Liposome nanovesicle-based elisa method and application

Abstract

This invention discloses an ELISA method and its application based on liposome nanovesicles, belonging to the field of biotechnology. The invention prepares HRP-coated and biotinylated liposome nanovesicles using phospholipids, sterol derivatives, and biotinylated polyethylene glycol phospholipids as raw materials. These nanovesicles are highly sensitive to acidic environments of pH 3-6, specifically recognizing streptavidin. Under acidic conditions, they cleave to release HRP and catalyze substrate color development. Using these nanovesicles as tracers, an ELISA is established based on the principle of antigen-antibody competitive binding to achieve the detection of thiamethoxam residues, a neonicotinoid insecticide. The method of this invention has a detection limit as low as 0.24 µg / L for thiamethoxam, exhibiting high sensitivity, simple operation, economy, speed, and high-throughput screening capabilities. It is suitable for the detection of thiamethoxam residues in agricultural products, soil, and water bodies, solving the technical problem of low sensitivity in traditional ELISA methods.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an ELISA method and its application based on liposome nanovesicles. Background Technology

[0002] Thiamethoxam is a neonicotinoid insecticide widely used to control pests such as aphids, thrips, and leafhoppers, but it has attracted global attention due to its ecotoxicity. Increasing evidence suggests it can adversely affect non-target organisms, especially pollinators like bees. To address the impact of thiamethoxam on bees, the European Union (EU) banned its use in outdoor agriculture in 2018, and the US Environmental Protection Agency (EPA) requires it to be used only for foliar or seed treatment. Meanwhile, many countries have established maximum residue limits (MRLs) for thiamethoxam. For example, China's GB2763-2026 sets the MRLs for vegetables at 0.01-2 mg / kg and for fruits at 0.05-5 mg / kg, while the US sets MRLs for vegetables at 0.06-3 mg / kg and for fruits at 0.05-8 mg / kg. Despite these regulations, thiamethoxam residues continue to be detected in groundwater, agricultural soils, and among bees. Therefore, developing a sensitive and accurate rapid detection method for thiamethoxam to achieve high-throughput screening is of great significance.

[0003] Currently, the detection methods for thiamethoxam can be divided into two main categories: chromatography and rapid detection methods. Chromatography includes gas chromatography, gas chromatography-tandem mass spectrometry, liquid chromatography, and liquid chromatography-tandem mass spectrometry, which are currently the most authoritative pesticide detection methods. However, these methods are expensive, time-consuming, labor-intensive, highly technical, and have limited accessibility. Rapid detection methods, such as ELISA (Enzyme-Linked Immunosorbent Assay), can achieve real-time, rapid, accurate, and high-throughput analysis of analytes on-site. They can complement instrument detection methods for different detection needs. However, their widespread application is limited by two fundamental factors: (1) the immobilization process of enzymes and antibodies may reduce the efficiency of the immune reaction due to potential steric hindrance and biomolecular denaturation; (2) the inherent 1:1 molar ratio between enzymes and antibodies limits the amplification of the catalytic signal, which poses a serious challenge to the trace detection of pollutants. Therefore, it is necessary to improve the traditional ELISA to enhance the detection performance of the immunoassay method. Summary of the Invention

[0004] The purpose of this invention is to overcome the low sensitivity of traditional ELISA in the prior art and to provide an ELISA method and its application based on liposome nanovesicles. At the same time, it provides a method for preparing the liposome nanovesicles used in this method and the application of the nanovesicles and analytical method in pesticide residue detection, so as to achieve high sensitivity, rapid and high throughput detection of neonicotinoid insecticides, especially thiamethoxam, and provide a new technical means for ensuring the quality and safety of agricultural products.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A liposome-based ELISA method uses biotin-labeled liposomes coated with horseradish peroxidase (HRP) as tracers to detect pesticide residues based on the principle of antigen-antibody competitive binding. The liposome nanovesicles are composed of phospholipids, sterol derivatives, and biotinylated polyethylene glycol phospholipids forming a bilayer structure. They are highly sensitive to acidic environments of pH 3-6. The biotin on the surface can specifically recognize streptavidin. Under acidic conditions, they cleave to release HRP and catalyze the color development of the substrate. The pesticide is a neonicotinoid insecticide.

[0006] In a further embodiment, the neonicotinoid insecticide is thiamethoxam; the phospholipid is 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine; the sterol derivative is cholesterol succinate monoester; and the biotinylated polyethylene glycol phospholipid is phosphatidylethanolamine-polyethylene glycol 2000-biotin, with a molar ratio of 70:20:10.

[0007] In a further embodiment, the preparation steps of the liposome nanovesicles are as follows: S1: Phospholipids, sterol derivatives, and biotinylated polyethylene glycol phospholipids are dissolved in chloroform, and the solvent is removed by vacuum evaporation to form a lipid membrane; S2: Ultrapure water containing HRP is added to the lipid membrane to hydrate it; S3: The mixture is sonicated until turbid, and biotin-labeled HRP-coated liposomes are extruded through a polycarbonate membrane; S4: Uncoated HRP is removed by dialysis with a polycarbonate membrane, using phosphate buffer as the dialysate; S5: The liposome nanovesicle powder is freeze-dried and stored at −20 °C for later use.

[0008] In a further embodiment, the amount of chloroform used in step S1 is 3 mL; the amount of ultrapure water used in step S2 is 20 mL, of which the mass of HRP is 25 mg; the pore size of the polycarbonate membrane in step S3 is 100 nm; and the pore size of the polycarbonate membrane in step S4 is 30 nm.

[0009] Further solutions, including specific testing procedures, include: Coating and washing: Dilute the pesticide coating antigen with 50 mM sodium carbonate buffer, add 100 μL to each well of a 96-well microplate, incubate for 2 hours, and then wash 5 times with PBS containing 0.05% Tween 20. Blocking and incubation: Add 200 μL of 5% skim milk powder solution to the microplate to block non-specific binding sites for 2 hours. After washing, add 50 μL of pesticide standard solution / sample pretreatment solution and 50 μL of biotinylated pesticide monoclonal antibody to each well in sequence, and incubate for 1 hour. To bind the tracer: Add 100 μL of 2.5 μg / mL streptavidin solution and incubate for 30 min, then add 100 μL of the liposome nanovesicles at 1 mg / mL and continue incubating for 30 min. Colorimetric reaction: Adding TMB substrate solution causes liposome nanovesicles to lyse and release HRP, which catalyzes the color development of the substrate; Termination and detection: Add 50 μL / well 2 M sulfuric acid solution to terminate the enzymatic reaction, measure the optical density at 450 nm, and determine the pesticide residue content in the sample based on the optical density value.

[0010] Further optimization of the detection system was achieved using the following experimental conditions: Na⁺ concentration of 0.14 M and acetonitrile volume fraction of 5%; the detection limit for thiamethoxam using this method was 0.24 μg / L.

[0011] In a further embodiment, the sample is an agricultural product, and the pretreatment steps for the agricultural product are as follows: the agricultural product is homogenized, 10 mL of acetonitrile is added to 10 g of sample as the extraction solvent, shaken for 5 min, sonicated for 10 min, allowed to stand for 5 min, centrifuged at 4000 rpm for 5 min, and the supernatant is taken and diluted to obtain the sample pretreatment solution.

[0012] In a further embodiment, liposome nanovesicles are formed by a bilayer stable structure of 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine, cholesterol succinate monoester, phosphatidylethanolamine-polyethylene glycol 2000-biotin in a molar ratio of 70:20:10, with HRP encapsulated internally and biotin-labeled on the surface. These nanovesicles can specifically recognize streptavidin and can cleave and release the internal HRP under acidic conditions of pH 3-6. The released HRP can catalyze the color development of the TMB-H2O2 substrate.

[0013] The above-mentioned liposome nanovesicles are used in the detection of pesticide residues, wherein the pesticide is the neonicotinoid insecticide thiamethoxam, and the application scenario is the high-throughput detection of thiamethoxam residues in agricultural products, soil or water.

[0014] In a further embodiment, the agricultural product is a fruit or vegetable product, and the liposome nanovesicles are used as tracers during detection, and the above-mentioned ELISA method is used to detect thiamethoxam residue.

[0015] The present invention has the following beneficial effects: The ELISA method based on liposome nanovesicles and related liposome nanovesicles involved in this invention significantly improves detection sensitivity, with a detection limit for thiamethoxam as low as 0.24 μg / L, effectively solving the core problem of the 1:1 molar ratio of enzyme to antibody limiting catalytic signal amplification in traditional ELISA methods; the biotin on the surface of the liposome nanovesicles can specifically recognize streptavidin, and it only cleaves and releases HRP under acidic conditions of pH 3-6 to catalyze substrate color development, resulting in excellent detection specificity; This method requires no expensive or sophisticated instruments, is simple to operate, economical, and fast, and can achieve real-time on-site detection and high-throughput screening. The reagents are low-cost and easy to distribute. The related liposome nanovesicles are freeze-dried into powder and stored at −20 °C, which is convenient for storage and transportation and has good stability. At the same time, the nanovesicles and analytical methods of this invention have a wide range of applications. They can accurately detect thiamethoxam residues in agricultural products, soil, and water. In actual agricultural product testing, the recovery rate meets the pesticide residue detection standards, and the accuracy is high. It can effectively complement instrumental detection methods such as chromatography and has broad application prospects in the fields of agricultural product quality and safety testing and agricultural environmental monitoring. Attached Figure Description

[0016] Figure 1 Schematic diagram of liposome nanovesicles; Figure 2 Transmission electron microscopy (TEM) images of liposome nanovesicles (A) and catalytic activity identification (B). Figure 3 Optimization of ELISA conditions: streptavidin and liposome nanovesicles (AB), salt particle concentration, and acetonitrile content (CD); Figure 4 This is a schematic diagram illustrating the ELISA detection principle based on liposome nanovesicles. Figure 5 The standard curve for liposome-based ELISA detection of thiamethoxam. Detailed Implementation

[0017] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. Example 1: Preparation of liposome nanovesicles

[0018] ① Dissolve 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine, cholesterol succinate monoester, phosphatidylethanolamine-polyethylene glycol 2000-biotin in 3 mL of chloroform in a molar ratio of 70:20:10. Then evaporate the solvent under vacuum to form a thin lipid film.

[0019] ② Add 20 mL of ultrapure water containing 25 mg HRP to the above lipid membrane to hydrate the membrane.

[0020] ③ The mixture was sonicated until turbid, and biotin-labeled HRP-coated liposomes were generated using a micro extruder with a 100 nm polycarbonate membrane.

[0021] ④ Remove unencapsulated HRP by dialysis using a 30 nm polycarbonate membrane. The dialysis buffer is phosphate buffer.

[0022] ⑤ Freeze-drying yields biotin-labeled HRP-coated liposome nanovesicle powder, as shown in the schematic diagram below. Figure 1 As shown, store at −20℃ for later use. Example 2: Identification of the structure and catalytic activity of liposome nanovesicles

[0023] Its structure was identified using transmission electron microscopy, such as... Figure 2 As shown in Figure A, the liposome nanovesicles have a uniform structure with an average particle size of 127 ± 14 nm, exhibiting a quasi-spherical morphology and a thin shell.

[0024] like Figure 2 As shown in Figure B, under acidic conditions, the optical density of the TMB solution (curve a), the TMB solution containing H₂O₂ (curve b), the TMB solution with liposome nanovesicles (curve c), and the solution containing liposome nanovesicles and H₂O₂ (curve d) remained unchanged. However, when liposome nanovesicles were mixed with TMB solution and H₂O₂ (curve e), a significant color change from colorless to blue was observed, demonstrating the specificity of liposome nanovesicles for substrate catalysis under acidic conditions. Example 3: Screening of ELISA experimental conditions based on liposome nanovesicles

[0025] Signal-to-noise ratio and immunoassay sensitivity were used as screening criteria for optimizing experimental conditions, including streptavidin concentration, liposome nanovesicle dosage, salt particle concentration, and acetonitrile content. Figure 3 As shown, 2.5 µg / mL streptavidin and 1 mg / mL liposome nanovesicles yielded a higher signal-to-noise ratio; therefore, 2.5 µg / mL streptavidin and 1 mg / mL liposome nanovesicles were determined to be the optimal dosage in the experiment. Additionally, 0.14 M Na... + Higher sensitivity can be achieved with 5% acetonitrile, therefore 0.14 M Na was determined in the experiment. + The optimal dosage is 5% acetonitrile.

[0026] Example 4: The operation steps of ELISA based on liposome nanovesicles are as follows: Taking thiamethoxam as an example, the thiamethoxam-coated antigen was diluted to 0.16 µg / mL with 50 mM sodium carbonate buffer, and 100 µL was added to each well of a 96-well ELISA plate. After incubation for 2 hours, the plate was washed 5 times with PBS containing 0.05% Tween 20. To block non-specific binding sites in the ELISA plate, the plate was blocked with 200 µL of 5% skim milk powder solution (m / v) for 2 hours. After washing 5 times, 50 µL of thiamethoxam standard solution or sample pretreatment solution and 50 µL of 0.63 µg / mL biotinylated thiamethoxam monoclonal antibody were added to each well, and the plate was incubated for 1 hour. Subsequently, 100 µL of SA solution was added and the plate was incubated for 30 min. Then, 100 µL of 1 mg / mL biotinylated HRP-coated liposome nanovesicles were added and the plate was incubated for another 30 min. TMB substrate solution was added to lyse the liposomes, releasing HRP and catalyzing the substrate color development. The enzymatic reaction was terminated by adding 50 µL of 2 M H₂SO₄, and the optical density (OD) was measured at 450 nm. 450 ).

[0027] Example 5: Sensitivity of ELISA based on liposome nanovesicles A 1000 mg / L thiamethoxam standard solution was serially diluted with buffer solution to a concentration gradient of 250–0.05 µg / L. The buffer solution was used as a negative control. The relationship between absorbance and thiamethoxam concentration is as follows: Figure 5 As shown, with a 10% inhibition concentration as the detection limit, the calculated value is 0.24 µg / L.

[0028] Example 6: ELISA based on liposome nanovesicles for the detection of thiamethoxam in samples, the steps of which are as follows: Taking cowpea samples as an example, the cowpeas were homogenized using a high-speed blender. 10 g of the homogenate was accurately weighed into a 50 mL centrifuge tube, and 10 mL of acetonitrile was added. The mixture was shaken for 5 min, sonicated for 10 min, allowed to stand for 5 min, and then centrifuged at 4000 rpm for 5 min. The supernatant was diluted and the detection was performed according to the steps in Example 4. Recovery experiments with actual samples confirmed the high accuracy of this method.

[0029] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An ELISA method based on liposome nanovesicles, characterized in that, Using HRP-coated and biotin-labeled liposome nanovesicles as tracers, pesticide residue detection is achieved based on the principle of antigen-antibody competitive binding; The liposome nanovesicles are composed of phospholipids, sterol derivatives, and biotinylated polyethylene glycol phospholipids forming a bilayer structure. They are highly sensitive to acidic environments of pH 3-6. The biotin on the surface can specifically recognize streptavidin. Under acidic conditions, they cleave to release HRP and catalyze the color development of the substrate. The pesticide is a neonicotinoid insecticide.

2. The method according to claim 1, characterized in that, The neonicotinoid insecticide is thiamethoxam; the phospholipid is 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine; the sterol derivative is cholesterol succinate monoester; the biotinylated polyethylene glycol phospholipid is phosphatidylethanolamine-polyethylene glycol 2000-biotin; the molar ratio of the above three substances is 70:20:

10.

3. The method according to claim 1, characterized in that, The preparation steps of the liposome nanovesicles are as follows: S1: Phospholipids, sterol derivatives, and biotinylated polyethylene glycol phospholipids are dissolved in chloroform, and the solvent is removed by vacuum evaporation to form a lipid film; S2: Add ultrapure water containing HRP to the lipid membrane to hydrate the membrane; S3: The mixture is sonicated until turbid, and then extruded through a polycarbonate membrane to obtain biotin-labeled HRP-coated liposomes; S4: Remove unencapsulated HRP by dialysis using a polycarbonate membrane; the dialysate is phosphate buffer. S5: The liposome nanovesicle powder was obtained by freeze-drying and stored at −20 °C for later use.

4. The method according to claim 3, characterized in that, In step S1, the amount of chloroform used is 3 mL; in step S2, the amount of ultrapure water used is 20 mL, of which the mass of HRP is 25 mg; in step S3, the pore size of the polycarbonate membrane is 100 nm, and in step S4, the pore size of the polycarbonate membrane is 30 nm.

5. The method according to claim 1, characterized in that, The specific steps for pesticide residue testing include: Coating and washing: Dilute the pesticide coating antigen with 50 mM sodium carbonate buffer, add 100 μL to each well of a 96-well microplate, incubate for 2 hours, and then wash 5 times with PBS containing 0.05% Tween 20. Blocking and incubation: Add 200 μL of 5% skim milk powder solution to the microplate to block non-specific binding sites for 2 hours. After washing, add 50 μL of pesticide standard solution / sample pretreatment solution and 50 μL of biotinylated pesticide monoclonal antibody to each well in sequence, and incubate for 1 hour. To bind the tracer: Add 100 μL of 2.5 μg / mL streptavidin solution and incubate for 30 min, then add 100 μL of 1 mg / mL of the liposome nanovesicles and continue incubating for 30 min. Colorimetric reaction: Adding tetramethylbenzidine substrate solution causes liposome nanovesicles to lyse and release HRP, catalyzing the color development of the substrate; Termination and detection: Add 50 μL / well 2 M sulfuric acid solution to terminate the enzymatic reaction, measure the optical density at 450 nm, and determine the pesticide residue content in the sample based on the optical density value.

6. The method according to claim 5, characterized in that, The optimized experimental conditions for the detection system are: Na⁺ concentration 0.14M, acetonitrile volume fraction 5%.

7. The method according to claim 5, characterized in that, The sample is an agricultural product. The pretreatment steps for the agricultural product are as follows: the agricultural product is homogenized, 10 mL of acetonitrile is added to 10 g of sample, shaken for 5 min, sonicated for 10 min, and then allowed to stand for 5 min. After centrifugation at 4000 rpm for 5 min, the supernatant is taken and diluted to obtain the sample pretreatment solution.

8. The method according to any one of claims 1 to 7, characterized in that, The liposome nanovesicles are composed of a stable bilayer structure formed by 1,2-dioleoyl-SN-glycerol-3-phosphoethanolamine, cholesterol succinate monoester, phosphatidylethanolamine-polyethylene glycol 2000-biotin in a molar ratio of 70:20:

10. The nanovesicles are coated with HRP and have biotin labeling on the surface. These nanovesicles can specifically recognize streptavidin and can be cleaved and release the internal HRP under acidic conditions of pH 3-6. The released HRP can catalyze the color development of the TMB-H2O2 substrate.

9. The application of the method according to any one of claims 1-8 in pesticide residue detection.

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