are measured, and the fluorescence signal difference is recorded. With the logarithm of thiamethoxam concentration as the abscissa and the fluorescence signal difference as the ordinate, a standard curve is constructed.

[0027] Step 7, Verification of the specificity of the sensor system: Different types of pesticide compounds are selected to repeat the detection process, and the output fluorescence signal intensity is recorded.

[0028] Preferably, the mass ratio of yttrium chloride hexahydrate, gadolinium chloride hexahydrate, ytterbium chloride hexahydrate, and holmium chloride hexahydrate described in Step 1 is: 0.1164 g∶0.0892 g∶0.062 g∶0.0006 g, methanol∶oleic acid∶octadecene = 2 mL∶6 mL∶14 mL; the first heating temperature is: 150 - 170 °C, and the heating time is 30 min; the mass ratio of ammonium fluoride and sodium hydroxide is: 0.2944 g∶0.2 g, and the methanol is 20 mL; the two-stage water bath conditions are: 50 °C water bath for 40 min, 70 °C water bath for 30 min; the second heating temperature is 290 - 310 °C, and the heating time is 1 h; the oven temperature is 60 °C, and the time is 8 h.

[0029] Preferably, the ratio of alendronic acid: deionized water described in Step 2 is 50 mg: 6 mL, oil-soluble upconversion nanoparticles∶chloroform∶ethanol = 200 mg∶10 mL∶4 mL; the acidic condition is pH = 2 - 3, and the stirring time is 30 min; the oven temperature is 60 °C, and the time is 8 h.

[0030] Preferably, the ratio of water-soluble upconversion fluorescent nanoparticles: glutaraldehyde described in Step 3 is 4 mg: 0.7 mL, and the complementary strand is 30 μL (concentration 100 μM).

[0031] Preferably, in step 4, the sodium acetate anhydrous: 1,6 - hexanediamine: ferric chloride hexahydrate: ethylene glycol = 2 g: 6.4 g: 1.0 g: 45 mL. The first oven heating temperature is 198 °C and the time is 6 h; the second oven heating temperature is 60 °C and the time is 8 h.

[0032] Preferably, in step 5, the magnetic nanoparticles: PBS: glutaraldehyde solution = 4 mg: 2 mL: 1 mL; streptavidin is 0.5 mL with a concentration of 1 mg / mL; the aptamer is 40 μL (concentration 100 μM).

[0033] Preferably, in step 6, the up - conversion nanoparticles modified with complementary strand: magnetic nanoparticles modified with aptamer = 50 μL (concentration 2 mg / mL): 140 μL (concentration 2 mg / mL); the addition amount of thiamethoxam is 0 - 102.4 ng / mL.

[0034] Preferably, in step 7, except for thiamethoxam, the other pesticide compounds are 2,4 - D, dinotefuran, deltamethrin, clothianidin, acetamiprid, imidacloprid, and their concentrations are all 0.5, 2, 10 ng / mL.

[0035] The present invention discloses the following technical effects:

[0036] (1) The present invention discloses a method for monitoring the fluorescence signal of thiamethoxam in food. Based on up - conversion fluorescent nanoparticles and magnetic nanoparticles, a steady - state specific thiamethoxam detection system is constructed, and its detection principle diagram is as Figure 1 shown;

[0037] (2) The specific detection system constructed by the present invention shows a high selectivity for thiamethoxam, can eliminate the interference of other pesticide compounds, can achieve the specific detection of thiamethoxam residues in food, overcomes the deficiencies of traditional detection methods, and has important significance for ensuring food safety.

[0038] (3) The linear range of the concentration of thiamethoxam and the fluorescence signal intensity established by the present invention is 0.4 - 102.4 ng / mL, with a wide linear detection range. The detection limit LOD is 0.08 ng / mL, which can well meet the high - sensitivity detection of thiamethoxam residues in food and has good versatility.

[0039] (4) By constructing a specific thiamethoxam detection system, the present invention realizes the high - specificity and high - sensitivity detection of thiamethoxam residues in food, has a wide detection linear range and a low detection limit, so that this method has good practical prospects. Description of the Drawings

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0041] Figure 1 Transmission electron microscopy image of the water-soluble upconversion nanoparticles prepared in Example 1;

[0042] Figure 2 Transmission electron microscopy image of the aptamer-modified magnetic nanoparticles prepared in Example 1;

[0043] Figure 3 Fluorescence signal spectrum of the detection system at different thiamethoxam concentrations in Example 1;

[0044] Figure 4 Fluorescence detection standard curve at different thiamethoxam concentrations in Example 1;

[0045] Figure 5 Fluorescence signal intensity of the detection system with different pesticide compounds in Example 1. Detailed implementation manners

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0047] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0048] In the present invention, unless otherwise specified, the percentage content for solid-liquid mixtures and solid-solid mixtures refers to the mass percentage, and for liquid-liquid mixtures, it refers to the volume percentage.

[0049] In the present invention, unless otherwise specified, the percentage concentration refers to the final concentration. The final concentration refers to the proportion of the added component in the system after adding the component.

[0050] The temperature parameters in the present invention, unless otherwise specified, allow both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument.

[0051] The reagents, equipment, and consumables used in the present invention are all conventional reagents, equipment, and consumables, which are purchased from commercial companies. Unless otherwise specified, the experimental methods used in the present invention are all conventional experimental methods or the experimental methods recommended in the manufacturer's instructions.

[0052] Aptamer: A class of single-stranded DNA or RNA molecules obtained by in vitro selection techniques (such as SELEX), which can bind to target molecules (such as proteins, small molecules, or cell surface markers) with high specificity and high affinity through a specific three-dimensional structure. The thiamethoxam aptamer DNA sequence used in the present invention is 5'-biotin-TATGTTCTTAACTGGTCGTCCTGTGAGCCGATCACTAGATAATTAGGAT-3'.

[0053] Complementary strand: Refers to the two anti-parallel nucleotide strands formed by base complementary pairing (A-T, C-G) in the DNA double helix structure. During DNA replication, the separation of complementary strands is a key step to ensure the accurate transmission of genetic information. The complementary strand sequence used in the present invention is 5'-NH2-ATCCTAATTATCTAGTGATCGGCTCACAGGACGACCAGTTAAGAACATA-3'.

[0054] The aptamer or its complementary strand used in the following examples was purchased from Shanghai Sangon Biotech Co., Ltd.

[0055] The following further describes the specific embodiments of the present invention in detail with reference to the accompanying drawings of the specification.

[0056] Example 1:

[0057] The present invention discloses a method for detecting thiamethoxam upconversion fluorescence based on magnetic separation, and the specific steps are as follows:

[0058] Step 1, prepare oil-soluble upconversion nanoparticles:

[0059] Dissolve yttrium chloride hexahydrate (0.1164 g), gadolinium chloride hexahydrate (0.0892 g), and ytterbium chloride hexahydrate (0.062 g) in methanol (2 mL). Then mix the mixture with 6 mL of oleic acid and 14 mL of 1-octadecene. Under nitrogen protection, heat the mixture to 160 °C and then continuously stir magnetically for 30 min until completely dissolved. Then, add a methanol solution of sodium hydroxide (0.2 g) and ammonium fluoride (0.2944 g). Methanol is completely volatilized in a water bath at 50 °C for 30 min and then completely volatilized in a water bath at 70 °C. After completely removing methanol, quickly heat the mixture to 300 °C, react for 1 h, and then cool to room temperature. Wash the synthesized product (i.e., oil-soluble UCNPs) with a mixture of cyclohexane and ethanol and place it in an oven at 60 °C for 8 h.

[0060] Step 2, Prepare amino-modified water-soluble upconversion particles:

[0061] Accurately weigh 25.0 mg of alendronic acid and dissolve it in 3 mL of deionized water. Then add 50 mg of the oil-soluble upconversion particles prepared in Step 1, 5 mL of chloroform, and 2 mL of ethanol, and ultrasonically treat for 30 min. Adjust the pH value of the mixed solution to 2 - 3 with hydrochloric acid solution (1 mol / L) and react for 1 h under magnetic stirring. After the reaction, wash the synthesized product with a mixture of ethanol and water (concentration ratio: 3:1). After washing, bake the alendronic acid-modified UCNPs in an oven at 60 °C for 8 h. As Figure 1 shown, it can be seen from the transmission electron microscope image of the prepared water-soluble upconversion nanoparticles that the upconversion nanoparticles are ellipsoidal in shape, with a diameter of about 48 nm, and there is no obvious aggregation between the nanoparticles, indicating that the nanoparticles have good dispersibility in aqueous solution.

[0062] Step 3, Prepare complementary strand-modified upconversion particles:

[0063] Accurately weigh 4 mg of the upconversion particles prepared in Step 2, dissolve them in 2 mL of PBS buffer solution and ultrasonically treat for 30 minutes. Then add 0.6 mL of glutaraldehyde solution and react for 2 h under magnetic stirring. Add 30 μL of complementary strand cDNA with a concentration of 100 μM to the solution, and then place the reaction on a shaker at 37 °C overnight. Subsequently, centrifuge the solution (8000 rpm / min, 3 min), discard the supernatant, and wash 2 - 3 times with PBS buffer solution. Finally, disperse the washed cDNA-UCNPs into PBS buffer solution and store them in a refrigerator at 4 °C.

[0064] Step 4, Prepare amino-modified magnetic nanoparticles:

[0065] Weigh 2 g of anhydrous sodium acetate, 6.4 g of 1,6-hexanediamine and 1.0 g of ferric chloride hexahydrate and disperse them in 45 mL of ethylene glycol solution. Heat the solution in a water bath until it becomes a clear and transparent solution. Subsequently, transfer the solution to a polytetrafluoroethylene liner and bake it in an oven at 198 °C for 6 hours. After baking, cool it to room temperature, magnetically separate the black solid by an external magnetic field, and wash it with deionized water to obtain amino-modified magnetic nanoparticles. Bake the nanoparticles in an oven at 60 °C for 8 hours and store them in powder form.

[0066] Step 5, prepare aptamer-modified magnetic nanoparticles:

[0067] Weigh 4 mg of the magnetic nanoparticles obtained in Step 4, dissolve them in 2 mL of PBS and then sonicate. Then add 1 mL of glutaraldehyde solution and react under magnetic stirring. After the reaction, add 0.5 mL of streptavidin solution (concentration: 1 mg / mL) and react under magnetic stirring. Subsequently, add 40 μL of aptamer (100 μM) to the reacted solution and react overnight at room temperature. Then, magnetically separate the solution and retain the precipitate, and wash it with PBS. Finally, disperse the washed apt-MNPs in PBS buffer and store them in a refrigerator at 4 °C. As Figure 2 shown, it can be seen from the transmission electron microscope image of the aptamer-modified magnetic nanoparticles that the magnetic nanoparticles present irregular squares with a diameter of about 28 nm and no obvious aggregation.

[0068] Step 6, construct an upconversion / magnetic nanoparticle fluorescence biosensor system: Dilute the complementary strand-modified upconversion nanoparticles obtained in Step 3 and the aptamer-modified magnetic nanoparticles obtained in Step 5 to 2 mg / mL for standby; pipette 50 μL of the upconversion solution and 140 μL of the magnetic nanoparticle solution, react at room temperature for 30 min, and after the reaction, measure and record the fluorescence signal value. Subsequently, add standard thiamethoxam solutions with different concentrations (0 - 102.4 ng / mL) to the solution, react for 20 min, and then measure and record the fluorescence signal value of the solution.

[0069] Reaction system optimization:

[0070] First step, incubate the aptamer-modified magnetic nanoparticle solution with different addition amounts (100, 120, 140, 160, 180, 200 μL) with 50 μL of the complementary strand-modified upconversion solution for a sufficient long time. After incubation, measure the fluorescence signal value. The optimal addition amount of the aptamer-modified magnetic nanoparticle solution is obtained as 140 μL.

[0071] Step 2: According to the results obtained in Step 1, incubate 140 μL of the aptamer-modified magnetic nanoparticle solution and 50 μL of the complementary strand-modified upconversion solution for 5, 10, 20, 30, 40, and 50 min, and measure the fluorescence signal values at different incubation times. The optimal incubation time for the magnetic nanoparticle solution and the upconversion solution is 30 min.

[0072] Step 3: According to the results obtained in Step 2, add 210 μL of the thiamethoxam standard solution to the incubated solution, and react for 5, 10, 15, 20, 25, and 30 min. Measure the fluorescence signal values at different reaction times. The optimal reaction time for the above mixed solution and the thiamethoxam standard solution is 20 min.

[0073] Step 4: Construction of the detection standard curve for different concentrations of thiamethoxam: First, incubate 50 μL of the upconversion solution and 140 μL of the magnetic nanoparticle solution for 30 min. Then, add 210 μL of thiamethoxam standard solutions with concentrations of 0, 0.4, 1.6, 3.2, 6.4, 12.8, 25.6, 51.2, and 102.4 ng / mL respectively. After reacting for 20 min, measure the fluorescence values. The fluorescence signal value when the thiamethoxam concentration is 0 is denoted as I0, and the fluorescence signal values at other thiamethoxam concentrations are denoted as I1. Take the difference in fluorescence signals ΔI (ΔI = I0 - I1) before and after the presence of thiamethoxam as Y, and establish a standard curve with the logarithm of the thiamethoxam concentration as X, obtaining the thiamethoxam standard curve y = 8854.68x + 13322.89, R 2 = 0.9924. Calculate the detection limit as 0.08 ng / mL according to the formula LOD = 3S0 / K (S0 is the relative standard deviation of ten repeated blank experiments, and K is the slope of the standard curve). As Figure 3 shown, the figure is the fluorescence signal spectrogram of the detection system at different thiamethoxam concentrations. It can be seen from Figure 3 it that as the thiamethoxam concentration increases, the fluorescence intensity at 544 nm gradually decreases. This is because thiamethoxam will preferentially capture the aptamer, causing the upconversion nanoparticles to fall off the magnetic nanoparticle surface and the fluorescence intensity to decrease. Figure 4 In it, the abscissa is the logarithm of the thiamethoxam concentration, and the ordinate is the difference in fluorescence intensity before and after the addition of thiamethoxam. The linear regression equation of the standard curve is y = 8854.68x + 13322.89, R 2 = 0.9924. The detection limit (LOD, F0 - 3SD) and the detection range are 0.08 ng / mL and 0.4 to 102.4 ng / mL respectively.

[0074] Step 5, verifying the specificity of the system: To evaluate the specificity and anti-interference ability of the fluorescence biosensor system based on upconversion / magnetic nanoparticles designed in this example, the system was applied to the detection of other pesticide compounds (2,4-D, dinotefuran, deltamethrin, imidaclothiz, acetamiprid, imidacloprid), see Figure 5 . The results showed that only when thiamethoxam was added to the system did the fluorescence signal value change significantly, and the addition of other pesticide compounds did not cause a significant change in the fluorescence signal of the system. Thus, it can be seen that the constructed sensor system has high specificity and high selectivity for thiamethoxam.

Claims

1. A thiamethoxam upconversion fluorescence detection method based on magnetic separation, characterized in that The following steps are involved: (i) reacting alendronic acid with oil-soluble upconversion nanoparticles to prepare water-soluble upconversion nanoparticles; (ii) adding the water-soluble upconversion nanoparticles to a glutaraldehyde solution, stirring, and then adding a complementary chain to obtain complementary chain-modified upconversion fluorescent nanoparticles; (iii) adding the amino-modified magnetic nanoparticles to the glutaraldehyde solution, stirring, adding the streptavidin solution and the aptamer to react, and then separating through an external magnetic field and retaining the precipitate to obtain the aptamer-modified magnetic nanoparticles; (iv) mixing the aptamer-modified magnetic nanoparticles and the complementary chain-modified upconversion fluorescent nanoparticles to carry out a ligation reaction to obtain a detection solution, then preparing thiamethoxam solutions of different concentrations, adding the thiamethoxam solutions of different concentrations to the detection solutions, respectively, measuring the fluorescence signal values ​​of the thiamethoxam solutions of different concentrations and the fluorescence signal values ​​of the solution without the addition of the thiamethoxam solution, and establishing a standard curve of the linear relationship between the thiamethoxam solution concentration and the fluorescence intensity based on the measured fluorescence intensity.

2. The detection method according to claim 1, characterized in that: The preparation method of the oil-soluble up-conversion nanoparticles comprises: dissolving yttrium chloride hexahydrate, gadolinium chloride hexahydrate, ytterbium chloride hexahydrate and holmium chloride hexahydrate in a methanol solution, then adding oleic acid and 1-octadecene to mix, and continuously stirring under nitrogen protection until the mixture is completely dissolved, adding a methanol solution of sodium hydroxide and ammonium fluoride, heating until the methanol is completely removed, rapidly heating to 300° C. to form a crystalline phase, then cooling to room temperature, and washing to obtain the oil-soluble up-conversion nanoparticles.

3. The detection method according to claim 1, characterized in that: The preparation method of the water-soluble upconversion nanoparticles further comprises: dissolving alendronic acid and the oil-soluble upconversion particles in deionized water and chloroform respectively, mixing the two, adding ethanol, ultrasonically treating until completely dissolved, adjusting the pH to 2-3 with a hydrochloric acid solution, stirring until the reaction is completed, and washing with a mixed solution of ethanol and water to obtain the water-soluble upconversion nanoparticles.

4. The detection method according to claim 1, characterized in that: The preparation method of the complementary chain modified upconversion fluorescent nanoparticles further comprises: adding the water-soluble upconversion nanoparticles into a phosphate buffer solution and stirring evenly, then adding a glutaraldehyde solution, stirring until the reaction is complete, then adding the complementary chain, reacting at room temperature, centrifuging after the reaction is complete, and retaining the precipitate to obtain the complementary chain modified upconversion fluorescent nanoparticles.

5. The detection method according to claim 1, characterized in that: The preparation method of the amino-modified magnetic nanoparticles comprises: dissolving anhydrous sodium acetate, 1,6-hexanediamine and ferric chloride hexahydrate in an ethylene glycol solution, heating to a clear transparent solution, baking, cooling to room temperature, and magnetically separating through an external magnetic field to obtain amino-modified magnetic nanoparticles.

6. The detection method according to claim 1, characterized in that: The preparation method of the aptamer-modified magnetic nanoparticles further comprises: adding the amino-modified magnetic nanoparticles to a phosphate buffer solution and stirring evenly, then adding a glutaraldehyde solution, stirring until the reaction is complete, adding a streptavidin solution, and then adding the aptamer to react, and after the reaction is complete, separating and retaining the precipitate through an external magnetic field to obtain the aptamer-modified magnetic nanoparticles.

7. The detection method according to claim 2, characterized in that: In the preparation method of the oil-soluble upconversion nanoparticles, the mass ratio of yttrium chloride hexahydrate, gadolinium chloride hexahydrate, ytterbium chloride hexahydrate and holmium chloride hexahydrate is 0.1164:0.0892:0.062:0.0006, the volume ratio of methanol:oleic acid:octadecene is 2:6:14; the mass ratio of ammonium fluoride and sodium hydroxide is 0.2944:0.

2.

8. The detection method according to claim 3, characterized in that: The mass volume ratio of alendronic acid:deionized water is 50 mg:6 mL, and the mass volume ratio of oil-soluble up-conversion particles:trichloromethane:ethanol is 200:10:

4.

9. The detection method according to claim 5, characterized in that: The mass volume ratio of anhydrous sodium acetate: 1,6-hexanediamine: ferric chloride hexahydrate: ethylene glycol is 2:6.4:1.0:

45.

10. The detection method according to claim 1, characterized in that: The mass volume ratio of the water-soluble upconversion nanoparticles:glutaraldehyde is 4:0.7, and the mass volume ratio of the amino-modified magnetic nanoparticles:glutaraldehyde solution is 4:1.

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